environment interactions (ibempa)

Transcripción

II IBEROAMERICAN CONFERENCE ON
BENEFICIAL PLANT - MICROORGANISM
- ENVIRONMENT INTERACTIONS
(IBEMPA)
XIV NATIONAL MEETING OF THE SPANISH
SOCIETY OF NITROGEN FIXATION (SEFIN)
XXVI LATIN AMERICAN MEETING ON
RHIZOBIOLOGY (ALAR)
III SPANISH-PORTUGUESE CONGRESS ON
NITROGEN FIXATION
“Microorganisms for future agriculture"
Organizers
Spanish Society of Nitrogen Fixation (SEFIN)
Latin American Society Rhizobiology (ALAR)
University of Seville
ISBN-10: 84-616-6794-8
ISBN-13: 978-84-616-6794-9
Sponsors
AMC Chemical
Bruker
Catering Sevilla
Coitand
C. Viral SL
Kimitec
Laboratorios Biagro
Metabolon
OpGen
ResBioAgro
Sistemas genómicos
Symborg
Thermo Fisher Scientific
Welcome address
Dear colleagues,
On behalf of the Organizing Committee for II IBEMPA Meeting, it is our great pleasure and
honour to welcome you to the II Iberoamerican Conference on Beneficial Plant Microorganism
Environment Interactions (IBEMPA), XIV National Meeting of the Spanish Society of Nitrogen
Fixation (SEFIN), XXVI Latin American Meeting on Rhizobiology (ALAR) and III SpanishPortuguese Congress on Nitrogen Fixation.
This meeting will provide the opportunity to share and discuss latest scientific news on
genomic, biochemical, ecological and agronomic aspects of diazotrofic microorganisms or
beneficial bacteria and their interaction with plants. We hope you agree that the selected
topics and speakers are of great interest and exceptional quality.
The meeting program includes six sessions plus the opening and closing keynote conferences.
Each session includes keynotes speakers, oral communications and poster presentations.
Before the beginning and in parallel to the congress three satellite workshops and a meeting
have also been organized: “Omic-technologies Workshop” and “Workshop FABATROPIMED”
(1st September), “Inoculant production Workshop” (2nd September) and the meeting on
"Microorganisms for Agriculture Future" (4th September). We encourage all delegates to
participate actively on all these activities. Besides, several attractive social activities have been
programmed.
We would like to thank all institutions and companies that contributed to the organization of
this congress, very specially to the University of Sevilla, the Spanish Society of Nitrogen
Fixation and the Latin American Society of Rhizobiology.
We hope that all delegates will enjoy during their stay in Sevilla and will found an excellent
atmosphere to discuss about scientific matters, new collaborations and future directions in the
field of the beneficial microorganisms and their association with plants. We also hope that you
discover the hospitality of the people of Sevilla and have some free time for visiting this
beautiful city. We look forward to welcome you in Seville in September 2013, for what we
hope will be an enjoyable meeting both scientifically and socially.
Yours sincerely,
Manuel Megías
President of the Organizing Committee
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Committees
Honor Committee
S.A.R. la Infanta Doña Elena.
Presidente del Comité de Honor.
Excmo. Sr. D. Miguel Arias Cañete.
Ministerio de Agricultura, Alimentación y
Medioambiente.
Excma. Sra. Dª Carmen Vela Olmo.
Ministerio de Economía y Competitividad.
Secretaría de Estado de Investigación Desarrollo
e Innovación
Excmo. Sr. D. Luis Planas Puchades.
Consejería Agricultura, Pesca y Medioambiente.
Excmo. Sr. D. Antonio Ávila Cano.
Consejería de Economía, Innovación, Ciencia y
Empleo.
Excmo. Sr. D. Juan Ignacio Zoido Álvarez.
Alcaldía de Sevilla.
Excmo. Sr. D. Fernando Rodríguez Villalobos.
Diputación de Sevilla.
Excmo. y Mgco. Sr. D. Antonio Ramírez de Arellano
López.
Universidad de Sevilla.
Excmo. y Mgco. Sr. D. Juan Manuel Suárez Japón.
UNIA.
Sra. Dª Teresa Millán Valenzuela.
Asociación Española de Leguminosas.
Ilmo. Sr. D. José Luís García Palacios.
Caja Rural del Sur.
Ilmo. Sr. D. Antonio Vergel Román.
COITAND.
Sr. D. Joaquín Moya-Angeler Cabrera.
CTA.
Ilma. Sra. Dª María Cinta Castillo Jiménez.
Doñana21.
Sr. D. Francisco Casero Rodríguez.
Valor Ecológico.
Organizing Committee
President:
Dr. Manuel Megías.
Universidad de Sevilla. España.
Vice presidents:
Dr. Eulogio Bedmar
(President of SEFIN).
Estación Experimental del Zaidín. CSIC. España.
Dr. Alicia Arias
(President of ALAR).
Instituto de Investigaciones Biológicas Clemente
Estable (IIBCE), Uruguay.
Dr. Francisco Merchán.
Dr. María Jesús Delgado.
Dr. Dulce Nombre Rodríguez.
IFAPA. Las Torres-Tomejil. España.
Dr. F. Javier Ollero.
Dr. Teresa Cubo.
Treasurers:
Secretary:
Web-Master:
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Scientific Committee
Dr. José E. Ruíz.
Dr. M. Rosario Espuny.
Dr. Eduardo Leidi.
Dr. Federico Sánchez.
Dr. Yaakov Okon.
Dr. Esperanza Martínez.
Dr. Doris Zúñiga.
Dr. Emanuel de Souza.
Dr. Marcia Toro.
Dr. Eduardo Ortega.
Dr. Maribel Parada.
Dr. Fabio L. Olivares.
Dr. Elena Beyhaut.
Dr. Rafael Rivilla.
Dr. Isabel V. Castro.
Dr. María J. Soto.
Dr. Raúl Rivas.
Dr. Milagros León.
Dr. Nuria Ferrol.
Dr. Manuel Becana.
Dr. Fernando González.
Dr. José Antonio Herrera.
Dr. José Antonio Lucas.
Dr. Eloisa Pajuelo.
Dr. Pedro F. Mateo.
Instituto de Recursos Naturales y Agrobiología. CSIC. España.
Instituto de Biotecnología. UNAM. Mexico.
The Hebrew University. Jerusalem. Israel.
Centro de Ciencias Genómicas. UNAM. México.
Universidad Nacional Agraria La Molina. Perú.
Universidad Federal de Parana. Brasil.
Universidad Central de Venezuela. Venezuela.
Universidad de la Habana. Cuba.
Universidad de la Frontera. Chile.
Universidad Estatal del Norte Fluminense. Brasil.
INIA. Uruguay.
Universidad Autónoma de Madrid. España.
Instituto Nacional de Investigaciones Biológicas. Portugal.
Universidad de Salamanca. España.
Universidad de la Laguna. España.
Estación Experimental de Aula Dei. CSIC. España.
Universidad de León. España.
Universidad de Granada. España.
Universidad San Pablo CEU. España.
Universidad de Sevilla y IFAPA-Las Torres-Tomejil. España.
Universidad de Salamanca. España.
Honor Scientific Committee
Dr. José Olivares.
Dr. Claudino Rodríguez-Barrueco.
Dr. M. Rosario de Felipe.
Dr. José M. Barea.
Dr. Eugenio M. Ferreira.
Dr. Roberto Racca.
Dr. Alicias Godeas.
Dr. Lillian Frioni.
Dr. Carlos Labandera.
Dr. Fabio Pedrosa.
Dr. María Valdés.
Dr. Eduardo Schroeder.
Dr. Pedro Balatti.
Dr. Gabriel Faveluques.
Dr. João Ruy Jardim Freire.
Dr. Avilio Antonio Franco.
España.
España.
España.
España.
Portugal.
Argentina.
Argentina.
Uruguay.
Uruguay.
Brasil.
Mexico.
Puerto rico.
Argentina.
Argentina.
Brasil.
Brasil.
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Local Organizing Committee
Dr. Ignacio Rodríguez.
Dr. Pilar Tejero.
Dr. Ramón Bellogín.
Dr. Ana Buendía.
Dr. José María Vinardell.
Dr. Miguel Ángel Rodríguez-Carvajal.
Dr. Francisco Javier López-Baena.
Dr. Miguel Ángel Caviedes.
Dr. María Camacho.
IFAPA-Las Torres-Tomejil. España.
Dr. Carolina Sousa.
Dr. M. Carmen Márquez.
Omic-Tecnologies Workshop Organizing Committee
Dr. Antonio M. Gil.
Dr. Francisco Martínez.
Dr. O. Mario Aguilar.
Universidad Nacional de La Plata. Argentina.
Inoculant Production Workshop Organizing Committee
Dr. Francisco Temprano.
IFAPA-Las Torres-Tomejil. España.
Dr. Enrique Moretti.
Laboratorios Biagro. Argentina.
Dr. Mohammed Dary.
ResBioAgro, SL. España.
Dr. Antonio Lagares.
BIOFAG. Universidad Nacional de La Plata. Argentina.
Dr. Mariangela Hungria.
EMBRAPA. Brasil.
Dr. Juan Sanjuán.
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General information
Venue
The meeting will be held in the Lecture Theatre of the Facultades de Ciencias del Trabajo y
Derecho of the University of Seville (http://www.derecho.us.es) and Hotel NH Central
Convenciones
(http://www.nh-hoteles.es/nh/es/hoteles/espana/sevilla/nh-centralconvenciones.html)
Language
Spanish and Portuguese are the official languages of the congress. English is the scientific
language.
Registration and information desk
The registration will be held on 2 September from 11:00 to 18:00 h. and during the meeting
period at the desk located on the convention hall of the hotel. Information about the congress,
social activities, etc. can also be obtained at the registration desk during the meeting. The
official travel agency of the congress, CAC Travel ([email protected]), will be
available at the registration desk for accommodation, touristic and travel information, etc.
Lunch and coffee-breaks
The lunch on 3, 5 and 6 September will be held at the hotel restaurant. These lunchs and
coffee-breaks are included on the registration fee for meeting participants and accompanying
persons.
Oral presentations
The keynote speakers have 20 minutes for their presentations plus 5 minutes for questions (25
minutes total), while the offered oral communications will be presented during 15 minutes
plus 5 minutes for questions (20 minutes total). We encourage speakers to prepare their
presentations according to the time assigned. Session Chairs have been asked to ensure that
speakers restrict themselves to the allowed time; they have been advised to adhere most
strictly to this policy. The meeting rooms are prepared with a PC (Powerpoint 2010) and
projector. Macintosh is not available. Please note that we are unable to allow the use of
personal laptops. Please bring your presentation in a memory stick (using the USB port in the
computer) and load it on the meeting computer. Congress staff will be prepared to assist you.
All presentations must be loaded preferably on Monday 2 September.
Poster sessions
Poster boards are located at Salón Marbella and Salón Málaga on the convention area of the
hotel. Posters will be on display throughout Tuesday morning (3 September) up to late
Thursday evening (5 September) with snacks and drinks. The dimensions of the poster are: 130
cm (height) by 90 cm (width). The organization will provide the materials necessary for
mounting the posters on the boards. First authors of posters from Sessions I, V y VI should be
present beside their own panel during the Poster Session on Tuesday 3 September (from 20:30
v
to 22:00 h). First authors of posters from Sessions II, III and IV should be present beside their
own panel during the Poster Session on Thursday 5 September (from 20:30 to 22:00 h).
Poster discussion sessions will take place at the Salones Almenara, Almería and Alanda on the
convention area of the hotel from 18:00 to 19:30 h, on Tuesday 3 September (Poster Sessions
I, V y VI) and Thursday 5 September (Poster Sessions II, III and IV).
Social events
The social program includes:
Musical performance at 19:40 h, on Monday 2 September following the opening ceremony
and opening conference at the Lecture Theatre of the Facultades de Ciencias del Trabajo y
Derecho of the University of Seville.
Get Together at 21:30 h, on Monday 2 September at the Patios of the Facultades de Ciencias
del Trabajo y Derecho of the University of Seville.
Visit to the Guadalquivir marshes, on Wednesday 4 September. Buses will departure from
Hotel NH Central Convenciones at 10:00 h (front door). It will include a visit to the rice fields of
Seville (from 11:00 to 13:00 h) and tasting of typical products of the Guadalquivir marshes
(from 13:00 to 15:30 h).
Visit to the University of Seville (front door, c/ San Fernando), on Wednesday 4 September at
19:00 h.
Tapas Tour, on Wednesday 4 September at 20:30 h.
Closing dinner, on Friday 6 September at 22:00 h. at the “Hípica Pedro Macías” a riding centre
served by Catering Sevilla in the arena of a bullring. (http://www.hipicapedromacias.es). It is
not included with the registration. Its estimated price will be 45€. Buses will departure from
Hotel NH Central Convenciones at 21:30 h.
Accompanying persons
The registration as accompanying participants includes Get together, lunches and coffee
breaks, snakcs and drinks during poster sessions, Cultural visits and visit to the rice fields of the
Guadalquivir marshes of Seville.
Badge policy
Delegates are requested to exhibit the congress badges all the time during the scientific and
social events.
Internet connection
Wireless internet service will be available in the congress venue.
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Liability and insurance
The organizers are not able to take responsibility whatsoever for injury or damage involving
persons and property during the congress.
Mobile phones
Participants are requested to keep their mobile phones switched off in the session rooms.
Time
Sevilla time is the Central European time zone (Greenwich Mean Time + 2:00), i.e. one hour
more than in Lisbon or London.
Climate and clothing
Sevilla enjoys a typically Mediterranean climate throughout the year. September in Sevilla is
not as hot as August but is still extremely warm. The average daily temperature may reaches
32 °C (about 90 °F) at noon and 18 °C (64 °F) at night. The average daily sunshine is 9 hours a
day with average rainfall being just 24 mm over 2 days. This means that the chance of rain is
extremely unlikely, and you can still bring light clothing.
Environment
Hotel NH Central Convenciones includes this congress on its ECO-Meeting programme, which
includes efficient use of water and energy, environmentally friendly materials, low fair-trade
coffee and voluntary commitment of CO2 emissions.
Travel agency
The official travel agency is CAC Travel, Calle Virgen de la Victoria 12, 41011 Sevilla, Spain;
phone: +34 955 099 142; email: [email protected]
The address of the organization is:
Department of Microbiology, Faculty of Biology, University of Sevilla, Avenida de la Reina
Mercedes s/n, 41012-Sevilla, Spain; phone: +34 954 557 117; fax: +34 954 557 830; e-mail:
[email protected], http://congreso.us.es/ibempa
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Meeting program
MONDAY, 2 SEPTEMBER
11:00 - 18:00
Registration (Hotel NH Central Convenciones)
LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO, UNIVERSITY
OF SEVILLE
Opening Session.
Introduced by Antonio Gil-Serrano (Universidad de Sevilla, Sevilla, España).
18:15 - 18:20
18:20 - 19:15
Welcome address.
Keynote Conference:
Quorum sensing: a double-edge sword in bacterial-plant interactions.
Miguel Cámara.
Centre for Biomolecular Sciences, University of Nottingham. Nottingham. Reino Unido.
The speaker will be introduced by Ildefonso Bonilla Mangas (Universidad Autónoma de Madrid,
Madrid, España).
19:20 - 19:40
19:40 - 20:15
20:15 - 21:30
21:30 - 23:00
Presentation of the Congress picture “Uniendo pueblos”.
Musical Performance.
Opening Ceremony.
Get together Patios of Facultades de Ciencias del Trabajo y Derecho,
University of Seville.
TUESDAY, 3 SEPTEMBRE
LECTURE THEATRE OF THE FACULTADES DE CIENCIAS DEL TRABAJO Y DERECHO. UNIVERSITY
OF SEVILLE.
Session I
Ecology, diversity and evolution of microorganisms beneficial to plants.
Chairpersons:
Dr. Fábio B. Reis Jr (EMBRAPA Cerrados, Brasil).
Dr. María Luisa Izaguirre (IVIC, Venezuela).
09:00 - 09:25
SI-P-1
Diversidad y evolución de las comunidades microbianas en la rizosfera tras un
incendio forestal.
Fernández-López, M.
Estación Experimental del Zaidín, CSIC. Granada. España.
09:25 - 09:55
SI-P-2
Species and symbiovars within Mesorhizobium: diversity and host range.
Laranjo, M.
Instituto de Ciências Agrárias e Ambientais Mediterránicas, Universidade de Évora, Évora,
Portugal.
10:00 - 11:30
Oral Communications
SI-CO-1
Diversidad de hongos micorrizógenos arbusculares bajo situaciones contrastantes
de fertilización fosfatada.
*
García, S., Rodríguez Blanco, A. , Pezzani, F.
Facultad de Agronomía. Universidad de la República. Av. Garzón 780. Montevideo. Uruguay.
viii
SI-CO-2
Prospecting metal resistant plant-growth promoting rhizobacteria for
rhizoremediation of metal contaminated estuaries using Spartina densiflora.
Andrades-Moreno, L., del Castillo, I., Redondo-Gómez, S., Mesa, J., Caviedes M.A.,
Pajuelo, E., Rodríguez-Llorente, I.D.*
Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla. España
SI-CO-3
Symbiovar loti-type genes are widely spread across different chromosomal
backgrounds, corresponding to up to nine Mesorhizobium genospecies nodulating
Cicer canariense.
*
Pérez-Yépez, J. , Armas-Capote, N., Martínez-Hidalgo, P., Velázquez, E., PérezGaldona, R., Martínez-Molina, E., León-Barrios, M.
Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife. España.
SI-CO-4
Molecular phylogeny and phenotypic characterization of salt tolerant Sinorhizobia
nodulating Phaseolus filiformis in Northern Mexico.
Rocha, G., Medina, A., Carreño, R., Bustillos, R., Contreras, J.L., Villegas, M.C.,
Chaintreuil, C., Dreyfus, B., Le Queré, A., Munive, J.A. *
Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de
Puebla, Mexico.
11:30 - 12:00
General discussion.
Coffee break.
Session II
Genetics and genomics of beneficial microorganisms and associated plants.
Chairpersons:
Dr. Fabricio Cassan (Univ. Nacional de Río Cuarto, Argentina).
Dr. Marta Martín (Univ. Autónoma de Madrid, España).
12:00 - 12:25
SII-P-1
Genomics of host specificity in the Rhizobium-legume symbiosis.
Imperial, J.
ETS de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP),
Universidad Politécnica de Madrid, Madrid, España.
12:30 - 12:55
SII-P-2
Genomic insights into the rhizosphere lifestyle of rhizobia.
Ormeño-Orrillo, E.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca,
Morelos, México.
13:00 - 14:30
Oral Communications
SII-CO-1
First analyses of the genomic sequence of the soybean symbiont Sinorhizobium
fredii HH103.
*
Vinardell, J.M. , Göttfert, M., Becker, A., Acosta-Jurado, S., Baena, I., Blom, J., Bonilla,
I., Buendía, A.M., Crespo-Rivas, J.C., Goesmann, A., Jaenicke, S., Krol, E., Lloret, J.,
McIntosh, M., Margaret, I., Pérez-Montaño, F., Schneiker-Bekel, S., Serranía, J.,
Szczepanowski, R., Zehner, S., Pühler, A., Ruiz-Sainz, J.E., Weidner, S.
Departamento de Microbiología, Universidad de Sevilla, España.
SII-CO-2
Identificación y caracterización del regulador maestro del flagelo lateral de
Bradyrhizobium japonicum USDA 110.
*
Mongiardini, E.J., Quelas, J.I., Lodeiro, A.R.
IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina.
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SII-CO-3
Pyrosequencing reveals the presence of diverse bacterial genera which have not
previously described to soil and rhizosphere.
Lagos, L. *, Jorquera, M., Maruyama, F., Mora, M.L.
Scientific and Technological Bioresource Nucleus, Universidad de La Frontera, Temuco, Chile.
SII-CO-4
Temporal profile of nif gene expression in Azotobacter vinelandii: effect of nifA
mutation.
Navarro-Rodríguez, M.*, Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M.
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid. Madrid.
España
14:45 - 16:30
General discussion.
Lunch at Hotel NH Central Convenciones.
HOTEL NH CENTRAL CONVENCIONES
17:00 - 18:00
Meeting of the Latin American Society of Rhizobiology.
18:00 - 19:30
Poster Discussion Sessions I, V and VI.
Chairpersons:
Dr. Milagros León (Univ. de la Laguna, España).
Dr. Maribel Parada (Univ. de la Frontera, Chile).
19:30 - 20:30
Corner business innovation I.
Chairpersons:
Dr. Alvaro Peix (IRNASA, CSIC, España).
Dr. Khalid Alki (AMC Chemical, S.L., España).
Participants:
Sistemas Genómicos, España.
Bruker, España.
Corporación Tecnológica de Andalucía, España.
20:30 - 22:00
Posters Session I, V and VI (with snacks and drinks).
WEDNESDAY, 4 SEPTEMBER
09:00 - 13:00
13:00 - 15:30
19:00 - 20:30
20:30
Visit to the rice fields of Seville.
Tasting of typical products of the Guadalquivir marshes.
Visit to the University of Seville.
Tapas Tour
THURSDAY, 5 SEPTEMBER
OF SEVILLE
Session III
Symbiotic plant/microbe Interactions.
Chairpersons:
Dr. Carmen Quinto (Instituto de Biotecnología, UNAM, México).
Dr. Maria do Carmo Catanho Pereira de Lyra (Instituto Agronômico de PernambucoIP, Brasil).
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09:00 - 09:25
SIII-P-1
Priming plant defences by beneficial soil microorganisms.
Pozo-Jiménez, M.J.
09:30 - 09:55
SIII-P-2
Una visión general del papel de los polisacáridos superficiales de Sinorhizobium
fredii en su simbiosis con la soja y otras leguminosas.
Ruiz Sainz. J.E.
Departamento de Microbiología. Facultad de Biología. Universidad de Sevilla. España
10:00 - 11:30
Oral Communications
SIII-CO-1
Boron deficiency affects rhizobia cell surface polysaccharides.
Quijada, N.M., Cerda, M.E., Abreu, I.*, Pérez de Nanclares, M., Bonilla, I., Bolaños, L.
Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid. Madrid.
SIII-CO-2
Threalose is a key carbon regulatory player in the Legume-Rhizobium symbiosis.
Sánchez, F.*, Barraza, A., Estrada-Navarrete, G.,Quinto, C., Merino, E.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad
Nacional Autónoma de México..
SIII-CO-3
RbohB-dependent reactive oxygen species regulates rhizobial infection, bacteroid
development and nitrogen fixation in common bean.
Arthikala, M.K., Montiel, J., Nava, N., Santana, O., Sánchez-López, R., Cárdenas, L.,
Quinto, C.*
Nacional Autónoma de México.
SIII-CO-4
Quorum Sensing systems and flavonoids via NodD1 coordinate and regulate the
biofilm transition in Sinorhizobium fredii SMH12, which is necessary for a successful
root colonization of Glycine max cv Osumi.
Pérez-Montaño, F.*, Jiménez-Guerrero, I., Del Cerro, P., López-Baena, F.J., Ollero, F.J.,
Bellogín, R.A., Lloret, J., Espuny, M.R.
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. España
11:30 - 12:00
General discussion.
Coffee break.
Session IV
Associative interaction plant/ beneficial microbe.
Chairpersons:
Dr. José Miguel Barea (Estación Experimental del Zaidín, CSIC, Spain).
Dr. Verónica Reis (EMBRAPA-Agrobiología, Brasil).
12:00 - 12:25
SIV-P-1
Aplicaciones biotecnológicas de la interacción planta-microorganismos: salud,
medioambiente y agricultura.
Gutiérrez-Mañero, F.J.
Facultad de Farmacia. Universidad San Pablo CEU. Madrid. España.
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12:25 - 12:55
SIV-P-2
Aspectos bioquímicos y moleculares de la interacción Delftia-Rizobio-Alfalfa: un
lenguaje de señales químicas.
Castro-Sowinski, S.
Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
13:00 - 14:30
Oral Communications
SIV-CO-1
Evaluation of Micromonospora as a potential biocontrol agent.
*
Martínez-Hidalgo, P. , Martínez-Molina, E., Pozo, M.J.
Departamento de Microbiología y Genética, Universidad de Salamanca, España. GIR
"Interacciones mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC,
Salamanca. España.
SIV-CO-2
Movimiento y quimiotáxis en Pseudomonas fluorescens F113, influencia en una
colonización competente de la rizosfera de las plantas.
Baena, I., Muriel, C., Martín, I., Jalvo, B., Hernanz, A., González de Heredia, E.,
Barahona, E., Navazo, A., Redondo-Nieto, M., Martínez-Granero, F., Lloret, J., Rivilla,
R., Martín, M.*
Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid.
SIV-CO-3
Caracterización de la interacción Delftia-Sinorhizobium en alfalfa.
Morel, M.A.*, Cagide, C., Dardanelli, M.S., Castro-Sowinski, S.
Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable
(IIBCE), Av. Italia 3318, Montevideo, Uruguay.
SIV-CO-4
Estrés salino, calidad nutricional y comportamiento postcosecha, en lechuga
inoculada con Azospirillum.
Fasciglione, G.*, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A.
Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC
276 (7620) Balcarce, Argentina.
General discussion.
HOTEL NH CENTRAL CONVENCIONES
17:00 - 18:00
Meeting of the Spanish Society of Nitrogen Fixation
18:00 - 19:30
Poster Discussion Sessions II, III and IV.
14:45 - 16:30
Chairpersons:
Dr. Fernando González (Univ. de León, España).
Dr. José Antonio Lucas (Univ. CEU San Pablo, España).
19:30 - 20:30
Corner business innovation II.
Chairpersons:
Dr. Jesús González (Univ. de Granada, España).
Dr. Mohamed Dary (ResBioAgro, S.L., España).
Participants:
Total Biotecnología, Brasil.
ResBioAgro, España.
ACM Chemical, España.
Simborg, España.
20:30 - 22:00
22:00
Posters Session II, III and IV (with snacks and drinks).
Removal of posters.
xii
FRIDAY, 6 SEPTEMBER
OF SEVILLE
Session V
Physiology and biochemistry of beneficial microorganisms and associated
plants
Chairpersons:
Dr. Manuel Sánchez (Univ. de Navarra, Spain).
Dr. Carmen Lluch (Univ. de Granada, Spain).
09:00 - 09:25
SV-P-1
Fijación de nitrógeno en leguminosas en entornos variables: el bacteroide, el
nódulo y la planta.
César Arrese Igor.
Universidad Pública de Navarra. Pamplona, España.
09:30 - 09:55
SV-P-2
Molecular physiology of nickel and cobalt homeostasis in Rhizobium
leguminosarum.
José Manuel Palacios Alberti
Escuela Técnica Superior de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de
Plantas (CBGP), Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid, España
10:00 - 11:30
Oral Communications
SV-CO-1
Engineering a hydrogen biosensor in the nitrogen-fixing strain Rhodobacter
capsulatus.
Gónzalez de Heredia, E.M.*, Barahona, E., Jiménez-Vicente, E., Echavarri-Erasun, C.,
Rubio, L.M.
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus de
Montegancedo, Pozuelo de Alarcón 28223, Madrid.
SV-CO-2
MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.
Rodríguez-Haas, B., Finney, L., Kryvoruchko, I., González-Melendi, P., Udvardi, M.,
Imperial, J., González-Guerrero, M.*
Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid.
Campus de Montegancedo, Pozuelo de Alarcón, Madrid, España.
SV-CO-3
Selección de líneas de garbanzo tolerantes a la salinidad empleando caracteres de
producción de biomasa y funcionamiento nodular como indicadores.
Gómez, L.A.*, Vadez, V., Vaillhe, H., Pernot, C., Drevon, J.J.
Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros. La
Habana, Cuba.
SV-CO-4
Estrés abitotico induce cambios en las señales de comunicación y en la interacción
temprana entre maní y rizobacterias.
Cesari, A., Paulucci, N., García, M., Dardanelli, M.S. *
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales, Universidad Nacional de Río Cuarto, Argentina.
General discussion.
11:30 - 12:00
Coffee break.
xiii
Session VI
Inoculants in agriculture and the environment.
Chairpersons:
Dr. Antonio M. de Ron (Misión Biológica de Galicia, MBG-CSIC).
Dr. Yeda Mendes (EMBRAPA-Cerrados, Brasilia, Brasil).
12:00 - 12:25
SVI-P-1
Efficient soil microbial consorcia: a new dimension for sustainable agriculture and
environmental sustainability.
Cruz, C.
Universidade de Lisboa, Lisboa. Portugal.
12:30 - 12:55
SVI-P-2
Oportunidades e ameaças à contribuição da fixação biológica do nitrogênio em
leguminosas no Brasil.
Nogueira, M.A.
Embrapa Soja, Londrina, Paraná, Brazil.
12:00 - 14:30
Oral Communications
SVI-CO-1
Selenium biofortification in wheat plants by co-inoculation of selenobacteria strains
and arbuscular mycorhizal fungy for obtaining enriched selenium foods.
Durán, P.*, Mora, M.M.
Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and
Biotechnological Bioresource Nucleus. Universidad de la Frontera, Temuco-Chile.
SVI-CO-2
Evaluación de la degradación de tamo de arroz por microorganismos
lignocelulolíticos.
Gutiérrez-Rojas, I.*, Pedraza-Zapata, C., Sánchez-Rodríguez, L., Moreno-Sarmiento, N.
Instituto de Biotecnología, Ciudad Universitaria, Edificio Manuel Ancizar, Universidad Nacional
de Colombia, Bogotá, D.C., Colombia. Departamento de Microbiología, Facultad de Ciencias,
Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e Industrial (GBAI),
Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
SVI-CO-3
Soil microbial phosphorus, phosphorus availability and crop yield of upland ricecommon bean intercropping under organic and mineral fertilizer inputs on ferrallitic
soil of Malagasy highlands.
Henintsoa, M.*, Andriamananjara, A., Razakatiana, A.T.E., Larvy Delarivière, J.,
Rabeharisoa, L., Becquer, T.
Laboratoire des Radioisotopes (LRI), University of Antananarivo, Madagascar. Laboratoire de
Microbiologie de l’Environnement (LME), Centre National de Recherches sur l’Environnement
(CNRE), Antananarivo, Madagascar. Institut de Recherche pour le Développement (IRD), UMR
Eco&Sols, c/o LRI, Madagascar.
SVI-CO-4
Sistema uruguayo de fiscalización de inoculantes: complementación
interinstitucional y aplicación de tecnologías de la información.
Beyhaut, E*, Barlocco, C., Altier, N., Mayans, M.
INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay. 2 División Control de Insumos, DGSSAA.
M.G.A.P, Av. Millán. Montevideo, Uruguay.
14:45 - 16:30
General discussion.
xiv
OF SEVILLE
Closing Session
18:30 - 19:30
Award “Antonio J. Palomares” Conference.
Simplicity turned out complicated: How FixK2, a key regulator of genes for
symbiosis, is exquisitely controlled by different means.
Mesa, S.
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín,
Consejo Superior de Investigaciones Científicas, Granada, Spain.
The speaker will be introduced by Eulogio Bedmar (SEFIN President).
19:30 - 19:40
“Bernardo Leicach - BIAGRO” awards ceremony.
19:40 - 20:40
Closing Conference.
Uma década de ouro se aproxima para a microbiologia do solo: expectativas da
pesquisa, da indústria, dos agricultores e da sociedade.
Mariangela Hungría.
Embrapa Soja, Londrina, Paraná, Brazil
The speaker will be introduced by Tomás Ruíz Argüeso (Centro de Biotecnología y Genómica de
Plantas, Universidad Politécnica de Madrid, España).
20:40
21:30
Closing ceremony
Buses departure for closing dinner (Hotel NH Central Convenciones)
22:00
Closing dinner. “Hípica Pedro Macías”
xv
Floor plan
xvi
Contents
Opening Session
Miguel Cámara.
2
Centre for Biomolecular Sciences, University of Nottingham. Nottingham NG7 2RD. Reino Unido).
Session I. Ecology, diversity and evolution of microorganisms beneficial to plants.
SI-P-1
Diversidad y evolución de las comunidades microbianas en la rizosfera tras un
incendio forestal.
Fernández-López, M.
7
SI-P-2
Species and symbiovars within Mesorhizobium: diversity and host range.
Laranjo, M.
11
Instituto de Ciências Agrárias e Ambientais Mediterránicas, Universidade de Évora, Évora,
Portugal.
SI-CO-1
Diversidad de hongos micorrizógenos arbusculares bajo situaciones contrastantes
de fertilización fosfatada.
García, S., Rodríguez Blanco, A.*, Pezzani, F.
15
SI-CO-2
rhizoremediation of metal contaminated estuaries using Spartina densiflora.
Andrades-Moreno, L., del Castillo, I., Redondo-Gómez, S., Mesa, J., Caviedes M.A.,
Pajuelo, E., Rodríguez-Llorente, I.D.*
17
Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor
García González 2, 41012 Sevilla, Spain.
SI-CO-3
Symbiovar loti-type genes are widely spread across different chromosomal
backgrounds, corresponding to up to nine Mesorhizobium genospecies nodulating
Cicer canariense.
Pérez-Yépez, J., Armas-Capote, N., Martínez-Hidalgo, P., Velázquez, E., PérezGaldona, R., Martínez-Molina, E., León-Barrios, M.*
19
Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife.
España.
SI-CO-4
Molecular phylogeny and phenotypic characterization of salt tolerant Sinorhizobia
nodulating Phaseolus filiformis in Northern Mexico.
Rocha, G., Medina, A., Carreño, R., Bustillos, R., Contreras, J.L., Villegas, M.C.,
Chaintreuil, C., Dreyfus, B., Le Queré, A., Munive, J.A. *
21
Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de
Puebla, Mexico.
SI-CP-01
Phylogenetic diversity of bradyrhizobia nodulating cowpea (Vigna unguiculata L.
Walp) in Extremadura (Spain).
Bejarano, A.*, Ramírez-Bahena, M.H., Velázquez, E., Peix, A.
23
SI-CP-02
MALDI-TOFF MS, a tool for diversity analysis and detection of novel rhizobial
species nodulating Phaseolus vulgaris.
*
Flores-Félix, J.D. , García-Fraile, P., Sánchez-Juanes, F., Ramírez-Bahena, M.H.,
Ferreira, L., Mulas, D., Rivas, R., González-Andrés, F., Peix, A., González-Buitrago,
J.M., Velázquez, E.
25
xvii
SI-CP-03
Análisis de la biodiversidad de microorganismos en nódulos de Lotus corniculatus.
Marcos-García, M., Menéndez, E., Celador-Lera, L., Rivera, L.P., Martínez-Molina, E.,
Mateos, P.F., Velázquez, E., Rivas, R.*
27
SI-CP-04
Where do PGPR interact with plants? An essential determination to develop
effective agricultural inputs based on microorganisms.
Mulas, R., Mulas, D.*, Menéndez, E., Rivera, L., Mateos, P., González-Andrés, F.
29
SI-CP-05
Biodiversity of endophytic bacteria and mycorrhizal fungi in roots of
pepper(Capsicum annuum L.) in León province (Spain).
*
Barquero, M., Velázquez, E., Terrón, A. , González-Andrés, F.
31
SI-CP-06
Diversidad de los rizobios que nodulan Medicago marina de regiones del Odiel y
San Fernando.
Alías-Villegas, C.*, Bellogín, R.A., Camacho, M., Cubo, M.T., Temprano, F., Espuny,
M.R.
33
SI-CP-07
Tolerancia a pH y salinidad extremos y propiedades simbióticas de aislamientos de
rhizobia de Medicago marina L.
Alías-Villegas, C., Espuny, M.R., Bellogín, R.A., Cubo, M.T., Camacho, M., Temprano,
*
F.
35
SI-CP-08
Estudio de la Biodiversidad microbiana en suelos de las Marismas del Odiel y de
las Minas de Rio Tinto.
Ruíz-Carnicer, A., Alías-Villegas, C., Bellogín, R.A., Manyani, H., Temprano, F.,
Espuny, M.R., Camacho, M.*
37
SI-CP-09
Componentes biológicos en el suelo de dos plantaciones similares de ciruelo en
producción ecológica y convencional.
Daza, A.*, Pérez-Romero, L.F., Arroyo, F.T., García-Galavís, P.A., Camacho, M.,
Santamaría, C.
39
SI-CP-10
Relative abundance of denitrification genes and diversity of bacterial denitrifiers
in sediments with different nitrate concentration.
Correa-Galeote, D., Tortosa, G., Bedmar, E.J. *
41
SI-CP-11
Experimental and modelling approach to the legume-Rhizobium interaction: test
of plant-host sanctions in co-inoculated plants with fixing and non-fixing strains.
Marco, D.E.*, Talbi, C., Bedmar, E.J.
43
SI-CP-12
Identification of rhizobial strains nodulating cultivated grain legumes in Egypt.
Zahran, H.H.*, Chahboune, R., Bedmar, E.J., Abdel-Fattah, M., Yasser, M.M.,
Mahmoud, A.M.
45
SI-CP-13
Genetic diversity and biogeography of rhizobial genospecies nodulating wild
chickpea Cicer canariense on La Palma (Canary Is.).
Armas-Capote, N., Pérez-Yépez, J., Martínez-Hidalgo, P., Garzón-Machado, V., del
Arco-Aguilar, M., Velázquez, E., León-Barrios, M.*
47
SI-CP-14
Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium
meyeni Walp.) de suelos altoandinos del Perú.
*
Chumpitaz, C. , Ogata, K., Santos, R. Zúñiga, D.
49
SI-CP-15
Análisis de la presencia natural de micorrizas en cultivos de algodón inoculados
con Bacillus sp. y/o Bradyrhizobium sp.
Valencia, C.*, Toro, M., Zúñiga, D.
51
SI-CP-16
Poblaciones microbianas con potencial PGPR en la rizósfera de cultivos de papas
nativas amargas del altiplano peruano.
Ramos, E.*, Santos, R., Velezmoro, C., Zúñiga, D.
53
xviii
SI-CP-17
Capacidad PGPR de cepas de Bacillus y Pseudomonas aisladas de la rizosfera de
aguaymanto (Physalis peruviana L.).
Cumpa, A.*, Flores, L., Chumpitaz, C., Ogata, K., Zúñiga, D.
55
SI-CP-18
Phenotypic and molecular characterization of Lotus parviflorus nodule bacteria.
*
Soares, R., Lorite, M.J., Videira e Castro, I., Sanjuán, J.
57
SI-CP-19
Contribution of root nodule bacteria for the sustainability of “montado” (cork oak)
ecosystem.
Fernández, C., Soares, R., Barrento, M.J., Machado, H., Gomes, A.A., Videira e
*
Castro, I.
59
SI-CP-20
Estudio del carácter endófito de bacterias del género Pantoea aisladas de los
cultivos de arroz de la Marisma del Guadalquivir.
Megías, E., Benitez, C., Diaz-Olivares, I., Ollero, F.J., Megías, M. *
61
SI-CP-21
El género Pantoea en los cultivos de arroz de la Marisma del Guadalquivir:
diversidad y posible descripción de una nueva especie.
Megías, E.*, Fernández, M., Reis Junior, F.B., Márquez, M.C., Ollero, F.J., Megías, M.
63
SI-CP-22
Bacterial biodiversity within the genus Enterobacter in paddies of Guadalquivir
marshes.
*
Fernández, M. , Megías, E., Reis Junior, F.B., Megías, M.
65
SI-CP-23
Isolation and characterization of a novel bacterium isolated from paddies of
Guadalquivir marshes.
Márquez, M.C.*, Fernández, M., Megías, E., Merchán, F.
67
SI-CP-24
Biodiversidad y performance simbiótica de bacterias noduladoras de soja
alóctonas de suelos de Argentina.
Covelli, J.M.*, López, M.F., Arrese-Igor, C., Lodeiro, A.R.
69
SI-CP-25
Búsqueda y caracterización de bacterias promotoras del crecimiento vegetal en
rizosfera de plantas nativas antárticas.
Fernández Garello, P., Braga, L., Senatore, D., Lagurara, P., Yanes, M.L., Vaz, P.,
Azziz, G., Bajsa, N.*
71
SI-CP-26
Caracterización de las comunidades de hongos micorrícico-arbusculares asociadas
a metalofitas en suelos contaminados con Cu.
Cornejo, P.*, García, S., Vidal, C., Meier, S., Borie, F.
73
SI-CP-27
Screening for phosphate solubilising rhizobia from faba bean in Marrakech region
field cultures.
Maghraoui, T.*, Domergue, O., Oufdou, K., Lahrouni, M., Galiana, A., Sanguin, H.,
Drevon, J.J., de Lajudie, P.
75
SI-CP-28
Estudio de la dinámica poblacional binaria de rizobacterias en Lupinus mediante
citometría de flujo.
Ruiz Palomino, M., Probanza, A.*
77
SI-CP-29
Utilização da técnica de MLSA em estudos taxonômicos e filogenéticos com
Bradyrhizobium e sua importância na descrição de novas espécies.
*
Delamuta, J.R.M. , Ribeiro, R.A., Hungria, M.
79
SI-CP-30
Mycorrhizal fungal identity as determinant of functional assemblage of plantgrowth promoting bacteria in the rhizosphere.
Meleiro, A.I.*, Carvalho, L., Correia, P., Melo, J., Carolino, M., Cruz, C.
81
SI-CP-31
Diversity and stress tolerance in rhizobia from Parque Chaqueño region of
Argentina nodulating Prosopis alba.
Chávez Díaz, L., González, P., Rubio, E., Melchiorre, M. *
83
xix
SI-CP-32
Condiciones de estrés hídrico o salino modifican la composición y capacidad
formadora de biofilm de comunidades bacterianas aisladas de rizósfera de alfalfa.
Bogino, P., Abod, A., Nievas, F., Santoro, V., Vicario, J. *, Giordano, W.
85
SI-CP-33
Supervivencia y capacidad formadora de biofilms por rizobacterias.
*
Abod, A. , Bogino, P., Vicario, J., Giordano, W.
87
SI-CP-34
Caracterización morfológica de rizobios asociados a cultivos de arveja (Pisum
sativum L.), chocho (Lupinus mutabilis S.), fréjol (Phaseolus vulgaris L.), haba
(Vicia faba L.) y vicia (Vicia atropurpurea) en suelos del Ecuador.
*
Carpio, M.J., Paucar, B.M. , Alvarado, S.P.
89
SI-CP-35
Bacterias con actividad ACC desaminasa en huertos de aguacate en Michoacán,
México.
Chávez-Bárcenas, A.T.*, Hernández-Valdés, E.F., Bárcenas-Ortega, A.E., LozunaLópez, F., García-Saucedo, P.A., Olalde-Portugal, V.
91
SI-CP-36
Caracterização genotípica de bactérias diazotróficas isoladas de plantas de
Brachiaria brizantha e B. humidicula no estado de Roraima, Amazônia, Brasil.
*
Chalita, P.B., Cunha, E.N., Zilli, J.E., Silva, K.
93
Session II. Genetics and genomics of beneficial microorganisms and associated plants.
SII-P-1
Imperial, J.
96
ETS de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de Plantas (CBGP),
Universidad Politécnica de Madrid, Madrid, España.
SII-P-2
Ormeño-Orrillo, E.
100
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca,
Morelos, México.
SII-CO-1
First analyses of the genomic sequence of the soybean symbiont Sinorhizobium
fredii HH103.
Vinardell, J.M.*, Göttfert, M., Becker, A., Acosta-Jurado, S., Baena, I., Blom, J.,
Bonilla, I., Buendía, A.M., Crespo-Rivas, J.C., Goesmann, A., Jaenicke, S., Krol, E.,
Lloret, J., McIntosh, M., Margaret, I., Pérez-Montaño, F., Schneiker-Bekel, S.,
Serranía, J., Szczepanowski, R., Zehner, S., Pühler, A., Ruiz-Sainz, J.E., Weidner, S.
103
Departamento de Microbiología, Universidad de Sevilla, Spain.
SII-CO-2
Identificación y caracterización del regulador maestro del flagelo lateral de
Mongiardini, E.J.*, Quelas, J.I., Lodeiro, A.R.
105
SII-CO-3
Pyrosequencing reveals the presence of diverse bacterial genera which have not
previously described to soil and rhizosphere.
Lagos, L. *, Jorquera, M., Maruyama, F., Mora, M.L.
107
SII-CO-4
Temporal profile of nif gene expression in Azotobacter vinelandii: effect of nifA
mutation.
*
Navarro-Rodríguez, M. , Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M.
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus
de Montegancedo, Pozuelo de Alarcón 28223, Madrid.
xx
109
SII-CP-01
Singularidades adaptativas del genoma de Pseudomonas fluorescens F113.
Comparación con otras cepas del género Pseudomonas.
Redondo-Nieto, M.*, Martínez-Granero, F., Muriel, C., Martín, M., Rivilla, R.
111
SII-CP-02
AmrZ es un regulador transcripcional global en Pseudomonas fluorescens F113.
*
Martínez-Granero, F., Redondo-Nieto, M., Vesga, P., Martín, M., Rivilla, R.
113
SII-CP-03
The rkp-2 region of Sinorhizobium fredii HH103 is involved in LPS and EPS
production.
Acosta-Jurado, S.*, Crespo-Rivas, J.C., Murdoch, P.S., Rodríguez-Carvajal, M.A., RuizSainz, J.E., Vinardell, J.M.
115
SII-CP-04
Study of the quorum sensing Sin system of Sinorhizobium fredii HH103.
*
Crespo-Rivas, J.C. , Pérez-Montaño, F., Acosta-Jurado, S., Payán-Bravo, L., McIntosh,
M., Meyer, S., Becker, A., Ruiz-Sainz, J.E., Vinardell, J.M.
117
SII-CP-05
Lotus japonicus Gifu and L. burttii responses to inoculation with a collection of S.
fredii HH103 mutants affected in symbiotic signals.
Rodríguez-Navarro, D.N., Jin, H., Kawaharada, Y., Sandal, N., Andersen, S.U.,
Stougaard, J., Ruiz-Sainz, J.E.*
119
SII-CP-06
Analyses of the Sinorhizobium fredii HH103 and USDA257 secretomes in the
presence and absence of the flavonoid genistein.
Yao, W., Thomas, J.R., Jiménez-Guerrero, I., López-Baena, F.J.*, Ruiz-Sainz, J.E.,
Vinardell, J.M.
121
SII-CP-07
Ocorrência e caracterização de bactérias isoladas de nódulos de amendoinzeiro
(Arachis hypogaea L.) em solos paranaenses, Brasil.
*
Andrade, D.S. , Cardoso, J.D., Pertinhez, G.N., Saturno, D.F., Lovato, G.M., Nomura,
R.B.G., Hungria, M.
123
SII-CP-08
The Type 3 secretion system effector NopP from Ensifer (=Sinorhizobium) fredii
HH103 is phosphorylated by a soybean kinase.
Calero, B.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Monreal, J.A., Ollero, F.J.,
López-Baena, F.J.
125
SII-CP-09
A yeast-based array to study the function of Ensifer (=Sinorhizobium) fredii HH103
Type 3 secretion system effectors in symbiosis.
Ollero, F.J.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Mesa, B., Medina, C., LópezBaena, F.J.
127
SII-CP-10
Implicación del gen bgvA en la formación de biofilm por Rhizobium tropici
CIAT899.
Del Cerro, P.*, Ollero, F.J., Megías, M., Bellogín, R.A., Guasch-Vidal, B., PérezMontaño, F., Espuny, M.R.
129
SII-CP-11
Identification and characterization and of a soybean kinase that phosphorylates
the Type 3 secretion system effector NopL from Ensifer (=Sinorhizobium) fredii
HH103.
*
Jiménez-Guerrero, I. , Pérez-Montaño, F., Ollero, F.J., Monreal, J.A., Cubo, M.T.,
López-Baena, F.J.
131
SII-CP-12
Control de movilidad y producción de EPS I en Sinorhizobium meliloti: nuevas
funciones asignadas al sistema NtrY/X.
Calatrava*, N., Nogales, J., Ameztoy, K., van Steenbergen, B., Soto, M.J.
133
SII-CP-13
Auxotrophy accounts for nodulation impairment in a Sinorhizobium meliloti
mutant defective for meso-diaminopimelate biosynthesis.
García-Rodríguez, F.M., Ortigosa, A., Millán, V., Toro, N., Martínez-Abarca, F.*
135
xxi
SII-CP-14
Genomic analysis of Azospirillum brasilense Az39 the most extensively used strain
for inoculant production in Argentina.
Cassán, F.*, Rivera Bottia, D., Molina, R., Revale, S., Vazquez, M., Spaepen, S.,
Vanderleyden, J., Perticari, A.
137
SII-CP-15
Two-dimensional proteomic reference map of Bradyrhizobium diazoefficiens
strain CPAC 7 (=SEMIA 5080).
Gomes, D.F.*, Batista, J.S.S., Hungria, M.
139
SII-CP-16
Emprego da metodologia de MLSA em avaliações de filogenia e taxonomia de
rizóbios: estudo com Rhizobium spp. microssimbiontes de feijoeiro (Phaseolus
vulgaris L.).
*
Ribeiro, R.A. , Delamuta, J.R.M., Hungria, M.
141
SII-CP-17
Relative expression of hypothetical protein-related genes for Bradyrhizobium
diazoefficiens CPAC 7.
Gomes, D.F., Rolla-Santos, A.A.P.*, Batista, J.S.S., Hungria, M.
143
SII-CP-18
PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host
plant.
*
Jorrín, B. , Imperial, J.
145
SII-CP-19
Incorporación estable y expresión del gen de la diguanilato ciclasa PleD* en el
genoma de bacterias que interaccionan con plantas.
Romero-Jiménez, L.*, Rodríguez, D., Prada-Ramírez, H., Gallegos, M.T., Sanjuán, J.,
Pérez-Mendoza, D.
147
SII-CP-20
Identification of the gene responsible for a non-nodulating phenotype in chickpea.
Ali, L., Madrid, E., Sánchez, R., Temprano, F., , Gil, J., Rubio, J., Millan, T.*
149
SII-CP-21
Functional characterization of Ensifer meliloti denitrification genes.
Torres, M.J.*, Rubia, M.I., Coba de la Peña, T., Pueyo, J.J., Bedmar, E.J., Delgado, M.J.
151
SII-CP-22
Genome sequence of Herbaspirillum rubrisubalbicans M1, an endophytic
diazotroph and mild phytopathogen of sugarcane.
Souza, E. M.*, Chubatsu, L., Cardoso, R.A., Raittz, R.T., Weiss, V.A., Monteiro, R.A.,
Faoro, H., Wassem, R., Baura, V.A., Balsanelli, E., Huergo, L., Muller-Santos, M.,
Tadra-Sfeir, M., Cruz, L.M., Pedrosa, F.O.
153
SII-CP-23
Sinorhizobium meliloti differentially expressed non-coding RNAs modulating
nitrogen fixaion and cell cycle progression.
Robledo, M.*, Frage, B., Schlüter, J.P., Becker, A.
155
Session III. Symbiotic plant/microbe Interactions.
SIII-P-1
Priming plant defences by beneficial soil microorganisms.
Pozo-Jiménez, M.J.
158
SIII-P-2
Una visión general del papel de los polisacáridos superficiales de Sinorhizobium
fredii en su simbiosis con la soja y otras leguminosas.
Ruiz Sainz. J.E.
162
Departamento de Microbiología. Facultad de Biología. Universidad de Sevilla.
SIII-CO-1
Quijada, N.M., Cerda, M.E., Abreu, I.*, Pérez de Nanclares, M., Bonilla, I., Bolaños, L.
xxii
166
SIII-CO-2
Thehalose is a key carbon regulatory player in the Legume-Rhizobium symbiosis
Sánchez, F.*, Barraza, A., Estrada-Navarrete, G., Quinto, C., Merino, E.
168
Nacional Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210,
Cuernavaca, Morelos, México.
SIII-CO-3
RbohB-dependent reactive oxygen species regulates rhizobial infection, bacteroid
development and nitrogen fixation in common bean.
Quinto, C.*
170
Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271,
México.
SIII-CO-4
Quorum Sensing systems and flavonoids via NodD1 coordinate and regulate the
biofilm transition in Sinorhizobium fredii SMH12, which is necessary for a
successful root colonization of Glycine max cv Osumi.
*
Pérez-Montaño, F. , Jiménez-Guerrero, I., Del Cerro, P., López-Baena, F.J., Ollero,
F.J., Bellogín, R.A., Lloret, J., Espuny, M.R.
172
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina
Mercedes, 6. 41012 - Sevilla, Spain.
SIII-CP-01
Proteolytic control of the Bradyrhizobium japonicum transcriptional regulator
FixK2.
Fernández, N.*, Bonnet, M., Stegmann, M., Maglica, Z., Weber-Ban, E., Hennecke,
H., Bedmar, E.J., Mesa, S.
174
SIII-CP-02
Effect of temperature on ectomycorrhizal fungi associated with Pinus sylvestris L.
in organic vs mineral soils.
Gómez-Gallego, T., García-Rabasa, S., Flores-Rentería, D., Rincón, A.*
176
SIII-CP-03
Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco
soils.
Missbah El Idrissi, M., Guerrouj, K., Pérez-Valera, E., Abdelmoumen, H.*, Bedmar,
E.J.
178
SIII-CP-04
Interaction between Arbuscular Mycorrhizal Fungi and rhizobia on the growth of
subclover under Mn toxicity: The role of Extraradical Mycelium.
Alho, L.*, Carvalho, M., Goss, M.J., Brito, I.
180
SIII-CP-05
Autochthonous drought-tolerant arbuscular mycorrhizal fungi and bacteria can
increase nutrients acquisition and alleviate drought stress in lavandula plants.
Armada, E., Roldán, A., Azcón, R.*
182
SIII-CP-06
Análisis filogenético de la diversidad de hongos formadores de micorrizas
arbusculares presentes en los distintos tipos de propágulos asociados a especies
de plantas características del Parque Natural Sierra de Baza (Granada, España).
Varela-Cervero, S., López-García, A., Berrio, E., Barea, J. M., Azcón-Aguilar, C.*
184
SIII-CP-07
Influencia de la composición de la cubierta vegetal sobre los hongos micorrícicoarbusculares y la estabilización de un suelo degradado en un ecosistema
mediterráneo.
Cornejo, P., Ferrol, N., Barea, J.M.*, Azcón-Aguilar, C.
186
SIII-CP-08
Salt tolerance evaluation in Casuarina glauca: impact on the photosynthetic
apparatus.
Batista-Santos, P.*, Graça I., Semedo J., Lidon, F., Alves, P., Scotti, P., Pais, I., Ribeiro,
A.I., Ramalho, J.C.
188
xxiii
SIII-CP-09
Rhizosphere symbiots valorisation: Common bean-rhizobia symbiosis adaptation
to P deficiency in Ain Temouchent agro-ecosystem in Algeria.7
Benadis, Ch.*, Bekki, A., Lazali, M., Drevon, J.J.
190
SIII-CP-10
The genotypic variability of beans affects the growth parameters, the nodulation
and the microorganisms which initiate nodulation.
Benadis, Ch., Bekki, A.*, Abed, N.H., Ouzane, H., Irekti, H.
192
SIII-CP-11
Phosphoenol pyruvate phosphatase transcript in nodule cortex of Phaseolus
vulgaris.
*
Bargaz, A. , Drevon, J.J., Lazali, M., Ghoulam, C.
194
SIII-CP-12
P-deficiency increases phytase activity and O2 uptake per N2 reduced in Phaseolus
vulgaris L.
Lazali, M.*, Abadi, J., Ounane, S.M., Bargaz, A., Pernot, C., Drevon, J.J.
196
SIII-CP-13
Effect of inoculation with selected rhizobia on symbiotic nitrogen fixation and
Faba bean grains yield in north Tunisia agro-ecosystems.
Sifi, B.*, Maazaoui, H., Drevon, J.J.
198
SIII-CP-14
Integrating soil microbial community context in plant response to mycorrhizal
symbionts.
*
Carvalho, L. , Correia, P., Meleiro, A.I., Carolino, M., Dionisio, F., Cruz, C.
200
SIII-CP-15
Identificación y caracterización de sistemas de transporte de hierro en el hongo
micorrícico arbuscular Rhizophagus irregularis.
Tamayo, E., Ferrol, N.*
202
SIII-CP-16
Structure of the exopolysaccharide isolated from Sinorhizobium fredii HH103.
Rodríguez-Carvajal, M.A.*, Acosta-Jurado, S., Rodríguez-Navarro, D.N., Crespo-Rivas,
J.C., Margaret, I., Sanjuán, J., Soto, M.J., Vinardell, J.M., Ruiz-Sainz, J.E., Gil-Serrano,
A.
204
SIII-CP-17
First evidence for interlinked control of surface motility and biofilm formation in
Sinorhizobium meliloti.
Amaya-Gómez, C.V., Hirsch, A.H., Soto, M.J.*
206
SIII-CP-18
Symbiotic phenotype of different rhizobial species on Lotus japonicus Gifu and
Lotus burttii.
Rodríguez-Navarro, D.N.*, Temprano, F., Velázquez, E., Ruiz-Sainz, J.E.
208
SIII-CP-19
Los efectores secretados a través del T3SS suprimen la respuesta de defensa en
soja inducida por Ensifer (=Sinorhizobium) fredii HH103.
Cubo, M.T.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Ollero, F.J., López-Baena, F.J.
210
SIII-CP-20
Pode Bradyrhizobium japonicum, em combinação com diferentes densidades de
plantio de soja alterar componentes do rendimento?
*
de Luca, M.J. , Nogueira, M.A., Hungria, M.
212
SIII-CP-21
Effects of glyphosate-resistant gene and herbicides on biological nitrogen fixation
symbiotic efficiency and grain productivity of soybean in Brazil.
*
Hungria, M., Mendes, I.C., Nakatami, A.S., Reis-Junior, F.B., Fernandes, M.F.
214
SIII-CP-22
Diversidade genética de bactérias que colonizam nódulos radiculares de Phaseolus
vulgaris L. cultivado em campo e em casa de vegetação.
*
Oliveira-Francesquini, J.P. , Hungria, M., Glienke, C., Kava-Cordeiro, V., GalliTerasawa, L.
216
SIII-CP-23
Symbionts of Mimosa spp. in ultramafic soils in central Brazil.
Reis Junior, F.B.*, James, E.K., Hertel Júnior, C.R., Lopes, A.A.C., Alves, M.R.P., Souza,
L.M., Baura, V.A., Mendes, I.C., Andrade, L.R.M.
218
xxiv
SIII-CP-24
Cloning, Expression and Characterization of a Chitinase class III from Casuarina
glauca nodules.
Graça, I.*, Liang, J., Guilherme, M., Ferreira-Pinto, M., Ribeiro, A., Pereira A.S,
220
SIII-CP-25
Negative short-term salt effects on the soybean-B. japonicum interaction and
partial reversion by calcium addition.
Muñoz, N., Rodríguez, M., Robert, G., Lascano, R. *
222
SIII-CP-26
Expresión de supresores de muerte celular en raíces en cabellera de soja: efectos
bajo condiciones de estrés y en la interacción con Bradyrhizobium japonicum.
*
Robert, G. , Muñoz, N., Lascano, R.
224
SIII-CP-27
Efecto del contenido de taninos condensados (TC) sobre la formación de nódulos
en especies del género Lotus.
Escaray, F.J.*, Estrella, J., Paolocci, F., Damiani, F., Ruiz, O.A.
226
SIII-CP-28
Alteraciones en el proceso de nodulación en ausencia y presencia de fuente de
nitrógeno externa de un mutante deficiente en el transporte de nitrato de la
leguminosa Lotus japonicus.
García-Calderón, M.*, Pal’ove-Balang, P., Pérez, C.M., Betti, M., Marquez, A.J.
228
SIII-CP-29
Phylogenetic diversity and structure of sebacinoid fungi associated with plant
communities along an altitudinal gradient.
Garnica, S.*, Riess, K., Bauer, R., Oberwinkler, F.
230
SIII-CP-30
Grain yield of cowpea in Ghana responds to Rhizobium inoculation.
Atakora, W.K., Guimarães, A.P.*, Fosu, M., Boddey, R.M., Xavier, G.R.
232
SIII-CP-31
Pseudomonas fluorescens enhances photosynthesis and improves bioactive
profiles and antioxidant potential in blackberry (Rubus sp. Var. Lochness) in field
conditions.
García-Seco, D., Bonilla, A., Algar, E., García-Villaraco, A., Gutiérrez Mañero, F.J.*,
Ramos Solano, B.
234
SIII-CP-32
Improving opium poppy yield through MAMPs from selected beneficial bacterial
strains in field trials.
Bonilla, A., Garcia-Seco, D., Muñoz Ledesma, F.J., Lucas, J.A.*, Ramos Solano, B.,
Gutiérrez Mañero, F.J.
236
SIII-CP-33
Rhizobial cellulase CelC2 and its Ensifer homolog play an important role in plant
and nodule development.
Menéndez, E.*, Robledo, M., Velázquez, E., Martínez-Molina, E., Rivas, R., Mateos,
P.F.
238
SIII-CP-34
Ocorrência de fungos micorrízicos arbusculares na cultura da palma no semiárido
de Pernambuco.
Mergulhão, A.C.E.S.*, Silva, M.L.R.B., Figueroa, C.S., Lyra, M.C.C.P.
240
SIII-CP-35
Bactérias diazotróficas associadas a cladódios de palma: solubilização de fosfato
inorgânico e tolerância à salinidade.
Silva, M.L.R.B.*, Figueroa, C.S., Mergulhão, A.C.E.S., Lyra, M.C.C.P.
242
SIII-CP-36
Genotypic characterization of natural rhizobial populations isolated from pea and
lentil in two eco-climatic subhumid and semi-arid zones in Algeria.
Riah, N.*, Djekoun, A., Heulin, K., Laguerre, G.
244
SIII-CP-37
Native bradyrhizobia isolated from Lupinus mariae-josephae possess an essential
T3SS for symbiosis.
Durán, D., Pastor, V., Zehner, S., Göttfert, M., Imperial, J., Rey, L., Ruiz-Argüeso, T.*
246
xxv
SIII-CP-38
Which phosphatases may contribute to P use efficiency for N2 fixation in the
rhizobial symbiosis with legumes?
Drevon, J.J.*, Amenc, L. , Pernot, C., Abadie, J., Blair, M., Lazali, M., Bargaz, A.,
Ghoulam, C., Ounane, S.M., Zaman-Allah, M.
248
SIII-CP-39
nod gene inducers released by Mimosa flocculosa seeds and its effects on
common bean growth and yield under Brazilian cerrado soil conditions.
Mercante, F.M.*, Otsubo, A.A., Cunha, C.O.
250
SIII-CP-40
Diversidade de fungos micorrízicos arbusculares e colonização do solo em sistema
de cultivo consorciado cafeeiro (Coffea arábica L.) e seringueira (Hevea
brasiliensis).
Colozzi-Filho, A.*, Bugatti, E.P., Garbossi, A., Scaramal, A., Machineski, O., Carrenho,
R.
252
SIII-CP-41
Effect of mycorrhizal inoculation on wheat and barley genotypes differing in Altolerance growing at phytotoxic Al level.
Meier, S., Curaqueo, G., Seguel, A., Aguilera, P., Cornejo, P., Borie, F.*
254
SIII-CP-42
Origin of arbuscular mycorrhizal fungi determines plant development and
resource distribution between Phaseolus vulgaris L. and Oriza sativa in
intercropping.
Ramanankierana, H.*, Rasamiarivelo, A.V., Razafimbelo, T., Becker, T., Razakatiana,
A.T.E., Manitriniaina, H., Rabeharisoa, L., Randriambanona, H., Drevon, J.J.,
Duponnois, R.
256
SIII-CP-43
Asociación Española de Leguminosas.
De Ron, A.M., De la Cuadra, C., Millán, T*
258
Session IV. Associative interaction plant/beneficial microbe.
SIV-P-1
Aplicaciones biotecnológicas de la interacción planta-microorganismos: salud,
medioambiente y agricultura.
Gutiérrez-Mañero, F.J.
261
Facultad de Farmacia. Universidad San Pablo CEU. Madrid. España.
SIV-P-2
Aspectos bioquímicos y moleculares de la interacción Delftia-Rizobio-Alfalfa: un
lenguaje de señales químicas.
Castro-Sowinski, S.
265
Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.
SIV-CO-1
Martínez-Hidalgo, P.* , Martínez-Molina, E., Pozo, M.J.
269
Departamento de Microbiología y Genética, Universidad de Salamanca, España. GIR
"Interacciones mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC,
Salamanca. España.
SIV-CO-2
Movimiento y quimiotáxis en Pseudomonas fluorescens F113, influencia en una
colonización competente de la rizosfera de las plantas.
*
R., Martín, M.
xxvi
271
SIV-CO-3
Morel, M.A.*, Cagide, C., Dardanelli, M.S., Castro-Sowinski, S.
273
Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable
(IIBCE), Av. Italia 3318, Montevideo, Uruguay.
SIV-CO-4
Estrés salino, calidad nutricional y comportamiento postcosecha, en lechuga
inoculada con Azospirillum.
Fasciglione, G.*, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A.
275
Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC
276 (7620) Balcarce, Argentina.
SIV-CP-01
Bioprospecção de Bactérias Isoladas de Milho para Promoção de Crescimento de
Plantas.
Ikeda, A.C.*, Szilagy-Zecchin, V.J., Hungria, M., Kava-Cordeiro, V., Glienke, C., GalliTerasawa, L.V.
277
SIV-CP-02
Caracterización de una metilobacteria aislada de la superficie del grano de arroz.
Gallego Parrilla, J.J., Alías-Villegas, C., Díaz-Olivares, I.M., Gutiérrez Alcántara, R.,
Madinabeitia-Peiró, N., Bellogín, R.A.*, Espuny, M.R.
279
SIV-CP-03
Resistencia a metales pesados de bacterias aisladas de leguminosas de las
Marismas del Odiel y del entorno del Río Tinto.
Lara-Dampier, V., Acera-Mateos, P., Alías-Villegas, C., Gómez-Cárdenas, A.J.,
Temprano, F., Camacho, M., Bellogín, R.A., Espuny, M.R.*
281
SIV-CP-04
Comparación de las características microbiológicas del suelo en huertos de
aguacate bajo manejos orgánico y convencional.
Bárcenas-Ortega, A.E.*, Chávez-Bárcenas, A.T., García-Saucedo, P.A., OlaldePortugal, V., Tulais-Alvarado, C.A., Zavala-Gómez, A.
283
SIV-CP-05
Efecto de la inoculación foliar y radicular de bacterias PGPR en el cultivo de
Aguaymanto (Physalis peruviana).
Flores, L., Ogata, K.*, Zúñiga, D.
285
SIV-CP-06
Bacillus sp. y hongos micorrícicos para el control biológico del nemátodo
Meloidogyne incognita en el cultivo de Aguaymanto (Physalis peruviana).
Isla, F.*, Carbonell, E., Ogata, K., Zúñiga, D.
287
SIV-CP-07
Identificación de dos bacterias diazótrofas asociadas a una Brassicaceae (Lepidium
meyeni Walp.) de suelos altoandinos del Perú.
*
Chumpitaz, C., Ogata, K., Santos, R., Zúñiga, D.
289
SIV-CP-08
Selección de cultivos rizobianos aislados de nódulos de leguminosas de diferentes
regiones del Perú con capacidad de promover el crecimiento de lechuga, páprika y
tomate.
Soriano-Bernilla, B.*, Prado-Chávarry, G., Zavaleta-Verde, D., Valdez-Nuñez, R.
291
SIV-CP-09
Promoção de crescimento de tomate (Solanum lycopersicum L.) estimulado por
Bacillus amyloliquefaciens FZB42.
Szilagyi-Zecchin, V.J.*, Ruaro, L., Mógor, A.F.
293
SIV-CP-10
Efecto de volátiles emitidos por la bacteria bCT34 en la promoción de crecimiento
de Arabidopsis thaliana.
Parada, M.*, Quiroz, A., Parra, L., Mendoza, D., Fincheira, P.
295
SIV-CP-11
Functionality of phosphate solubilizing bacteria isolated from the rhizosphere of
papaya plants under conventional and organic farming systems.
*
Melo, J. , Azevedo-Junior, R.R. , Eutropio, F.J., Correira, P., Carvalho, L. , Meleiro, A.I.,
Teixeira, M.S., Carolino, M. , Ramos, A.C., Cruz, C.
297
xxvii
SIV-CP-12
Plant-growth promoting Sphingomonas sp. associated with annual ryegrass.
Dourado, A.C., Castanheira, N., Cortés Pallero, A., Delgado Rodriguéz, A.I., Alves,
P.I.L., Barreto Crespo, M.T., Fareleira, P.*
299
SIV-CP-13
Paenibacillus species isolated from Pisum sativum nodules in Lanzarote present
several in vitro plant growth promotion mechanisms.
Ramírez-Bahena, M.H., Flores-Félix, J.D., Tejedor, C., León-Barrios, M., Peix, A.*,
Velázquez, E.
301
SIV-CP-14
Efecto de rizobacterias en el enraizamiento de miniestacas en dos clones híbridos
de Eucalyptus spp.
González-Candia, P.*, Sossa, K., Rodríguez, F., Sanfuentes, E.
303
SIV-CP-15
Azoarcus sp. CIB, un nuevo endófito del arroz que degrada anaeróbicamente
hidrocarburos aromáticos.
Fernández, H., Prandoni, N., Fernández-Pascual, M., Morcillo, C., Fajardo, S., Díaz, E.,
Carmona, M.*
305
SIV-CP-16
Impacts of rice-bean intercropping on soil microbial activity and the development
of rice plants.
*
Razakatiana, A.T.E. , Henintsoa, M., Becquer, T., Ramanankierana, H., Baohanta,
H.R., Raherimandimby, M., Rabeharisoa, L., Duponnois, R.
307
SIV-CP-17
Efeito de bactérias PRPG com indução de ácido salicílico em plântulas de duas
cultivares de palma forrageira (Opuntia e Nopalea).
Lyra, M.C.C.P.*, Pérez-Montaño, F., Jiménez-Guerrero, I., Madinabeitia, N., DiazOlivares, I.M., Gutiérrez-Alcántara, R., Ollero, F.J.
309
SIV-CP-18
Análisis cualitativo y cuantitativo de vitamina C en fresas, en presencia de
microorganismos endófitos.
Aranda-Alonso, C.*, Rodríguez-Carvajal, M.A., Ollero, F.J., Megías, M., Gil-Serrano, A.
311
SIV-CP-19
Occurrence of rhizobacteria with PGPR activities in different ecosystems and agroecosystems of northern, central and southern Chile.
Barra, P., Jorquera, M., Acuña, J.*, Lagos, L., Marileo, L., Inostroza, N., Mora, M.L.
313
SIV-CP-20
Efecto de plaguicidas sobre la actividad biológica de la cubierta vegetal (L.
perenne) de un sistema de biopurificación denominado lecho biológico.
Diez, M.C.*, Gallardo, F.
315
Session V. Phisiology and biochemistry of beneficial microorganisms and associated
plants.
SV-P-1
Fijación de nitrógeno en leguminosas en entornos variables: el bacteroide, el
nódulo y la planta.
César Arrese Igor.
318
SV-P-2
leguminosarum.
José Manuel Palacios Alberti
Escuela Técnica Superior de Ingenieros Agrónomos y Centro de Biotecnología y Genómica de
Plantas (CBGP), Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid, España
xxviii
319
SV-CO-1
Engineering a hydrogen biosensor in the nitrogen-fixing strain Rhodobacter
capsulatus.
Rubio, L.M.
323
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Campus
de Montegancedo, Pozuelo de Alarcón 28223, Madrid.
SV-CO-2
MtNramp1 mediates iron import in rhizobia-infected Medicago truncatula cells.
Rodríguez-Haas, B., Finney, L., Kryvoruchko, I., González-Melendi, P., Udvardi, M.,
*
Imperial, J., González-Guerrero, M.
325
Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid.
Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España.
SV-CO-3
Selección de líneas de garbanzo tolerantes a la salinidad empleando caracteres de
producción de biomasa y funcionamiento nodular como indicadores.
Gómez, L.A.*, Vadez, V., Vaillhe, H., Pernot, C., Drevon, J.J.
327
Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros.
La Habana, Cuba.
SV-CO-4
Estrés abitotico induce cambios en las señales de comunicación y en la interacción
temprana entre maní y rizobacterias.
329
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y
Naturales, Universidad Nacional de Río Cuarto, Argentina.
SV-CP-01
Movilidad en cepas de rizobios nodulantes de maní.
Vicario, J.*, Dardanelli, M.S., Giordano, W.F.
331
SV-CP-02
Effects of phosphorus and nitrogen level fertility on growth parameters of Faba
bean inoculated with rhizobia.
Maazaoui, H.*, Sifi, B., Drevon, J.J.
333
SV-CP-03
Phytate mineralising bacteria increase in the rhizosphere of nodulated common
bean (Phaseolus vulgaris) under P deficiency.
Maougal, R.T.*, Brauman, A., Plassard, C., Abadi, J., Djekoun, A., Drevon, J.J.
335
SV-CP-04
Is the contrast between nodulated recombinant imbred lines of common bean
(Phaseolus vulgaris L.) linked with available phosphorus in soil ? A multi-local field
test in Mediterranean conditions.
Alkama, N.*, Jaillard, B. , Ounane, S.M., Ounane, G., Drevon, J.J.
337
SV-CP-05
Phosphoenol pyruvate phosphatase transcript in nodule cortex of Phaseolus
vulgaris.
*
Bargaz, A. , Lazali, M., Ghoulam, C., Drevon, J.J.
339
SV-CP-06
Phytate-mineralizing rhizobia from Vicia faba symbiosis in an agro-ecosystem of
south of France.
*
Domergue, O. , Chouayekh, H., Abadie, J., Amenc, L., Pernot, C., de Lajudie, P.,
Galiana, A., Drevon, J.J.
341
SV-CP-07
Daily simulation of plant and microbial transfers of carbon between the soil and
the atmosphere under wheat-faba bean intercropping.
*
Ibrahim, H. , Hatira, A., Blavet, D., Drevon, J.J., Pansu, M.
343
SV-CP-08
Effect of phosphorus deficiency on some agrophysiological and biochemical
parameters in faba bean (Vicia faba)-rhizobia symbiosis.
Makoudi, B., Mouradi, M., Kabbadj, A., Farissi, M., Drevon, J.J., Ghoulam, C.*
345
SV-CP-09
Acúmulo de açúcares e fixação biológica de nitrogênio em genótipos de soja sob
restrição hídrica.
*
Cerezini, P., Pípolo, A.E., Hungria, M., Nogueira, M.A.
347
xxix
SV-CP-10
Mannose Rich N-Glycoproteins synthesized during the development of the
Rhizobium-pea symbiosis are potential boron ligands.
Abreu, I., Valiente, R., Poza, L., Quijada, N.M., Reguera, M., Bonilla, I., Bolaños, L. *
349
SV-CP-11
Regulation of cellulose-like polysaccharide synthesis in Sinorhizobium meliloti.
*
Baena, I., Valiente, E., Bonilla, I., Martín, M., Rivilla, R., Lloret, J.
351
SV-CP-12
Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in
Rhizobium leguminosarum.
Albareda, M.*, Manyani, H., Rey, L., Brito, B., Ruiz-Argüeso, L., Imperial, J., F. Pacios,
L., Palacios, J.M.
353
SV-CP-13
Quorum Sensing is essential for an effective symbiosis in R. leguminosarum
UPM791.
Sánchez-Cañizares, C., Ruiz-Argüeso, T., Imperial, J., Palacios, J.M. *
355
SV-CP-14
DmeRF system is required for nickel and cobalt resistance in Rhizobium
leguminosarum bv. viciae.
Rubio-Sanz, L.*, Prieto, R.I., Palacios, J.M., Brito, B.
357
SV-CP-15
Efectos de la variación de la transpiración sobre la fijación biológica de nitrógeno y
el transporte de metabolitos a larga distancia en guisante.
*
Aldasoro Galán, J. , Arrese-Igor, C.
359
SV-CP-16
Importancia de la relación fuente/sumidero en la inhibición de la fijación de
nitrógeno.
García y García, K.*, Arrese-Igor, C.
361
SV-CP-17
Implicación de la trehalosa-6-fosfato en la respuesta a sequía de nódulos de judía.
*
Hidalgo-García, A. , Argandoña, M., González, E.M., Arrese-Igor, C., Vargas, C.,
Delgado, M.J.
363
SV-CP-18
Study of sulphur assimilation and sulphur sources used by Rhizobium
leguminosarum strain 3841 in free-living and symbiotic conditions.
Muñoz-Azcárate, O.*, Karunakaran, R., Arrese-Igor, C., Poole, P.S.
365
SV-CP-19
Towards the optimization of hydrogen production by nitrogenase: In vitro directed
evolution of Rhodobacter capsulatus molybdenum nitrogenase.
Barahona, E.*, González de Heredia, E.M., Rubio, L.M.
367
SV-CP-20
The FdxN protein is required for the biosynthesis and activity of the NifDK
nitrogenase components in Azotobacter vinelandii.
Jiménez-Vicente, E.*, Poza-Carrión, C., Navarro-Rodríguez, M., Rubio, L.M.
369
SV-CP-21
Two-Hybrid System in the nitrogen-fixing bacterium Azotobacter vinelandii.
Moreno-Urbano, E.*, López-Torrejón, G., Rubio, L.M.
371
SV-CP-22
Boron deficiency in Anabaena PCC7120: potential boron ligands.
*
Bonilla, I. , Abreu, I., Bolaños, L.
373
SV-CP-23
Metabolismo antioxidante inducido por imazamox en distintos órganos de judía
en simbiosis con Rhizobium tropici.
*
García-Garijo, A., López, M., Palma, F., Tejera, N.A., Lluch, C.
375
SV-CP-24
Implicación de las poliaminas en la respuesta a la salinidad de nódulos de
Phaseolus vulgaris.
*
Cobos, L., López-Gómez, M. , Hidalgo, J., Iribarne, C., Lluch, C.
377
SV-CP-25
Pantoea eucalypti M91 favorece la adquisición de hierro y mejora la performance
de Lotus japonicus, ecotipo Gifu, bajo condiciones de estrés alcalino.
Campestre, M.P., Castagno, L.N., Estrella, M.J., Ruiz, O.A. *
379
xxx
SV-CP-26
Caracterización de aldehído oxidasas en la simbiosis Medicago truncatula-Ensifer
meliloti en condiciones de estrés por cadmio.
Coba de la Peña, T.*, Pallol, B., Pérez, E., Pueyo, J.J., Lucas, M.M.
381
SV-CP-27
Response of the symbiotic system Medicago truncatula-Ensifer meliloti to
cadmium stress: Ionome and ion transporters.
Fedorova, E., Coba de la Peña, T., Pueyo, J.J., Lucas, M.M. *
383
SV-CP-28
Nonsymbiotic and truncated hemoglobins of legumes: biochemical
characterization and subcellular localization.
*
Sainz, M., Pérez-Rontomé, C., Ramos, J., Mulet, J.M., James, E.K., Becana, M.
385
SV-CP-29
Producción y actividad nodular de dos variedades de alfalfa (Europa y Aragón) en
condiciones de CO2 elevado.
Kizildeniz, T., Erice, G., Sanz-Sáez, A., Aguirreolea, J., Sánchez-Díaz, M., Irigoyen, J.J.*
387
SV-CP-30
Análisis de la diversidad de polihidroxialcanoato-sintasas (PhaC) en
Quelas, J.I.*, Pérez-Giménez, J., Mongiardini, J., Carriño, J., Parisi, G., Lodeiro, A.R.
389
SV-CP-31
La mutación de ntrB en Bradyrhizobium japonicum afecta su crecimiento aeróbico
en nitrato pero no en amonio.
*
López, M.F. , Lamelza, F., Lodeiro, A.R., López García, S.L
391
SV-CP-32
Application of calcium, phosphorus and antioxidants do not promote tolerance to
high solar irradiance in Rhizobium-nodulated Phaseolus vulgaris L.
Izaguirre-Mayoral, M.L.*, Silvera, J., Rodríguez, M.
393
SV-CP-33
Localization and dynamics of the succinoglycan translocon in Sinorhizobium
meliloti cells.
Rivero, M.R., Medeot, D.B., Contreras-Moreira, B., Liaudat, J.P., Ferrari, W., Rossi, F.,
Fischer, S.E., Becker, A., Jofré, E.*
395
SV-CP-34
Implication of NRT2.5 and NRT2.6 genes in growth promotion response of
Arabidopsis by Phyllobacterium brassicacearum STM196.
Kechid, M.*, Desbrosses, G., Varoquaux, F., Djekoun, A., Touraine, B.
397
SV-CP-35
Micorrizas arbusculares: estado actual del conocimiento y perspectivas en
pastizales de Uruguay.
Pezzani, F.*
399
SV-CP-36
The potential of NIR and MIR spectroscopy to predict the P retention capacity of
Malagasy and Brasilan uplands soils.
Ramaroson, H.V.*, Marchão, R.L., Vendrame, P.R.S., Razafimahatratra, H.,
Fanjaniaina, M.L., Andriamalaza, S., Rakotondrazafy, A.F.M., Razafimbelo, T.,
401
SV-CP-37
Avaliação da produção de ácido indolacético por bactérias do gênero Azospirillum
isoladas de pastagens nativas do Pantanal Sul-Mato-Grossense.
Souza, M.S.T., Brasil, M.S.*, Reis Junior, F.B.
403
SV-CP-38
La respuesta de la expresión fitasa nodular a bajo fósforo varía según aislados de
rizobios en la simbiosis Phaseolus vulgaris L.
Morales Valdés, A.N.*, Gómez, L.A. , Amenc, L., Gregorio, C., Drevon, J.J.
405
SV-CP-39
Oxidases of diterpene phytohormone biosynthesis in Bradyrhizobium japonicum
bacteroids.
Rojas, M.C.*, Mendez, C., Baginsky, C., Hedden, P., Carú, M.
407
SV-CP-40
Effects of salts levels on phosphorus availability in non calcareous soil.
Hamdi, W.*, Seffen, M., Deleporte, P., Drevon, J.J., Blavet, D., Gérard, F.
409
SV-CP-41
Bacterial acid phosphatase activity and localization in Phaseolus vulgaris nodules.
Amenc, L.*, Drevon, J.J.
411
xxxi
SV-CP-42
Responses of soil catabolic functions in cereal-legume intercrops.
Wahbi, S., Baudoin, E., Maghraoui, T., Sanguin, H., Oufdou, K., Hafidi, M., Galiana,
A., De Lajudie, P., Prin, Y., Duponnois, R. *
413
SV-CP-43
Managing a pluridisciplinary data base: Integrating knowledge to engineer the
complexity of microbe-soil-plant interactions in FabaTropiMed agro-ecosystems
Soto, P.*, Blavet, D., Balboa, A., Zanetto, A., Drevon, J.J., Desclaux, D.
415
Session VI. Inoculants in agriculture and the environment.
SVI-P-1
Efficient soil microbial consorcia: a new dimension for sustainable agriculture and
environmental sustainability.
Cruz, C.
418
Universidade de Lisboa, Lisboa. Portugal.
SVI-P-2
Oportunidades e ameaças à contribuição da fixação biológica do nitrogênio em
leguminosas no Brasil.
Nogueira, M.A.
422
Embrapa Soja, Londrina, Paraná, Brazil.
SVI-CO-1
Selenium biofortification in wheat plants by co-inoculation of selenobacteria
strains and arbuscular mycorhizal fungy for obtaining enriched selenium foods.
Durán, P.*, Mora, M.M.
426
Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and
Biotechnological Bioresource Nucleus. Universidad de la Frontera, Temuco-Chile.
SVI-CO-2
Evaluación de la degradación de tamo de arroz por microorganismos
lignocelulolíticos.
Gutiérrez-Rojas, I.*, Pedraza-Zapata, C., Sánchez-Rodríguez, L., Moreno-Sarmiento,
N.
428
Instituto de Biotecnología, Ciudad Universitaria, Edificio Manuel Ancizar, Universidad
Nacional de Colombia, Bogotá, D.C., Colombia. Departamento de Microbiología, Facultad de
Ciencias, Laboratorio de Biotecnología Aplicada, Grupo de Biotecnología Ambiental e
Industrial (GBAI), Pontificia Universidad Javeriana, Bogotá, D.C., Colombia.
SVI-CO-3
Soil microbial phosphorus, phosphorus availability and crop yield of upland ricecommon bean intercropping under organic and mineral fertilizer inputs on
ferrallitic soil of Malagasy highlands.
Henintsoa, M.*, Andriamananjara, A., Razakatiana, A.T.E., Larvy Delarivière, J.,
430
Laboratoire des Radioisotopes (LRI), University of Antananarivo, Madagascar. Laboratoire de
Microbiologie de l’Environnement (LME), Centre National de Recherches sur l’Environnement
(CNRE), Antananarivo, Madagascar. Institut de Recherche pour le Développement (IRD), UMR
Eco&Sols, c/o LRI, Madagascar.
SVI-CO-4
Sistema uruguayo de fiscalización de inoculantes: complementación
interinstitucional y aplicación de tecnologías de la información.
*
Beyhaut, E. , Barlocco, C., Altier, N., Mayans, M.
432
INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay. 2 División Control de Insumos, DGSSAA.
M.G.A.P, Av. Millán 4703, Montevideo, Uruguay.
SVI-CP-01
Análise de crescimento e acúmulo de nutrientes pela variedade RB92579 de cana
de açúcar plantada em um Argissolo com e sem inoculação e aplicação de Nfertilizante.
Reis, V.M.*, de Oliveira, R.P., Schultz, N., de Araújo, A.P., Urquiaga, S.
434
SVI-CP-02
Effect of Maize-common bean intercropping on nitrogen uptake.
Latati, M., Pansu, M., Drevon, J.J., Ounane, S.M. *
436
xxxii
SVI-CP-03
Leguminous and cereal associations in the Sudano-sahelian agro-systems of
Burkina Faso: agronomic and socio-economic determinants.
Zongo, K.F.*, Hien, E., Drevon, J.J., Nacro, H.B.
438
SVI-CP-04
Emisión del gas de efecto invernadero óxido nitroso por nódulos de soja.
*
Tortosa, G., Bedmar, E.J., Mesa, S., Delgado, M.J.
440
SVI-CP-05
Development of biofertilisers and biocontrol agents with autochthonous
microorganisms in Spain and Dominican Republic.
Mulas, D., Díaz Alcántara, C., Barquero, M., Marcano, I., Urbano, B., Terrón, A.,
*
Velázquez, E., González-Andrés, F.
442
SVI-CP-06
Soybean yield and nodule occupancy as a function of yearly inoculation in the
Brazilian Cerrados.
Mendes, I.C.*, Reis Junior, F.B., Hungria, M.
444
SVI-CP-07
Recuperação e sobrevivência de Bradyrhizobium em sementes de soja tratadas
com fungicidas e inseticidas
Ferreira, E.*, Nogueira, M.A., Fukami, J., Gundi, J.S., Terassi, F.S., Conceição, R.,
Hungria, M.
446
SVI-CP-08
Influence of nitrate fertilization on Hg uptake and oxidative stress parameters in
alfalfa plants cultivated in two different Hg-polluted soils.
Carrasco-Gil, S., Aguareles, A., Leralta, D., Sobrino-Plata, J.*, Millán, R., Hernández,
L.E.
448
SVI-CP-09
Gluconacetobacter diazotrophicus enhances growth in initial stages of Ilex
paraguariensis development.
Dos Santos, K . C. G . , Lopez, D.S.H., Perez, L.M., Roberti, L., Figueredo, I.E., Schmid,
P.G., Peres, C.K., Rojas, C.A.*
450
SVI-CP-10
Nuevas herramientas metodológicas para la evaluación de inoculantes destinados
al tratamiento de leguminosas.
Penna, C.*, Massa, R., Cabrini, P., Scarabel, D., de Araujo, S.C., Cassan, F.
452
SVI-CP-11
Selección de cepas de rhizobia tolerantes a mercurio por su capacidad de
bioacumulación y su efectividad en leguminosas arbustivas.
Ruiz-Díez, B., Fajardo, S., Quiñones, M.A., López, M.A., Higueras, P., FernándezPascual, M.*
454
SVI-CP-12
Genetically modified rhizobia (GMR) for phytostabilization of copper in polluted
soils.
*
Pajuelo, E. , Delgadillo, P., Doukkali, J., Pérez-Palacios, B., Caviedes, M.A., RodríguezLlorente, I.D.
456
SVI-CP-13
Utilización de biorrollos vegetales en cultivos de olivar para la reducción de las
pérdidas de nitratos por escorrentía.
*
Pesciaroli, C. , Rodríguez García, E., Rodelas, B., Contreras-Medrano, V., GarcíaMartínez, F.J., González-Martínez, J., González-López, J., Osorio Robles, F.
458
SVI-CP-14
Soybean Biological Nitrogen Fixation in Brazil, inoculant quality 2010- 2011.
Beneduzi, A.*, Bangel, E.V., Alvarenga, S.M., Ferreira, S., Bertolo, F., Silva, G., Freire,
J.R.J.
460
SVI-CP-15
Ensayo en diferentes cultivos y condiciones de un biofertilizante en base a
Pseudomonas spp y Azospirillum spp.
Rosas, S.*, Neiderhauser, M., Bettera, C., Bosch, E.
462
SVI-CP-16
Mistura polimérica como veículo na formulação de inoculante rizobiano para
feijão-caupi.
*
Xavier, G.R. , Silva Júnior, E.B., Fernandes Júnior, P.I., Oliveira, P.J., Rumjanek, N.G.,
Boddey, R.M.
464
xxxiii
SVI-CP-17
Substrate-based vs commercial inocula of arbuscular mycorrhizal fungi in lettuce.
Garmendia, I., Mangas, V.J.*
466
SVI-CP-18
Algunas experiencias con inoculantes de hongos Glomeromycota y bacterias
solubilizadoras de roca fosfórica en sistemas agrícolas venezolanos.
*
Toro, M. , Mora, E., López-Hernández, D.
468
SVI-CP-19
Plant-growth promotion and biocontrol properties of Colombian Streptomyces
spp. isolates to control bacterial rice diseases.
Suárez-Moreno, Z.R., Vergara, D.I., Vinchira-Villarraga, D.M., Castellanos, L., Ramos,
*
F.A., Degrassi, G., Guarnaccia, C., Venturi, V., Moreno-Sarmiento, N.
470
SVI-CP-20
Microbial consortia of mycorrhizal fungi and plant-growth promoting
rhizobacteria: a tool to increase maize nutrient use efficiency.
Correia, P.*, Carvalho, L., Meleiro, A.I., Reis, A., Costa, F., Delgado, M., Mesquita, L.,
Dores, J.M., Patanita, M., Castro Pinto, J., Varennes, A., Carolino, M., Cruz, C.
472
SVI-CP-21
Mycelial inoculant production of the ectomycorrhizal fungus Amanita caesarea.
Daza, A., Camacho, M., Romero de la Osa, L., Santamaría, C.*
474
SVI-CP-22
Organic selenium forms from selenobacteria strains able to enhance selenium in
wheat grain.
*
Mora, M.L. , Acuña, J.L., Durán, P., Demanet, R.
476
SVI-CP-23
Efecto de metales pesados en el crecimiento de bacterias aisladas del compost de
lodos de depuradora.
Vela Cano, M.*, Castellano Hinojosa, A., Fernández Vivas, A., Martínez Toledo, M.V.
478
SVI-CP-24
Capacidad de nodulación y fijación de nitrógeno de cepas nativas de
Bradyrhizobium sp. en el cultivo de guandul (Cajanus cajan, L.) bajo ambiente
controlado, en República Dominicana.
Monegro, F., Sosa, R., Vicioso, A.F., Pimentel, A., González-Andrés, F., Díaz Alcántara,
C.A.*
480
SVI-CP-25
Mycorrhizal inoculation in organic farming can improve fruit quality.
Aguirrebengoa, M., Rivero, J., García, J.M., Azcón-Aguilar, C., López Raez, J.A., Pozo,
M.J.*
482
SVI-CP-26
Efecto del herbicida MCPA y flumetsulam en bacterias nitrificantes en suelos
volcánicos tratados con urea.
Palma, G.*, Jorquera, M., Marileo, G., Briceño, G., Demanet, R., Mora, M.L.
484
SVI-CP-27
Produção de biomassa e fixação biológica de nitrogênio em Flemingia
macrophylla.
Abboud, A.C.S.*, Salmi, A.P.
486
SVI-CP-28
A construção de uma rede de promoção do benefício da fbn através dos
inoculantes: uma proposta metodológica de parceria público privado.
Amâncio, C.*, Amâncio, R., Garofolo, A.C., Xavier, G.R., Hungria, M., Cipriano, R.,
Balbinot Jr, A., Motta, R.
488
SVI-CP-29
FP7 REFERTIL Project: results on Input materials and Compost on Month 24.
*
*
González Granados, I. , Segura Muñoz, I., Estrada de Luis, B.I., García García, R.
490
xxxiv
Award “Antonio J. Palomares” Conference
Simplicity turned out complicated: How FixK2, a key regulator of genes for symbiosis, is
exquisitely controlled by different means.
Mesa, S.
493
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior
de Investigaciones Científicas, Granada, Spain.
Closing Conference
Uma década de ouro se aproxima para a microbiologia do solo: expectativas da pesquisa, da
indústria, dos agricultores e da sociedade.
Mariangela Hungría.
497
Embrapa Soja, Londrina, Paraná, Brazil
Indexes
Author index
506
Participants
519
xxxv
Opening Session
Opening Session
Cámara, M.*
Centre for Biomolecular Sciences, School of Life Sciences, University of Nottigham, Nottingham NG7
2RD.
*
[email protected]
Quorum sensing: a universal signaling language
Bacteria are highly social organisms capable of sophisticated co-operative behaviours
mediated via chemical communication or ‘quorum sensing’ (QS). This is a gene
regulatory mechanism usually defined as cell population density dependent regulation
and is mediated via self-generated extracellular signal molecules (Figure 1) (Joint et al.,
2007; von Bodman et al., 2008; Williams et al., 2007). These low molecular weight
compounds or ‘autoinducers’ activate or repress QS target genes once a critical
threshold concentration of signal has been reached. The key components of any QS
‘module’ are the QS signal synthase, the signal receptor and the signal molecule (Figure
2) ( Williams et al., 2007).
γ-Butyrolactones
N-Acylhomoserine Lactones
Many bacteria possess several
HO
O
interacting QS gene regulatory
OH
O
CH
modules employing multiple
AI-2
2-Alkyl-4signal molecules. Such QS
Cyclic Pep des
Quinolones
HO OH
HO
O
systems are often subject to
B
O HO
DSF
O
positive
feedback
CH
Furanones
CSP-1
(autoinduction) which at high
C18-FAME
-EMRLSKFFRDFILQRKKpopulation densities results in
a marked increase in QS signal
Gram-posi ve
Gram-nega ve
molecule (QSSM) production
and consequently enhanced
expression of the target
gene(s) (Figure 2). Bacterial
Farnesol
Oxylipids
cell-to-cell
communication
does not however only occur
at high cell population
Figure 1. Chemical structures of a range of quorum
sensing signal molecules in eukaryotes and prokaryotes
densities and QS has been
adopted as a generic term for
the description of any bacterial intercellular communication involving diffusible signal
molecules. Bacteria employ a chemically diverse range of molecules as QSSMs some
of which interact with receptors at the cell surface while others act following
internalization ((Joint et al., 2007; Williams et al., 2007).
R1
R2
O
H
O
N
H
HO
O
H
3
O
O
R1
N
H
O
R2
COOH
O
OH
S
S
H Tyr
3
Ser Thr
N
H
O
Phe
Me
NH
Asp
Ile
2HN
PROKARYOTES
O
O
COOH
H3CO
EUKARYOTES
Until the early 1990’s, N-acylhomoserine
lactone
(AHL)mediated regulation of gene
expression was considered to be
interesting but restricted to just a
few
esoteric
bioluminescent
marine bacteria related to Vibrio
fischeri.
However, the Gramnegative plant pathogen Erwinia
carotovora was unexpectedly
Figure 2: Typical prototype of quorum sensing cascade
2
Opening Session
discovered to use N-3-oxohexanoyl-homoserine lactone (3OC6-HSL) to control both
the production of a carbapenem antibiotic and virulence. Mutants which could not
synthesize 3OC6-HSL were avirulent in a plant model of infection, and virulence could
be restored in these mutants by the addition of synthetic 3OC6-HSL. This discovery
was followed by a major surge of interest in the role of AHLs in bacterial gene
expression. A family of AHL signal molecules has now been identified which varies
predominantly in the presence or absence of an acyl chain substituent group at C3 (oxoor hydroxy-) and the length of the N-acyl side chain. In addition, genes encoding
homologues of the V. fischeri LuxI and LuxR proteins have identify from many
different Gram-negative associated with human, animals and plants. Since this initial
discoveries, quorum sensing signal molecules of a novel nature have been identified not
only in Gram negative and positive bacteria but also in eukaryotic organisms such as
yeast (Figure 1) ( Williams et al., 2007)
Quorum sensing and cross-talk QSSMs have been found in different environments from
the human host during infection to marine biofilms providing evidence that QS is active
in vivo situations. These molecules not only affect the expression the biology of the
organisms that make them but can also affect the behaviour of other organisms from
different species and kingdom. Figure 3 illustrates some examples of crosscommunication.
Cross-talk bacteria-bacteria
While most investigations of QS signal transduction pathways have focused on the
response of the producer to its own signal, in natural environments and in mixed
infections, QS signal molecules are likely impact on the behavior of other microorganisms which either produce similar signals or very different signals. In the latter
case, the P. aeruginosa QS signals 3-oxo-C12-HSL has been shown to inhibit the
growth of Staphylococcus aureus at concentrations above 30 µM whereas at sub-growth
3
Opening Session
inhibitory concentrations inhibited the expression of the staphyloccal agr-mediated
quorum sensing system, enhancing the expression of colonisation factors but switching
off the production of host-damaging toxins (Qazi et al., 2006).
Cross-talk bacteria-yeast
Cross-communication has also been found between bacteria and yeast using QSSMs.
One example is the interactions observed between P. aeruginosa and Candida albicans
which can co-exist in certain environments The QSSM 3-oxo-C12-HSL from P.
aeruginosa can suppres filamentation in C. albicans without affecting fungal growth
whereas the sesquiterpene farnesol used as a QSSM in C. albicans can interfere with QS
in P. aeruginosa (Cugini et al., 2007; Cugini et al., 2010). Current research is looking at
how wide spread these mechanisms of cross-talk between eukaryotes and prokaryote
using QSSMs are.
Cross-talk bacteria eukaryotic cells
A significant amount of work has shown that QSSM molecules can have a serious
impact in the mammalian host by altering immune responses, producing strong
cardiovascular effects resulting in the potential increase in blood flow to the site of
infection and disrupting tight cell junction to enable invasion. This multiple impact
facilitate the survival of pathogenic bacteria under hostile environment of the host
(Williams and Cámara, 2009).
Cross-talk between bacteria and plants
It has been demonstrated that QS is required for the successful colonisation of plant
hosts by bacteria. The best studied QSSMs in this relationship are AHLs. In plantassociated Gram-negative bacteria, AHLs are used by symbiotic, pathogenic, and
biological control strains to regulate a wide range of phenotypes including virulence,
rhizosphere competence, conjugation, the secretion of hydrolytic enzymes, and the
production of antimicrobial secondary metabolites. Very importantly, AHLs play a key
role in bacterial-plant cross-signaling, and some plants have been shown to be able to
reprogram gene expression in the presence of these bacterial molecules. In general
AHLs with short N-acyl chains can promote plant growth by inducing a change in the
hormonal balance between indole acetic acid and cytokinin whereas some AHLs with
longer N-acyl chain tend to modulate root development and root hair formation
although the mechanisms behind this are still not well understood. Finally some AHLs
with long N-acyl chains have been shown to induce resistance against microbial
pathogens. Interestingly, plants have develop the ability to interfere with AHL-QS
systems by producing small molecules which can interfere with bacterial QS-mediated
mechanisms (Gonzalez and Venturi, 2013). The close relationship between QS
producers and the plants reach such level that a new class of AHL signals have been
found which require the plant precursors for its biosynthesis. These AHLs are pcoumaroyl-AHLs. The precursor molecule p-coumarate is not synthesized by the
bacterium but is provided by the plant and it can be found in the plant environment
suggesting that bacteria can only synthesise these signals in the proximity of the plant
(Schaefer et al., 2008).
Cross-talk bacteria-algae
The green seaweed Ulva is the most common macro-alga contributing to biofouling of
man-made surfaces throughout the world with enormous reproductive potential
releasing vast quantities of motile zoospores from each thallus. Zoospores were shown
4
Opening Session
to preferentially settle on top of AHL-producing bacterial biofilms and hence using
these signal molecules as a cue for the selection of sites for attachment (Joint et al.,
2002). The mechanisms responsible for this seems to involve a process of chemokinesis
which affects the swimming behaviour of the zoospores through the induction of an
increase in intracellular Ca2+ levels. Interestingly, bacteria producing AHL degrading
enzyme within microbial biofilms on rocks can interfere with the settlement of
zoospores in mixed biofilms showing a direct correlation between the levels of AHLs,
degradation activity and spore settlement (Tait et al., 2009). Similarly, reduction of
AHL stability by the pH of sea water can affect spore settlement (Tait et al., 2005).
Very recently it has been demonstrated that epiphytic bacteria associated with Ulva can
interfere with the germination and growth of Ulva zoospores and that this interference is
mediated via the production of AHLs by these bacteria, revealing an additional role for
AHLs per se in the interactive relationships between marine bacteria and Ulva
zoospores (Twigg et al., 2013).
In conclussion, quorum sensing is a very versatile bacterial language with an important
impact on the relationship between bacterial species and between bacterial
communitites and their hosts. Whilst these relationship can sometimes result in very
postive symbiosis, in some instances it can have a negative efffect on the host.
Understanding better this interactions will be paramount in may areas of research
including agriculture.
REFERENCES
Cugini, C., et al. (2007). Mol. Microbiol. 65: 896-906.
Cugini, C., et al. (2010). Microbiology 156: 3096-3107.
Gonzalez, J.F., and Venturi, V. (2013). Trends Plant Sci, 18: 167-174.
Joint, I., et al. (2007). Philos. T. Roy. Soc. B 362: 1115-1117.
Joint, I., et al. (2002). Science 298: 1207.
Qazi, S., et al. (2006). Infect. Immun. 74: 910-919.
Schaefer, A.L., et al. (2008). Nature 454: 595-599.
Tait, K., et al. (2005). Environ. Microbiol. 7: 229-240.
Tait, K., et al. (2009). Environ. Microbiol. 11: 1792-1802.
Twigg, M., et al. (2013). Environ. Microbiol. (in press).
von Bodman, S.B., et al. (2008). J. Bacteriol. 190: 4377-4391.
Williams, P., and Cámara, M. (2009). Curr. Opin. Microbiol. 12: 182-191.
Williams, P., et al. (2007). Philos. T. Roy. Soc. B 362: 1119-1134.
5
Session I
Ecology, diversity and evolution
microorganisms beneficial to plants
of
Session I
SI-P-1
Diversidad y evolución de las comunidades microbianas en la rizosfera
tras un incendio forestal.
Fernández-López, M.1*, Fernández-González, A.J.1, Cobo-Díaz, F.J.1, Villadas, P.J.1,
Tringe, S.G.2, Toro, N.1
1
Departamento de Microbiología del Suelo, Estación Experimental del Zaidín, CSIC, calle Profesor
Albareda 1, 18008 Granada, España. 2 Joint Genome Institute, Dept. of Energy, Walnut Creek, California,
USA.
* [email protected]
Ecología microbiana y nuevas tecnologías de secuenciación masiva.
Si como científicos pioneros de los estudios de diversidad microbiana podemos citar a
Louis Pasteur y a Robert Koch, en los de ecología microbiana no podemos dejar de
mencionar a Vladimir I. Vernadsky, Segei Winogradsky y Martinus Beijerinck. El
primero de ellos definió los conceptos de ciclos biogeoquímicos y el de biosfera, el
segundo trabajó en los ciclos biogeoquímicos de S, Fe y N, así como desarrolló el
concepto de quimioautotrofía, y por su parte Beijerinck ideó los cultivos de
enriquecimiento, trabajó en el ciclo del N y aisló los microorganismos responsables de
este proceso. A pesar de que sus inicios están en el siglo XIX, el desarrollo real de la
ecología microbiana se ha producido gracias a las técnicas de biología molecular. Así no
podemos olvidar los trabajos de Carl Woese en la década de los años 70 del siglo XX, a
partir de los que se estableció el gen 16S rRNA como herramienta con validez
taxonómica y filogenética para identificar microorganismos. La introducción de estas
técnicas moleculares ha permitido realizar estimaciones realistas sobre la diversidad de
microorganismos presentes en el suelo, constituyendo un tópico citar lo mucho que
desconocemos. Así se ha citado ampliamente que los microorganismos constituyen la
mayor biodiversidad del planeta (Gans et al., 2005; Rusch et al., 2007) y comprenden la
mayor parte de la biomasa de los organismos que lo habitan con 10 3-104 kg de masa
microbiana por hectárea de superficie del suelo (Fierer et al., 2007), que sólo hemos
sido capaces de cultivar el 1 % del total de microorganismos; o que en 1 gramo de suelo
podemos encontrar entre 103 y 104 especies diferentes, representando una abundancia
total de 109 procariotas por gramo de suelo (Gans et al., 2005, Torsvik et al., 1990). De
lo expuesto anteriormente se deduce que el que un microorganismo pueda ser cultivado
es más una excepción que la regla. Todo esto nos lleva a concluir que debemos aplicar
técnicas moleculares a los estudios de las comunidades microbianas, si queremos tener
una visión realista de lo que está ocurriendo en el medio ambiente.
En este contexto, Handelsman y colaboradores acuñaron el término
“metagenoma” para definir el conjunto de los genomas de todos los organismos
presentes en un determinado hábitat. En su primera acepción, se consideró el
metagenoma como el resultado de extraer el ADN de todo un ecosistema para clonarlo
en complejas genotecas de ADN recombinante (librerías de ADN ambiental).
Posteriormente estas genotecas se sometían a protocolos de búsqueda de compuestos de
interés o de identificación de genes que aporten información sobre las rutas metabólicas
activas en el ecosistema, el funcionamiento potencial del ecosistema (Hall, 2007). Estas
aproximaciones metodológicas también se han aplicado al estudio de la rizosfera tanto
de plantas con interés agronómico como forestal (Fernández-López et al., 2013). Hasta
ahora, el éxito de estos métodos metagenómicos dependía en gran medida de la
capacidad de clonar fragmentos de ADN de suelo suficientemente grandes en los
vectores apropiados y de su capacidad para expresar la información genética en
hospedadores heterólogos de forma eficiente (Rondon et al., 2000).
7
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Sin embargo el desarrollo de las tecnologías de secuenciación masiva (NGS) a partir del
año 2005 nos permite obviar la construcción de estas genotecas para pasar a secuenciar
directamente el ADN ambiental extraído del suelo, es decir sin necesidad de clonarlo en
ningún tipo de vector. El desarrollo vertiginoso de estas tecnologías de secuenciación
masiva es el que nos ha llevado a una nueva era, permitiendo la aparición de
aproximaciones de tipo post-genómico (Medini et al., 2008). Al realizar la
secuenciación de forma directa se evitan los sesgos motivados por el propio proceso de
clonación y obtenemos una visión más realista de los procesos que ocurren en el suelo.
Estos nuevos métodos de secuenciación basados en distintas técnicas y puestos a punto
por diferentes compañías, tienen en común su bajo coste y su alto rendimiento: el gran
número de lecturas que ofrecen en un periodo corto de tiempo. Así la pirosecuenciación
454 Titanium Plus de Roche permite obtener 1 millón de lecturas de 800 bp, la
secuenciación por síntesis de Illumina nos ofrece 1.500 millones de lecturas de 2 x 100
bp o la secuenciación por ligación SOLID rinde 171 millones de lecturas de 70 bp
(Zhang et al., 2011). Una alternativa es la aplicación de estas técnicas a la secuenciación
de amplicones de genes de interés, como puede ser el 16S rRNA, de esta manera se
puede conocer la diversidad presente en las comunidades microbianas con una cobertura
superior al 90 %.
Por otra parte las tecnologías NGS han permitido que se empiece a desarrollar la
“metatranscriptómica”. En esencia, esta aproximación consiste en la extracción del
ARN total presente en el suelo, para posteriormente sintetizar un cDNA mediante el
empleo de oligonucleótidos cebadores al azar y proceder a su secuenciación mediante
cualquiera de las técnicas NGS. A diferencia de la metagenómica que nos ofrece una
visión del catálogo general de especies y de procesos funcionales que pueden estar
presentes y desarrollarse en un ecosistema, la metatranscriptómica, nos ofrece una
visión de las especies y procesos metabólicos que están activos. Por tanto este doble
abordaje nos permite describir tanto el potencial de cada ecosistema (metagenoma),
como los organismos procariotas y procesos que actúan en un momento determinado
(metatranscriptoma). Esta aproximación se ha aplicado para el estudio de distintos
ecosistemas incluyendo el océano abierto o columnas de agua del mismo (Stewart et al.,
2010), suelos, suelos agrícolas, compost, muestras fecales y orales de mamíferos, y
aguas residuales (McGrath et al., 2008), así como en suelos forestales (Baldrian et al.,
2012).
Bosque mediterráneo e incendios forestales.
Donde las condiciones ambientales lo permiten, los bosques son la etapa clímax en la
sucesión de un ecosistema, presentando al mismo tiempo la menor diversidad de
especies y la mayor especialización de las mismas. Esto hace que pequeños cambios
ambientales puedan tener un gran impacto sobre estas formaciones. En los ecosistemas
de tipo mediterráneo las comunidades vegetales están expuestas a unas condiciones
climáticas que pueden considerarse como extremas ya que alternan periodos de heladas
invernales, sequía y alta temperatura estival, y ocasionales lluvias torrenciales. Además
en un escenario de cambio climático como el que se prevé para la zona mediterránea,
con una temperatura mayor y una precipitación menor, catástrofes como los incendios
forestales se verán incrementadas. En España, la superficie afectada por incendios
forestales alcanzó una media de 127.209 ha al año en el periodo comprendido entre los
años 2000-09, incluyendo en esta superficie bosques, matorral y formaciones herbáceas
(http://www.magrama.gob.es/es/biodiversidad/temas/defensa -contra-incendiosforestales/estadisticas-de-incendios-forestales/). En la cuenca mediterránea, la
intervención humana promoviendo el pastoreo, las talas o las quemas controladas ha
8
Session I
SI-P-1
dado lugar al desarrollo de una vegetación específica y adaptada a estas condiciones.
Entre esta vegetación podemos destacar a la encina, Quercus ilex subsp. ballota, que
tiene capacidad de rebrotar desde la raíz tras un incendio forestal. La encina es la
quercínea que ocupa una mayor extensión en España (96.579 Km2), aunque sus bosques
son muy escasos ya que en su mayoría han sido manejados hasta la actual fisonomía de
dehesas (Felicísimo et al., 2011). Se trata de un árbol o arbusto perenne ampliamente
distribuido por toda la región mediterránea, apareciendo desde el nivel del mar hasta los
1.700 m de altitud en exposiciones no umbrías, con climas desde frío semiárido hasta
mediterráneo húmedo templado, aunque prevalece en zonas áridas con precipitaciones
estivales bajas y máximas elevadas, siendo indiferente al tipo de suelo.
En el mes de Septiembre del año 2005 se produjo un incendio forestal en el Parque
Nacional de Sierra Nevada, que afectó 3.416,74 has. (Gómez-Zotano et al., 2010).
Aunque la cubierta vegetal más afectada en este incendio fue la de matorral, hay que
señalar que la quema de vegetación forestal constituyó casi el 21% del total, estando
localizada principalmente en los términos municipales de Lanjarón y Lecrín. El encinar
afectado supuso el 1,56% del total de la vegetación, con una superficie de 53,41 has;
pero si sumamos los distintos tipos de cubiertas vegetales (tomillar, espinal, piornal,…)
afectadas que presentaban encinas en su composición, este porcentaje se eleva al
12,06% lo que se traduce en una superficie de 412 has quemadas (Gómez-Zotano et al.,
2010).
Microbiota rizosferica tras un indendio forestal.
Las altas temperaturas alcanzadas en un incendio forestal inducen cambios inmediatos
en las propiedades físicas y químicas del suelo, cuya intensidad depende de la gravedad
del incendio y del tipo de suelo (Certini 2005). Estos cambios más las condiciones
ambientales post-incendio determinarán los cambios de las características biológicas del
suelo como la biomasa y la actividad microbiana (Wang et al., 2012). Además se
produce una alteración de la estructura de las comunidades microbianas con un
incremento de las bacterias formadoras de esporas más resistentes a las altas
temperaturas (Yeager et al., 2005; Bárcenas-Moreno et al., 2011). Hay que tener en
cuenta que después de un incendio forestal, de forma efímera, hay una ganancia neta de
Nitrógeno (N) disuelto. Así se ha concluido que en las zonas mediterráneas hay un
incremento del Carbono orgánico del suelo y del N total después del incendio, pero no
de la mineralización de N (Wang et al., 2012), por lo que se incrementa la posibilidad
de pérdidas de este elemento debido a lixiviación y a una menor actividad. Las bacterias
del suelo son componentes esenciales del ciclo biogeoquímico del N, pero han sido
poco estudiadas en las condiciones del suelo posteriores a un incendio forestal (Goodale
and Aber, 2001).
En nuestro grupo estamos trabajando en estas condiciones en la rizosfera de especies
forestales, en concreto en encinas. Para estudiar la diversidad y la evolución de las
comunidades microbianas hemos optado por la aplicación de técnicas de secuenciación
masiva, tanto 454 Titanium como Illumina. Así, la rizosfera de encinas afectadas y no
afectas por el incendio forestal del año 2005, la hemos muestreado 3 y 6 años después.
El ADN total obtenido del suelo ha sido tratado con 2 aproximaciones:
a) Amplificación del gen 16S rRNA y pirosecuenciación para determinar la
diversidad microbiana de forma exhaustiva.
b) Secuenciación directa del ADN mediante el sistema Illumina para determinar
las variaciones del ciclo del N por efecto del incendio.
El detalle y las implicaciones de estos resultados se discutirá en la presentación.
9
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SI-P-1
AGRADECIMIENTOS
Este trabajo ha sido financiado por el Organismo Autónomo Parques Nacionales del
Ministerio de Medio Ambiente y Medio Rural y Marino (proyecto OAPN 21/2007), por
la Junta de Andalucía (proyecto de excelencia P08-CVI-03549) y por el programa
Consolider-Ingenio (CSD 2009-00006). AJFG es beneficiario de una beca FPU del
Ministerio de Innovación y Ciencia, y JFCD de una beca predoctoral de la Junta de
Andalucía.
BIBLIOGRAFÍA
Baldrian, P., et al. (2012). ISME J. 6: 248-258.
Bárcenas-Moreno, G., et al. (2011). Biol. Fert. Soils 47: 261-272.
Certini, G. (2005) Oecologia 143: 1-10.
Felicísimo, Á.M., et al. (2011). Impactos, vulnerabilidad y adaptación al cambio climático de la
biodiversidad española. 1. Flora y vegetación. Ministerio de Medio Ambiente y Medio Rural y
Marino, Madrid.
Fernández-López, M., et al. (2013). En: Encyclopedia of Metagenomics. K.E. Nelson, S. Highlander, F.
Rodríguez-Valera
y
B.A.
White
(eds.).
http://www.springerreference.com
DOI:
10.1007/SpringerReference_304477.
Fierer, N., M. et al. (2007). Appl. Environ. Microbiol. 73: 7059-7066
Gans J, M. et al. (2005). Science 309: 1387-1390.
Gómez-Zotano, J., et al. (2005). Cuadernos Geográficos 37: 205-214.
Goodale, C.L., and J.D. Aber (2001) Ecol. Appl. 11: 253-267.
Hall, N. (2007) J. Exp. Biol. 209: 1518-1525.
McGrath, et al. (2008).J. Microb. Meth. 75: 172-176.
Medini D, D. et al. (2008). Nature Rev. Microbiol. 6: 419-430
Rondon M.R., P.R. et al. (2000). Appl Environ Microbiol 66:2541-2547.
Rusch, D.B., et al. (2007). PLoS Biol. 5: e77.
Stewart, F.J., et al. (2010). ISME J 4: 896-907.
Torsvik V, et al. (1990). Appl. Environ. Microbiol. 56: 782-787.
Wang, Q., et al.. (2012). Forest Ecol. Manag. 271: 91-97.
Yeager, C.M., et al. (2005). Appl. Environ. Microbiol. 71: 2713-2722.
Zhang, J., et al. (2011). J. Genet. Genomics 38: 95-109.
10
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SI-P-2
Species and symbiovars within Mesorhizobium: diversity and host
range.
Laranjo, M.1, 2*, Oliveira, S.1
1
ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Portugal
IIFA-Instituto de Investigação e Formação Avançada, Universidade de Évora, Portugal
*
[email protected]
2
ABSTRACT
The genus Mesorhizobium includes species able to nodulate a wide variety of legumes.
Our aim was to investigate the evolutionary relationships among mesorhizobia species
that nodulate the same host plant. A multilocus sequence analysis (MLSA) phylogeny
of the Mesorhizobium genus showed a higher resolution compared to the 16S rRNA
gene phylogeny. The phylogenies of symbiosis genes, such as nodC, are not congruent
with the phylogenies based on core genes, reflecting rhizobial host range and legume
phylogeny. This suggests that Mesorhizobium species are able to exchange symbiosis
genes by lateral gene transfer (LGT), thus acquiring the ability to nodulate a new host.
Phylogenetic analyses of the Mesorhizobium genus based on core or accessory genes
reveal complex evolutionary relationships and a high genomic plasticity, rendering the
Mesorhizobium genus as a good model to investigate rhizobia genome evolution and
adaptation to different host plants.
INTRODUCTION
Rhizobia are the root nodule bacterial symbionts of legumes, which fix atmospheric N2
in a process known as biological nitrogen fixation. All described rhizobial species
belong to ten genera of Alpha- and three genera of Betaproteobacteria. Mesorhizobia
nodulate a wide variety of legumes, such as chickpea (Sprent, 2009). There are 25
Mesorhizobium species. Additionally, five more species are listed in “IJSEM-Papers in
Press”. This genus includes a non-rhizobium-M. thiogangeticum (Ghosh & Roy, 2006).
Biovars have been described in bacteria as a group of strains distinguishable from others
of the same species on the basis of physiological or biochemical characters. In rhizobia,
biovars have been used to distinguish symbiotically distinct groups within a single
species (Rogel et al., 2011). These symbiovars may belong to different species due to
lateral transfer of symbiosis genes (Laranjo et al., 2008).
A Mesorhizobium MLSA phylogeny based on the 16S rRNA gene, the ITS region
(16S–23S rRNA) and five core genes: atpD (ATP synthase F1, β subunit), dnaJ (DnaJ
chaperone), glnA (glutamine synthetase I), gyrB (DNA gyrase β subunit) and recA
(recombinase A) is presented. Moreover, phylogeny of the symbiosis gene nodC (Nacetylglucosaminyltransferase) is compared with a legume phylogeny based on the
chloroplast maturase K (matK) gene (Wojciechowski et al., 2004).
MATERIAL AND METHODS
Phylogenetic analyses were conducted with MEGA5 using the maximum likelihood
method. The best-fitting evolutionary model of nucleotide substitutions was determined.
A bootstrap confidence analysis was performed on 100 replicates. Azorhizobium
caulinodans was used as outgroup.
RESULTS AND DISCUSSION
The 16S rRNA gene based phylogeny including all 25 species is shown in Figure 1.
Four species groups (A to D) can be considered and at least two species have an
11
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SI-P-2
undefined position, namely M. albiziae and M. chacoense. M. thiogangeticum has a
clearly separate position from other mesorhizobia.
Mesorhizobium mediterraneum UPM-Ca36
93 Mesorhizobium temperatum SDW 018
Mesorhizobium muleiense CCBAU 83963
47
86
Mesorhizobium robiniae CCNWYC 115
Mesorhizobium caraganae CCBAU 11299
70 Mesorhizobium gobiense CCBAU 83330
A
Mesorhizobium metallidurans STM 2683T
59
Mesorhizobium tarimense CCBAU 83306
Mesorhizobium tianshanense A-1BS
Mesorhizobium tamadayense Ala-3
41
70
Mesorhizobium huakuii IFO 15243
Mesorhizobium opportunistum WSM2075
Mesorhizobium amorphae ACCC19665
21
61
Mesorhizobium septentrionale SDW 014
B
Mesorhizobium plurifarium LMG 11892
95 Mesorhizobium silamurunense CCBAU 01550
Mesorhizobium albiziae CCBAU 61158
95
96
Mesorhizobium australicum WSM2073
Mesorhizobium shangrilense CCBAU 65327
100
90
Mesorhizobium ciceri UPM-Ca7
C
Mesorhizobium loti LMG 6125
Mesorhizobium chacoense PR5
Mesorhizobium alhagi CCNWXJ12-2
40
100
Mesorhizobium camelthorni CCNWXJ40-4
D
Mesorhizobium thiogangeticum SJT
Azorhizobium caulinodans ORS 571
0.01
Figure 1. Mesorhizobium maximum likelihood phylogeny of the 16S rRNA gene (25 species). Tamura 3parameter model with a discrete Gamma distribution and invariant sites was used.
The 16S rRNA gene phylogeny was compared to a MLSA phylogram of seven loci
(16S rRNA, ITS, atpD, dnaJ, glnA, gyrB and recA) (Figure 2).
46 Mesorhizobium tarimense
100 Mesorhizobium tianshanense
100
48
100
73
43
Mesorhizobium gobiense
Mesorhizobium mediterraneum
Mesorhizobium temperatum
Mesorhizobium metallidurans
Mesorhizobium amorphae
100
Mesorhizobium septentrionale
74
Mesorhizobium caraganae
100
Mesorhizobium ciceri
Mesorhizobium loti
100
56
60
Mesorhizobium australicum
Mesorhizobium huakuii
Mesorhizobium opportunistum
Mesorhizobium plurifarium
Mesorhizobium chacoense
Azorhizobium caulinodans
0.5
Figure 2. MLSA maximum likelihood phylogeny for the Mesorhizobium genus (15 species). Tamura 3parameter model with a discrete Gamma distribution and invariant sites was used.
The resulting MLSA phylogeny with a subgroup of 15 species is highly resolved, with
all branches having high bootstrap support (Laranjo et al., 2012).
Although most Mesorhizobium species show relatively low sequence divergence at core
loci, there are certain groupings of species that are consistently recovered both in the
16S rRNA gene phylogeny and in the MLSA phylogeny, namely M. mediterraneum/M.
12
Session I
SI-P-2
temperatum, M. gobiense/M. tarimense/M. tianshanense, M. loti/M. ciceri, M.
amorphae/M. septentrionale, as had been observed before between phylogenies of
different core loci (Alexandre et al., 2008; Laranjo et al., 2012). This suggests that there
is little horizontal gene transfer of core loci between these groupings. However, it is not
clear that every species within each group is genetically isolated. On the other hand,
there are some species within the Mesorhizobium clade which have a poorly resolved
phylogenetic position, such as M. albiziae, M. chacoense and M. thiogangeticum,
among others.
Contrary to Rhizobium and Sinorhizobium, which carry symbiosis plasmids,
Bradyrhizobium and Mesorhizobium species harbour their symbiosis genes clustered in
the chromosome. In Mesorhizobium strains symbiosis genes are most commonly located
in chromosomal symbiosis islands (Kaneko et al., 2000; Nandasena et al., 2007) and
rarely in plasmids (Wang et al., 1999; Zhang et al., 2000). These islands have been
shown to be mobile in Mesorhizobium, but not in Bradyrhizobium.
100
100
Mesorhizobium australicum
Mesorhizobium opportunistum
Mesorhizobium metallidurans
77
Mesorhizobium ciceri
36
99
100
Cicer arietinum
Mesorhizobium mediterraneum
Mesorhizobium loti
26
Lotus
Mesorhizobium tarimense
Mesorhizobium amorphae
14
44
Biserrula pelecinus
Mesorhizobium chacoense
Amorpha fruticosa
Prosopis
Mesorhizobium temperatum
13
97
Mesorhizobium septentrionale
86
Mesorhizobium gobiense
91
Mesorhizobium tianshanense
Glycyrrhiza
Astragalus
Mesorhizobium caraganae
Mesorhizobium huakuii
Azorhizobium caulinodans
Sesbania
0.1
Figure 3. Mesorhizobium genus maximum likelihood nodC based phylogeny (15 species). Tamura 3parameter model with invariant sites was used. Legume hosts are indicated on the right-hand side.
nifH (data not shown) (Laranjo et al., 2008) and nodC (Figure 3) phylogenies are
congruent and evidence a close relationship among rhizobial strains nodulating the same
host, as suggested before (Laguerre et al., 2001).
LGT of symbiosis genes between the different species in the soil seems to be the most
plausible hypothesis to explain incongruence between phylogenies based on symbiosis
(e. g. nodC) and core genes (e. g. 16S rRNA).
Furthermore, phylogeny of symbiosis genes is compared to host legume phylogeny
(Figure 4) based on the chloroplast encoded maturase K gene (matK) (Wojciechowski et
al., 2004).
13
Session I
SI-P-2
100
100
Astragalus americanus
Biserrula pelecinus
41
Oxytropis deflexa
Cicer arietinum
68
Alhagi maurorum
100
Caragana arborescens
88
100
Glycyrrhiza lepidota
Robinia pseudoacacia
100
Sesbania vesicaria
96
Anthyllis vulneraria
36
100
100
Lotus japonicus
Clitoria ternatea
Amorpha fruticosa
Prosopis glandulosa
100
79
Acacia greggii
Albizia julibrissin
Oxalis tomentosa
0.02
Figure 4. Maximum likelihood legume phylogeny based on the matK gene (16 legume species). General
time reversible (GTR) model with a discrete Gamma distribution was used.
In conclusion, rhizobia with different chromosomal backgrounds may carry similar
symbiosis genes, explaining how the same legume host is nodulated by several different
rhizobium species.
However, it is possible that the transfer of symbiosis genes is limited to certain donorrecipient species combinations, which would mean that certain core genome features are
needed to support particular symbiovars (Laranjo et al., 2012).
ACKNOWLEGEMENTS
This work was supported by project (PTDC/BIA/EVF/4158/2012) from Fundação para a Ciência e a
Tecnologia (FCT). This work is funded by FEDER Funds through the Operational Programme for
Competitiveness Factors - COMPETE and National Funds through FCT under the Strategic Project PEstC/AGR/UI0115/2011. M. Laranjo acknowledges a Post-Doc fellowship (SFRH/BPD/27008/2006) from
FCT.
REFERENCES
Alexandre, A., et al. (2008). Int. J. Syst. Evol. Microbiol. 58: 2839-2849.
Ghosh, W., and Roy, P. (2006). Int. J. Syst. Evol. Microbiol. 56: 91-97.
Kaneko, T., et al. (2000). DNA Res. 7: 331-338.
Laguerre, G., et al. (2001). Microbiology 147: 981-993.
Laranjo, M., et al. (2008). FEMS Microbiol. Ecol. 66: 391-400.
Laranjo, M., et al. (2012). Syst. Appl. Microbiol. 35: 359-367.
Nandasena, K.G., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 1041-1045.
Rogel, M.A., et al. (2011). Syst. Appl. Microbiol. 34: 96-104.
Sprent, J.I., (2009). Legume Nodulation: A Global Perspective. Oxford, UK: Wiley-Blackwell.
Wang, E.T. et al. (1999). Int. J. Syst. Bacteriol. 49: 51-65.
Wojciechowski, M.F., et al. (2004). Am. J. Bot. 91, 1846-1862.
Zhang, X. X. et al. (2000). Appl. Environ. Microbiol. 66: 2988-2995.
14
Session I
SI-CO-1
Diversidad de hongos micorrizógenos arbusculares bajo situaciones
contrastantes de fertilización fosfatada.
García, S., Rodríguez Blanco, A*, Pezzani, F.
* [email protected]
RESUMEN
Los pastizales de Uruguay presentan bajos niveles de P por lo que son frecuentes los
mejoramientos extensivos que implican fertilización fosfatada. Este trabajo tiene como
objetivo determinar el efecto de la fertilización fosfatada sobre la diversidad de hongos
micorrizógenos arbusculares (HMA) asociados a dos especies de gramíneas de los
pastizales de Uruguay. El trabajo se basa en un ensayo con dos niveles de fertilización a
largo plazo y parcelas de pastizal natural (PN) sin fertilizar. En base a resultados de
composición florística y colonización micorrícica se seleccionaron las especies nativas
Paspalum dilatatum y Coelorhachis selloana para el estudio de diversidad de las
comunidades de HMA utilizando la técnica Terminal Restriction Fragment Length
Polymorphism (T-RFLP). Se presentan resultados correspondientes al primer muestreo
(invierno). Se detectaron diferencias significativas en los índices de riqueza y de
diversidad entre los niveles de fertilización fosfatada en las comunidades de HMA
asociadas a C. selloana. El análisis de cluster demostró una similitud entre todas las
comunidades mayor al 55% y formó 2 grupos: uno incluyó las comunidades asociadas a
P. dilatatum fertilizadas y sin fertilizar y a C. selloana sin fertilización, mientras que en
el otro cluster se separaron las comunidades asociadas a C. selloana con fertilización
fosfatada. Estos resultados evidenciarían que los efectos de la fertilización sobre la
diversidad de HMA que colonizan las plantas serían dependientes de la especie vegetal.
INTRODUCCIÓN
Uno de los beneficios más importantes que recibe la planta por los hongos
micorrizógenos arbusculares (HMA) es que estos aumentan la absorción de nutrientes,
principalmente P que se encuentra poco disponible para las plantas (Smith and Read,
2008). Los pastizales de Uruguay presentan bajos niveles de P por lo que son frecuentes
los “mejoramientos extensivos” de los pastizales que implican fertilización fosfatada
(Hernández et al., 1995). Según un relevamiento florístico realizado en nuestro sitio de
estudio, luego de 10 años de aplicada la fertilización con P, la especie Paspalum
dilatatum no modificó su frecuencia relativa en la comunidad en relación al incremento
de P, mientras que Coelorhachis selloana disminuyó su frecuencia relativa, viéndose
aumentadas las frecuencias de especies invasoras como Cynodon dactylon y Lolium
multiflorum (Pezzani et al., 2012) De acuerdo a la bibliografía, la fertilización fosfatada
puede tener efectos negativos sobre la colonización micorrícica arbuscular (Covacevich
et al., 1995; Aguilar et al., 2004). Para evaluar esto, durante los años 2011 y 2012, se
realizaron muestreos estacionales de varias especies de plantas y suelos. Los resultados
de colonización radicular por HMA muestran diferencias entre las especies nativas e
invasoras. P. dilatatum presentó altos porcentajes de colonización por HMA en todos
los tratamientos, aunque los mayores valores fueron observados en el PN y los menores
en parcelas con fertilización alta. C. selloana presentó porcentajes menores de
colonización por HMA en tratamientos con altos niveles de fertilización con P. El
potencial micorrícico (esporas en el suelo) fue afectado negativamente por la
fertilización fosfatada (Pezzani et al., 2012). Conocer la diversidad de los HMA en los
pastizales, sería un aporte complementario al conocimiento sobre estas interacciones ya
que no existen antecedentes sobre este tema en Uruguay.
15
Session I
SI-CO-1
El objetivo de este trabajo es determinar el efecto de la fertilización fosfatada sobre la
diversidad de hongos micorrizógenos arbusculares (HMA) asociados a dos especies de
gramíneas nativas de los pastizales de Uruguay.
MATERIAL Y MÉTODOS
El trabajo se basa en un ensayo instalado en el año 1996 que consiste en dos niveles de
fertilización (media: 30 kg ha -1 de P2O5 anuales y alta: 60 kg ha-1 de P2O5 anuales) y
parcelas de pastizal natural (PN) sin fertilizar. Los tratamientos se realizaron en bloques
al azar de 2 ha cada uno. Para el estudio de la diversidad de HMA que colonizan las
plantas se seleccionaron las especies nativas P. dilatatum y C. selloana. Se realizaron
dos muestreos de plantas (invierno y verano). Se extrajeron plantas de ambas especies
de cada parcela, se formaron muestras compuestas de raíces y se extrajo el ADN. Para
evaluar la diversidad se utilizó la técnica Terminal Restriction Fragment Length
Polymorphism (T-RFLP) siguiendo la metodología descripta por Mummey and Rillig
(2006). Se utilizaron los primers FLR3-FAM y FLR4, específicos para
Glomeromycotas, y la enzima de restricción MboI. Los análisis multivariados se
realizaron utilizando el programa PAST y se consideraron los picos cuya abundancia
relativa fue >1%.
RESULTADOS Y DISCUSIÓN
Se presentan resultados correspondientes al primer muestreo. El número de TRFs
(biotipos) en las comunidades de HMA varió entre 11 y 19. Las plantas presentaron 7
TRFs (biotipos) en común, mientras que otros TRFs fueron característicos de algunos
tratamientos. En las comunidades de HMA asociadas a C. selloana se detectaron
diferencias significativas entre los niveles de fertilización fosfatada tanto en el índice de
riqueza como en los índices de diversidad, mientras que estas diferencias no se
observaron en las comunidades asociadas a P. dilatatum. El análisis de cluster demostró
una similitud entre todas las comunidades mayor al 55% y formó 2 grupos. Uno de los
clusters incluyó las comunidades asociadas a plantas de P. dilatatum fertilizadas y sin
fertilizar y a plantas de C. selloana sin fertilización, mientras que en el otro cluster se
separaron las comunidades asociadas a C. selloana con fertilización fosfatada. Estos
resultados estarían demostrando que la fertilización fosfatada en las plantas de C.
selloana provocó un cambio en la estructura de las comunidades de HMA que las
colonizan, mientras que ese cambio no se observó en P. dilatatum. Para profundizar y
confirmar estos resultados se está analizando el segundo muestreo (verano).
BIBLIOGRAFÍA
Aguilar, C., et al. (2004). In: Memorias de la XX Reunión del grupo Técnico Regional del Cono Sur en el
Mejoramiento y Utilización de los Recursos Forrajeros del Area Subtropical y Tropical-Grupo
Campos. Salto, Uruguay. Pp. 281-282.
Covacevich, F., et al. (1995). Ciencia del Suelo. 13: 47-51.
Hernández, J., et al. (1995). Boletín de Investigaciones N°43. Facultad de Agronomía. Montevideo.
Mummey, D.L., and Rillig, M.C. (2006). Plant Soil 288: 81-90.
Pezzani F., et al. (2012). Resumen de la presentación oral en el VII Symposium Nacional y IV Reunión
Iberoamericana de la Simbiosis Micorrízica. 27-30 mayo 2012. Xalapa. México. p. 115.
Smith, S.E., Read, D.J (2008). Arbuscular Mycorrhizal. En: Mycorrhizal Simbiosis. Elsevier. 11-145.
16
Session I
SI-CO-2
rhizoremediation of metal contaminated estuaries using Spartina
densiflora.
Andrades-Moreno, L.1, del Castillo, I.2, Redondo-Gómez, S.1, Mesa, J., Caviedes,
M.A.2, Pajuelo, E.2, Rodríguez-Llorente, I.D.2*
1
Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla. Sevilla,
Spain. 2 Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla, c/ Profesor
García González 2, 41012 Sevilla, Spain.
* [email protected]
ABSTRACT
The total bacterial population in the rhizosphere of Spartina densiflora grown on the
Tinto and Piedras river estuaries, with different levels of metal contamination, was
analyzed by PCR-DGGE. 22 different cultivable bacterial strains were isolated from the
rhizosphere of S. densiflora grown on the Tinto river estuary. 70% of the strains showed
one or more PGP properties and high resistance to Cu. A bacterial consortium with PGP
properties and high multiresistance to heavy metals was selected for further
experiments. This consortium has probed to increase seed germination and to protect for
fungus contamination.
INTRODUCTION
In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the
most polluted areas by heavy metals in the world, S. densiflora grows over sediments
with high concentrations of heavy metals (Sáinz et al., 2004). Cambrollé et al. (2008)
found that heavy metals accumulated at different rates in S. densiflora tissues and
around its roots, concluding that these species could be used for phytoremediaton (either
phytoextraction or phytostabilization) of estuarine sediments. Although the importance
of the rhizosphere community for plant development in contaminated soils has been
recognized, nothing is known about the bacterial population that colonizes the root of S.
densiflora grown in contaminated soils.
PCR-DGGE analysis.
PCR-DGGE analysis was performed as described in del Castillo et al. (2012).
Bacterial isolation, identification and PGP properties.
Isolation, identification and evaluation of PGP properties of the isolated bacteria were
done as described in Andrades-Moreno (2012).
PCR-DGGE analysis.
The biodiversity of bacteria present both in the rhizosphere of S. densiflora
(rhizosediment) and in naked soil (sediment) in two estuaries was analysed by DGGE
(Figure 1A). Results indicated that the highest similarity corresponded to samples
coming from the estuary of the Tinto river, suggesting that soil contamination
influences bacterial population in a higher extent than the presence of the plant (Figure
1B).
17
Session I
SI-CO-2
Figure 1. PCR-DGGE analysis. (A) Analysis of bacterial populations present in the sediments (S) and the
rhizosediments (Rs) of S. densiflora grown in the estuaries of the Tinto (T) and Piedras (P) rivers. (B)
Dendrogram showing the similarity between the DGGE profiles corresponding to the four samples based
on the Dice coefficient.
Bacterial identification and PGP properties.
22 different cultivable bacterial strains were isolated from the rhizosphere of Spartina
densiflora grown on the Tinto river estuary. 16S rDNA was partially sequenced to
determine species affiliation. Copper resistance was determined, ranging from 1 to 13
mM. 7 strains were able to fix nitrogen, 10 strains produced siderophores, 10 strains
were able to solubilize phosphate and only one strain produced auxins. Based on these
characteristics a bacterial consortium comprising 3 strains were selected (Table 1).
Table 1. Resistance towards Cu and plant-growth promotion properties of the bacterial consortium
selected from the isolated stains.
Strain
Pseudomonas composti SDT3
Aeromonas aquariorum SDT13
Bacillus thuringiensis SDT14
Cu (mM)
13
7
5
PGP properties
Siderophores and phosphate solubilization
Siderophores, phosphate and auxins
Siderophores, nitrogen fixation, antifungal
Increased seed germination induced by the bacterial consortium.
Seeds of S. densiflora were germinated in presence or absence of the bacterial
consortium. After two weeks, seed germination was observed. Percentage of
germination was higher in seeds inoculated with the bacterial consortium, 62% vs 32%.
In addition, fungal contamination appeared in 72% of the seeds germinated in absence
of the bacteria, while 40% of the inoculated seeds showed fungal contamination.
ACKNOWLEGMENTS
This work was supported by project RNM07274 of the Junta de Andalucía. J. Mesa was recipient of a
FPU grant by the Ministerio de Educación, Cultura y Deporte.
REFERENCES
Andrades-Moreno, L. (2012). Ph. D. Thesis, Universidad de Sevilla.
Cambrollé, J., et al. (2008). Mar. Pollut. Bull. 56: 2037-2042.
del Castillo, I., et al. (2012). Water Res. 46: 1723-1734.
Sáinz, X., et al. (2004). Environ. Int. 30: 556-557.
18
Session I
SI-CO-3
Symbiovar loti-type genes are widely spread across different
chromosomal backgrounds, corresponding to up to nine
Mesorhizobium genospecies nodulating Cicer canariense.
Pérez-Yépez, J.1, Armas-Capote, N.1, Martínez-Hidalgo, P.2, Velázquez, E.2, PérezGaldona, R.1, Martínez-Molina, E.2, León-Barrios, M.1*
1
Departamento de Microbiología y Biología Celular. Universidad de La Laguna. Tenerife.
Departamento de Microbiología y Genética. Universidad de Salamanca.
* [email protected]
2
ABSTRACT
The phylogeny based on nodulation genes showed that the rhizobia nodulating Cicer
canariense harboured sequences belonging to three different symbiotic lineages. Most
isolates showed sequences closely related to symbiovar (sv.) loti which were widely
distributed among up to nine Mesorhizobium genospecies. Another two smaller groups
of isolates belonged to symbiotypes more restricted to particular species. Differences in
N2-fixation capability were noted between strains from different symbiotypes.
INTRODUCTION
Cicer canariense is a wild chickpea endemic to the Canary Islands. The rhizobia
nodulating this legume are found to be highly diverse, belonging or closely related to
Mesorhizobium ciceri (the typical symbiont of the domesticated C. arietinum), M.
caraganae, M. opportunistum, M. australicum, M. shangrilense, M. amorphae and the
M. metallidurans/M. tianshanense/M. tarimense/M. gobiense cluster. The nodC
phylogeny has been shown to correlate with the legume host range. Thus, in this work
we have sequenced the nodC genes from representative strains of nine mesorhizobium
genospecies to build a phylogeny which reflects the symbiovars of the C. canariense
symbionts.
Sequencing of nodC genes. The nodC genes using the pair of primers nodCF/nodCI as
described (Laguerre et al., 2001) or the pair nodCMesoF and nodCMesoR as described
(Rivas et al., 2007). The PCR-amplified products were purified (Qiaquick extraction kit,
Qiagen) and sequenced in an ABI3730XL (Macrogen, Inc.) or alternatively in a Genetic
Analyzer 3500 (Applied Biosystems; Servicio de Genómica, Universidad de La
Laguna).
Phylogenetic analyses. Sequence alignments (ClustalW) and phylogenetic analyses
were conducted using the MEGA 5.0 software package (Tamura et al., 2011). The
phylogenetic trees were inferred by the Neighbour-joining method (NJ) using Kimura’s
2-parameter model. Confidence levels of the tree branches were estimated with 1000
bootstrap replications. The sequences of rrs genes were compared with those of
bacterial type-strains using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim
et al., 2012).
Infectivity tests. Sterilized and germinated seeds were inoculated with a high cell density
suspension of the corresponding bacteria grown on YEM broth. Inoculated seedlings
were grown in vermiculite containing a N-free nutrient solution. Non-inoculated plants
were used as negative controls. Nitrogen fixation capacity was deduced from plants and
nodules appearance.
19
Session I
SI-CO-3
The phylogeny based on nodC gene sequences resolved the mesorhizobia nodulating C.
canariense into three symbiotic lineages: two symbiotypes correlating with symbiovars
ciceri and loti, while the third symbiotype has not been previously described.
Surprisingly, most isolates carried nodC sequences highly related (95%) to reference
strains from the symbiovar loti, as M. loti NZP 2213T type strain and MAFF303099.
The simbiovar loti-type sequences were the most widely distributed among the isolates,
since they were detected in nine chromosomal backgrounds of different Mesorhizobium
species. A small number of isolates had nodC and nodA sequences identical to those of
symbiovar ciceri (such as M. ciceri UPM-CaT type strain) and were only detected in
isolates with an M. ciceri-chromosome. Another group of M. caraganae and M.
opportunistum isolates presented sequences divergent from other previous known nodC
gene sequences. This could be a specific symbiovar for C. canariense, whose closest
relatives were a congruent clade of seven Mesorhizobium species (type strains of M.
tianshanense, M. gobiense, M. caraganae, M. temperatum, M. septentrionale, M.
shangrilense and M. tamadayense) which shared the highest similarities in the range of
84.6-87.0%.
These results indicated that C. canariense is a promiscuous legume that can be
nodulated by several genomic species and symbiotypes, which make it necessary to
determine the combination of core and symbiotic genes producing the most effective
symbiosis. Our preliminary results indicate that the isolates with symbiovar loti-type
genes produced well developed plants.
ACKNOWLEDGEMENTS
Supported by Ministerio de Medio Ambiente y Medio Rural y Marino, Organismo Autónomo de Parques
Nacionales (Ref. 111/2010).
REFERENCES
Jarabo-Lorenzo A., et al. (2000). Syst. Appl. Microbiol. 23: 418-425.
Laguerre, et al. (2001). Microbiolgy 147: 981-993.
Lorite, M.J., et al. (2010). Syst. Appl. Microbiol. 33: 282-290.
Rivas, R., et al. (2007). Lett. Appl. Microbiol. 44: 412-418.
Tamura, K., et al. (2011). Meth. Mol. Biol. Evol. 28: 2731-2739.
20
Session I
SI-CO-4
Molecular phylogeny and phenotypic characterization of salt tolerant
Sinorhizobia nodulating Phaseolus filiformis in Northern Mexico.
Rocha, G.1, Medina, A.1, Carreño, R.1, Bustillos, R.1, Contreras, J.L.2, Villegas, M.C.3,
Chaintreuil, C.4, Dreyfus, B.4, Le Queré, A.5, Munive, J.A.1*
1
Centro de Investigaciones en Ciencias Microbiológicas, Benemérita Universidad Autónoma de Puebla,
Mexico. 2 Facultad de Arquitectura, Benemérita Universidad Autónoma de Puebla, Benemérita
Universidad Autónoma de Puebla, Mexico. 3 Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, Mexico. 4 Laboratoire des Symbioses Tropicales et Méditerranéennes, Unité Mixte
de Recherche (113) CIRAD, IRD, Université Montpellier 2, SupAgro, USC INRA, France. 5 Laboratoire
de Microbiologie et Biologie Moléculaire, Faculté des Sciences, Université Mohammed V-Agdal, Rabat,
Morocco.
* [email protected]; [email protected]
ABSTRACT
The aim of the present work was to establish the phylogenetic relationships of a
collection of salt tolerant sinorhizobia, nodulating P. filiformis in northern Mexico, by
Multilocus Sequence Analysis (MLSA). This collection was phenotypically and
genetically characterized. P.filiformis is nodulated by two groups of sinorhizobia, one of
them related to S. meliloti, and the other one related to a new Sinorhizobium species,
closely related to S. saheli. It was apparent that there is no strict correlation between the
chromosomal genotype of a strain and its salt tolerance.
INTRODUCTION
The genus Phaseolus comprises around 50 species, all indigenous to the Americas.
Among this, P. lunatus, P. vulgaris, P. polyanthus, P. coccineus and P. acitifolius were
domesticated by prehispanic civilizations and are widely used for human consumption.
Beans, like other legume plants, can establish symbiotic relationships with nitrogen
fixing soil bacteria called rhizobia. These bacteria invade root tissues and induce the
formation of specialized structures known as nodules, where they fix atmospheric
nitrogen. Between a diversity of bean nodulating bacteria, Rhizobium etli is the
predominant species in Mexico; although some other rhizobia species have been
described as symbionts of Phaseolus vulgaris where bean cultures have been
introduced. The number of rhizobia species capable of nodulating bean shows that this
legume is a promiscuous host, with a high diversity of interactions RhizobiumPhaseolus.
A number of rhizobia strains were isolated from Phaseolus filiformis, a wild bean native
to the southwestern United States and northern Mexico. Its common names include
slimjim bean, slender-stem bean, and Wright's phaseolus. Strains were identified by
16SrDNA sequence analysis using primers designed by Normand et al. (1991) and Sy et
al. (2001). MLSA was performed by sequence analysis of housekeeping genes: recA
(modified from Gaunt et al., 2001), dnaK (Stepkowsky et al., 2003), gyrB and rpoB
(Martens et al., 2008), atpD and glnII (Vinuesa et al., 2005). Salt tolerance was
determined using 8.5 x 10 -4, 0.4, 0.6, 0.8 and 1M of NaCl in YMA medium, and at
greenhouse level. This collection was also phenotypically and genetically characterized
by carbon source utilization analysis and by restriction patterns produced by ARDRA
(using four endonucleases AluI, CfoI, HinfI and NheI) and REP-PCR (Versalovic et al.,
1991), using the software FreeTree.
21
Session I
SI-CO-4
Strains were identified as belonging to genus Sinorhizobium, varying in their tolerance
to salt-stress. Seven strains were intolerant to 0.4M NaCl, and only two sinorhizobia
strains were tolerant to high levels of salinity (1M); indicating that salt-tolerance is not
related to their ecological origin. Greenhouse test showed the salt-tolerance induced in
plants inoculated with the most salt-tolerant sinorhizobia strain EMM451. Genotypic
characterization showed 4 different genotypes. The phylogenetic relationships of
sinorhizobia collection by MLSA, using 6 chromosomal loci, showed that species P.
filiformis is nodulated by two groups of sinorhizobia, one of them related to S. meliloti
(S. meliloti EMM456), and the other one related to a new Sinorhizobium species,
closely related to S. saheli (Figure 1). There is no strict correlation between the
chromosomal genotype and the salt tolerance. Salt-tolerance determinants must reside
on extrachromosomal elements that may be subject to lateral transfer and
recombination.
100
100
S. meliloti
S. meliloti EMM456 (P. filiformis)
78
100
S. arboris
100
51
100
60
100
100
100
88
100
87
S. kostiense
S. saheli
27
99
S. medicae
100
34
S. meliloti
Sinorhizobium sp. (Phaseolus filiformis)
S. terangae
S. fredii/S. xinjiangense
S. adhaerens
Rhizobium spp.
47
M loti MAFF303099
0.02
Figure 1. Maximum likelihood species tree inferred from the concatenated alignment recA-dnaK-gyrBrpoB-atpD genes, showing the phylogenetic relationships of Sinorhizobium sp. (P. filiormis) strains and
Sinorhizobium species. The bar represents the number of expected substitutions per site. Bootstrap values
calculated for 1,000 replications are indicated.
ACKNOWLEGMENTS
This work was granted by ECOS-NORD M08-A02.
REFERENCES
Gaunt, M., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 2037-2048.
Normand, P., et al. (1991). Int. J. System. Bacteriol. 46: 1-9.
Martens, M., et al. (2008). Int. J. Syst. Evol. Microbiol. 58: 200-214.
Martens, M., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 489-503.
Stepkowski, T., et al. (2003). Syst. Appl. Microbiol. 26: 483-494.
Silva C., et al. (1999). Mol. Ecol. 8: 277-287.
Sy A., et al. (2001). J. Bacteriol. 183: 214-220.
Versalovic, J., et al. (1991). Nucl. Acids Res. 24: 6823-6831.
Vinuesa, P., et al. (2005). Mol. Phyl. Evol. 34: 29-54.
22
Session I
SI-CP-01
Phylogenetic diversity of bradyrhizobia nodulating cowpea (Vigna
unguiculata L. Walp) in Extremadura (Spain).
Bejarano, A.1*, Ramírez-Bahena, M.H.1, 2, Velázquez, E.2, 3, Peix, A.1, 2
1
Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Salamanca, Spain. 2 Unidad Asociada
Grupo Interacción Planta-Microorganismo USAL-IRNASA-CSIC. 3 Departamento de Microbiología y
Genética, Universidad de Salamanca (USAL), Spain.
* [email protected]
ABSTRACT
Vigna unguiculata, a grain legume worldwide cultivated but poorly cropped in Spain, is
a good alternative over other legumes to be included into the mediterranean diet and has
a great agronomic interest by its resistance to soil acidity, drought and high
temperatures. Cowpea establishes symbiosis mainly with Bradyrhizobium in most of
regions, and this rhizobia-legume interaction has been studied in Asia, Africa and
America. However up to date there are no data about rhizobia nodulating V. unguiculata
in Europe. In this work we analysed the phylogenetic diversity of slow-growing
rhizobia isolated from nodules of this legume in Extremadura through the analysis of
their 16S rRNA genes and 16S-23S intergenic regions. The results showed that unlike
in other continents, V. unguiculata is mainly nodulated in Spain by B. cytisi and B.
canariense and by several new phylogenetic lineages within genus Bradyrhizobium.
INTRODUCTION
Vigna unguiculata (cowpea) is cultivated in concrete regions in Spain where the main
production is located in Extremadura region. Cowpea contains high proportions of
protein (20-30%), and dietary fiber (20-35%) being of great interest in human and
animal nutrition (Fontenele-Urano-Carvalho et al., 2005). Therefore it is a good
alternative to be included into the mediterranean diet whose beneficial effects in human
health has been widely proven (Sofi et al., 2010). Moreover, this legume is
agronomically very interesting since it is very resistant to soil acidity, drought and high
temperatures as well as for its ability to establish nitrogen fixing symbiosis with
rhizobia mostly belonging to genus Bradyrhizobium. The endosymbionts of Vigna
unguiculata have been studied in Africa, Asia and America, but no data about those
present in European soils are available.
Therefore the main objective of this study was to analyse the phylogenetic diversity of
bradyrhizobia nodulating Vigna unguiculata in a soil from Extremadura in which this
legume is cultivated and to compare the Spanish cowpea nodulating strains with those
isolated from other continents.
Vigna unguiculata plants were used as trap plant in an acidic soil from Extremadura and
the isolation of rhizobia was performed following the method of Vincent (1970) on
YMA plates.
RAPD patterns were obtained as was previously described using the primer M13 (Rivas
et al., 2006). The mathematical analysis of the patterns was performed using
BioNumerics™ software (Applied Maths, Belgium).
Amplification and analysis of the 16S rRNA gene and ITS region was performed as
previously described (Velázquez et al., 2010).
23
Session I
SI-CP-01
About fifty slow-growing strains of Bradyrhizobium were isolated from Vigna
unguiculata nodules which were grouped by using RAPD fingerprinting. After the
mathematical analysis of the obtained patterns a representative strain from groups with
similarity values lower than 80% among them was selected for 16S rRNA gene and ITS
analyses.
These analyses allowed the identification of several Bradyrhizobium species in V.
unguiculata nodules as well as the detection of some putative novel species within this
genus.
The identified species were B. canariense and B. cytisi, which are reported here by the
first time in V. unguiculata nodules. Some strains cluster within strain B. japonicum
BGA-1, of uncertain taxonomic status since the strains from this group are divergent to
that formed by the type strain of B. japonicum USDA 6T.
Some other strains formed independent branches in both 16S and ITS phylogenetic trees
and then they could constitute new species within genus Bradyrhizobium.
A comparison with the strains nodulating Vigna in other continents showed that our
Spanish strains belong to different groups than those isolated in other countries with the
exception of B. canariense from which a single strain isolated in Japan has been found
in nodules of V. unguiculata.
Considering that V. unguiculata from tribe Phaseolae is indigenous of South Africa
Transvaal region (Paludosi and Ng, 1997), these results suggest an adaptation of this
legume to be nodulated by Bradyrhizobium species nodulating Genisteae species in
mainland Spain (Velázquez et al., 2010).
ACKNOWLEDGMENTS
MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF.
REFERENCES
Fontenele-Urano-Carvalho, A. et al. (2012). J. Food Compos. Anal. 26: 81-88.
Paludosi, S., and Ng, N.Q. (1997). Advances in Cowpea Research. Pp. 1-12. Edited by B.B. Singh, D.R.
Mohan Raj, and K.E. Dashiell, L.E.N. Jackai. Nigeria: Copublication of International Insitute of
Troplical Agriculture (IITA) and Japan International Research Center of Agricultural Siences
(JIRCAS).
Rivas, R., et al. (2006). Plant Soil 287: 23-33.
Sofi, F., et al. (2010). Am. J. Clin. Nutr. 92: 1189-1196.
Velázquez, E., et al. (2010). Antonie Van Leeuwenhoek. 97: 363-376.
Vincent J.M. (1970). A Manual for the Practical Study of Root-Nodule. pp. 1-13. Edited by J.M. Vincent.
Blackwell Scientific Publications, Oxford (United Kingdom).
24
Session I
SI-CP-02
MALDI-TOFF MS, a tool for diversity analysis and detection of novel
rhizobial species nodulating Phaseolus vulgaris.
Flores-Félix, J.D.1*, García-Fraile, P.1, Sánchez-Juanes, F.2, 3, 4, Ramírez-Bahena,
M.H.5, 6, Ferreira, L.2, 3, 4, Mula, D.7, Rivas, R.1, 6, González-Andrés, F.7, Peix, A.5, 6,
González-Buitrago, J.M.2, 3, 4, Velázquez, E.1, 6
1
Departamento de Microbiología y Genética, Universidad de Salamanca (USAL), Spain. 2 Unidad de
Investigación. Hospital Universitario de Salamanca, Spain. 3 Departamento de Bioquímica y Biología
Molecular. Universidad de Salamanca (USAL), Spain. 4 Instituto de Investigación Biomédica. Salamanca,
Spain. 5 IRNASA-CSIC, Salamanca, Spain. 6 Unidad Asociada Grupo Interacción Planta-Microorganismo
USAL-IRNASA-CSIC. 7Instituto de Medio Ambiente y Recursos Naturales, Universidad de León, Spain.
* [email protected]
ABSTRACT
In this work we analysed by MALDI-TOF MS (Matrix-Assisted Laser Desorption
Ionization-Time-of-Flight Mass Spectrometry) a collection of endosymbionts from P.
vulgaris which is nodulated by different genera and species of rhizobia. These
endosymbionts were isolated in different regions from the Northwest to the Southest of
Spain. The results of MALDI-TOF MS analysis showed that they belonged to
Rhizobium and Ensifer but despite a 53% of strains were correctly identified, the
remaining 47% of strains was not identified with any of species present in the
Rhizobiaceae database. This is a frequent result when the isolates of legume nodules are
analysed and grouping techniques are necessary to finish the identification schemes.
After the mathematical analysis of spectra from P. vulgaris isolates the dendrogram
obtained showed that the unidentified strains presented similarity values lower than
70% with respect to the groups containing the identified ones. The strains belonging to
the same species presented similarity internal values higher to 70% allowing the
selection of strains for housekeeping gene analysis. The combination of MALDI-TOF
MS analysis and gene sequencing allowed the detection of several new species in
Phaseolus vulgaris nodules in Spain.
INTRODUCTION
Phaseolus vulgaris, indigenous of America, is currently the most cultivated legume
worldwide after soybean and establishes nitrogen-fixing symbiosis with several fast
growing species of genera Rhizobium and Ensifer (Velázquez et al., 2010). Therefore
the P. vulgaris-rhizobia symbiosis is a good model to evaluate the reliability of
MALDI-TOF MS for the identification of rhizobia at species level as well as for the
grouping of unidentified strains in order to select them for identification by gene
analyses. MALDI-TOF MS is a technique based on protein analysis that has been
recently proposed for differentiation of genera and species from family Rhizobiaceae
(Ferreira et al., 2011). It has been previously proven that this technique is useful for
identification of legume nodules isolates belonging to already described species,
nevertheless frequently nodule isolates that do not belong to described species are
isolated.
The aim of this work was to analyze the suitability of spectra analysis to group the
unidentified rhizobial strains isolated from P. vulgaris at species level in order to select
strains for housekeeping gene analysis.
The isolates from P. vulgaris nodules growing in different Spanish locations were
obtained using the methodology described by Vincent (1970) on YMA plates. For
25
Session I
SI-CP-02
MALDI-TOF MS analysis and for DNA isolation the strains were incubated on TY
plates (Beringer, 1974) during 24h. The samples preparation, the performing of
MALDI-TOF MS analysis and that of recA and atpD genes were performed as was
previously described (Ferrerira et al., 2011).
After comparison of spectra obtained for P. vulgaris isolates with those present in the
family Rhizobiaceae database previously build in our laboratory (Ferreira et al., 2011)
about 53% of strains were identified as already described species of Rhizobium, such as
R. leguminosarum and R. giardinii, and Ensifer, such as E. fredii. The remaining strains
although were closely related to other species of these two genera, the score values were
lower than 2.0 indicating that they belong to undescribed species.
The mathematical analysis of the spectra showed that the identified strains clustered
with the type strains of the species in which were placed after MALDI-TOF MS
analysis forming groups with internal similarities higher than 70%. The unidentified
strains occupied branches or clusters with similarity values lower than 70% with respect
to the described species of genera Rhizobium and Ensifer.
The analysis of recA and atpD genes showed that the unidentified strains by MALDITOF MS belonged to groups phylogenetically divergent to those from the already
described species from family Rhizobiaceae. These strains were phylogenetically
related to R. leguminosarum, R. vallis, R. tibeticum, R. mongolense and Ensifer fredii.
Interesting, in León in soils commonly cultivated with P. vulgaris the predominant
species was R. leguminosarum. In Barco de Ávila, although this legume is commonly
cultivated and R. leguminosarum also was abundant, R. giardinii was frequently found.
In Salamanca, in a soil commonly cultivated with Lens culinaris but where P. vulgaris
is not cultivated, a new species related to R. leguminosarum was found. In Badajoz in a
soil not cultivated with P. vulagris, R. giardinii was found. Finally, in Sevilla, in a soil
not cultivated with P. vulgaris, we found two undescribed species related to R.
mongolense and E. fredii.
ACKNOWLEDGMENTS
This work was funded and supported by Junta de Castilla y León project SA183A11-2. MHRB is
recipient of a JAE-Doc researcher contract from CSIC, cofinanced by ERDF.
REFERENCES
Beringer, J.E. (1974). J. Gen. Microbiol. 84: 188-198.
Ferreira, et al. (2011). PLoS One. 6: e20223.
Velázquez, E., et al. (2010). Microbes for Legume Improvement. Khan, M.S., Zaidi, A., and Musarrat, J.
(eds). Dordrecht, The Netherlands: Springer. pp. 1-25.
Oxford : Blackwell Scientific Publications.
26
Session I
SI-CP-03
Análisis de la biodiversidad de microorganismos en nódulos de Lotus
corniculatus.
Marcos-García, M.1, Menéndez, E.1, Celador-Lera, L.1, Rivera, L.P.1, Martínez- Molina,
E.1, 2, Mateos, P.F.1, 2, Velázquez, E.1, 2, Rivas, R.1, 2*
1
Departamento de Microbiología y Genética. Universidad de Salamanca.
Universidad de Salamanca (USAL)-CSIC (IRNASA).
*
[email protected]
2
Unidad Asociada de I+D
RESUMEN
Los nódulos de plantas de Lotus corniculatus procedentes de la localidad salmantina de
Carbajosa de la Sagrada nos ha proporcionado una elevada biodiversidad de
microorganismos, capaces de vivir en el interior de estos nichos seguros junto con el
endosimbionte natural de esta planta, Mesorhizobium loti. Las cualidades fenotípicas
que presentan varias de estas cepas, algunas de las cuales se encuentran dentro de los
géneros Micromonospora y Lysinibacillus, las convierte en potenciales bacterias
promotoras del crecimiento vegetal y susceptibles de ser utilizadas como coinoculantes.
INTRODUCCIÓN
La adaptabilidad a diferentes presiones ambientales que presentan las especies del
género Lotus, las convierte en plantas muy importantes desde un punto de vista
económico, ya que se utilizan como cultivos altamente productivos en sistemas de
pastoreos en una amplia gama de paisajes, incluyendo algunos sometidos con frecuencia
a ambientes y condiciones del suelo extremas. En concreto, la especie L. corniculatus se
considera una de las leguminosas forrajeras más importantes después de la alfafa
(Medicago sativa) y del trébol blanco (Trifolium repens), siendo su valor nutricional
similar o incluso superior a la de especies como: L. uliginosus (Schkuhr.), L. tenuis
(Waldst et kit.) y L. subbiflorus (Lagasca). Además, es una buena candidata para la
restauración y la fitorremediación de ambientes degradados. Esta especie es, con toda
probabilidad, la especie de Lotus más comúnmente empleada en restauraciones
ecológicas de los suelos afectados por la deficiencia de nutrientes, la salinidad, la sequía
o los contaminantes. Conocer la diversidad y la abundancia de los microorganismos que
nodulan o que ocupan los nódulos en las especies de Lotus, es un enfoque valioso para
una mejor selección y aplicación de las cepas como inoculantes. Estas cepas no sólo
deben ser eficientes en la fijación del nitrógeno, como ocurre para las especies de los
rhizobia, sino que también deben ser capaces de competir favorablemente con los
microorganismos nativos de la planta y la rizosfera circundante. Los estudios realizados
hasta la fecha sobre nódulos de L. corniculatus, se han centrado principalmente en las
especies del género Mesorhizobium de forma aislada. Conocer la existencia de una
mayor diversidad de especies en el interior de estos nichos ecológicos específicos y
seguros en cuanto a la presencia de patógenos, abre un abanico de posibilidades en lo
que se refiere a la búsqueda de nuevas bacterias promotoras del crecimiento vegetal en
plantas no leguminosas.
MATERIAL Y MÉTODOS
Se obtuvieron 90 aislados que se agruparon utilizando técnicas moleculares como los
TP-RAPD. Se secuenció el gen ribosómico 16S de diversos representantes, utilizando
para ello los primers 27F y 1522R. El análisis de las secuencias se llevó a cabo con el
programa BLASTn. Estas secuencias también se compararon con las cepas tipo
utilizando el programa Eztaxon 2.0. Para el análisis filogenético se utilizaron los
programas Clustal X y Mega 2.1. Para determinar la potencial capacidad de promoción
27
Session I
SI-CP-03
del crecimiento vegetal de cada uno de los aislados se hicieron distintas pruebas de
caracterización fenotípica. La solubilización de fosfato fue evaluada en medio YED-P
de acuerdo con Peix et al. (2009). La producción de sideróforos se evaluó utilizando el
medio de cultivo M9-CAS-AGAR, que es una modificación del medio utilizado por
Schwyn y Neilands, donde se observan halos de distintas tonalidades. La determinación
cualitativa de la producción de celulosa se llevó a cabo utilizando el medio de cultivo
YMA convencional con 25mg/l de rojo congo. La detección de la actividad celulolítica,
se determinó utilizando como sustrato carboximetil celulosa sódica, CMC (viscosidad
media, Sigma), un compuesto soluble derivado de la celulosa, utilizado para la
valoración de la actividad 1,4-β-glucanásica. La determinación de la formación de
biofilms se llevó cabo según Fujishige et al. (2006) con algunas modificaciones. Se
realizaron dos ensayos: en placas de microtitulación de poliestireno y en portaobjetos
(vidrio).
El análisis del gen ribosómico 16S nos mostró una amplia diversidad de especies,
siendo las cepas mayoritarias las de la especie Mesorhizbium loti, el endosimbionte
natural de L. corniculatus. Dentro del género Mesorhizobium también se aislaron tres
especies más: M. australicum, M. amporhae y M. gobiense. Por otra parte, según los
datos de secuenciación, aislamos especies no encontradas hasta ahora en nódulos de L.
corniculatus y que pertenecen a los géneros: Micromonospora, Dermacoccus,
Arthrobacter, Metylobacterium, Acinetobacter, Lysinibacillus y Paenibacillus. En este
trabajo, aislamos un total de 90 cepas del interior de nódulos de Lotus corniculatus,
obteniendo cepas con capacidad de solubilizar fosfato, promover la síntesis de indol
acético, producir sideróforos y celulosa, tener actividad celulolítica y ser capaces de
formar biofilms. Esta planta, por tanto, tiene una gran diversidad de microorganismos
endofíticos asociados a ella, capaces de mantener una actividad altamente beneficiosa,
influyendo de forma directa en el crecimiento de las plantas y sus funciones, no sólo en
su actividad cómo planta forrajera sino también como fitorremediadora de suelos en
condiciones insalubres. Podemos decir que el potencial que presenta L. corniculatus,
hacen de ella una planta con nuevas y amplias posibilidades de estudio y por supuesto,
el posible potencial de sus microorganismos endófitos como bacterias promotoras del
crecimiento vegetal, nos plantea la posibilidad de aplicarlos a otras especies vegetales,
incluidas las no leguminosas.
AGRADECIMIENTOS
Este trabajo está financiado por el proyecto SA183A11-2 de la Junta de Castilla y León y por el proyecto
AGL2011-29227 del Ministerio de Ciencia e Innovación. Marta Marcos agradece a la Fundación Miguel
Casado San José la financiación de su beca.
BIBLIOGRAFÍA
Fujishige, N.A., et al. (2006). Bot. J. Linnean Soc. 150: 79-88.
Peix, A., et al. (2009). Syst. Appl. Microbiol. 32: 334-341.
28
Session I
SI-CP-04
Where do PGPR interact with plants? An essential determination to
develop effective agricultural inputs based on microorganisms.
Mulas, R.1, Mulas, D.2*, Menéndez, E.3, Rivera, L.3, Mateos, P.3, González-Andrés, F.1
1
Instituto de Medio Ambiente y Recursos Naturales, Universidad de León, León, Spain. 2 Fertiberia S.A.,
Dpto de I+D+i, Huelva, Spain. 3 Universidad de Salamanca. Dpto de Microbiología y Genética,
Salamanca, Spain.
*
[email protected]
ABSTRACT
Although PGPR are extensively used in agriculture, they are just becoming popular in
Europe now, and this is because the strict regulations (Regulation EC 2003/2003 about
fertilisers and Directive 91/414/EEC about plant protection products) and the lack of
constancy in their effectiveness in some cases. Studying where they locate at a
rhizospheric level can provide valuable information to design agricultural inputs based
on microorganisms. This works shows how a phosphate-solubilizing Rhizobium sp
strain attaches to barley roots by observing transformed cells (fluorescent) under
confocal microscopy. The cells were observed on the root surface, and no endophytic
evidence was found for this strain. However, several studies have shown presence of
Rhizobium spp. as endophytes in cereal roots, such as rice or maize, indicating that the
characteristics of the symbiosis depend on the strain and not on the taxon.
INTRODUCTION
Bacteria that improve plant nutritional status (PGPR) are gaining acceptance amongst
farmers in Europe as an additional input, since a number of studies have shown the
feasibility or their use with cereals, legumes, industrial and horticultural crops.
However, little is known about the exact location of PGPR in the rhizosphere. Knowing
if the bacterium act as an endophyte or stays in the rhizoplane is interesting because it
can lead the strategy followed to develop products based on PGPR. Confocal
microscopy was first successfully used to view Rhizobium cells in Lucerne by Gage et
al. (1996), and since then it is a useful tool to determine the position of the cells in the
root. This study aimed at determining the location of a PGPR (Rhizobium sp phosphatesolubilizer) with barley (Hordeum vulgare L.) in the rhizosphere, in order to design the
best way of delivering the PGPR to this extensive winter crop.
A Rhizobium strain belonging to the collection of the University of León, was
transformed with the pHC60 plasmid (containing a GFP) harboured by Escherichia coli
S17.1 (Simon et al., 1983) by means of biparental mating. The transformed strain was
inoculated on barley plants, whose seeds had been previously surface-sterilized (ethanol
70% 1 min, NaClO 7% 7 min).
First test: barley plantlets (48 h after seeding) were inoculated with 250 L of the
bacterium culture (107 CFU mL-1). Ten days after inoculation (DAI), plant roots were
observed by using confocal microscopy to determine the plant root colonization.
Second test: barley plants were inoculated by immersion in the bacterium culture for 30
min. Ten DAI, plant roots were observed and the number of bacterial cells present
within the roots was determined by the following procedure: surface-sterilized roots
(ethanol 70% 1 min, NaClO 7% 5 min) were smashed and the root internal tissue was
inoculated on YMA medium plates at the dilutions 10-1, 10-3 and 10-5. A control was
used by placing sterile roots over YMA without smashing, which indicated the sterility
of the root surface.
29
Session I
SI-CP-04
Third test: barley plants were immersed (3 h) in a bacterium suspension in saline
solution (108 CFU mL-1). The same observations were done as previously, but the
sterilization was less aggressive (NaClO 5% 5 min) to avoid sterilization within the root
tissue.
With the endophytic isolates from the root that showed a colony growth similar to
rhizobia, RAPD profiles were obtained using the PCR primer M-13 as described by
Huey and Hall (1989). This method allowed discriminating amongst bacterial strains
within the same species, thus indicating whether the isolates were the inoculated strain
or not.
The transformed Rhizobium sp was observed with confocal microscopy, showing an
association with the plant roots, but it was not possible to determine whether it
happened on the root surface or endophytically (Figure 1). Regarding to the root tissue,
no colonies were found in the second test. However, the less aggressive sterilization of
the third test, allowed bacterial growth from inside the root, although no external growth
was detected. The M13-RAPD profiles indicated that the isolates were different from
the inoculated Rhizobium sp, which hints the possibility for the seed to harbor other
endophytic bacteria from the early stages, without evidence of endophytic rhizobia.
Nonetheless, other studies have shown rhizobium-root endophytic association in nonlegume crops (i.e. Gutiérrez-Zamora and Martínez-Romero, 2001; Yanni et al., 2010;
López-López et al., 2010). As a conclusion, the presence of the rhizobia inside the roots
is strain-dependent and, therefore it is needed to determine the location of each bacterial
strain in the rhizosphere. This information is crucial to design agricultural inputs (such
as fertilizers, inoculants and other biostimulants) with efficient performance, and thus,
improved nutritional status of the crops.
Figure 1. Confocal microscopy images of the first (a), second (b) and third tests (c). Whereas the first test
shows presence of bacteria, the second and the third tests show a higher number of bacteria in the barley root.
ACKNOWLEDGEMENTS
This work has been funded by Fertiberia S.A., Junta de Castilla y León (Research Projects) and the
Spanish Ministry of Economy (INNPACTO project IPT-2011-1283-060000, co-funded by the EUFEDER).
REFERENCES
Gage, D.J., et al. (1996). J. Bacteriol. 178: 71597166.
Gutiérrez-Zamora, M.L., and Martínez-Romero, E. (2001). J. Biotechnol. 91: 117-126.
Huey, B., and Hall, J. (1989). J. Bacteriol. 171: 2528–2532.
López-López, A., et al. (2010). Syst. Appl. Microbiol. 33: 322-327.
O'Hara, G.W., et al. (1989). Appl. Environ. Microbiol. 1870-1876.
Yanni, Y.G., et al. (2010). Plant Soil 336: 129-142.
30
Session I
SI-CP-05
Biodiversity of endophytic bacteria and mycorrhizal fungi in roots of
pepper (Capsicum annuum L.) in León province (Spain).
Barquero, M.1, Velázquez, E.1, Terrón, A.2*, González-Andrés, F.2
1
Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain. 2 Instituto de
Medio Ambiente y Recursos Naturales, Universidad de León, León, Spain.
*
[email protected]
ABSTRACT
Eigthy one endophytic PGPR strains and 9 AMF isolates were obtained from roots of
pepper in the PGI “Fresno-Benavente”, and characterized to be used like biofertilisers.
Bacteria were analysed in vitro regarding several plant growth promotion properties, in
order to pre-select strains for in field selection. The identification of the bacteria is a key
aspect as only safe taxa can be used. In our case an ARDRA test following TP-RAPD
were carried out in order to group the strains prior to the sequentation of the gene 16S
rRNA for identification. The AMF isolates belonged to the species Glomus mosseae.
INTRODUCTION
Second generation biofertilisers (Mulas et al. 2013) consist on mixes of different Plant
Growth Promoting Rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF),
which as a result of a complex mix of mechanisms have one or several of the following
effects: improve plant nutrition, stimulates plant growth and overcome plant stresses
(Dobbelaere et al., 2003, Singh et al., 2011). The use of biofertilisers in agriculture is an
increasing practice, as they improve the efficiency in the use of the mineral nutrients
from the soil, reducing nutrient losses an improving the crops yield for reduced rates of
chemical fertilisers. By the other hand, rhizobacteria and AMF may play a role like
microbial control agents and can be used for the replacement of chemicals according to
the EC Regulation 1107/2009. One key point for the success of microorganisms like
biofertilisers is the use of native isolates (Mulas et al., 2011, 2013). Castilla y León is a
Spanish region with an outstanding agri-food sector. The pepper (Capsicum annuum L.)
from Fresno and Benavente, is a product with high added value, labeled with a
Protected Geographic Indication (PGI), and for this reason started a research project to
develop biofertilisers. Here we present the results of the prospection of native
microorganisms and the preliminary characterization of the biodiversity encountered,
the first stage to develop an autochthonous biofertiliser.
Endophitic bacteria were isolated from surface sterilized roots of pepper crops collected
in the in 3 locations and 3 soils per location, inside the PGI region. Crushed roots were
plated onto TSA medium and purified. Clones were discarded on the basis of the RAPD
(primer M13) profiles. Pure strains were analysed for several activities in vitro:
Ca3(PO3)2 solubilization; siderophore production; IAA production; ACC deaminase
activity; control of Phitopthora capsicii. The identification of the isolates was based on
the sequentiation of the gen 16S rRNA and comparison with the ezTaxon database
(ezbiocloud.net). Previously to the sequentiation, isolates were grouped on the basis of
and Amplified Ribosomal DNA Restriction Analysis (ARDRA). Each group typically
consists on several taxa, so the TP-RAPD (Rivas et al., 2001) profiles within each
ARDRA group were obtained in order to group the isolates by recognised taxa and to
sequentiate only one strain from each group. AMF were isolated from the rhizosphere
of the same plants, using leak (Allium porrum) as trap plant in sterile sand. After this
first stage, from each soil 10 spores of the same morphology were used to inoculate a
31
Session I
SI-CP-05
leak plant in order to produce inoculum. One spore per soil was used for DNA
amplification with the primers of Lee et al. (2008), sequencing the obtained band and
comparing it with databases with BLAST-N (Altschut et al., 1990).
Figure 1. In vitro test for biocontrol of Phytophtora capsici by bacteria.
Eighty one different PGPR’s strains were isolated from the locations of Fresno de la
Vega, San Cristobal de Entreviñas and Micereces. None of the isolated strains was
phosphate solubilizer. Regarding the IAA production, the values ranged from 1.25
µg/ml to 8.93 µg/ml. However only 12 strains from the 81 tested, produce more than 3.0
µg/ml, and half of the 81 produced less than 2.0 µg/ml. The production of IAA was
medium-low, compared with the best producers isolated in tropical and subtropical
areas. The production of -Ketobutirate showed a broad range from nearly 0 moles mg
prot-1 h-1 up to 247, showing a continuous variation within this range. Compared to other
works (i.e. Siddikee et al., 2010), the higher values observed in our collection are up to
100 time higher that the higher values of the mentioned work and the lower values are
similar in both cases. About siderophores production, more than 50% of the isolates
produced them. The isolates belonged to Bacillus pumilus, Bacillus amyloliquefaciens,
Pesudomonas geniculata and other species were discarded like Bacillus cereus (human
pathogen) and Pseudomonas corrugata (plant pathogen). Five out of the 81 strains
controlled Phytophtora capsicii (Figure 1) four belonging to B. pumilus and the last to
B. cereus which was discarded. All the AMF isolated belonged to Glomus mosseae
(Nicol. and Gerd.) Gerd. and Trappe, in contrast to the isolates in tree crops in the same
area, which usually belonged to Glomus Intraradices.
The selection of the PGPR strains to be tested in microplots in field conditions was
based on the level of expression of plant growth promotion properties in vitro, and in
the taxonomic identification, as only safe taxa can be used for biofertilisers.
AKNOWLEDGEMENTS
This work has been funded by Junta de Castilla y León (Project LE029A10-2) and Agencia Española de
Cooperación Internacional para el Desarrollo (AECID) (Project A1/035364/11). M. Barquero was
granted by AECID.
REFERENCES
Altschul, S.F., et al. (1990). J. Mol. Biol. 215: 403-410.
Lee, J., et al. (2008). FEMS Microbiol. Ecol. 65: 339-349.
Mulas, D., et al. (2011). Soil Biol. Biochem. 43: 2283-2293.
Mulas, D., et al. (2013). Beneficial plant-microbe interactions: Ecology and applications. CRC Press.
Rivas, R., et al. (2001). Electrophoresis 22: 1086-1089.
Siddikee, M.A., et al. (2010). J. Microbiol. Biothechnol. 20: 1577-1584.
Singh J.S., et al. (2011). Agr. Ecosyst. Environ. 140: 339-353.
32
Session I
SI-CP-06
Diversidad de los rizobios que nodulan Medicago marina de regiones
del Odiel y San Fernando.
Alías-Villegas, C.1*, Bellogín, R.A.1, Camacho, M.2, Cubo, M.T.1, Temprano, F.2,
Espuny, M.R.1
1
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes 6,
41012-Sevilla. Spain. 2 Centro Las Torres-Tomejil (IFAPA), Apartado Oficial, Alcalá del Río, Sevilla.
Spain.
* [email protected]
RESUMEN
La diversidad de los rizobios que nodulan Medicago marina en las Marismas del Odiel
y San Fernando, zonas con suelos sometidos a estrés abiótico por pH y salinidad, se ha
estimado usando 14 aislamientos de plantas de San Fernando y 18 de las Marismas del
Odiel. Tras eliminar redundancias de los perfiles PCR-ERIC se obtuvieron 12
aislamientos diferentes, 7 de las Marismas del Odiel y 5 de San Fernando.
La secuenciación del gen ARNr 16s mostró que todas las cepas pertenecen al género
Sinorhizobium, estando estrechamente relacionadas con S. meliloti LMG 6133T. Para
estudiar si son distintas cepas de S. meliloti se han realizado RFLP del gen nod C y de la
región ITS. Según el perfil RFLP de nod C, se han obtenido dos grupos diferenciados (I
y II), con las enzimas MspI y RsaI, pero éstos no coinciden exactamente con las áreas
geográficas donde se han recogido las muestras y no corrobora que todos los
aislamientos sean S. meliloti, puesto que al digerir con MspI el perfil de S. meliloti
coincide con el grupo II y al digerir con RsaI en perfil de S. meliloti coincide con el
grupo I. Por otra parte, según el perfil RFLP de la región ITS, todos los aislamientos
mostraron el mismo perfil que S. meliloti, con excepción del aislamiento ORT17 que
mostró un perfil distinto con la enzima MspI.
INTRODUCCIÓN
La agricultura actual sufre la sobreexplotación de los recursos y el empleo masivo de
fertilizantes, lo que hace preciso la recuperación de suelos que presentan algún tipo de
estrés abiótico por pH o salinidad extremos que imposibilitan el cultivo. Así, se planteó
la búsqueda de bacterias simbióticas fijadoras de nitrógeno atmosférico en ambientes
con estos tipos de estrés, con el objetivo de introducir las parejas simbióticas rizobioleguminosa más adecuadas a los parámetros físico-químicos de los suelos a recuperar.
Con este fin se ha iniciado el estudio de los simbiontes de M. marina aislada de dos
zonas, las Marismas del Odiel (Huelva) y la zona dunar de San Fernando (Cádiz)
Además, dada la relativa abundancia de esta leguminosa en las playas de San Fernando
una vez determinados los mejores simbiontes, podría ser utilizada para la estabilización
de sistemas dunares.
MATERIAL Y MÉTODOS
Para el aislamiento de bacterias, los extractos de los nódulos previamente desinfectados
(hipoclorito de sodio al 5% durante 4 minutos) fueron sembrados sobre placas de YMA
con Rojo Congo (25 mg/L). La PCR-ERIC se hizo utilizando los cebadores consenso
ERIC 1R y ERIC 2, siguiendo el protocolo descrito por Versalovic et al. (1991).
La amplificación del gen ARNr 16S se realizó con los cebadores 616F y 1522R. Los
fragmentos del gen ARNr 16s amplificados fueron secuenciados por Stab-Vida
(Portugal) usando los cebadores 519F, 907R, 616F y 1522R. La identificación de las
secuencias se realizó empleando la base de datos EzTaxon-e. Para el análisis de datos,
se realizaron alineamientos con el programa Clustal X (Thomson et al., 1997). Los
33
Session I
SI-CP-06
análisis filogenéticos y de evolución molecular se hicieron usando el programa MEGA
versión 4 (Tamura et al., 2007); con tres algoritmos diferentes para realizar el árbol,
máximum-likelihood, (Felsenstein, 1981), maximum-parsimony (Kluge y Farris, 1969)
y neighbour-joining (Saitou y Nei, 1987).
El gen nod C se amplificó con los cebadores nodCF2 y nodCI (Laguerre et al., 2001) y
el método de Laguerre et al. (1994). El producto de PCR se digirió independientemente
con los enzimas MspI y RsaI. La región ITS se amplificó con FGPS1490 y FGPS132
(Laguerre et al., 1996) y según el método de Vinuesa et al. (1998). El producto de PCR
se digirió independientemente con MspI y AluI.
Del aislamiento de los nódulos de las plantas de M. marina se obtuvieron 32 clones que,
tras el empleo de la PCR-ERIC, se agruparon en 12 perfiles distintos, 7 de las Marismas
del Odiel y 5 de las Dunas de San Fernando. La secuencia del gen del ARNr 16S ha
identificado todos los aislamientos como S. meliloti; la mayor parte de ellos con
porcentajes de identidad que oscilan entre el 99,3 y 100%. Tan solo uno de los
aislamientos de San Fernando (SF 1.2) muestra un porcentaje de identidad del 98%. Los
árboles filogenéticos construidos muestran la relación de todos los aislamientos con S.
meliloti y la proximidad de los diferentes aislamientos entre sí.
Los perfiles RFLP con MspI y RsaI del gen nodC agrupan a los aislamientos en dos
tipos de perfil no coincidentes entre ellos. Los perfiles RFLP de la región intergénica
ITS muestran que todos los aislamientos presentan el mismo perfil que S. meliloti,
excepto el aislamiento ORT17 al ser digerido con MspI.
Los resultados obtenidos muestran que los aislamientos de M. marina, pertenecen al
género Sinorhizobium y están estrechamente emparentados con S. meliloti, según nos
indican los porcentajes de similitud del gen ARNr 16S y los árboles filogenéticos, pero
que existen diferentes grupos de cepas que difieren de la cepa tipo de S. meliloti en el
patrón de los fragmentos de restricción del gen nodC. Además, los dos perfiles
obtenidos por RFLP no se corresponden con la diferente localización geográfica de los
aislamientos.
AGRADECIMIENTOS
Este trabajo ha sido financiado por el proyecto de Excelencia P10-AGR5821 de La Junta de Andalucía.
Cynthia Alías Villegas disfruta una beca predoctoral del mismo programa.
BIBLIOGRAFÍA
Laguerre, G., et al. (1994). Appl. Environ. Microbiol. 60: 56-63.
Versalovic, et al. (1991). Nucl. Acids Res. 19: 6923-6831.
Vinuesa, P., et al. (1998). Appl. Environ. Microbiol. 64: 2096-2104.
34
Session I
SI-CP-07
Tolerancia a pH y salinidad extremos y propiedades simbióticas de
aislamientos de rhizobia de Medicago marina L.
Alías-Villegas, C.1, Espuny, M.R.1, Bellogín, R.A.1, Cubo, M.T.1, Camacho, M.2,
Temprano, F.2*
1
41012-Sevilla. España. 2 IFAPA-Centro Las Torres-Tomejil. Carretera Sevilla-Alcalá del Río km. 12,2.
41200 Alcalá del Río (Sevilla), España.
* [email protected]
INTRODUCCIÓN
Medicago marina L. es una leguminosa que crece en suelos arenosos o gravosos en
numerosos lugares del litoral de la Península Ibérica y de la cuenca mediterránea
(Castroviejo et al., 2000). Esta planta podría tener interés para fijar las dunas costeras o
servir de planta pionera para facilitar la colonización posterior de los suelos por otras
plantas. Es capaz, como muchas leguminosas, de formar nódulos y establecer simbiosis con
bacterias del suelo para fijar nitrógeno molecular. Poco se sabe de las características de los
rhizobia capaces de nodular M. marina y sería de interés conocer sus propiedades
simbióticas con otras especies de Medicago o géneros afines y fisiológicas como
tolerancia a pH y salinidad extremos.
MATERIAL Y MÉTODOS
Se aislaron rhizobia de nódulos de dos poblaciones de plantas de M. marina, una de las
marismas costeras de los ríos Tinto y Odiel (37º 10´ 28,5´´ N, 6º 55´51,5 W), en la provincia
de Huelva y otra de la playa de San Fernando (36º 28´52,5´´ N, 6º 15´49,4´´ W) en Cádiz.
Los nódulos se esterilizaron en superficie y su contenido se sembró en placas con medio
sólido de extracto de levadura manitol (ELM), según la técnica habitual (Vincent, 1970). Se
realizaron mediante PCR y desarrollo en geles de agarosa perfiles ERIC de ADN para
diferenciar los aislamientos (de Bruijn, 1992) tomando de cada grupo similar un aislamiento
representativo. Los aislamientos ORT11, 12, 13, 15, 16, 17, 18 y 19 proceden de Huelva y
ORT51, 52, 53, 54 y 55 son originarios de Cádiz.
Para estudiar el crecimiento a distintos pH se utilizó medio líquido de ELM estabilizando el
pH en valores entre 5 y 8,5 con tampones adecuados. Para la tolerancia a salinidad se usó el
mismo medio líquido con concentraciones crecientes de cloruro sódico desde 300 mM hasta
900 mM. Los aislamientos se crecieron en tubos con 3 mL de medio, inoculando cada tubo
con 50 μL de un cultivo crecido hasta fase estacionaria. Hubo tres repeticiones por cepa
bacteriana. Después de 4 días de crecimiento en agitación (180 rpm, 28 ºC) se evaluó el
crecimiento midiendo la D.O. de los cultivos a 600 nm.
Para la comprobación de las propiedades simbióticas de los aislamientos se utilizaron
semillas de M. marina, M. sativa (cv. Aragón), M. polymorpha (cv. Santiago), M.
truncatula, M. murex, M. arabiga, M. minima, M. orbicularis y Melilotus indicus. Las
semillas se esterilizaron previamente en superficie con etanol al 70% (30 s.) e hipoclorito
sódico al 5% (6 min.) y posterior lavado (6 veces) con agua estéril. Las semillas,
germinadas en agar agua (10%), se sembraron en tubos de 20 x 200 mm, con una tira de
papel de filtro como soporte y 15 mL de medio de cultivo sin nitrógeno (Rigaud y Puppo,
1975). Las plantas se inocularon, a las 24 h de la siembra, con 0,1 mL de una suspensión
concentrada de 108 células/mL de cada cepa bacteriana (2 repeticiones/cepa y planta). Las
plantas se crecieron en invernadero entre 16 ºC (noche) y 25 ºC (día) y con un fotoperíodo
de 14 h de luz. Después de 2 meses se observó la nodulación (presencia y tamaño de
nódulos) y la efectividad de la fijación por comparación con los controles de las plantas no
inoculadas.
Como referencia se utilizaron las cepas LMG6133T de Sinorhizobium meliloti, LMG19920T
o M19-1 de S. medicae e ISLU16 de Bradyrhizobium sp. (Lupinus).
35
Session I
SI-CP-07
El crecimiento de los aislamientos de M. marina a distintos pH en el rango 5-8,5 mostró un
patrón similar, entre ellos y con las cepas de referencia de S. meliloti y S. medicae. Ninguna
de las cepas creció a pH 5, mientras que a pH 5,5 mostraron algo de crecimiento y a pH 7, 8
y 8,5 crecieron satisfactoriamente. La cepa ISLU16 de Bradyrhizobium sp., sin embargo,
creció bien a pH 5 y también a 5,5, 7 y 8, viéndose disminuido el crecimiento a pH 8,5.
La tolerancia a salinidad de los distintos aislamientos fue muy parecida y semejante a la de
S. meliloti: todas las cepas crecieron bien hasta con 700 mM de NaCl, poco a 800 mM y
nada a 900 mM. La cepa tipo de S. medicae no creció en medios con 800 mM de NaCl.
ISLU16 fue incapaz de crecer en medios con más de 300 mM de NaCl.
En la Tabla 1 se muestran los resultados de nodulación y fijación de los aislamientos con las
distintas especies de medicagos y con Melilotus indicus. Su comportamiento simbiótico fue
muy semejante entre ellos y muy parecido al de la especie tipo de S. meliloti y muy
diferente al de S. medicae (Garau et al., 2005). Con las especies M. truncatula, M. minima y
M. orbicularis la efectividad fijadora de nitrógeno de los aislamientos fue variable.
Tabla 1. Nodulación y fijación de nitrógeno de aislamientos de Medicago marina con algunas especies de
Medicago y Melilotus indicus1.
Bacteria
1
M.
marina
M..
sativa
M.
polymorpha
M.
truncatula
M.
murex
M.
arabiga
M.
minima
M.
orbicularis
M.
indicus
ORT11
nod+fix+ nod+fix+
Ab.
nod+fix+
Ab.
Ab.
nod+fix+
nod+fix-
nod-
ORT12
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
Ab.
ORT13
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+ nod+fix+
nod-
ORT15
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix-
nod+fix+
Ab.
ORT16
nod+fix+ nod+fix+
Ab.
nod+fix-
nod-
Ab.
nod+fix-
nod+fix+
Ab.
ORT17
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+ nod+fix+
ORT18
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix-
nod+fix-
Ab.
ORT19
nod+fix+ nod+fix+
Ab.
nod+fix+
Ab.
Ab.
nod+fix-
nod+fix-
nod-
ORT51
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
nod-
ORT52
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
Ab.
ORT53
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
Ab.
ORT54
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
Ab.
ORT55
nod+fix+ nod+fix+
Ab.
nod+fix-
nod-
Ab.
nod+fix+
nod+fix-
nod-
S.
meliloti2
nod+fix+ nod+fix+
Ab.
nod+fix-
Ab.
Ab.
nod+fix+
nod+fix-
Ab.
S.
medicae3
nod+fix-
nod+fix+
nod+fix+
nod+fix+
nod+fix-
nod+fix
nod+fix+ nod+fix+ nod+fix+ nod+fix+
+
T
Ab.: Abultamientos blancos en las raíces. 2 Cepa LMG19920 . 3 Cepa M19-1.
AGRADECIMIENTOS
Este trabajo se ha realizado gracias a la financiación de los proyectos P10-AGR5821 de La Junta de
Andalucía y RM2010-00014-00-00 del INIA.
BIBLIOGRAFÍA
Castroviejo, S., et al.(Eds.) (2000). Flora Iberica. VII(II). Real Jardín Botánico. Madrid. p. 755.
de Bruijn, F. (1992). Appl. Environ. Microbiol. 58: 2180-2187.
Garau, G., et al. (2005). Plant Soil.176: 263-277.
Rigaud, J., Puppo, A. (1975). J. Gen. Microbiol. 88: 223-228.
Vincent, J.M. (1970). A manual for the practical study of root nodule bacteria. IBP Handbook No. 5.
Balckwell Sci. Pub. Oxford (Reino Unido)
36
Session I
SI-CP-08
Estudio de la biodiversidad microbiana en suelos de las Marismas del
Odiel y de las Minas de Rio Tinto.
Ruíz-Carnicer, A.1, Alías-Villegas, C.2, Bellogín, R.A.2, Manyani, H.1, Temprano, F.3,
Espuny, M.R.2, Camacho, M.3*
1
ResBioagro. 3ª planta Edificio Citius. Reina Mercedes, 4. Sevilla. 2 Departamento de Microbiología,
Facultad de Biología, Universidad de Sevilla. Sevilla. 3 IFAPA Centro Las Torres-Tomejil. Sevilla.
* [email protected]
RESUMEN
Se ha hecho un estudio de la diversidad microbiana presente en suelos altamente
contaminados de las Marismas del Odiel y de las Minas de Río Tinto para determinar
cómo afectan distintos factores (acidez y salinidad) a las poblaciones bacterianas e aislar
aquellas especies mayoritarias en cada uno de ellos. De 300 aislamientos realizados, se
han seleccionado aquellos que poseen alguna característica promotora del crecimiento
de la planta (PGP) o alguna actividad enzimática útil en la industria. Los resultados
muestran la gran biodiversidad presente en las distintas zonas, así como la selección de
28 bacterias altamente prometedoras.
INTRODUCCIÓN
El impacto de la actividad humana influye de forma notable en la degradación de los
estuarios. Entre estos, el estuario de Huelva está considerado como uno de los más
contaminados de Europa (Sainz et al., 2004; Ruiz et al., 2008) debido, por una parte al
aporte altamente ácido que recibe de los Ríos Tinto y Odiel tras recorrer gran parte de la
Faja Pirítica Ibérica y por otra, a los vertidos provenientes del polo industrial onubense.
Hasta ahora se han hecho numerosos estudios sobre la influencia de estos vertidos sobre
la flora y fauna de la zona (Mateos-Naranjo et al., 2011; Sánchez, 2006), especialmente
tras los intentos de recuperación llevados a cabo en parte del estuario (Rubio et al.,
2001), pero los estudios acerca de las comunidades microbianas son escasos o nulos. En
este trabajo se ha comparado, mediante la técnica de la electroforesis en gel con
gradiente de desnaturalización (DGGE), las poblaciones bacterianas presentes en tres
zonas diferenciadas del estuario: zona del Dique Juan Carlos (suelo básico, no salino),
zona recuperada (suelo básico, salino) y zona del Rio Tinto (suelo ácido, no salino).
Además, se han aislados una serie de bacterias que pueden ser en utilizadas tanto en los
procesos de restauración (aplicadas como inoculantes) como en distintos procesos
industriales.
MATERIAL Y MÉTODOS
Toma de muestras: se tomaron muestras de suelo rizosférico de varias sub-parcelas de
cada zona de muestreo. Se mantuvieron en frio hasta su procesamiento.
Aislamiento de bacterias y pruebas bioquímicas.
Se llevaron a cabo utilizando los métodos descritos por Tsavkelova et al. (2007), con
ligeras modificaciones.
Extracción de ADN de suelo.
Se realizó siguiendo el protocolo del kit PowerSoilTM DNA Isolation Kit. Se comprobó
el resultado de la extracción en un gel de agarosa al 1%.
Electroforesis en gradiente desnaturalizante (DGGE).
Se hizo siguiendo el protocolo descrito por Heuer et al. (1997) con un gradiente de
agentes desnaturalizantes comprendido entre el 55% y el 70%. El posterior análisis de
las bandas se realizó con el programa Quantity One.
37
Session I
SI-CP-08
De las 300 cepas aisladas se han seleccionados 28 en base a la determinación de las
siguientes propiedades:
 Actividades enzimáticas: lipasa, proteasa, celulasa y amilasa
 Características como PGP: producción de ácido indol-acético, de sideróforos,
solubilización de fosfatos y actividad ACC (1-aminociclopropano carboxilato)
desaminasa
 Características fisiológicas: tolerancia a salinidad y a pH
De las 28 cepas seleccionadas, 15 proceden de Rio Tinto, 12 del Dique Juan Carlos y
tan solo 1 procede de la zona de recuperación, lo que parece indicar que las zonas más
contaminadas son lugares más apropiados para el aislamiento de bacterias con alguna
característica deseable.
La mayoría de los aislamientos son capaces de crecer a concentraciones de 600 mM de
NaCl, a un amplio rango de pH (de 4.5 a 9) y poseen una o dos actividades enzimáticas.
Sólo uno de ellos (RBA-OR120) resultó positivo en las cuatro actividades. Respecto a
las propiedades como PGP, la mayoría de las cepas exhibió al menos dos de ellas,
presentando 5 de los aislamientos todas las estudiadas.
En cuanto a la diversidad bacteriana de las distintas zonas, en cada punto de muestreo
aparecieron entre 25-30 especies distintas, existiendo una mayor heterogeneidad entre
los distintos puntos en la zona del Rio Tinto (Figura 1). Por otra parte, en términos
globales, la zona que presentó una mayor diversidad bacteriana fue la zona recuperada,
donde se observaron más de 70 especies distintas.
Figura 1. DGGE correspondiente a
la zona de Rio Tinto, con 13 puntos
de muestreo.
AGRADECIMIENTOS
Este trabajo ha sido financiado por el Proyecto de Excelencia P10-AGR5831 de la Junta de Andalucía y
la beca del mismo programa de la Junta de Andalucía de Cynthia Alias Villegas
BIBLIOGRAFÍA
Heuer, H., et al. (1997). Appl. Environ. Microbiol. 63: 3233-3241.
Mateos-Naranjo, E., et al. (2011). Ecotox. Environ. Safe. 74: 2040-2049.
Rubio, J.C., et al. (2001). III Congreso Forestal Español. Tomo: 3CFE04-061-T4: pág. 392-395.
Ruiz, F., et al. (2008). Mar. Pollut. Bull. 56: 1258-1264.
Sainz, A., et al. (2004). Environ. Int. 30: 557-566.
Sánchez, M., et al. (2006). Arch. Fur Hydrobiologie 166: 199-223.
Tsavkelova, E.A., et al. (2007). Microbiol. Res. 162: 69-76.
38
Session I
SI-CP-09
Componentes biológicos en el suelo de dos plantaciones similares de
ciruelo en producción ecológica y convencional.
Daza, A. *, Pérez-Romero, L.F., Arroyo, F.T., García-Galavís, P.A., Camacho, M.,
Santamaría, C.
IFAPA Centro “Las Torres-Tomejil”, 41200-Alcalá del Río (Sevilla), España.
* [email protected]
RESUMEN
Se han analizado la composición biológica del suelo en dos plantaciones de ciruelo
japonés (Prunus salicina Lindl.), en un suelo franco limoso, una manejada en
producción ecológica y la otra en producción convencional. Las poblaciones de
bacterias y hongos cultivables han sido el doble en el suelo ecológico, que contuvo
también poblaciones superiores de rizobios. El grado de micorrización de las raíces de
ciruelo y de la herbácea Conyza bonariensis, común en ambos suelos, ha sido elevada y
no ha mostrado diferencias significativas. Las poblaciones de lombrices fueron
alrededor de tres veces más abundantes en el suelo ecológico.
INTRODUCCIÓN
La Agricultura Ecológica ha crecido exponencialmente en todo el mundo en las dos
últimas décadas. Su defensa se basa tanto en aspectos relacionados con la calidad y
sanidad de los productos como en los beneficios medioambientales que dicha práctica
agraria conlleva. En este estudio se ofrecen datos que indican que determinados
componentes vivos del suelo, claves para su sostenibilidad y fertilidad, se ven
favorecidos por el manejo ecológico
MATERIAL Y MÉTODOS
El estudio se ha desarrollado en dos parcelas experimentales de 5500 m2 cada una de la
finca experimental del IFAPA Centro “Las Torres-Tomejil” en Alcalá del Río (Sevilla).
Desde su plantación, en enero de 2005, una parcela está en producción ecológica y la
otra en producción convencional. La fertilización en la parcela ecológica ha sido a base
de estiércol de origen animal (3-4 kg m-2) y la siembra de cubiertas vegetales
conteniendo leguminosas. Las poblaciones bacterianas y fúngicas cultivables se han
cuantificado en los medios sintéticos Nutrient Agar y PDA, respectivamente. Las
poblaciones de rizobios se determinaron por el método del número más probable. El
grado de micorrización en raíces de ciruelo (Prunus salicina Lindl.) y la especie
herbácea Conyza bonariensis L. se determinó según Smith y Read (1997). Las
lombrices se calcularon tomando muestras de suelo de 0,5 m2 y 40 cm de profundidad.
Se ha constatado que las cubiertas vegetales de leguminosas aportaron cantidades
importantes de los principales macronutrientes (Tabla 1).
39
Session I
SI-CP-09
Tabla 1. Aportación bruta en materia seca y principales macroelementos de las diferentes cubiertas
vegetales utilizadas en años sucesivos en la plantación ecológica de ciruelos
Tipo de cubierta
kg/ha
Materia seca
N
P
K
Glycine max (pre-plantación)
10.060
320
34
206
Vicia faba
6.320
180
14
185
Brassica napus + Vicia sativa
2.500
70
8
75
Vegetación espontánea
2.913
56
9
75
Vicia sativa + Avena sativa
8.320
180
19
220
Vicia faba
10.353
225
24
274
Con relación a las poblaciones de bacterias y hongos, a partir del año 2008 se viene
observando de forma consistente que éstas son aproximadamente dos o tres veces más
abundantes en el suelo ecológico. En la Figura 1 se representa el periodo 2011-2013.
Número de hongos por gramo de suelo seco
Número de bacterias por gramo de suelo seco
400000
350000
120000000
100000000
300000
250000
80000000
Ecológico
60000000
Convencional
40000000
20000000
Ecológico
200000
150000
100000
Convencional
10/02/2013
10/01/2013
10/12/2012
10/11/2012
10/10/2012
10/09/2012
10/08/2012
10/07/2012
10/06/2012
10/05/2012
10/04/2012
10/03/2012
10/02/2012
10/01/2012
10/11/2011
10/02/2013
10/01/2013
10/12/2012
10/11/2012
10/10/2012
10/09/2012
10/08/2012
10/07/2012
10/06/2012
10/05/2012
10/04/2012
10/03/2012
10/02/2012
10/01/2012
10/12/2011
10/11/2011
10/12/2011
50000
0
0
Figura 1. Poblaciones de bacterias y hongos en los suelos ecológico y convencional en el periodo 20112013.
En ocasiones se sembró también una muestra de la cubierta vegetal en la parcela
convencional, para comparar la nodulación y las poblaciones de rizobios. Los datos
obtenidos con Vicia faba y Vicia sativa se presentan en la Tabla 2.
Tabla 2. Número y peso seco de nódulos correspondientes a 12 plantas en los suelos ecológico y
convencional
Especie
Tratamiento
Nº nódulos
Peso nódulos
NMP
(g)
(Nº rizobios/g suelo)
Convencional
105
0,403
499
Vicia faba
Ecológico
1094
1,888
10.429
Vicia sativa
Convencional
Ecológico
92
143
0,157
0,254
nd
nd
Se ha observado un elevado grado de micorrización (>70%) en las raíces de ciruelo y de
la herbácea Conyza bonariensis, pero sin diferencia significativa entre tratamientos. Sin
embargo, sí existieron claras diferencias en las poblaciones de lombrices, tres o cuatro
veces más abundantes en el suelo ecológico.
AGRADECIMIENTOS
Los autores agradecen la financiación recibida del INIA (Proyecto RTA2010-00046-00-00)
BIBLIOGRAFÍA
Smith S.E., and Read, D.J. (1997). Mycorrhizal Symbiosis. Academic Press, San Diego.
40
Session I
SI-CP-10
Relative abundance of denitrification genes and diversity of bacterial
denitrifiers in sediments with different nitrate concentration.
Correa-Galeote, D., Tortosa, G., Bedmar, E.J.*
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín,
CSIC, Granada, España. Apartado Postal 419, 18080-Granada.
*
[email protected]
INTRODUCTION
Denitrification is the biological process by which nitrate is sequentially reduced to
dinitrogen gas (N2) via the intermediate compounds nitrite (NO2-), nitric oxide (NO) and
nitrous oxide (N2O). Respiratory nitrate reduction is catalysed by a membrane-bound
(Nar) or periplasmic (Nap) nitrate reductase encoded by the narGHIJ or the napABC
operon, respectively. The reduction of nitrite to nitric oxide is carried out by two
structurally different, but functionally similar, enzymes, the Cu-containing and the cd1containing nitrite reductase encoded by the nirK and nirS genes, respectively. The last
step of denitrification is the reduction of nitrous oxide and is catalysed by nitrous oxide
reductase encoded by the nosZ gene. Denitrifiers include more than 60 genera of
Bacteria, and some Archaea and Fungi, and can represent up to 5% of the total soil
microbial community (Henry et al., 2006). Denitrification is one of the main branches
of the global N cycle, and is responsible for the return of fixed nitrogen to the
atmosphere. It also accounts for significant losses of fertilizer nitrogen from agricultural
soils (Philippot et al., 2006) and contributes to modify the global atmospheric
chemistry, essentially through the greenhouse effect and destruction of the Earth’s
ozone layer through stratospheric nitric oxide production. Here we describe differences
in the abundance and in the biodiversity of two sampling sites with contrasting nitrate
content.
Sampling sites.
Two sites, the lagoon of el Acebrón (S1) and la Cañada creek (S2), representing the
sites with the lowest (0.27 ± 0.05 mg NO3−/kg sediment) and highest (32.77 ± 1.95 mg
NO3−/kg sediment) nitrate concentration along la Rocina stream, which feeds Marisma
del Rocio in Doñana National Park (South West, Spain), were selected for sampling.
Sediment samples were taken as indicated earlier (Tortosa et al., 2011) in April and
October years 2008, 2009 and 2010. Samples were placed on ice while returned to the
laboratory and then stored at -80 ºC until use.
DNA extraction.
DNA was extracted from 250 mg of each subsample according to Correa-Galeote et al.
(2013a).
Quantification of the denitrification-associated microbial community.
The size of the denitrifier community was estimated by quantitative, real-time PCR
(qPCR) of narG, napA, nirK, nirS and nosZ gene fragments using reaction mixtures,
primers and thermal cycling conditions described previously (Correa-Galeote et al.,
2013a). The total bacterial community was quantified using 16S rRNA gene as
molecular marker as described by Correa-Galeote et al. (2013b).
Clone library construction and DNA sequencing.
nosZ amplicons from samples taken in April and October 2009 and 2010 from sites S1
and S2 were purified using the QIAquick PCR purification kit (Quiagen) and cloned in
Escherichia coli JM109 using the pGEM-T Easy cloning kit. Cells were screened by
PCR using the vector primers Sp6 and T7 (Invitrogen). Nucleotide sequences of clones
41
Session I
SI-CP-10
containing inserts of the expected size were determined at the DNA Sequencing Service
of Estación Experimental del Zaidín, CSIC, Granada, Spain. Nucleotide sequences were
compared to entries in GenBank using BlastN (Altschul et al., 1990).
Relative abundance of the denitrification genes.
For the 3 years under study, regardless of the sampling dates, the relative abundance of
the narG and napA genes in sediments taken at S2 was higher than that in sediments
sampled at S1. Whereas the relative contributions of narG to the total eubacterial
community in April and October were similar at S1, there was a significant difference
between April and October at S2. However, there were no significant differences in
relative abundance of the nirK gene between S1 and S2 for the three years under study.
Whereas values of relative contribution of nirK were similar at S1, the contribution was
higher in October than in April at S2. The percentage of the nirS gene was also higher
in S2 than in S1. Despite differences in the relative contribution of the nirS gene both
for years and sampling dates, the contribution of nirS to the total eubacterial community
was higher in October than in April at S1 and also at S2. For the 3 years under study,
the relative abundance of nosZ was higher in S2 than in S1, regardless of the sampling.
Whereas no differences in the relative contribution of nosZ to the total eubacterial
community were found between the months of April and October at S1, there was a
statistically significant difference between April and October at S2. Taken together, our
data show that relative abundance of the narG, napA, nirS, nirK and nosZ genes was
15.54%, 8.96%, 6.86%, 5.72% and 0.26%, respectively.
Biodiversity of bacterial denitrifiers.
For the 2 years under study, when sequences of the nosZ gene from the eight clone
libraries were compared, the number of operational taxonomic units (OTUs) found in
sediments from S1 were 29 for samples taken in both April 2009 and 2010, and 27 and
29 for samples taken in October 2009 and 2010, respectively. Similarly, the number of
OTUs in sediments from S2 collected in April 2009 and 2010 were 25 and 26,
respectively. However, the number of OTUs in sediments taken at S2 in October 2009
and 2010 were 35 and 34, respectively. Nitrate concentration in sediments from S2 was
always higher in October (47 and 34 mg NO3 -/kg in years 2009 and 2010, respectively)
than in April (31 and 26 mg NO3-/kg in years 2009 and 2010, respectively). Thus, it is
possible that increase in the number of OTUs could be due to the higher nitrate
concentration. The most abundant genera in sediments were Acidovorax, Leptothrix,
Azospirillum, Paracoccus and Bradyrhizobium.
ACKNOWLEDGEMENTS
This work was supported by ERDF-cofinanced grants P09-RNM-4746 from Consejería de Economía,
Innovación y Ciencia (Junta de Andalucía, Spain).
REFERENCES
Correa-Galeote, D., et al. (2013a). FEMS Microbiol. Ecol. 83: 340-351.
Correa-Galeote, D., et al. (2013b). J. Metagenomics doi:10.4303/mg/235702
Henry, D., et al. (2006). Appl. Environ. Microbiol. 72: 5181-5189.
Philippot, L., et al. (2006). In: Molecular Approaches to Soil, Rhizosphere and Plant Microorganisms
Analysis. Cooper JE and Rao JR, eds. CABI International, Cambridge. Pp. 146-164.
Tortosa, G., et al. (2011). Ecol. Eng. 37: 539-548.
42
Session I
SI-CP-11
Experimental and modelling approach to the legume-Rhizobium
interaction: test of plant-host sanctions in co-inoculated plants with
fixing and non-fixing strains.
Marco, D.E.1*, Talbi, C.2, Bedmar, E.J.2
1
Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba and CONICET, Argentina. 2
Departmento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín,
Agencia CSIC, Granada, Spain.
* [email protected]
ABSTRACT
We tested the plant host sanction hypothesis using soybean plants co-inoculated with
two rhizobial strains, a normally N2 fixing strain and a mutant derivative that lacks
nitrogenase activity but has the same nodulation abilities. We found no evidence of
functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability
in co-inoculated plants.
INTRODUCTION
The origin and persistence in nature of symbiotic interactions is difficult to explain
since the existence of exploitative, cheating' partners that could erode the interaction is
common. Host sanctions against non N2 fixing, cheating symbionts have been proposed
as a force stabilizing mutualism in legume-Rhizobium symbiosis (Denison, 2000).
Penalizations would include decreased nodular rhizobial viability and/or early nodule
senescence in nodules occupied by cheating rhizobia.
We analyze the ecological and evolutionary stability of Rhizobium-legume symbiosis
when "cheating" strains are present, using a combination of experiments and modelling.
Co-occupation of the same nodule by strains with different fixation abilities is an
important source of concern in cultivated legumes (Rolfe and Gresshoff, 1980). Effects
of co-occupation of nodules by non-fixing rhizobia would be diluted by fixing rhizobia
occupying the same nodule (Denison, 2000). We performed experiments with soybean
plants co-inoculated with two rhizobial strains, a normally N2 fixing strain and an
isogenic non-fixing, cheating mutant derivative that lacks nitrogenase activity but has
the same nodulation abilities.
Bacterial strains and inoculum preparation. Mutant derivatives BJD321 (nopB-lacZuidA, Zehner et al., 2008) and A3 (nifH::Tn5, Nod+ Fix-; Hahn et al., 1984) strains
derived from Bradyrhizobium japonicum USDA110 were grown in PSY medium
(Regensburger and Hennecke, 1983) and used for inoculations (10 9 cells/ml). Coinocula were prepared by mixing bacterial solutions containing similar number of
colony forming units (CFUs). Strain A3 lacks nitrogenase activity but shows similar
infection and nodule formation levels respect to USDA110 and BJD321.
Plant experimental setting. Seeds of soybean (Glycine max) cv. Williams were surfacesterilized, germinated and sowed in Leonard jar assemblies. Seedlings (2/jar) were
inoculated independently with 1 ml of a bacterial suspension made from cultures of
each BJD321, A3 and a mixture of each strain. Jars were periodically supplied with a
sterile N2-free nutrient solution. Plants were placed in a growth chamber under 600
µEm-2 s-1 photosynthetically active radiation, at 25/18 °C day/night temperature, and
16/8 h photoperiod. Four weeks after inoculation nodules of each plant were collected.
Determination of nodule occupancy and viable rhizobial counts. Collected nodules were
individually surface sterilized using Cl2Hg (2.5%), manually crushed, homogenized and
43
Session I
SI-CP-11
resuspended in Tris-HCL mannitol buffer containing. Each crushed nodule was smeared
on PSY plates supplemented with selective antibiotics depending on the strain to
determine if the nodule was occupied by BJD321, A3 or both strains. To determine
rhizobial viability, appropriate serial dilutions from each nodule of another set of
homogenized nodules were plated (three replicates per dilution) in PSY supplemented
with selective antibiotics depending on the strain. Plates were incubated at 28 °C for a
week or until no further growth was detected, and CFUs were counted. Plant dry weight
and N content were determined in plants that had been heated at 60 ºC for 48 h.
Plants with all nodules occupied by non-fixing rhizobia were not able of maintaining
good vegetative conditions as plants with co-occupied or exclusively occupied nodules
with fixing rhizobia, and ultimately they died due to N starvation about 5 weeks after
inoculation. Thus, comparisons were performed using plants co-inoculated and plants
inoculated only with strain BJD321. In co-inoculated plants nodule co-occupation did
not differ (36.35 % BJD321, 33.32 % BJD321 and 27.28 % A3, χ 2 = 6.00, p = 0.199, n
= 66). Co-inoculated plants and plants only inoculated with strain BJD321 did not differ
in dry weight (Kolmogorov-Smirnov Z = 0.707, p = 0.699, n = 6). Total plant N did also
not differ between treatments (Kolmogorov-Smirnov Z = 1.299, p = 0.068, n = 6).
Nodule mass did not differ between co-inoculated plants and plants inoculated with
BDJ321 only (χ2 = 1.66, p = 0.56, n = 66). Number of CFUs did not differ between cooccupied nodules and BDJ321 or A3 single-occupied nodules in co-inoculated plants
(Figure 1).
Figure 1. Number of CFUs in cooccupied nodules or singleoccupied nodules in co-inoculated
plants.
No evidence of functioning plant host sanctions to cheater rhizobia based on nodular
rhizobia viability in co-inoculated plants was found. These experimental results will be
incorporated to the mathematical model (Marco et al., 2009) to check for plant
population persistence in presence of cheating rhizobia.
ACKNOWLEDGEMENTS
We thank Michael Goffert for B. japonicum BJD321 and Hans-Martin Fischer for A3 strains.
DM was granted with a Sabbatical leave from the Ministry of Education of Spain.
REFERENCES
Denison, R.F. (2000). Amer. Nat. 156: 567-576.
Hahn, M., et al. (1984). Plant Mol. Biol. 3:159-168.
Marco, D.E., et al. (2009). J. Theor. Biol. 259: 423-433.
Regensburger, B., and Hennecke, H. (1983). Arch. Microbiol. 135: 103-109.
Rolfe, B.G., and Gresshoff, P.M., 1980. Austr. J. Biol. Sci. 33, 491-504.
Zehner, S., et al. (2008). Mol. Plant Microbe Interact. 21: 1087-1093.
44
Session I
SI-CP-12
Identification of rhizobial strains nodulating cultivated grain legumes
in Egypt.
Zahran, H.H.1*, Chahboune, R.2, Bedmar, E.J.2, Abdel-Fattah, M.1, Yasser, M.M.1,
Mahmoud, A.M1.
1
Department of Botany and Microbiology, Faculty of Science, University of Beni-Suef, Beni-Suef 62511,
Egypt. 2 Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín,
Agencia Estatal Consejo Superior de Investigaciones Científicas (EEZ-CSIC). Apartado Postal 419,
18080-Granada, Spain.
*
[email protected]
INTRODUCTION
Members of the Leguminosae (Fabaceae), comprising 17,000 to 19,000 species, play an
important ecological role, with representatives in nearly every terrestrial biome on
Earth. These plants are best characterized by their ability to establish N2-fixing
symbiotic associations with Alphaproteobacteria of the genus Azorhizobium,
Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer (for reviews see Rivas et al.,
2009; Velázquez et al., 2010) collectively referred to as rhizobia. Other non-rhizobial
genera have been shown to nodulate legumes (for a review see Velázquez et al., 2010),
and they can also be nodulated by Betaproteobacteria of the genus Burkholderia (for
reviews see Angus et al., 2010; Bontemps et al., 2010; Gyaneshwar et al., 2011).
During the infection process, an exchange of molecular signals occurs between the two
partners, leading to the formation of root nodules, where nitrogen fixation takes place.
In Egypt, the grain legumes chickpea (Cicer arietinum), lentil (Lentils esculentus),
common bean (Phaseolus vulgaris), pea (Pisum sativum) and broad bean (Vicia faba)
are widely used for people consumption. Cultivated all along the River Nile, no data
exist on the bacterial diversity within nodules formed on those legume crops. In this
regard, a culture collection of more than 50 rhizobial-like strains isolated from root
nodules of those legumes were obtained whose phenotypic characteristics and
nodulation capacity have been published (Zahran et al., 2012). Our research had the aim
of identifying the strains isolated from nodules formed on agriculturally-grown
chickpeas, lentils, common beans, peas and broad beans, and to identify them on the
basis of their 16S rRNA gene phylogenies.
Isolation of bacteria from nodules and culture conditions
Nodules (10-12/plant) were collected from roots of healthy C. arietinum, L. esculentus,
P. vulgaris, P. sativum and V. faba plants agriculturally-grown in the Beni-Suef
Governorate (Egypt). They were surface-sterilized and crushed in a drop of sterile
water. The resulting suspension was streaked onto Petri dishes containing yeast extractmannitol (YEM) medium supplemented with 0.025 g/l Congo Red. Colonies forming
units were selected as indicated earlier (Chahboune et al., 2011).
DNA extraction and PCR amplifications
For DNA extraction and PCR amplifications, genomic DNA was isolated as described
previously (Chahboune et al., 2011). Repetitive extragenic palindromic (REP)polymerase chain reactions (PCR) were performed using primers REPIR-I and REP2-I
according to de Bruijn (1992). PCR amplifications of 16S rRNA gene fragments were
carried out using the two opposing primers 41f and 1488r as previously reported
(Chahboune et al., 2011). Amplification products were purified and subjected to cycle
sequencing using the same primers as for PCR amplification. The obtained sequences
45
Session I
SI-CP-12
were compared to the GenBank database using the BLAST program and also with the
sequences held in EzTaxon-e server (Kim et al., 2012). Phylogenetic trees were
prepared as indicated earlier (Chahboune et al., 2011). Nitrogenase activity was
determined as acetylene-dependent ethylene production as described previously (Zahran
et al., 2012).
A total of 54 bacterial strains, 12 from P. sativum, 11 from each C. arietinum and V.
faba, and 10 from each L. esculentum and P. vulgaris, respectively, were isolated from
extracts of nodules from healthy, agriculturally-grown plants in Beni-Suef Governorate
(Egypt). According to the results, the 54 isolates showed 15 different REP-PCR
patterns. The nearly complete sequence of the 16S rRNA gene from a representative
strain of each REP pattern revealed they all were members of the family Rhizobiaceae
of the Alphaproteobacteria. Of all the 15 strains, 12 were classified into the genus
Rhizobium and 3 into the Mesorhizobium group. The maximum likelihood phylogenetic
tree and EzTaxon-e analysis inferred from the 16S rRNA genes sequences indicated that
strains BSPV2, BSPV7, BSPS4, BSVF2, BSVF5, BSVF9, BSLE4 and BSLE10
clustered with R. leguminosarum USDA 2370T with identity values higher than 99.6%,
and that strains BSPS7, BSPS10 and BSPV9 grouped with R. pisi DSM 30132T, R. etli
CFN 42T and R. mesosinicum CCBAU 25010T, respectively, each of them with identity
values higher than 99.8%. Also, the strain BSCA1 clustered with M. amorphae ACCC
19665T, and strains BSCA8 and BSCA9 did with M. robiniae CCNWYC 115T, the all
three strains with 100% identity for the 16S rRNA gene sequences of their
corresponding type strains. The 15 rhizobial strains identified in this study nodulated
their original host plants. Nodules fixed N2, with values of nitrogenase activity,
determined as acetylene-dependent ethylene production, varying from 51 nmol C2H4
plant-1 h-1 in C. arietinum nodulated by strain BSCA8 to 480 nmol C2H4 plant-1 h-1 in P.
vulgaris inoculated with strain BSPV2.
ACKNOWLEDGEMENTS
This study was supported by ERDF-cofinanced grant RNM4746 from Consejería de Economía,
Innovación y Ciencia (Junta de Andalucía, Spain). A. M. Mahmoud thanks the Egyptian Government for
a Partnership and Ownership Initiative (PAROWN) grant to support his stay at Department of
Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Agencia Estatal CSIC, Granada,
Spain.
REFERENCES
Angus, A.A., et al. (2010). Mol. Ecol. 19: 28-23
Bontemps, C., et al., (2010). Mol. Ecol. 19: 44-52.
Chahboune, R., et al. (2011). Syst. Appl. Microbiol. 34: 440-445
De Bruijn, F.J. (1992). Appl. Environ. Microbiol. 58: 2180-2187.
Gyaneshwar, P., et al. (2011). Mol. Plant Microbe Interact. 24: 1276-1288.
Kim, O.S., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 716-721.
Rivas, R., et al. (2009). Rev. Microbiol. Insight, 2: 251-269.
Velázquez, E., et al. (2010). In: Proteobacteria: Phylogeny, metabolic diversity and ecological effects.
Sezenna, M.L. (ed). Nova Science Publishers Inc. New York, USA, pp37-56.
Zahran, H.H., et al. (2012). Aust. J. Basic Appl. Sci., 6: 571-583.
46
Session I
SI-CP-13
Genetic diversity and biogeography of rhizobial genospecies
nodulating wild chickpea Cicer canariense on La Palma (Canary Is.).
Armas-Capote, N.1, Pérez-Yepez, J.1, Martínez-Hidalgo, P.2, Garzón-Machado, V.3, del
Arco-Aguilar, M.3, Velázquez, E.2, León-Barrios, M.1*
1
Departamento de Microbiología y Biología Celular. 2 Departamento de Microbiología y Genética,
Universidad de Salamanca. 3 Departamento de Biología Vegetal (Botánica). Universidad de La Laguna.
Tenerife. Spain.
* [email protected]
ABSTRACT
We have characterized a collection of one hundred and thirteen rhizobial strains that
nodulate Cicer canariense and show the distribution of the bacterial genotypes at ten
locations on the island of La Palma (six inside Caldera de Taburiente National Park and
four in areas to the northwest and south of the Park). Analysis of the 16S rRNA gene
classified these isolates within the genus Mesorhizobium and distinguished nine
genotypes. The five predominant genotypes belonged or were close to reference strains
of M. caraganae (32 isolates), M. tianshanense/M. metallidurans/M. gobiense (29
isolates), M. opportunistum (14 isolates) M. ciceri (12 isolates) and M. tamadayense (11
isolates). M. tamadayense isolates were exclusive at one location outside the Park. The
M. tianshanense and M. ciceri groups predominated in planted plots and showed scarce
genetic diversity at infraspecific level. The M. caraganae and M. opportunistum groups
were widely distributed inside and outside the Park, though they predominated in
natural populations, and were genetically diverse.
INTRODUCTION
Cicer canariense is a perennial wild chickpea endemic to the Canary Islands, the only
native Cicer species in the archipelago. It is found almost exclusively on La Palma, in
the dry mesocanarian bioclimatic belt characteristic of pine woodland (Pinus
canariensis). Natural populations of C. canariense are fragmented and contain a low
number of individuals, and currently it is catalogued as “endangered” (EN) in the Red
List of the Spanish Vascular Flora (Moreno, 2008), according to the IUCN (2001)
criteria. Herbivores have probably been the most important threat for C. canariense
(mouflons and rabbits on Tenerife, and Barbary sheep, goats and rabbits on La Palma)
(Garzón-Machado et al., 2010). In this study we investigated for the first time the
genetic diversity of the rhizobia nodulating C. canariense in natural and reinforced
populations on La Palma (Canary Islands).
Random Amplified Polymorphic DNA (RAPD) fingerprints were obtained as previously
described (Ramírez-Bahena et al., 2012).
Restriction Fragment Length Polymorphism of PCR-amplified 16S rRNA (rrs) genes
(16S-RFPL). The rrs genes were amplified, restricted and analysed as described
(Jarabo-Lorenzo et al., 2000).
Sequencing of rrs genes. The rrs genes were amplified as above described and the
purified products (Qiaquick extraction kit, Qiagen) were sequenced in an ABI3730XL
(Macrogen, Inc.) or in a Genetic Analyzer 3500 (Servicio de Genómica, ULL).
Phylogenetic analyses were conducted as described (Lorite et al., 2011). The sequences
of rrs genes were compared with those of bacterial type strains using the EzTaxon-e
server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012).
47
Session I
SI-CP-13
A collection of one hundred and thirteen rhizobia that nodulate C. canariense were
characterized. Isolations were done from populations at ten sampling sites on La Palma,
which included six locations with natural and planted populations within the Caldera de
Taburiente National Park and another four localities with natural populations outside the
Park.
The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP
analysis and sequencing of the 16S rRNA genes. 16S-RFLP classified the isolates
within the genus Mesorhizobium and distinguished nine different ribotypes. Four
branches were minority genotypes (3-5 isolates), whereas another five contained the
predominant genotypes that clustered with reference strains M. tianshanense/M.
metallidurans/M. gobiense (M. tianshanense-group) M. caraganae, M. opportunistum,
M. ciceri and M. tamadayense. The 16S sequences confirmed homogeneity for M.
ciceri, M. opportunistum and M. tamadayense RFLP-groupings. They also resolved
further internal divergence within the two larger groups (M. caraganae and M.
metallidurans) outlining two novel Mesorhizobium species. RAPD fingerprints were
used to estimate the genetic diversity at infraspecies level, showing that highly similar
isolates belonged to the same RFLP group and were in general recovered from the same
location.
The geographical distribution of these genotypes varied among the different locations.
Some genotypes predominated or were restricted to a single location, whereas others
were widely distributed. Isolates from the M. tianshanense-group were detected at six
locations, although 23 out of 29 isolates were recovered from the same single location.
The M. ciceri-genotype isolates were detected at four sampling sites but predominated
at two neighbouring locations. The M. tamadayense genotype showed infraspecific
diversity, despite being exclusive to a single location outside the Park. Isolates from the
M. caraganae and M. opportunistum groups were widely distributed inside and outside
the Park, although they predominated in natural populations and were genetically
diverse.
ACKNOWLEGMENTS
Supported by the Ministerio de Medio Ambiente y Medio Rural y Marino, Organismo Autónomo de
Parques Nacionales (Ref. 111/2010).
REFERENCES
Garzón-Machado, V., et al. (2010). Biol. Conserv. 143: 2685-2694.
Jarabo-Lorenzo, A., et al. (2000). Syst. Appl. Microbiol. 23: 418-425.
Lorite, M.J., et al. (2010). Syst. Appl. Microbiol. 33: 282-290.
Moreno, J.C. coord. (2008). Lista Roja 2008 de la flora vascular española. Ministerio de Medio Ambiente
y Medio Rural y Marino, y Sociedad Española de Biología de la Conservación de Plantas, Madrid. 86
pp.
Ramírez-Bahena, M.H., et al. (2012). Syst. Appl. Microbiol. 35: 334-341.
48
Session I
SI-CP-14
Identificación de dos bacterias diazótrofas asociadas a una
Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú.
Chumpitaz, C. *, Ogata, K., Santos, R., Zúñiga, D.
Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso, Dpto. Biología, Universidad
Nacional Agraria La Molina. Lima12, Perú. www.lamolina.edu.pe/lmt
* [email protected]
RESUMEN
Se amplificó el gen nifH de la dinitrogenasa reductasa en dos cepas, LMTZ064-109 y
LMTZ064-119, asociadas a la rizosfera de Lepidium meyenii Walp. Los aislados
pertenecen a los géneros Stenotrophomonas y Rahnella, respectivamente, siendo ambas
Gram negativas, cocobacilos y oxidasa negativa. La aparición de un producto de
aproximadamente 370 pb perteneciente al gen nifH confirmó la capacidad fijadora de
nitrógeno molecular de ambas cepas.
INTRODUCCIÓN
La maca (L. meyenii Walp.) es una Brassicaceae que habita en regiones altoandinas del
Perú entre los 3700-4500 msnm. Es muy apreciada por sus cualidades nutricionales y
medicinales (Tovar, 2001). Al ser un cultivo muy extractivo genera un
empobrecimiento de los suelos, disminuyendo los rendimientos (Zúñiga, 2009). Los
diazótrofos, por otro lado, son un grupo de microorganismos capaces de fijar N 2 a
formas más asimilables en diferentes ambientes. El gen nifH de la Fe-proteína de la
nitrogenasa (enzima responsable del proceso de fijación) es usado como un indicador
determinante de esta capacidad (Zehr et al., 2003). El objetivo de este estudio fue
identificar la capacidad fijadora de N2 de los aislados bacterianos asociados al cultivo de
maca.
MATERIAL Y MÉTODOS
Reactivación y caracterización morfológica y bioquímica de bacterias conservados.
Amplificación BOX PCR de acuerdo a Versalovic et al. (1991).
Amplificación del gen nifH de acuerdo a Barua et al. (2011).
Análisis filogenético por medio del gen ARN ribosomal 16S según Weisburg et al.
(1991), usando la base de datos del Genbank (NCBI).
RESULTADOS Y DISCUSION
Un total de siete bacterias (LMTZ064-21, LMTZ064-30, LMTZ064-94, LMTZ064-95,
LMTZ064-109, LMTZ064-119 y LMTZ064-120) fueron seleccionadas de la colección
de bacterias promotoras de crecimiento vegetal (186 cepas) aisladas de suelos de
cultivo L. meyenni (Garcia, 2011). Estas cepas fueron caracterizadas morfológicamente
de acuerdo a su crecimiento en medio mineral sin nitrógeno (MMN-) por 48 horas a 28
°C. Las colonias tuvieron formas convexas, incoloras, de borde liso, mucosas y
diámetro entre 0.5-2 mm. Todas resultaron ser Gram negativas, cocobacilos y oxidasa
negativa.
De acuerdo al análisis BOX PCR se obtuvieron cinco perfiles distintos (Figura 1A), a
los cuales se les evaluó su capacidad fijadora de nitrógeno por medio de la
amplificación del gen nifH de la dinitrogenasa reductasa (Fe-proteína). Solo dos
aislados (LMTZ064-109 y LMTZ064-119) resultaron ser nifH positivos (Figura 1B).
49
Session I
SI-CP-14
A
B
A B
C C D
E
E
A B
C D E
nifH
370pb
Figura 1. A) Perfiles BOX A1R, A (21), B
(30), C (94, 95), D (109) y E (119, 120). M:
marcador 1 kb (fermentas). B) PCR nifH de
perfiles BOX diferentes. Cepas nifH+:
LMTZ064-109 y LMTZ064-119. CIAT899
Rizhobium tropici (control+). M: marcador 100
pb (Axygen).
Figura 2. Árbol filogenético en base al gen del
ARNr 16S mostrando la localización de los
aislados LMTZ064-109 y LMTZ064-119. Se
realizó un análisis Neighbor-joining con un valor
bootstrap calculado para 1.000 réplicas. Escala:
2% de secuencias divergentes.
De acuerdo al análisis filogenético realizado (Figura 2), se encontró que la cepa
LMTZ064-109 está relacionada a S. maltophila ATCC 13637T con 98%.
Stenotrophomonas spp. es un habitante común de suelos y plantas (Ryan et al., 2009),
especies de este género pueden actuar como promotores de crecimiento vegetal
mediante diferentes mecanismos como la fijación de nitrógeno y producción de
fitohormonas (Liba et al., 2006). Por otro lado, la cepa LMTZ064-119 tuvo un 100% de
similitud con Rahnella aquatilis HX2. Esta especie se reportó por primera vez como
una enterobacteria fijadora de nitrógeno presente en rizosferas de cultivos de maíz y
trigo (Berge et al., 1991) y su capacidad como PGPR en rizosfera de cultivos de soja y
papa (Kim et al., 1997; Kohashikawa, 2010) al producir fitohormonas y solubilizar
sales fosfato. En este trabajo se pudo determinar que las dos bacterias aisladas del
cultivo de maca fueron capaces de fijar nitrógeno molecular. Este parámetro es
importante a tener en cuenta para la producción de inoculantes bacterianos, ya que
además de reducir el uso de fertilizantes químicos, promueve el crecimiento de cultivos
de importancia económica dentro de una agricultura sustentable; mejorando a su vez la
calidad del suelo.
AGRADECIMIENTOS
Perú biodiverso GTZ-CONCYTEC, 2009. Procyt 309-2009-CONCYTEC. FDA Biol.111-UNALM.
BIBLIOGRAFÍA
Barua, M.S., et al. (2012). Microbiol. Res. 167: 95-102.
Berge, O., et al. (1991). Can. J. Microbiol. 37: 195-203.
Garcia, F. (2011). Tesis Doctoral. Universidad Nacional Agraria La Molina. Lima, Perú.
Kim, K,Y., et al. (1997). FEMS Microbiol. Lett. 153: 273-277.
Kohashikawa, N. (2010). Tesis Doctoral. Universidad Nacional Agraria La Molina. Lima, Perú.
Liba, C.M., et al. (2006). J. Appl. Microbiol. 101: 1076-1086.
Ryan, R.P., et al. (2009). Nature Rev. Microbiol. 7: 514-525.
Tovar, O. (2001). Publicación CONCYTEC. Lima 144 pp.
Weisburg, W.G., et al. (1991). J. Bacteriol. 173: 697-703.
Zehr, J.P., et al. (2003). Environ. Microbiol. 5: 539-554.
Zúñiga, D. (2009). Perú biodiverso GTZ-Concytec. Lima, Perú.
50
Session I
SI-CP-15
Análisis de la presencia natural de micorrizas en cultivos de algodón
inoculados con Bacillus sp. y/o Bradyrhizobium sp.
Valencia, C.1*, Toro, M.2, Zúñiga, D.1
1
Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Dpto. de Biología, Facultad de
Ciencias, Universidad Nacional Agraria La Molina, La Molina, Lima, Perú. Web:
www.lamolina.edu.pe/lmt. 2 Laboratorio de Estudios Ambientales, Instituto de Zoología y Ecología
Tropical, Facultad de Ciencias, Universidad Central de Venezuela.
* [email protected]
RESUMEN
Para el presente estudio, se extrajeron las raíces de cultivos de algodón (Gossypium
barbadense L.) inoculados con Bacillus sp. (B) y/o Bradyrhizobium sp. (Br), B + Br (I),
y los controles N+ (Nitrato de Potasio) y N- (Sin inocular). Para la tinción se realizó una
modificación optimizada del protocolo propuesto por Phillips & Hayman. Una vez
teñidas las raíces se calculó el porcentaje de Longitud de Raíz Colonizada (%LRC) para
determinar la influencia de las diferentes bacterias sobre la presencia natural de
micorrizas arbusculares (MA). Se observó que el %LRC (28.77%) disminuye cuando el
cultivo es fertilizado con nitrato de potasio, mientras que la colonización micorrícica se
promueve cuando las plantas son inoculadas con Bacillus sp., alcanzando en promedio
un 70.98%.
INTRODUCCIÓN
Las bacterias promotoras de crecimiento (PGPRs) están asociadas con la rizósfera de las
plantas y son capaces de ejercer un efecto benéfico en el crecimiento de plantas. Bajo
ciertas condiciones, estas bacterias son capaces de promover la germinación de esporas
de hongos y la elongación del tubo germinativo o incluso aumentar la densidad de la
colonización micorricica (Jaizme et al., 2006) Las MA son asociaciones ecológicamente
mutualistas que se establecen entre un selecto grupo de hongos (Glomeromycota) y la
gran mayoría de las plantas. Aproximadamente un 80% de las familias de plantas
existentes tienen la potencialidad de formar este tipo de asociación (Cuenca et al.,
2007). La distribución de micorrizas en el suelo puede ser afectada directamente por el
pH obteniéndose una buena germinación de esporas de MA en un intervalo amplio de
pH que va de 5 a 8 (Alvarado et al., 2004).
MATERIAL Y MÉTODOS
Las muestras fueron tomadas de un cultivo de algodón cosechado a los 180 días, el cual
había sido inoculado con B, Br y la interacción de ambos (I) en diferentes momentos: a
la siembra (M1), en la aparición del hipocotilo (M2) y en ambos momentos (M12)
(Zúñiga, 2011). El ensayo fue a nivel de invernadero y el sustrato utilizado para las
plantas fue una mezcla de suelo agrícola y arena (2:1). Las raíces fueron extraídas y
lavadas; para la tinción de raíces se utilizó el protocolo de Phillips y Hayman (1970)
con las modificaciones propuestas por Chávez et al. (2013). Las observaciones se
realizaron con un lente objetivo de 4x y se contaron los campos colonizados
(Sieverding, 1983). Los datos del %LRC se analizaron con el software estadístico
Statgraphics centurión.
El protocolo optimizado previamente para la tinción (Datos no mostrados) permitió
visualizar las características morfológicas de las MA (vesículas y arbúsculos) en raíces
de algodón (Figura 1). El análisis de varianza multifactorial (ANOVA) mostró que el
51
Session I
SI-CP-15
factor “tipo de inóculo” tuvo un efecto altamente significativo sobre el %LRC (p =
0.0000), mas no el factor “momento de inoculación”. Mediante la prueba de rangos
múltiples LSD, el tratamiento N+ afectó negativa y significativamente (28.77%) la
infección natural de los hongos micorrícicos, reportados por primera vez en el cultivo de
algodón en nuestro país (Figura 2). De acuerdo a lo indicado por Cornejo (2008), ello
podría deberse a la reducción del pH del suelo causada por la producción de H+
generado en el proceso de nitrificación. A su vez el control sin inocular N- presentó un
%LRC significativamente menor (53.81%) que las raíces inoculadas con B (70.98%), se
resalta que el porcentaje de infección en condiciones naturales es un indicador de que el
algodón es sensible a la infección por micorrizas. Sin embargo, cuando es inoculado con
la cepa de Bacillus sp. el %LRC se incrementa, esto es posible gracias a que las PGPRs
y las MA comparten la misma zona de infección y pueden crear una interacción que
favorece tanto el crecimiento de la planta como el aumento de la biomasa del hongo, en
contraste, otros autores señalan que no existe una interacción positiva entre las PGPRs y
las MA (Jaizme et al. 2006).
B
A
A
Porcentaje de colonización
Figura 1. Infección micorrícica en raíces de algodón inoculadas con Bradyrhizobium sp. A: Vesículas, B:
Hifas. Aumento 10X (Izquierda) y 4X (Derecha).
100
80
60
M1
40
M2
M12
20
0
B
Br
I
Tratamientos
N+
N-
Figura 2. Porcentaje de colonización para los tratamientos usados en la inoculación (B: Bacillus sp., Br:
Bradyrhizobium sp., I: Bacillus sp., + Bradyrhizobium sp. y dos controles N+: Con Nitrógeno y N-: Sin
Nitrógeno). Las series representan los momentos en los que se realizó la inoculación: a la siembra (M1),
aparición del hipocotilo (M2) y en ambos momentos (M12). Las barras señaladas con las mismas letras no
difieren significativamente (p ≤ 0.05).
AGRADECIMIENTOS
Protec-CONCYTEC-OAI N° 278-2009. FDA Biol. 111-UNALM.
BIBLIOGRAFÍA
Alvarado, A., et al. (2004). Agronomía Costarricense, 28: 89-100.
Chávez-Bárcenas, A.T., et al. (2013). African J. Microbiology Res. En prensa.
Cornejo, P., et al. (2008). Chilean J. Agric. Res. 68: 119-127.
Cuenca, G., et al. (2007). Interciencia, 32: 23-29.
Jaizme Vega, M., et al. (2006). Fruits, 61: 1-7.
Sieverding, E. (1983). Manual CIAT, Cali, 123 pp.
Zuñiga, D. (2011). Informe final Proyecto Protec-CONCYTEC, 2009.
52
Session I
SI-CP-16
Poblaciones microbianas con potencial PGPR en la rizósfera de
cultivos de papas nativas amargas del altiplano peruano.
Ramos, E.1*, Santos, R.1, Velezmoro, C.2, Zúñiga, D.1
1
Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Facultad de Ciencias.
Universidad Nacional Agraria La Molina. Lima 12, Perú. 2 Facultad de Industrias Alimentarias.
Universidad Nacional Agraria La Molina. Lima 12, Perú.
* [email protected]
RESUMEN
Se evaluaron las poblaciones microbianas de la rizosfera de papas nativas amargas de
las variedades locka (Solanum juzepczukii) y occucuri blanco (S. curtilobum) cultivadas
en Ilave - Puno (Perú), entre las coordenadas geográficas 16º06´00” y 69º38´00” de
latitud sur y oeste respectivamente y 3860 m.s.n.m.
INTRODUCCIÓN
Alrededor del 65% de la población andina económicamente activa se dedica a la
agricultura y la papa es el principal alimento básico, no gramíneo, de la dieta de sus
pobladores. Las papas nativas amargas (Solanum juzepczukii y S. curtilobum) pueden
crecer a altitudes de hasta 4200 m.s.n.m., donde los riesgos de pérdida de cosechas
debido a heladas son mucho mayores para papas de variedad “dulce”.
De acuerdo a la Dirección de Información Agraria de Puno, en la campaña 2010-2011,
este departamento cultivó alrededor de 46 966 ha de papa, con rendimientos promedio
de 9 641 kg/ha. Las estadísticas oficiales no diferencian áreas específicas para papas
dulces y amargas, sin embargo puede inferirse que alrededor del 26% son papas
amargas, obtenidas principalmente por producción orgánica.
Algunas poblaciones microbianas de papas han sido caracterizadas, sin embargo, a
pesar de la importancia de los cultivos de papas amargas en el Altiplano, pocos estudios
se han realizado al respecto. Así, el presente estudio pretende evaluar las poblaciones
microbianas de papas nativas amargas cultivadas en Ilave (Puno, Perú).
MATERIAL Y MÉTODOS
La zona de estudio se situó en la cuenca del río Ilave, de la provincia de El Collao, al
sur del departamento de Puno, con temperaturas que oscilan entre 1-15 °C y con
precipitación media anual de 620 mm. Se eligieron las comunidades andinas de
Jallamilla y Concahui. Se tomaron muestras de suelo rizosférico (franco arenoso, pH
5.1-5.5) de papas amargas de las variedades locka y occucuri blanco en 3 periodos de
evaluación: floración, tuberización y cosecha, en los meses de enero, abril y mayo del
2009 de manera respectiva. También se tomaron muestras de suelo no rizosférico.
Poblaciones microbianas de la rizosfera de papas amargas.
Se llevó a cabo la cuantificación de bacterias esporulantes mesófilas aerobias y
anaerobias (EMA y EMANA) y psicrófilas aerobias y anaerobias (EPA y EPANA),
mediante el método de APHA (1998). Los aerobios mesófilos viables (AMV),
Pseudomonas (PS), mohos y levaduras (MYL) fueron analizados según la ICMSF
(2000). Los diazotrofos (DIAZO) se ensayaron de acuerdo a Zapater (1975). Los datos
fueron sometidos al análisis comparativo de medias (prueba de Duncan, p≤0.05)
utilizando el programa R.
Caracterización molecular de los microorganismos aislados.
Se realizó mediante perfiles de amplificación BOX-PCR y secuenciamiento del gen
ARNr 16S, según las técnicas descritas por Versalovic et al. (1991).
53
Session I
SI-CP-16
Se observaron diferencias significativas en los grupos microbianos de las poblaciones
de EMA, EMANA, EPA, EPANA y DIAZO, según la época de muestreo, no
encontrándose diferencias según la comunidad ni variedad cultivada. La influencia
rizosférica solo se evidenció en las poblaciones de DIAZO de vida libre y PS. Así,
durante la floración, en los suelos cultivados, se registraron poblaciones de 10 5 NMP/g
PS, 107 NMP/g DIAZO y 105 UFC/g EMA (Figura 1).
Tabla 1.Clasificación filogenética de las cepas aisladas
de la rizosfera de papas amargas.
Figura 1. Poblaciones microbianas de
la rizosfera de papas amargas en tres
estados fenológicos del cultivo
Se observaron 25 diferentes perfiles de amplificación BOX-PCR entre los aislados
provenientes de la rizosfera de papas nativas amargas. Se identificó B. simplex NBRC
15720T, reportado en la literatura como secretor de auxinas y B. safensis FO-036bT, de
esporas altamente resistentes (Tabla 1). Otras cepas estuvieron relacionadas con B.
pumilus ATCC 7061T, conocido promotor de crecimiento vegetal, Brevibacillus
laterosporus DSM 25T, de uso potencial como controlador biológico y Lysinibacillus
parviboronicapiens BAM-582T. Se encontró además la presencia de cepas del género
Pseudomonas relacionadas a P. fragi ATCC 4973T que promueve la movilización de
fósforo, P. gessardii CIP 105469T que produce enzimas de gran resistencia al frío y P.
azotoformans IAM1603T de demostrada capacidad de fijación de nitrógeno. Las cepas
anteriormente mencionadas vienen siendo ensayadas para la evaluación de sus
propiedades PGPR.
AGRADECIMIENTOS
Proyecto PROCOM 255-2008-CONCYTEC-OAJ, Consorcio de productores de tunta “Los Aymaras”,
FDA Biol.111- UNALM.
BIBLIOGRAFÍA
APHA, AWWA, WPCF (1998). Standard Methods for Examination of Water and Waste Water. 20 ed.
ICMSF (2000). Microorganismos de los alimentos. Técnicas de análisis microbiológicos. 2 a ed.
Zapater, J. (1975). Anales científicos de la UNALM 13: 45-57.
54
Session I
SI-CP-17
Capacidad PGPR de cepas de Bacillus y Pseudomonas aisladas de la
rizosfera de aguaymanto (Physalis peruviana L.).
Cumpa, A. *, Flores, L., Chumpitaz, C., Ogata, K., Zúñiga, D.
Nacional Agraria La Molina. Lima-Perú. www.lamolina.edu.pe/lmt.
* [email protected]
RESUMEN
176 cepas, tanto de Bacillus como de Pseudomonas fueron aisladas del suelo y la
rizosfera del cultivo de aguaymanto (Physalis peruviana L.) proveniente de las regiones
de Lima y Junín en el Perú. Se realizaron dos pruebas de caracterización PGPR:
solubilización de fosfatos y antagonismos contra hongos. Además se realizó una prueba
de emergencia en semillas de aguaymanto. Las cepas Ba60 y Pa15 promovieron de
forma significativa la emergencia de las semillas de aguaymanto y la cepa Ba53
demostró una alto porcentaje de inhibición (59%) del hongo Fusarium sp.
INTRODUCCIÓN
Las bacterias promotoras de crecimiento vegetal o PGPR (por sus siglas en inglés)
otorgan múltiples beneficios a las plantas con las que interactúan, tales como el
incremento de la germinación, colonización de raíces, estimulación del crecimiento de
las plantas, control biológico, inducción de la resistencia a patógenos y mejoramiento en
la asimilación de agua y nutrientes (Barka et al., 2000). El objetivo de este trabajo fue
evaluar la capacidad PGPR de las bacterias aisladas y determinar su efecto en la
emergencia de semillas de aguaymanto.
MATERIAL Y MÉTODOS
Producción de ácido indol acético (Gordon y Webber, 1951)
Solubilización de fosfatos bicálcico y tricálcico (Nautiyal, 1999)
Pruebas de antagonismo (Ahmed et al., 2007 modificado).
Prueba de emergencia (Zúñiga, 2012)
Producción de ácido indol acético
Se evaluaron en total 176 cepas: 71 cepas de Bacillus sp. y 105 cepas de Pseudomonas
spp. aisladas de la rizosfera de aguaymanto. De todas las cepas, 86 (81.9%)
Pseudomonas spp. y 58 (81%) Bacillus sp. produjeron AIA. La producción más alta la
registraron las cepas Pa87 y Ba64, con valores de 54.31 y 44.56 ppm de ácido indol
acético respectivamente.
55
Session I
SI-CP-17
Solubilización de fosfatos bicálcico y tricálcico.
Tabla 1. Porcentaje de cepas analizadas, distribuidas por género y según su capacidad para solubilizar
fosfato
Solubilización de fosfatos
Medio
Cepas
No solubiliza
Bajaa
Mediab
Altac
Muy altad
Bicálcico
Tricálcico
Pseudomonas
40%
27.62%
12.38%
20%
0%
Bacillus
92.96%
7.04%
0%
0%
0%
Pseudomonas
70.48%
24.77%
2.86%
0%
0%
Bacillus
74.65%
23.94%
1.41%
0%
0%
a
b
c
d
h: halo de solubilización medido en cm; h<0.5, 0.5<h<1, 1<h<1.5, h>1.5
Emergencia(%)
Los mejores halos de solubilización de fosfato bicálcico se obtuvieron con las cepas
Pa15 y Pa85 (1.45cm y 1.5 respectivamente) y de tricálcico con las cepas Ba60 y
(0.7cm y 0.43cm respectivamente).
Prueba de antagonismo.
De entre las 40 cepas evaluadas la mejor fue la cepa Ba53 con un porcentaje de
inhibición del 59% (Figura 1). Es posible que el mecanismo empleado por estas
bacterias sea el de antibiosis con producción de metabolitos secundarios volátiles o
difusibles que pueden inhibir o restringir el crecimiento del patógeno (Rodríguez et al.,
2010).
Prueba de emergencia en semillas de aguaymanto (Physalis peruviana).
Se hallaron diferencias significativas en la emergencia de semillas de aguaymanto al
inocular la cepa Ba18 (T2) y la cepa Pa15 (T4) en comparación con el control sin
inóculo (T1) (Figura 2)
Cepas
Figura 2. Efecto de las cepas PGPR aisladas
en la emergencia de plántulas de
aguaymanto a nivel de laboratorio.
Figura 1. Cultivo dual de cepas Ba53, Ba54
y Ba56. Entre ellas, la cepa Ba53 presenta
un porcentaje de inhibición de 59%
AGRADECIMIENTOS
Este trabajo fue financiado por el proyecto Procyt-325-011-CONCYTEC-OAJ y la cuenta FDA-Biología, Lima-Perú.
BIBLIOGRAFÍA
Ahmed, H., et al. (2007). Biological Control 40: 97-106.
Barka, E., et al. (2000). FEMS Microbiol. Lett. 186: 91-95.
Gordon, S., and Weber, R. (1951). Plant Physiol. 26: 192-195.
Nautiyal, C. (1999). FEMS Microbiol. Lett. 170: 65-70.
Rodríguez, J. (2010). Revista de la Facultad Nacional Agraria de Medellín. 63: 5499-5509.
Zúñiga, D. (2012). Manual de Microbiología Agrícola. Universidad Nacional Agraria La Molina.
56
Session I
SI-CP-18
Phenotypic and molecular characterization of Lotus parviflorus nodule
bacteria.
Soares, R.1, 2, Lorite, M.J.2, Videira e Castro, I.1, Sanjuán, J.2*
1
2
Laboratory of Soil Microbiology, INIAV, Oeiras, Portugal. Department of Soil Microbiology and
Symbiotic Systems, Estación Experimental del Zaidín, CSIC, Granada, Spain.
* [email protected]
ABSTRACT
A total of thirteen root nodule bacterial isolates from Lotus parviflorus plants were
isolated from Estarreja, Portugal. Five isolates were slow growing strains and eight fast
growers. A high diversity was revealed by ERIC-PCR, grouping them in ten clusters at
75% similarity. The isolates were identified as bacteria belonging to the genera
Rhizobium, Bradyrhizobium, Sphingobacterium, Lysobacter and Pseudomonas. All
isolates nodulated at least one of the various Lotus hosts tested.
INTRODUCTION
The legume nodulating bacteria collectively known as Rhizobia, are able to establish
interactions with compatible plant species that culminate into the formation of nitrogen
fixing root nodules. It has been thought for long time that Lotus spp. would only form
specific symbioses with either slow-growing Bradyrhizobia or with intermediategrowing Mesorhizobia (Jarvis et al., 1982; Jordan, 1982), but recent studies have shown
that actually various genus of Rhizobia, such as Rhizobium, Mesorhizobium,
Bradyrhizobium, Sinorhizobium, Agrobacterim and Aminobacter can establish specific
interactions with Lotus spp. (Estrella et al., 2009; Lorite et al., 2010a, 2010b). In fact,
most Lotus species root nitrogen-fixing symbioses remain unexplored (Lorite et al.,
2010a, b). Our goal was to characterize L. parviflorus root nodule symbionts, which
have never been described previously.
Bacteria were isolated from root nodules of Lotus parviflorus wild plants growing in a
field of Estarreja, Portugal. The bacterial isolates were grown on Yeast Manitol Agar
(YMA) (Vincent, 1970), on YMA with congo red and YMA with Bromothymol Blue
(Somasegaran and Hoben, 1994).Total DNA was extracted following Estrella et al.
(2009). ERIC-PCR was performed following the procedure of Caetano-Anollés and
Gresshoff (1997) with De Brujin (1992) primers. 16S rRNA and glnII gene
amplifications were performed as described in Herrera-Cervera et al. (1999) and
Stepkowski et al. (2005), respectively. recA and atpD gene were amplified as described
in Gaunt et al. (2001). nodC and nifH genes were amplified as reported Laguerre et al.
(2001) and nodA following Moulin et al. (2004).
The amplified DNAs were extracted with the QIAEXII kit and sequenced with an
ABI373 automated sequencer. Plant nodulation assays were performed using L.
parviflorus PI415815, L. parviflorus PI283615, L. tenuis cv. INTA PAMPA, L. tenuis
cv. Esmeralda, L. corniculatus cv. São Grabiel, L. pedunculatus cv. Sunrise, grown on a
modified Agar Jensen medium. Three individual plants were inoculated with each
isolate; non-inoculated or KNO3 fertilised control plants were also included.
ERIC-PCR fingerprinting of the thirteen isolates showed the presence of ten different
clusters at 75% similarity, indicating a very high genetic diversity. Indeed, phylogenetic
analysis of the 16S rRNA gene sequences suggests that the isolates belong to either of 5
57
Session I
SI-CP-18
different genera: Rhizobium, Bradyrhizobium, Sphingobacterium, Lysobacter, and
Pseudomonas. A majority of the isolates belong to the Rhizobium/Agrobacterium
genera that are not typical Lotus symbionts and only one isolate was placed into the
Bradyrhizobium genus. Phylogenetic analyses of glnII, recA and atpD genes are in
progress to verify genus and species assignment. Since original L. parviflorus host
plants were wild, non-cultivated ones, seeds were not available and nodulation tests had
to be done on different Lotus spp. and genotypes. All isolates were able to nodulate at
least one of the Lotus plant hosts tested, and only the Bradyrhizobium Lp11 isolate
displayed a Fix+ phenotype in L. pedunculatus and L. parviflorus PI283615, although it
was Fix- in the other L. parviflorus genotype PI415815. All other bacteria-Lotus
combinations were Fix-, suggesting a very narrow host range of the L. parviflorus
isolates. This will need to be confirmed with additional host range tests together with
sequence analysis of symbiotic genes.
ACKNOWLEDGEMENTS
This work was supported by a BIOFAG network grant and by an ERASMUS mobility grant to R. Soares.
REFERENCES
Caetano-Anollés, G., and Gresshoff, P.M. (1997). DNA Markers Protocols, Applications, and Overviews.
Wiley-Liss, Inc.
Estrella, M.J., et al. (2009). Appl. Environ. Microbiol. 75: 1088-1098.
Gaunt, M.W., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 2037-2048.
Herrera-Cervera, J.A., et al. (1999). FEMS Microbiol. Ecol. 30: 87-97.
Jarvis, B.D.W., et al. (1982). Int. J. Syst. Bacteriol. 32: 378-380.
Jordan, D.C. (1982). Int. J. Syst. Bacteriol. 32: 136-139.
Lorite, M.J., et al. (2010a). Appl. Environ. Microbiol. 76: 4019-4026.
Lorite, M.J., et al. (2010b). Syst. Appl. Microbiol. 33: 282-290.
Moulin, L., et al. (2004). Mol. Phylogenet. Evol. 30: 720-732.
Somasegaran, P., and Hoben, H.J. (1994). Methods in legume-Rhizobium Technology. Paia: Univ.
Hawaii.
Stepkowski, T., et al. (2005). Appl. Environ. Microbiol. 71: 7041-7052.
Vincent, J.M. (1970). A Manual for the Practical Study of Root Nodule Bacteria. Oxford: Blackwell
Scientific.
58
Session I
SI-CP-19
Contribution of root nodule bacteria for the sustainability of
“montado” (cork oak) ecosystem.
Fernández, C.1, Soares, R.1, Barrento, M.J.2, Machado, H.2, Gomes, A.A.1, Videira e
Castro, I.1*
1
Laboratório de Microbiologia do Solo, UEISAFSV, INIAV,I.P., Oeiras, Portugal.
Sanidade Vegetal, UEISAFSV, INIAV,I.P., Oeiras, Portugal.
* [email protected]
2
Laboratório de
ABSTRACT
A total of fifty-three root nodule bacterial strains from Trifolium subterraneum plants
were isolated from soils of the Southern of Portugal and were subsequently
characterized. Besides nitrogen fixation, other important functions were also searched
such as solubilization of mineral phosphate, hydrolysis of plant polymers and
production of siderophores. Antagonistic activity against pathogenic disease caused by
Phytophtora cinnamomi was also investigated.
INTRODUCTION
Nitrogen is the primary nutrient limiting plant production in most natural ecosystems.
Legumes are important components of the strategy for increasing productivity and
sustainability of disturbed ecosystems, using symbiotic nitrogen fixation as a major
process of providing nitrogen to the soils. Therefore, legumes and their root nodule
bacteria, are considered as a powerful management tool for improving pastures yield in
the “montado” ecosystem (“dehesas” in Spain), which is an agroforestry system
associated with the exploitation of cork oaks.
In addition to nitrogen fixation, rhizobia can also promote the growth of plants either
directly through the solubilization of minerals, such as phosphorus, or indirectly as a
biocontrol agent by inhibiting the growth of pathogens. In Portugal, the decline caused
by Phytophthora cinnamomi is the main disease that in the last decades has led to the
widespread mortality of Quercus suber (Moreira and Martins, 2005). The aim of the
present work was to characterize and select root nodule bacteria from Trifolium
subterraneum plants grown in soils from Q. suber “montado” in different sanitary and
vegetative states in the Southern of Portugal (Grândola hill), in order to evaluate their
potential as plant growth promoters. So, besides nitrogen fixation, other important
functions were also searched, such as solubilization of mineral phosphate, hydrolysis of
plant polymers and production of siderophores, as well as antagonistic activity against
the P. cinnamomi.
For each soil sample, rhizobial natural population was estimated by the most probable
number (MPN) plant infection method using T. subterraneum cv. Clare as trap host. For
evaluating the effectiveness of rhizobial population, plants of T. subterraneum
inoculated with each soil were grown during 8 weeks in controlled environmental
conditions and the effectiveness was determined by the shoots dry weight.
A total of 53 root nodule bacterial strains were isolated on yeast manitol agar from
nodules of T. subterraneum (cv. Clare) grown in three different soils and subsequently
characterized. Biodiversity analysis was performed by ERIC-PCR fingerprinting (De
Bruijn, 1992). In vitro tests for determination phosphate-solubilization (Peix et al.,
2001), siderophore production (Perez-Miranda et al., 2007) and cellulase and pectinase
activities (Verma et al., 2001) were conducted qualitatively. Tests of antifungal activity
were performed in PDA plates inoculated first with fungus mycelium and two days later
59
Session I
SI-CP-19
with 3µl of each bacterial culture (grown for 48 h in TY liquid medium).
Tests with clovers inoculated separately with two strains of P. cinnamomi and two
strains of selected bacteria were performed in controlled environmental conditions,
during 8 weeks. Two strains of P. cinnamomi isolated from two different sites of
Grândola Hill, were used.
The size of the rhizobial population revealed great differences among the soil samples
tested, with values ranging from over 104 bact.g-1 of soil to less than10 bact.g-1 in soils
with low fertility. The results for nitrogen fixing capacity were variable. Values similar
to controls with nitrogen were observed only in one of the soil samples analyzed. In
soils with low fertility, the rhizobial population was ineffective (similar to controls
without nitrogen).
Analyzing the patterns obtained by ERIC-PCR, it was verified the existence of several
clusters, at 80% similarity, indicating a high genetic diversity among the population
studied. However, isolates from soils with low fertility were maintained almost
together.
Results obtained from plant growth promoting in vitro activities showed that within the
set of isolates, approximately more than half (60%) presented cellulase activity, 18%
could hydrolyse pectin, 12% were capable to solubilize mineral phosphate, but only 6%
were able to produce siderophores. These last isolates have also an antagonistic activity
against P. cinnamomi. This relationship may be important, since siderophore production
in iron stress conditions provides bacteria an added advantage, resulting in exclusion of
pathogens due to iron starvation.
The antagonistic activity of bacterial strains against P. cinnamomi was demonstrated by
the differences observed between the development of plants inoculated simultaneously
with the mycelium and rhizobia isolates and plants inoculated only with P. cinnamomi
which showed root damage and yellow leaves. These results indicate that root nodule
bacteria could have a potential as biological control agent against this disease.
ACKNOWLEDGEMENTS
This work was supported by Fundo Florestal Permanente. C. Fernandez is granted by Quercus Vprograma Leonardo da Vinci (FUNDECYT, España).
REFERENCES
Moreira, A.C., and Martins, J.M.S. (2005). For. Path. 35: 145-162.
Peix, A., et al. (2001). Soil Biol. Biochem. 24: 389-395.
Perez-Miranda, et al. (2007). J. Microbiol. Meth. 70: 2420-2428.
Verma, S.C., et al. (2001). J. Biotech. 91: 127-141.
60
Session I
SI-CP-20
Estudio del carácter endófito de bacterias del género Pantoea aisladas
de los cultivos de arroz de la Marisma del Guadalquivir.
Megías, E.1, Benitez, C.1, Diaz-Olivares, I.2, Ollero, F.J.2, Megías, M.1*
1
Universidad de Sevilla. Departamento de Microbiología y Parasitología.
Departamento de Microbiología.
*
[email protected]
2
Universidad de Sevilla,
RESUMEN
Muchas de las especies del género Pantoea se han descrito como bacterias endófitas en
diferentes plantas, incluidas las plantas de arroz. En este estudio se describe el carácter
endófito de aislados seleccionados de plantas de arroz y su relación con la movilidad de
la cepa y la producción de moléculas de homoseril-lactona. Se exponen los resultados
de cuatro cepas del género Pantoea respecto a la movilidad estudiada en medio sólido.
Todas presentan movilidad tipo swimming y producen AHL. Las cuatro cepas se
comportan como cepas endófitas, según los resultados observados en los ensayos de
reinoculación. Asimismo estudiamos la influencia de la movilidad y la producción de
AHLs en el carácter endófito.
INTRODUCCIÓN
El género Pantoea pertenece a la familia Enterobacteriaceae, y comprende actualmente
19 especies. Presentan una distribución ubicua y se han descrito como endófitos de
plantas de arroz (Mano et al., 2007; Okunishi et al., 2005; Sun et al., 2007; Verma et
al., 2001). La definición de endófito aceptada en la actualidad es la propuesta por
Hallman et al. (1997), que describe a los endófitos como bacterias que pueden ser
aisladas del interior de una planta o tejido vegetal esterilizado superficialmente, y no
provocan daños aparentes en la planta. Para acceder al interior de la planta los endófitos
utilizan como vía de entrada preferencial las zonas de aparición de las raíces
secundarias (Dong et al., 2003), aunque también pueden entrar a través de los estomas,
las lenticelas y heridas de la planta. Una vez en el interior de la planta los endófitos
pueden permanecer en un tejido específico, como el córtex radicular, o colonizar la
planta sistémicamente moviéndose a través de los vasos conductores o el apoplasto
(Hurek et al., 1994). A día de hoy se desconocen los mecanismos moleculares que
intervienen en el proceso de colonización y movimiento de la bacteria en el interior de
la planta. De este modo, el objetivo de este trabajo es determinar si la movilidad de las
bacterias y su capacidad de producir moléculas de comunicación del tipo AHLs, inciden
sobre la capacidad de colonización y movimiento de los potenciales endófitos.
MATERIAL Y MÉTODOS
Como material biológico se utilizaron 4 cepas previamente identificadas y clasificadas
por análisis filogenético como miembros del género Pantoea, se trata de las cepas
AMG-153 (P. dispersa), AMG-461 (P. deleyi), AMG-501 (P. ananatis) y AMG-521
(Pantoea sp.). Se siguió el protocolo descrito por Kearn and Losick (2003) para
determinar la movilidad de tipo swimming y de tipo swarming a las 48 horas. La
producción de AHLs de cada una de las cepas se calculó siguiendo el método descrito
por McClean et al. (1997).
Para describir el carácter endófito se llevaron a cabo ensayos de reinoculación en
condiciones controladas en el laboratorio, permitiendo de esta forma observar si las
bacterias se encontraban o no en el interior de la parte aérea de la planta de arroz. La
presencia de la cepa se confirmó mediante ensayos de BOX-PCR (Johnson and Clabots,
2000) frente al inóculo utilizado.
61
Session I
SI-CP-20
Se obtuvieron mutantes espontáneos inmóviles de las cepas AMG-501 y AMG-521,
mediante el uso de resistencias naturales a antibióticos como la rifampicina y la
espectinomicina. Para la obtención de mutantes no productores de AHLs se insertó el
plásmido pME6863 (Reimmann et al., 2002 ) en el interior de las cepas AMG-501 y
AMG-521, inhibiéndose la producción de moléculas de señalización poblacional. Se
comprobó la capacidad de colonización de los mutantes frente al silvestre, mediante
ensayos de reinoculación.
Todas las cepas analizadas presentaban movilidad de tipo swimming, cubriendo
prácticamente la totalidad de la placa a las 48 horas. En ninguna de las cuatro cepas se
observó movilidad de tipo swarming. Los resultados de producción de AHLs nos
muestran que todas las cepas producen AHLs en mayor o menor medida.
En los ensayos de reinoculación todas las cepas estaban presentes en el homogeneizado
de la parte aérea de la planta. En todos los casos se confirmó por BOX-PCR que la cepa
inoculada coincidía con las colonias recuperadas del interior de la planta. Se han
obtenido resultados preliminares en los ensayos de reinoculación destinados a
determinar la capacidad de colonización de la planta por parte de las cepas mutantes (en
movilidad y en producción de AHLs), frente a las cepas silvestres. Actualmente se están
llevando a cabo nuevos ensayos que confirmen los resultados observados.
En este trabajo hemos confirmado por ensayos microbiológicos la presencia de las
bacterias en el interior de los tejidos aéreos de la planta de arroz, pero no hemos llevado
a cabo ningún ensayo confirmativo por microscopía confocal o electrónica, uno de los
requisitos mencionados en la literatura (Reinhold-Hurek and Hurek, 1998) para poder
afirmar que una cepa es endófita. El objetivo futuro pasa por realizar este tipo de
ensayos de microscopía confocal en plantas de arroz inoculadas con cepas que
potencialmente actúan como endófitos.
Desde el punto de vista biotecnológico, y a la luz de las evidencias mencionadas en la
bibliografía, los endófitos pueden resultar interesantes al participar en diferentes
procesos relacionados con una posible mejora de las condiciones de la planta, tales
como el control de determinados patógenos (Pusey et al., 2011), la interacción con la
producción de hormonas por parte de la planta (Feng et al., 2006) o la capacidad de fijar
nitrógeno atmosférico (Loiret et al., 2004). Es por ello interesante determinar qué
factores intervienen en el proceso de colonización de las plantas y en el movimiento
bacteriano en el interior de las mismas.
BIBLIOGRAFÍA
Dong, Y., et al. (2003). Plant Soil 257: 49-59.
Feng, Y,. et al. (2006). J. Appl. Microbiol. 100: 938-945.
Hallman, J., et al. (1997). Can. J. Microbiol. 43: 895-914.
Hurek, T., et al. (1994). J. Bacteriology. 176: 1913-1923.
Johnson, J.R., and Clabots, C. (2000). Clin. Diagn. Lab. Immunol. 7: 258-264.
Loiret, F.G., et al. (2004). J. Appl. Microbiol. 97: 504-511.
Mano, H., et al. (2007). Microbes Environ. 22: 175-185.
McClean, K.H., et al. (1997). Microbiology 143: 3703-3711.
Okunishi, S., et al. (2005). Microbes Environ. 20: 168-177.
Pusey, P.L., et al. (2011). Phytopathology 101: 1234-1241.
Reimmann, C., et al. (2002). Microbiology 148: 923-932.
Reinhold-Hurek, B., and Hurek, T. (1998). Nuc. A. Sym. Series, 41: 95-98.
Sun, L., et al. (2007). Microbial Ecol. 55: 415-424.
62
Session I
SI-CP-21
El género Pantoea en los cultivos de arroz de la Marisma del
Guadalquivir: diversidad y posible descripción de una nueva especie.
Megías, E.1*, Fernández, M.1, Reis Junior, F.B.2, Márquez, M.C.1, Ollero, F.J.3, Megías,
M.1
1
Universidad de Sevilla. Departamento de Microbiología y Parasitología.
Universidad de Sevilla. Departamento de Microbiología.
*
[email protected]
2
Embrapa Cerrados.
3
RESUMEN
El género Pantoea se engloba dentro de la familia Enterobacteriaceae y presenta una
distribución ubicua, se han descrito algunas especies como endófitos de plantas de
arroz. De las cepas aisladas como endófitos de plantas de arroz de la Marisma del
Guadalquivir, 29 fueron clasificadas mediante la secuenciación del ARNr 16S como
pertenecientes al género Pantoea. Se realizo el análisis por MLSA basado en cuatro
genes housekeeping, atpD, gyrB, infB y rpoB, para clasificar las cepas dentro del
género. En este análisis se observó que 18 de los 29 aislados se encontraban
estructurados en un clado diferente próximo filogenéticamente a P. ananatis, P. allii, y
P. stewartii. Se postula que estos aislados podrían ser una especie nueva en el género, a
pesar de que la distancia filogenética con el grupo más cercano (P. ananatis), es
pequeña. Actualmente se están realizando ensayos de DDH (DNA-DNA hibridization)
con el fin de determinar si las 18 cepas dentro del clado problema pueden ser
consideradas como una nueva especie.
INTRODUCCIÓN
El género Pantoea se engloba dentro de la familia Enterobacteriaceae, y comprende
actualmente 19 especies. Presentan una distribución ubicua y se han descrito como
endófitos de plantas de arroz (Mano et al., 2007; Okunishi et al. 2005; Sun, et al., 2007;
Verma et al., 2001). Las evidencias encontradas en la literatura ponen de manifiesto la
capacidad de algunas cepas de este género para actuar en beneficio de la planta, bien
actuando como bacterias PGPR (Feng et al., 2006), bien como agentes de biocontrol
(Pusey et al., 2011). Esto las hace interesantes desde el punto de vista biotecnológico ya
que pueden tener diversas aplicaciones sobre diferentes cultivos, y en particular en los
cultivos de arroz.
El objetivo de este trabajo es determinar la diversidad de bacterias endófitas del género
Pantoea asociada a las plantas de arroz en los arrozales de la Marisma del Guadalquivir,
y encontrar cepas con posibles aplicaciones biotecnológicas.
MATERIAL Y MÉTODOS
A las cepas aisladas como endófitos se les extrajo el ADN (NucleoSpin® Tissue de MN)
y se amplificó el gen del ARNr 16s mediante los cebadores F27 y R1488 (Edwards et
al., 1989). Para la amplificación de los genes atpD, gyrB, infB y rpoB, se utilizaron
cebadores diseñados en nuestro laboratorio a partir de los descritos por Brady et al.
(2008). Los productos de PCR obtenidos se enviaron a secuenciar a la empresa Sistemas
Genómicos S.L. El tratamiento de las secuencias fue análogo para cada uno de los genes
trabajados y también se siguió la misma metodología para el concatenado de los 4 genes
housekeeping, base del análisis por MLSA. Cada paquete de genes se alineó mediante el
programa ClustalX 2.0 (Larkin et al.,2007) y los alineamientos fueron debidamente
editados mediante los programas Dambe (Xia et al., 2001) y BioEdit (Hall, 1999). Para
los análisis de las secuencias por Maximum-likelihood (ML), se eligió el modelo de
sustitución adecuado para cada set de datos mediante el programa jModelTest 2.1.1
63
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SI-CP-21
(Darriba et al., 2012). Los árboles filogenéticos se construyeron utilizando el programa
PhyML 3.0 (Guindon, 2010) partiendo de 200 árboles iniciales construidos en PAUP* y
un árbol construido por BioNJ (método habitual de inicio del programa PhyML). Se
eligieron los árboles con mejor lnL y se analizaron y editaron utilizando MEGA 5.0.
Para las pruebas bioquímicas se siguieron las indicaciones descritas por el fabricante en
el protocolo de las galerías Api 50CH (Biomerieux). Los resultados se analizaron
utilizando el programa Bionumerics.
El árbol filogenético obtenido del concatenado de genes housekeeping permite ubicar 9
de las 29 cepas analizadas en diferentes especies del género Pantoea. Dos de las cepas
analizadas se encontraban asociadas filogenéticamente al grupo externo, usado para
enraizar el árbol, por lo que estas dos, presuntamente asociadas al género Enterobacter,
se desecharon en posteriores análisis. El resto de las cepas, un total de 18, se agrupaban
en un único clado próximo a las especies P. ananatis, P. stewartii y P. allii. Este grupo
se repetía, con pequeños cambios en la filogenia, en los análisis gen a gen y en el
análisis del ARNr 16s. El análisis de los datos de consumo de azucares (pruebas Api
50CH) expresados en forma de matriz de distancias y dendrograma construido por
UPGMA, soporta los resultados obtenidos en el análisis filogenético, apareciendo el
clado formado por las 18 especies con la misma estructura que en los árboles
filogenéticos. Las cepas agrupadas en el clado problema presentan una distancia
filogenética pequeña en relación a la especie P. ananatis, siendo esto insuficiente para
considerar a este clado como una nueva especie. Brady et al (2008, 2009, 2010a, 2010b,
2011, 2012) estructura el género Pantoea, describe nuevas especies y reubica especies
anteriormente consideradas Pantoea en otros géneros, apoyándose en la hibridación
ADN-ADN para confirmar la descripción de las nuevas especies. Por otro lado, es
interesante mencionar la solidez de nuestros resultados filogenéticos en relación a
aquellos aportados por Brady et al en los trabajos arriba mencionados. En ellos no
mencionan los modelos de sustitución empleados y aplican rearreglos de rama basados
en Bootstrap, método mucho más lento y menos informativo que los rearreglos
mediados por algoritmos SH-like. En conclusión, los resultados de filogenia mostrados
en este trabajo están mejor adaptados a las nuevas corrientes en filogenia
computacional.
BIBLIOGRAFÍA
Brady, C.L., et al. (2008). Syst, Appl. Microbiol. 31: 447-460.
Brady, C.L., et al. (2009). Int. J. Syst. Evol. Microbiol. 59: 2339-2345.
Brady, C.L., et al. (2010a). Int. J. Syst. Evol. Microbiol. 60: 2430-2440.
Brady, C.L., et al. (2010b). Int. J. Syst. Evol. Microbiol. 60: 484-494.
Darriba, D., et al. (2012). Nature Methods 9: 772.
Edwards, U., et al. (1989). Nucl. Acid. Res. 17: 7843-7853.
Feng, Y., et al. (2006). J. Appl. Microbiol. 100: 938-945.
Guindon, S. (2010). Syst. Biol. 59: 307-321.
Hall, T.A. (1999). Nucleic Acid Symposium Series 41: 95-98.
Larkin, M.A., et al. (2007). Bioinformatics 23: 2947-2948.
Mano, H., et al. (2007). Microbes Environ. 22: 175-185.
Okunishi, S., et al. (2005). Microbes Environ. 20: 168-177.
Pusey, P.L., et al. (2011). Phytopathol. 101: 1234-1241.
Sun, L., et al. (2007). Microbial Ecol. 55: 415-424.
Xia, X., et al. (2001). J. Heredity 92: 371-373.
64
Session I
SI-CP-22
Bacterial biodiversity within the genus Enterobacter in paddies of
Guadalquivir marshes.
Fernández, M.1*, Megías, E.1, Reis Junior, F.B.2, Megías, M.1
1
Universidad de Sevilla, Dpto. de Microbiología y Parasitología. España 2 Embrapa Cerrados, Brasil.
* [email protected]
ABSTRACT
Associated with paddies there are many microbial communities that could be used as
biological agents in order to improve rice crops yields. Bacteria associated with rice
plants from different areas of paddies of Guadalquivir marshes were isolated and
studied focusing on Enterobacter. Using cultural and biochemical tests and 16S rDNA
gene sequencing we obtained 13 strains belonging to 5 different species of the genus
Enterobacter, and we observed that they were more common in areas irrigated with
river water than those irrigated with pit water. Now we are working on the remaining
strains isolated to identify them, studying by rpoB gene the strains already identified by
16S rDNA gene sequencing to complete our results and testing the plant growthpromoting capacities of our strains.
INTRODUCTION
The Guadalquivir marshes constitute an area placed in western Andalusia (Spain). This
area has approximately the 40% of Spain rice production.
Associated with paddies there are many microbial communities that could be used as
biological agents in order to improve rice crops yields, since these microorganisms are
able to carry out fundamental processes for rice plants.
The aim of this study is to isolate and identify bacteria present in rice plants grown in
the marshes of the Guadalquivir river, focusing on genus Enterobacter, which has been
described as one of the most commonly isolated genus of bacteria from rice plants
(Cottyn et al., 2001).
Rice plants of Puntal variety were collected in July 2011 from different areas in
Guadalquivir marshes, each of these areas with a different kind of irrigation: “Hato
ratón” area with pit water irrigation, “Los Alemanes” area with river water irrigation
and “La compañía” area with river water irrigation of better quality since this area is
closer to the river and water has not been recycled. Plants from each area were divided
in different zones: rhizosphere soil, roots and aerial part.
Roots and aerial part were surface sterilized with ethanol 70% and NaClO 5%. To
obtain the extract of each part, sterilized roots and sterilized aerial part along with NaCl
0.9% were triturated with Polytron PT 2100.
From each different sample 1 ml was pre-cultivated in TSB medium. Secondly, the
preculture was cultivated in Mossel enrichment medium for Enterobacteriaceae and
finally, that culture was seeded in MacConkey agar plates. Pink and red colonies were
selected and pure cultures were done form each of them.
These strains were subjected to IMViC (indole, methyl-red, Voges-Proskauer and
citrate tests) in order to identify if they belonged to the genus Enterobacter, and DNA
from those strains that were identified as Enterobacter was extracted and 16S rDNA
gene amplified by PCR with primers F27 and R1488 (Edwards et al., 1989). Then PCR
products were sequenced and analyzed with online servers Blast and EzTaxon (Chun et
al., 2007) to identify isolates.
65
Session I
SI-CP-22
We obtained a total of 61 isolates from the different parts of plants and different zones.
From “La Compañía” area we obtained: 18 isolates from soil samples, 5 isolates from
roots samples and 3 isolates from aereal part samples. From “Los Alemanes” area we
obtained: 15 isolates from soil samples, 2 isolates from roots samples and 3 isolates
from aereal part samples. From “Hato Ratón” area, we obtained 12 isolates from soil
samples and 3 isolates from roots samples.
32 isolates were positive for Enterobacter by the IMViC tests, and 21 of them have
been already identified by the 16S rDNA gene amplification as bacteria belonging to 5
different species of genus Enterobacter (Enterobacter ludwigii, E. aerogenes, E.
absuriae, E. hormaechei and E. cancerogenus) and other related genera.We will
complete these results by rpoB gene sequencing to make phylogenetic analysis.
Our results show that rice plants from paddies of Guadalquivir marshes constitute a
source of bacteria from several species of genus Enterobacter, and it can be observed
that this endophytic community of rice plants was influenced by paddy irrigation type,
being more abundant in areas irrrigated with river water than those irrigated with pit
water.
The presence of Enterobacter strains in the endosphere of rice plants also demonstrates
the ability of these bacteria to colonize rice plants, even the aereal part, suggesting that
they act as endophytes that could have several plant growth-promoting capacities that
we are now testing.
REFERENCES
Cottyn, B., et al. (2001). Phytopathol. 91: 282-292.
Edwards, U., et al. (1989). Nucl. Acid. Res. 17: 7843-7853.
Chun, J., et al. (2007). Int. J. Syst. Evol. Microbiol. 57: 2259-2261.
66
Session I
SI-CP-23
Isolation and characterization of a novel bacterium isolated from
paddies of Guadalquivir marshes.
Márquez, M.C.*, Fernández, M., Megías, E., Merchán, F.
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla.
*
[email protected]
ABSTRACT
During an investigation of the presence of bacteria associated with rice plant, we
isolated 32 strains from different areas of paddies of Guadalquivir marshes. The
phylogenetic relationships of these isolates were investigated performing a comparative
analysis of the sequences of the 16S rRNA. This study showed that one of these
isolates, designated as strain S.EP-T3-1, represents a novel sub line within the family
Enterobacteriaceae. Currently a polyphasic taxonomic approach of this strain is
performing to determine its exact taxonomic position.
INTRODUCTION
The family Enterobacteriaceae comprises a heterogeneous group of bacterial species
currently distributed among almost fifty genera. Many representatives are known to
exhibit plant-growth-promoting effects that include production of phytohormones,
fixation of atmospheric nitrogen and biocontrol activity (Götz et al., 2006; Ruppel and
Merbach, 1997; Ruppel et al., 1992;).
In this work, the phylogenetic relationships of 32 strains with potential plant growth
promoting capacities, has been investigated by comparative 16S rRNA analysis.
Besides, further studies based on a polyphasic characterization are carrying out with one
of these isolates, strain S.EP-T3-1, to confirm its precise taxonomic position as a novel
taxon within the family Enterobacteriaceae.
Bacterial strains.
A total of 32 strains were isolated from rice plant of Puntal variety in July 2011.
Klebsiella oxytoca CECT 860T was obtained from the Spanish Type Culture Collection
and used as reference for comparative phenotypic studies.
Sequence analysis.
The phylogenetic position of all the isolates was determined by almost-complete 16S
rRNA gene sequences analysis. Sequencing of the 16S rRNA genes was performed
according to Márquez et al. (2011). Phylogenetic analyses were carried out with the
ARB software package (Ludwig et al., 2004).
Phenotypic study.
Phenotypic characteristics of strain S.EP-T3-1 were observed on TSA after 24 h of
incubation at 37 ºC. Gram staining, motility, microscopic observations, and biochemical
analysis were performed according to standard laboratory procedure (Barrow and
Feltham, 2004). The API20E and API50CH (bioMerieux, Inc. Durham, NC) were used
to obtain the biochemical profile according to the manufacturers’ instructions. The G+C
content of DNA was determined by the thermal denaturation method described by
Marmur and Doty (1962).
Comparative 16S rRNA gene sequence analysis revealed that the 32 isolates allocate
into different genera of the family Enterobacteriaceae: Enterobacter, Klebsiella,
Citrobacter and Leclercia. From the results obtained in this study we observed that one
67
Session I
SI-CP-23
of these strains, designated as strain S.EP-T3-1, was loosely clustered to members of
this family, at the highest 16S rRNA similarity of 94% to Klebsiella oxytoca. In general,
organisms sharing less than 97.0% 16S rRNA gene sequence similarity do not have
DNA-DNA reassociation values greater than 70%, so DNA-DNA hybridization has not
been performed. The DNA G+C content of strain S.EP-T3-1 is 55.4 ± 0.9 mol%.
Well-isolated colonies of S.EP-T3-1 appear within 24 h of incubation at 37 ºC on TSA
medium. The colonies are cream, circular with entire margin and convex elevation.
They have a glistening texture due to capsule production. The size of a single colony of
an 18–24 h culture is about 0.15-0.2 cm in diameter. The isolate is capable of growing
over a temperature range of 10–43 ºC with an optimum of 37 ºC. The cells are Gramnegative straight rods (0.7-1 µm wide and 2-3 µm long) occurring singly and sometimes
in pairs. Biochemical tests have been performed on the isolate to confirm its taxonomic
position and to specifically differentiate it from K. oxytoca. In contrast to the type strain
of K. oxytoca, our isolate is motile and negative for indol test, nitrite reduction and
lactose fermentation. The API 20E and API50CH profiles are also clearly different
between both strains.
The data obtained up to now in the phenotypic and phylogenetic characterization
suggest that strain S.EP-T3-1 may constitute a novel taxon of the family
Enterobacteriaceae. Besides, we are carrying out the study of its chemotaxonomic
features in order to complete its characterization and confirm its precise taxonomic
position.
REFERENCES
Barrow, G.L., and Feltham, R.K.A. (2004). Cowan and steel’s manual for the identification of medical
bacteria, 3rd edn. Cambridge University Press, Cambridge.
Götz, M., et al. (2006). FEMS Microbiol. Ecol. 56: 207-18.
Ludwig, W., et al. (2004). Nucleic Acids Res. 32: 1363-71.
Marmur J., and Doty, P. (1962). J. Mol. Biol. 5: 109-18.
Márquez, M., et al. (2011). Syst. Appl. Microbiol. 34: 424-8.
Ruppel, S., et al. (1992). Plant Soil 145: 261-73.
Ruppel, S., and Merbach, W. (1997). Microbiol. Res. 152: 377-83.
68
Session I
SI-CP-24
Biodiversidad y performance simbiótica de bacterias noduladoras de
soja alóctonas de suelos de Argentina.
Covelli, J.M.1*, López, M.F.1, Arrese-Igor, C.2, Lodeiro, A.R.1
1
Ciencias del Medio Natural, Universidad Pública de Navarra, Navarra, España.
* [email protected]
2
Departamento de
RESUMEN
Aislamientos de bacterias noduladoras de soja tomados de cinco suelos de Argentina
mostraron una considerable biodiversidad genotípica agrupada por la acidez del suelo
de origen, e incluyeron rizobios de crecimiento rápido y bradirrizobios de crecimiento
lento. Los rizobios tendieron a ser malos fijadores de N 2, mientras que los bradirrizobios
tuvieron una performance similar o en ocasiones superior a las de Bradyrhizobium
japonicum E109 o USDA 110, usadas como referencia. La competitividad para nodular
frente a una E109 SmR/SpR fue en general baja, más particularmente la de los rizobios.
INTRODUCCIÓN
El cultivo de leguminosas más importante de la Argentina es la soja, cuya superficie
sembrada ha superado en 2012 los 19 millones de hectáreas. Gran parte de esta
superficie se inocula con B. japonicum para aprovechar la nodulación y fijación
biológica de N2 (FBN), pero en las zonas con historia previa de cultivo de soja a
menudo la inoculación no tiene efecto sobre el rendimiento. Esta falta de respuesta se
debe, en gran medida, a que los suelos donde se ha cultivado soja ya recibieron
inoculaciones en años anteriores y por lo tanto, cuentan con una población de rizobios
noduladores de soja derivados de dichos inoculantes (población alóctona).
Frecuentemente esta población alóctona ocupa la mayor parte de los nódulos en
desmedro del inoculante, lo cual restringe su impacto en la FBN global del cultivo
(López-García et al., 2009).
Un problema que surge en esta situación es si la calidad simbiótica de la población
alóctona se asemeja a la del inoculante. Esto no tiene por qué ser así, ya que la alta
performance de la FBN no es un carácter sujeto a selección positiva en el ambiente
edáfico. Por lo tanto, una vez que la población alóctona se establece en el suelo, puede
derivar genéticamente y/o intercambiar material genético con la población bacteriana
autóctona de forma tal de dar origen, en sucesivas generaciones, a una población cuya
calidad fijadora de N2 sea altamente heterogénea. Sin embargo, éste no parece ser el
caso de la performance para la nodulación: ya que el nódulo es un ambiente protector
para los rizobios, la capacidad de nodular sí puede estar sujeta a selección positiva en el
ambiente y por lo tanto, la población alóctona puede estar formada por genotipos
altamente competitivos para nodular.
En este trabajo hemos puesto a prueba las tres hipótesis, a saber: 1) la población
alóctona posee una significativa biodiversidad; 2) la población alóctona es variable en
su FBN, 3) la población alóctona es altamente competitiva para nodular.
MATERIAL Y MÉTODOS
El análisis de biodiversidad se llevó a cabo a través de la huella digital de ADN por
PCR con primers Box A1R (Versalovic et al., 1994), y posterior análisis con
GelCompare II.4.0 La FBN se estimó en plantas de soja DonMario 3010 cultivadas en
solución mineral libre de N (Lodeiro et al., 2000) a través del peso seco de nódulos
(PSN), el peso seco de la parte aérea (PSA) y la concentración de ureidos en hojas
69
Session I
SI-CP-24
(CUH) (Trijbels y Vogels, 1966) registrados al inicio de la floración. La competición
para la nodulación se midió coinoculando cada cepa en una relación 1:1 con LP 3018
(E109 SmR/SpR) a plantas de soja cultivadas como se describió, y registrando la
ocupación de nódulos por cada cepa alóctona (SmS o SpS) o LP 3018, a los 25 días postinoculación, en placas réplica con antibióticos selectivos (Althabegoiti et al., 2011).
Biodiversidad de la población alóctona: Hemos estudiado la distribución de genotipos
de 100 aislamientos obtenidos de cinco zonas del cinturón sojero de Argentina. La
mitad de los aislamientos provenían de nódulos obtenidos en el campo y la otra mitad
fueron obtenidos en laboratorio a partir de muestras de suelo. De este modo, estaban
representados, por un lado, los genotipos capaces de nodular en las condiciones
específicas de suelo y clima al momento de muestrear, y por otro lado, los genotipos
potencialmente capaces de nodular bajo condiciones ideales. Observamos una
considerable biodiversidad, con al menos 44 cepas diferentes en la colección de
aislamientos. Los genotipos se agruparon en ocho clados, pero el tipo de muestreo no
influyó en la distribución de genotipos entre los clados. Sin embargo, dicha distribución
estuvo significativamente sesgada por la zona de origen, y en particular, por la acidez
del suelo de origen, cuyos valores de pH iban de 6,1 a 4,8. Por lo tanto, confirmamos
que la población alóctona sufrió procesos de diversificación genética en el suelo.
Heterogeneidad en la FBN: Dado que la colección de aislamientos contenía tanto
rizobios de crecimiento rápido como bradirrizobios de crecimiento lento, elegimos
cinco cepas de rizobios y cinco de bradirrizobios para continuar con los estudios. Como
cepas de referencia utilizamos B. japonicum E109 y USDA 110. El PSN, el PSA y la
CUH mostraron correlación positiva estadísticamente significativa, con lo cual se
tomaron como representativos de la capacidad FBN. Esta capacidad divergió entre los
rizobios y los bradirrizobios. Mientras solo una de las cinco cepas de rizobios mostró
una performance FBN similar a las cepas de referencia, cuatro de las cinco cepas de
bradirrizobios se comportaron igual o mejor que las cepas de referencia. Por lo tanto,
confirmamos que la performance FBN de la población alóctona es heterogénea y
observamos que diverge entre rizobios y bradirrizobios.
Competitividad para la nodulación: En este caso, solo una de las cepas de rizobios y
dos de las de bradirrizobios compitieron igual que LP 3018, pero ninguna fue más
competitiva que dicha cepa de referencia. Dado que las 10 cepas elegidas al azar
representan aproximadamente el 25 % del total de cepas cuyos genotipos fueron
establecidos, no podemos asegurar que las cepas alóctonas son más competitivas que la
cepa E109, utilizada mayoritariamente en los inoculantes comerciales.
En este trabajo se evaluó la competitividad intrínseca de cada cepa, pero no la que
podría ejercer en su ambiente edáfico original. Por lo tanto, estos resultados indican que
las condiciones del ambiente original de la población alóctona tendrían una importante
gravitación para determinar la alta competitividad para nodular observada a campo.
AGRADECIMIENTOS
Este trabajo fue financiado por la Agencia Nacional de Promoción de la Investigación CientíficoTecnológica, la Universidad Nacional de La Plata y Barenbrug-Palaversich SA (todas de Argentina) y la
Dirección General de Investigación Científica y Técnica de España, proyecto AGL2011-30386-C02-01.
BIBLIOGRAFÍA
López-García, S.L., et al. (2009). Agron. J. 101: 357-363.
Versalovic, M., et al. (1994). Methods Mol. Cell Biol. 5: 25-40.
Lodeiro, A.R., et al. (2000). Plant Sci. 154: 31-41.
Trijbels, F., and Vogels, G.D. (1966). Biochim. Biophys. Acta. 113: 292-301.
Althabegoiti, M.J., et al. (2011). FEMS Microbiol Lett 319: 133-139.
70
Session I
SI-CP-25
Búsqueda y caracterización de bacterias promotoras del crecimiento
vegetal en rizosfera de plantas nativas antárticas.
Fernández Garello, P.1, Braga, L.1, Senatore, D.1, Lagurara, P.1, Yanes, M.L.1, Vaz, P.1,
Azziz, G.2 y Bajsa, N.1*
1
Laboratorio de Ecología Microbiana-Instituto de Investigaciones Biológicas Clemente Estable. Uruguay.
Laboratorio de Microbiología, Facultad de Agronomía - UDELAR. Uruguay.
* [email protected]
2
RESUMEN
En este trabajo se aislaron y caracterizaron bacterias rizosféricas a partir de las plantas
nativas antárticas Colobanthus quitensis y Deschampsia antarctica. Se estudiaron las
características promotoras del crecimiento vegetal de los aislamientos, encontrándose
producción de proteasas, sideróforos, celulasas y capacidad para solubilizar fosfato.
Algunos de los aislamientos presentaron varias actividades simultáneamente.
INTRODUCCIÓN
La Antártida posee uno de los ambientes terrestres más severos, caracterizado por bajas
temperaturas y falta de agua líquida, que limita la abundancia y actividad de organismos
terrestres. La vegetación terrestre nativa de la Antártida marítima incluye dos especies
de plantas vasculares: Colobanthus quitensis (clavel antártico) y Deschampsia
antarctica (pasto antártico). Dadas las condiciones ambientales adversas, es probable
que estas plantas presenten asociaciones con microorganismos que favorezcan su
crecimiento. El objetivo de este trabajo fue aislar y caracterizar bacterias promotoras del
crecimiento vegetal a partir de rizósfera de clavel y pasto antárticos.
MATERIAL Y MÉTODOS
Se colectaron plantas en varios sitios de la Isla Rey Jorge (Archipiélago Shetland del
Sur) en el verano de 2012 y 2013. A partir de suspensiones de raíces, utilizando medios
semiselectivos e incubando a 4 °C y 25 °C, se aislaron Pseudomonas fluorescentes
(King’s B), actinobacterias (almidón-caseína), bacterias heterótrofas (tripticasa-soja) y
hongos filamentosos (agar malta). Se evaluó su capacidad celulolítica (carboximetil
celulosa), proteolítica (medio con leche descremada), de producción de sideróforos
(CAS), solubilización de fosfato (NBRIP) y producción de biosurfactantes (ensayo de la
gota).
De 16 muestras de pasto y 17 de clavel se obtuvieron 667 aislamientos de heterótrofos,
546 de Pseudomonas fluorescentes, 125 de actinobacterias y 16 de hongos filamentosos
(Tabla 1). El 63% de los heterótrofos evaluados presentaron actividad proteolítica, el
61% mostró capacidad para solubilizar fosfato, el 41% produjo sideróforos, mientras
que sólo el 1% mostró actividad celulolítica. De las actinobacterias evaluadas, el 67%
presentó actividad celulolítica, el 3% mostró capacidad para solubilizar fosfato y el 15%
produjo sideróforos. No se obtuvieron actinobacterias capaces de producir proteasas
extracelulares ni biosurfactantes en las condiciones de cultivo ensayadas. Todas las
Pseudomonas fluorescentes evaluadas mostraron actividad proteolítica, en tanto que el
70% fue capaz de solubilizar fosfato (Figura 1). Considerando los aislamientos
evaluados hasta el momento, no se observaron diferencias en la proporciones con
actividad a ambas temperaturas. Se encontró un mayor porcentaje de actinobacterias y
heterótrofos productores de sideróforos aislados de clavel que de pasto, mientras que los
heterótrofos productores de proteasas fueron más abundantes en pasto que en clavel.
71
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SI-CP-25
Tabla 1. Colección de bacterias rizosféricas. Número de aislamientos de bacterias heterótrofas,
Pseudomonas fluorescentes y actinobacterias obtenidos a partir de pasto, clavel o suelo a las dos
temperaturas de incubación utilizadas.
nº aislamientos 4ºC
Muestra
nº aislamientos 25ºC
Pseudomonas
Actinobacterias
Heterótrofos
Pseudomonas
Actinobacterias
Pastos
153
172
23
157
203
42
Claveles
85
37
13
165
90
24
Suelo
57
6
9
50
53
14
TOTAL
295
215
45
372
346
80
Porcentaje de aislamientos que
presentaron actividad
Heterótrofos
100
80
60
40
20
0
actividad
proteolítica
Actinobacterias
actividad
celulolítica
producción de
sideróforos
Bacterias heterótrofas
solubilización de
fosfato
Pseudomonas fluorescentes
Figura 1. Actividades encontradas en los diferentes grupos bacterianos. Porcentaje de aislamientos de
actinobacterias, bacterias heterótrofas y Pseudomonas fluorescentes que presentaron las actividades
ensayadas (la actividad celulolítica en Pseudomonas no fue determinada hasta el momento).
Estos estudios se continuarán para analizar todos los aislamientos de la colección,
además de determinar otras propiedades tales como producción de fitohormonas,
fijación de nitrógeno, y capacidad de antagonismo in vitro. Los aislamientos que
resulten de interés serán identificados filogenéticamente.
AGRADECIMIENTOS
Financiación: Instituto Antártico Uruguayo y ANII (Beca de Iniciación a la Investigación a P.F.G.).
72
Session I
SI-CP-26
Caracterización de las comunidades de hongos micorrícicoarbusculares asociadas a metalofitas en suelos contaminados con Cu.
Cornejo, P.*, García, S., Vidal, C., Meier, S., Borie, F.
Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, P.O. Box 54-D,
Temuco, Chile.
* [email protected]
RESUMEN
En un ecosistema contaminado con Cu en la Zona Central de Chile (Valle de
Puchuncaví) se analizaron las comunidades de hongos formadores de micorrizas
arbusculares (HMA) y las características del suelo rizosférico de tres especies
metalofitas. Se observó la presencia de un ecotipo de Acaulospora presente en las tres
rizosferas, que representaba la práctica totalidad de esporas asociada a Oenothera
picensis, especie endémica que acumula importantes cantidades de Cu en su raíz y parte
aérea, y que puede representar una alternativa para realizar procesos de
biorremediación.
INTRODUCCIÓN
La minería del Cu representa en Chile la principal actividad económica, viéndose
también seriamente afectados los ecosistemas que se encuentran aledaños a los sitios de
producción, principalmente por la depositación de material enriquecido en metales y
lluvia ácida (Meier et al., 2012a). Bajo estas condiciones solamente unas pocas especies
de plantas pueden establecerse y crecer, conocidas como metalofitas, algunas de las
cuales pueden establecer simbiosis con HMA. Estos hongos favorecen la captación de
agua y nutrientes de estas plantas, y las protegen frente al estrés abiótico generado por la
presencia de éstos contaminantes en el ambiente, disminuyendo el estrés oxidativo de la
planta (Meier et al., 2011), o inmovilizando éstos contaminantes en sus estructuras de
resistencia, hifas o la glomalina que liberan en el suelo (Cornejo et al., 2008, 2013). En
este estudio se analizan las comunidades de HMA asociadas a la rizosfera de tres
metalofitas endémicas de la Zona Central de Chile, en el Valle de Puchuncaví, una zona
que ha recibido por varias décadas el impacto del funcionamiento de una fundición de
Cu.
MATERIAL Y MÉTODOS
Se recolectó suelo rizosférico de las metalofitas Baccharis linearis (Asteraceae),
Imperata condensata (Poaceae) y Oenothera picensis (Onagraceae), las que crecen de
forma natural en suelos contaminados (hasta 830 mg Cu kg -1) en el Valle de
Puchuncaví. Las esporas se extrajeron mediante tamizado húmedo y posterior
decantación en gradientes de sacarosa, recuperadas desde la suspensión, cuantificadas y
analizadas morfológica y molecularmente. Con las abundancias por cada ecotipo de
HMA encontrado, se caracterizó la diversidad de las comunidades con los índices de
riqueza (S`), equidad de Shannon-Wienner (H`) y Dominancia de Simpson (Λ). En el
suelo obtenido se determinó igualmente el pH, el P disponible, el porcentaje de
agregados estables al agua (AEA), las actividades microbianas dehidrogenasa (DH) y
fosfatasa ácida (P-asa), y la longitud del micelio micorrícico.
Se observó en todos los casos una fuerte acidificación del suelo, varias unidades por
debajo de los valores normales para la zona y suelo (alrededor de 7). Cornejo et al.
(2008) reportan para esta zona valores extremos de pH y de disponibilidad de Cu (más
73
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SI-CP-26
de 330 mg Cu kg-1) en el suelo rizosférico de I. condensata, que a pesar de lo anterior,
en este estudio presentó las comunidades de HMA más diversas, así como también los
mayores valores de micelio fúngico y AEA. Esto puede deberse a que también en estos
suelos es donde se han encontrado los mayores valores de glomalina, que permitirían
una modificación del medio ambiente en el cual se están desarrollando los HMA y otros
microorganismos. Por otra parte, estudios en esta especie han demostrado una gran
capacidad de exudación de ácidos orgánicos, especialmente cítrico, que posee una alta
capacidad para detoxificar el Cu libre en el medio (Meier et al. 2012b), por lo que las
plantas de I. condensata pueden hacer frente por varias vías al estrés ambiental.
Tabla 1. Principales resultados químicos, bioquímicos y de descripción de la comunidad de hongos
arbusculares asociados a plantas de especial interés ecológico del valle de Puchuncaví, Región de
Valparaíso, Chile.
Especie
pH1
P2
AEA3
PDH5
Mic.6
Esp.7
S`8
H`9
Λ10
4
asa
B. linearis
4.7
30.1
57.1
2.0
7.3
1.2
207
5.7
1.2
0.4
I.
4.5
49.4
89.4
2.6
6.3
4.6
148
5.3
1.4
0.2
condensata
O. picensis
5.0
24.1
66.2
2.2
11.1
2.5
582
2.3
0.1
0.9
1
pH suelo/agua en relación 2/5; 2 P según método Olsen (ppm); 3 Agregados estables al agua (%); 4 Actividad
Fosfatasa ácida (mg de PNP g-1 h-1); 5 Actividad dehidrogenasa (mg INTF g-1 h-1); 6 Longitud de micelio (m g-1); 7
Densidad de esporas (esporas en 100 g de suelo); 8 Riqueza de especies; 9 Índice de equidad de Shannon-Wienner;
10
Índice de dominancia de Simpson.
La alta dominancia en O. affinis se debe a la presencia mayoritaria (más de 90%) de un
ecotipo de Aculospora en la comunidad de HMA asociada a su rizosfera. Este ecotipo
también compone las comunidades de las otras plantas estudiadas, y se ha detectado por
medios moleculares usando PCR-TTGE (Cornejo et al., 2004) colonizando las raíces,
por lo que puede ser un candidato idóneo para obtener inoculantes que puedan mejorar
el establecimiento y crecimiento de especies micorrizables en suelos contaminados con
Cu. Sin embargo, los aspectos funcionales de la simbiosis necesitan un mayor grado de
comprensión, sobre todo por la capacidad que pudieran tener algunos HMA en
particular de producir sustancias como la glomalina, que sería un componente idóneo
para mejorar los procesos de fitoestabilización. En este sentido, existen indicios que la
glomalina se produce en mayor cantidad a altos niveles de estrés, por lo que ecotipos de
HMA no adaptados a estas condiciones igualmente pudieran ser una herramienta, si
genética y funcionalmente pudieran tener la capacidad de generar elevadas cantidades
de glomalina (Meier et al., 2012c)
AGRADECIMIENTOS
Estudio financiado por el Proyecto FONDECYT 1120890, CONICYT-Chile.
BIBLIOGRAFÍA
Cornejo, P., et al. (2004). FEMS Microbiol. Lett. 241: 265-270.
Cornejo, P., et al. (2008). Sci. Total Environ. 406:154-160.
Cornejo, P., et al. (2013). Soil Biol. Biochem. 57: 925-928.
Meier, S., et al. (2011). Appl. Soil Ecol. 48:117-124.
Meier, S., et al. (2012a). Crit. Rev. Environ. Sci. Technol. 42: 741-775.
Meier, S., et al. (2012b). Ecotox. Environ. Safe. 75: 8-15.
Meier, S., et al. (2012c). Appl. Soil Ecol. 61: 280-287.
74
Session I
SI-CP-27
Screening for phosphate solubilising rhizobia from faba bean in
Marrakech region field cultures.
Maghraoui, T.1, 2*, Domergue, O.2, 3, Oufdou, K.1, Lahrouni, M.1, Galiana, A.2, Sanguin,
H.2, Drevon, J.J.3, de Lajudie, P.2
1
Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences Semlalia, Cadi
Ayyad University, PO Box 2390, Marrakech, Morocco. 2 INRA, IRD, CIRAD, LSTM, Campus de
Baillarguet TA A82/J 34398 Montpellier. 3 INRA, Eco&Sols, 1 Place Viala, 34060 Montpellier, France.
*
[email protected]
ABSTRACT
Rhizobia and phosphorus (P)-solubilising bacteria are important to plant nutrition.
These microbes also play a significant role as plant growth-promoting rhizobacteria
(PGPR) in the biofertilization of crops. The present study was conducted in order to
select rhizobial strains adapted to low P, isolated from faba bean nodules in Morocco
and to investigate their ability to solubilise the mineral P. Our results revealed that
among 100 isolates, 15 are able to solubilising the mineral P using TCP test. 40 strains
were evaluated for their effect on the growth of faba plant and were genotypically
characterized. Preliminary results show contrasting effect of strains on nodulation rate
and plant growth.
INTRODUCTION
Low soil phosphorus availability is a major constraint for crops specially when they are
depending on symbiotic nitrogen fixation. Phosphorus is often present in soil as
insoluble complexes thus unavailable to plant. Phosphorus complexes with aluminum,
iron, silicium, and other metallic ions in acidic soil (Whitelaw, 2000) or with calcium
carbonate in alkaline soil (Gyaneshwar et al., 2002).
Some rhizobial strains, beneficial N2-fixing symbiotic partners of legumes, were
reported to solubilize both organic and inorganic phosphate complexes (Richardson,
2001; Alikhani et al., 2006; Jisha and Joseph, 2008). Using selecting rhizobia, as
phosphate-solubilizing microorganisms (PSM) as inoculums would have thus a dual
beneficial nutritional effect resulting both from P mobilization and N 2-fixation (Peix et
al., 2001). The current study was designed to select phosphate solubilising rhizobia in
order to circumvent phosphorus deficiency in soil in an environmentally-friendly, and
sustainable manner.
The rhizobial strains were isolated from nodules of faba bean plants in Marrakech
region according to Vincent (1970). We evaluated their P solubilising capacity using
four solid media and two phosphorus forms: NBRIY with CaHPO 4, NBRIY Ca3(PO4)2,
TCP NH4Cl and TCP KNO3 with Ca3(PO4)2.
The characterization of Rhizobium strains was performed by PCR amplification and
sequencing of 16s rRNA coding gene, recA, of nodD and pqqC gene encoding the
pyrroloquinoline quinone synthase C.
Infectivity and efficiency of rhizobial strains was studied on plants in hydroaeroponic
conditions.
We screened a collection of 100 rhizobial strains isolated from P deficient and P
sufficient soils of field cultivated with faba bean in the Houz region. 15 strains were
able to solubilise the two types of complex phosphorus on the four mediums used. The
75
Session I
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diameter of peripheral halo zone formed by these strains on NBRIY CaHPO 4 was larger
than the others media. Using the second source of phosphorus complex Ca 3(PO4)2, the
largest diameter were obtained on TCP NH4Cl medium (Figure 1).
Figure 1. Rhizobial isolates forming
peripheral halo zone on TCP NH4 medium.
Figure 2. Faba bean plants inoculated with
rhizobial strains selected, in hydroaeroponic
conditions.
The phosphate solubilising rhizobia strains were tested for their efficiency to grow with
faba bean plants under phosphorus deficiency in hydroaeroponic cultures (Figure 2).
The preliminary results indicate that inoculation with selected strains contribute to a
difference in root and shoot weight, plants size, and in nodulation. P and N content
determination in the different compartments of the plant is in progress.
16s rRNA and rec A gene sequences of twenty strains shows that they all belong to
Rhizobium leguminosarum. nodD and pqqC gene analysis are in progress
ACKNOWLEDGEMENTS
This work was supported by the Great Federative Project FABATROPIMED of Agropolis Foundation
under the reference ID 1001-009 and AVERROES scholarship programme provided by the EU for the
stay of Tasnime MAGHRAOUI in Montpellier.
REFERENCES
Alikhani, H.A., et al. (2006). Plant Soil 287: 35-41.
Arcand, M.M., Schneider, K.D. (2006). Ann. Acad. Bras. Ciênc. 78: 791-807.
Gyaneshwar, P., et al. (2002). Plant Soil 245: 83-93.
Hamdali, H. et al. (2008). Appl. Soil Ecol. 38: 12-19.
Valverde A., et al. (2007). First international Meeting on Microbial Phosphate solubiliszation. 273-276.
Vincent J.M. (1970). A manual for the practical study of root-nodule. Blackwell Scientific Publications,
Oxford.
Whitelaw, M.A. (2000). Adv. Agron. 69: 99-104.
Peix, A., et al. (2001). Soil Biol. Biochem. 33: 103-110.
76
Session I
SI-CP-28
Estudio de la dinámica poblacional binaria de rizobacterias en Lupinus
mediante citometría de flujo.
Ruiz Palomino, M.1, 2, Probanza, A.1*
1
Universidad San Pablo CEU. Facultad de Farmacia. Departamento de Biología.
Madrid. Departamento de Biología.
*
[email protected]
2
C. San Agustín
RESUMEN
El presente estudio analiza mediante Citometría de Flujo (CF) la dinámica de dos
poblaciones bacterianas (Bacillus P1C11 y Pseudomonas P1C10) en Lupinus albus L.,
creciendo en hidroponía. Se evalúa el crecimiento de ambas poblaciones solas o en
interacción doble a 3 diferentes proporciones. El estudio se completa sin el efecto de la
planta, en medios ricos (caldo nutritivo) y restrictivos (solución de Hoaglands) respecto
a fuentes de carbono.
INTRODUCCIÓN
El conocimiento de la dinámica e interacción entre poblaciones bacterianas rizosféricas
no sólo tiene interés básico, sino que también es imprescindible optimizar
biotecnologías aplicadas, como puede ser la introducción de PGPRs para
biorremediación, fitoprotección o la mejora de la producción. En este sentido hay
algunos estudios de interacción poblacional binaria (del Gallo et al., 1986) pero sin el
efecto planta. La CF es una técnica con buen rendimiento analítico en estudios de
ecología microbiana realizados en varios ambientes (Bergquist et al., 2009; Wang et al.,
2010) pero su aplicación es infrecuente en el caso de muestras de suelo/planta, aunque
rinde interesantes resultados (Müller y Nebe von Caron, 2010) singularmente en
estudios de rizosfera (Gamalero et al., 2004; Rochat et al., 2010).
MATERIAL Y MÉTODOS
La Figura 1 esquematiza del diseño de los experimentos y protocolo seguidos.
La CF (en concreto FC/FACS, Flow Cytometry/Fluorescence-Activated Cell Sorting) se
realizó con un equipo FACS CALIBUR (Becton Dickinson) usándose tampón PBS
como líquido envolvente, con una velocidad de fujo para contar 200 eventos s -1. Las
bacterias fueron enumeradas usando SSC (side angle-scattered light) y la emisión de
fluorescencia en verde (FL1, Fluorocrome light), tras tratarlas con el flourocromo
SYTO 13. La adquisición y análisis de los datos se realizó con el programa CellQuestn.
Dinámicas poblacionales en caldo nutritivo. En los ensayos de Bacillus/Pseudomonas
100:0 ó 0:100, se observa que a lo largo del tiempo analizado descienden los recuentos
de microorganismos, debido probablemente a que ha superado la fase estacionaria, y la
disminución de nutrientes impide el mantenimiento de dicha fase. En el caso de
aquellos cultivos mixtos de Bacillus/Pseudomonas 75:25 y 50:50, las tendencias de las
curvas de crecimiento son similares. La población de Bacillus en cualquier caso es
dominante frente a las Gram negativas, pero ambas poblaciones mantienen
aproximadamente los mismos valores iniciales. Sin embargo, en el caso de los cultivos
mixtos con un porcentaje inicial de Pseudomonas del 75%, se muestra claramente un
gran crecimiento de Bacillus, superando a término las primeras. En este caso (en
minoría), la población de Pseudomonas es menos competitiva que la población de
Bacillus. A término los niveles de cada población son análogos a los otros dos mixtos.
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SI-CP-28
Dinámicas poblacionales en solución de Hoagland. En al cultivo puro de Bacillus, se
mantiene la población, con oscilaciones menores. La CF muestra que una gran parte de
estas bacterias están esporuladas, por lo que mantiene un contingente poblacional
estacionario (e inactivo). En el cultivo puro de Pseudomonas, observamos el descenso
poblacional más dramático que en el del caso del caldo nutritivo. Las muestras
pertenecientes a cualquiera de las tres combinaciones ensayadas, tienen perfiles
similares a los del ensayo en caldo nutritivo: se mantienen estables, aunque atendiendo
a la localización de las partículas en las representaciones gráficas obtenidas en los
análisis (SSC y FL1) habría diferenciaciones en cuanto a la morfología y estado del
ciclo vegetativo de las dos cepas utilizadas.
Figura 1. Esquema del diseño de los experimentos y protocolo seguidos. Figura 2. Análisis de recuentos
bacterianos por citometría de flujo a los 4 días de cultivo, en cultivo hidropónico de Lupinus, en las
muestras con inoculación inicial Bacillus: Pseudomonas, 100:0, 75:25, 50:50, 25:75 y 0:100,
respectivamente. Eje X: dispersión de luz por tamaño de la partícula (SSC); eje Y: luz absorbida por el
fluorocromo SYTO 13 (FL1) en escala logarítmica. Los ejes internos separan las regiones de recuentos:
superior derecha (Bacillus) y superior izquierda (Pseudomonas) (100.000 eventos analizados)
Dinámicas poblacionales en cultivo hidropónico de Lupinus. En el cultivo de Bacillus
100% se mantiene durante los primeros 4 días una curva de población similar a la
obtenida en los estudios sin influencia de la planta. Pese a algunas oscilaciones
observamos aquí claramente la influencia de los exudados radicales en el medio de
cultivo, ya que se mantiene durante más tiempo y más activas (no esporuladas) el
contingente poblacional. En el caso del cultivo puro de Pseudomonas, se detectan
fluctuaciones, tal vez relacionables con pulsos cíclicos (circadiano) de exudación
radical. Las muestras pertenecientes a cualquiera de las tres combinaciones ensayadas
resultan en un dominio de Pseudomonas a término, con cierta densodependencia inicial
(Figura 2) y que relacionamos (entre otras razones) con la mayor capacidad
colonizadora de Pseudomonas.
BIBLIOGRAFÍA
Bergquist, P., et al. (2009). Extremophiles 13: 389-401.
del Gallo, M., et al. (1986). Plant Soil 90: 107-116.
Gamalero, E., et al. (2004). FEMS Microbiol. Ecol. 48: 79-87.
Müller, S., and Nebe Von Caron, G. (2010). FEMS Microbiol. Rev. 34: 554-587.
Rochat, L., et al. (2010). Mol. Plant Microbe Interact. 23: 949-961.
Wang, Y., et al. (2010). Trends Biotechnol. 28: 416-424.
78
Session I
SI-CP-29
Utilização da técnica de MLSA em estudos taxonômicos e filogenéticos
com Bradyrhizobium e sua importância na descrição de novas espécies.
Delamuta, J.R.M.1*, Ribeiro, R.A.2, Hungria, M.2
1
Programa de Pós-Graduação em Microbiologia; Universidade Estadual de Londrina, Bolsista CAPES.
Laboratório de Biotecnologia do Solo, Embrapa Soja, Londrina. Bolsistas CNPq.
* [email protected]
2
RESUMO
A metodologia de MLSA (Multilocus Sequence Analysis) tem sido cada vez mais
utilizada como ferramenta para se inferir a taxonomia, a filogenia e a evolução de
bactérias simbióticas fixadoras de nitrogênio, conhecidas como rizóbios. Como
exemplo, o emprego do MLSA foi decisivo para a recente descrição da nova espécie
Bradyrhizobium diazoefficiens por nosso grupo de pesquisa (Delamuta et al., 2013).
Neste estudo, utilizando-se a sequência concatenada dos genes atpD, glnII e recA, as
relações filogenéticas de 13 estirpes de Bradyrhizobium foram determinadas e os
resultados indicam a existência de possíveis novas espécies dentro do gênero.
INTRODUÇÃO
Estudos sobre taxonomia, diversidade e evolução de procariotos, incluindo rizóbios, têm
como base técnicas polifásicas, incluindo dados fenotípicos e genotípicos (Zhang et al.,
2012). Na última década, a análise de genes housekeeping utilizando a técnica de
MLSA (Multilocus Sequence Analysis) tem sido empregada com sucesso em estudos
sobre as relações taxonômicas e filogenéticas entre rizóbios, mostrando-se como uma
ferramenta poderosa para a descrição de novas espécies (Delamuta et al., 2013).
MATERIAL E MÉTODOS
Treze estirpes de Bradyrhizobium foram escolhidas de estudos anteriores (Delamuta et
al., 2012; Menna et al., 2009). Além da sequência do gene ribossomal 16S, três genes
housekeeping (atpD, glnII, e recA) foram utilizados na análise filogenética por MLSA,
usando-se parâmetros pré-definidos, com o modelo de distância Tamura-Nei e o
algoritmo de Neighbor-Joining. O suporte estatístico foi avaliado pela análise de
bootstrap, com 1.000 repetições.
RESULTADOS
A árvore construída com o gene 16S RNAr dividiu as estirpes de Bradyrhizobium em
dois grandes grupos (Figura 1A), com o primeiro (G-I) reunindo oito estirpes SEMIAs e
as estirpes tipo do grupo B. japonicum e o segundo grupo (G-II) incluindo as SEMIAs
6154, 6028, 6053, 6148 e 6145 e a estirpe tipo de B. elkani. Na Fig. 1A também fica
evidenciado que a posição taxonômica das estirpes não ficou claramente definida com
base apenas no gene 16S RNAr.
Para uma análise taxonômica mais refinada das estirpes, as sequências dos genes atpD,
glnII e recA foram concatenadas (Figura 1B). Com base no MLSA, a SEMIA 656 foi
agrupada com B. huanghuaihaiense CCBAU 23303T, o par de estirpes 6002-6144
agrupou com B. arachidis CCBAU 051107T e as SEMIAs 6028, 6053 e 6145
mostraram uma maior similaridade com B. pachyrhizi PAC 48T. Interessantemente, sete
estirpes ocuparam posições isoladas na árvore.
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Session I
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DISCUSSÃO
O gênero Bradyrhizobium é considerado ancestral de todos os rizóbios e tem sido
isolado de várias leguminosas (Lloret et al., 2005). Contudo, apesar de sua posição
evolutiva, poucas espécies estão atualmente descritas no gênero. Uma possível
explicação para isso é que a taxonomia moderna é baseada no gene 16S RNAr, cuja
sequência é extremamente conservada nesse gênero. Nesse contexto, a técnica de
MLSA está se mostrando cada vez mais útil nos estudos filogenéticos e uma prova disto
é a sua utilização, nos últimos anos, na descrição e/ou reclassificação de novas espécies
de Bradyrhizobium (Guerrouj et al., 2013; Wang et al., 2013), inclusive com a recente
descrição de B. diazoefficiens (Delamuta et al., 2013). Os resultados deste estudo
indicam que as sete estirpes que ocuparam posições isoladas na árvore concatenada
provavelmente representam novas espécies e confirmam que esta técnica é importante e
eficaz para revelar a diversidade ainda pouco explorada dentro do gênero
Bradyrhizobium e, certamente, de outras espécies fixadoras de nitrogênio de
importância econômica e ambiental.
B)
A)
Figura 1. Relações filogenéticas entre as estirpes de Bradyrhizobium deste estudo e as estirpes tipo com
base no (A) gene 16S RNAr e (B) nos genes housekeeping concatenados (atpD, glnII, e recA).
REFERÊNCIAS
Delamuta, J.R.M., et al. (2012). Braz. J. Microbiol. 43: 698-710.
Delamuta, J.R.M., et al. (2013). Int. J. Syst. Evol. Microbiol. (online).
Guerrouj, K., et al. (2013). Syst. Appl. Microbiol. 36: 218-223.
Lloret, L., et al. (2005). Rev. Latinoam. Microbiol. 47: 43-60.
Menna, P., et al. (2009). Int. J. Syst. Evol. Microbiol. 59: 2934-2950.
Wang, J.Y., et al. (2013). Int. J. Syst. Evol. Microbiol. 63: 616-624.
Zhang, Y.M., et al. (2012). PLoS ONE 7(9): e44936. doi:10.1371.
80
Session I
SI-CP-30
Mycorrhizal fungal identity as determinant of functional assemblage of
plant-growth promoting bacteria in the rhizosphere.
Meleiro, A.I.1*, Carvalho, L.1, Correia, P.1, Melo, J.1, 2, Carolino, M.1, Cruz, C.1
1
Centre for Environmental Biology, Faculty of Sciences, University of Lisbon. Campo Grande, Bloco
2
C2, 1749-016 Lisboa, Portugal. Enviromental Microbiology and Biotecnology Lab, University of Vila
Velha (UVV). Street Comissário José Dantas de Melo 21, 29102-770, Vila Velha, Espirito Santo State,
Brazil.
* [email protected]
ABSTRACT
We found that rhizospheric abundance and functionality of groups of plant-growth
promoting bacteria related with phosphorus and nitrogen nutrition can vary with
arbuscular mycorrhizal fungal taxa colonizing maize roots, which indicates that
mycorrhizal fungi can modulate bacterial functional diversity in the plant rhizosphere.
INTRODUCTION
The rhizosphere microbiome can modulate plant health and productivity, and
understanding how microbes are assembled in the rhizosphere can offer us new
strategies to maximize the sustainable production of food (Bisseling et al., 2009).
Arbuscular mycorrhizal fungi (AMF) are root symbionts enhancing plant nutrition, and
whilst recent evidences indicate that AM fungi can potentially shape total soil microbial
community composition by acting as large suppliers of carbon to the rhizosphere (Singh
et al., 2008; Drigo et al., 2010), little is known about their influence on the functional
diversity of microbial communities. In this work, we addressed whether there is a
specific mycorrhizal fungal selection for particular soil bacterial functional groups with
emphasis on phosphorus (P) solubilizing and nitrogen (N) fixing bacterial communities.
Rhizospheric soil samples were collected from greenhouse experiments where maize
plants were inoculated with one of five AMF species (belonging to Glomeraceae and
Gigasporaceae families), and with (1) a known soil community of six species of plantgrowth promoting rhizobacteria involved in P and N nutrition, or with (2) a whole soil
biotic community. P-solubilizing and N-fixing bacterial cell densities for each AMF
treatment in both soil communities were determined by culturing methods. The most
promising P-solubilizing bacteria were isolated from each AMF treatment, and their
functional P-solubilizing capacities were determined. Bacterial isolates identification
was evaluated by 16S rRNA Gene Sequencing.
AMF taxa significantly affect the rhizospheric densities of P-solubilizing and N-fixing
bacteria. When a known community consisting of P-solubilizer and N-fixing bacterial
species were added, two distinct groups of AMF species were found regarding Psolubilizing bacteria, one (AMF from Glomeraceae family) with highest bacterial
densities, while other (AMF from Gigasporaceae family) greatly decreasing bacterial
densities (Figure 1). N-fixing bacteria densities also varied according with AMF
identity; we found highest densities with one AMF species from the Glomeraceae
family, whereas lowest densities were observed in one AMF species from the
Gigasporaceae family (Figure 1). When AMF were inoculated into a whole soil
community, similar results were found (data not shown).
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Figure 1. Phosphorus-solubilizing and nitrogen-fixing bacterial densities in rhizospheric soils of maize
plants inoculated with an AMF species (species 1, 2 and 3 belong to Glomeraceae family, and species 4
and 5 to Gigasporaceae family) and with a known community of six PGPR consisting of P-solubilizing
and N-fixing bacteria (means ± sd).
Overall, the functional characterization of bacterial isolates varied with AMF taxa (data
not shown).
Our results indicate that functional assemblage of soil bacterial community is influenced
by AMF identity. This suggests an ongoing mycorrhizal fungal-bacterial interaction in
soil influencing plant productivity.
ACKNOWLEDGEMENTS
This work was supported by the project PTDC/AGR-PRO/115888/2009 and
SFRH/BPD/33633/2009 (to LC) of the Fundação para a Ciência e a Tecnologia of Portugal.
REFERENCES
Bisseling, T., et al. (2009). Science 324: 691.
Drigo, B., et al. (2010). Proc Natl. Acad. Sci. USA 107: 10938-10942.
Singh, B.K., et al. (2008). Environ. Microbiol. 10: 534-541.
82
grant
Session I
SI-CP-31
Diversity and stress tolerance in rhizobia from Parque Chaqueño
region of Argentina nodulating Prosopis alba.
Chávez Díaz, L.1, González, P.1, Rubio, E.2, Melchiorre, M.1, 3*
1
Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), Centro de Investigaciones
Agropecuarias-Instituto Nacional de Tecnología Agropecuaria (CIAP-INTA). 2 Instituto de Zoología y
Microbiología Agrícola (IMYZA), Instituto Nacional de Tecnología Agropecuaria (INTA). 3 Facultad de
Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba.
* [email protected]
ABSTRACT
The genetic diversity, symbiotic effectiveness, drought tolerance and Indol Acetic Acid
(IAA) production of indigenous rhizobial populations in the Parque Chaqueño of
Argentina able to nodulate Prosopis alba were assayed. The populations were sampled
at five locations from the Arid, Semi-arid and Humid Chaco in the Parque Chaqueño
region. A set of rhizobial strains able to nodulate P. alba was obtained and selected
based on their molecular diversity by BOX-PCR. The highest molecular variability was
observed in rhizobial isolates from Semi-arid Chaco. High level of indolic compound
production and tolerance to osmotic treatment were significantly correlated with water
restrictions of the environments where the strains belonged. The strains were identified
by 16S rDNA sequencing as belonging to the genera Mesorhizobium, Bradyrhizobium
and Sinorhizobium. To our knowledge, this is the first report of P. alba nodulation by
strains other than Mesorhizobium chacoense, which was already described for the
Parque Chaqueño.
INTRODUCTION
The Parque Chaqueño in Argentina is a very extensive region, subjected to abiotic
stresses commonly modifying biodiversity of soil microorganisms and their capacity to
establish symbiotic relationships. To our knowledge, there is no information about
rhizobium diversity in areas of different moisture in the Parque Chaqueño region, or
about the physiological characteristics of rhizobia that might contribute to improve
Prosopis nodulation, growth and survival. We hypothesize that rhizobia from Arid,
Semi-arid and Humid Chaco of the Parque Chaqueño region of Argentina, able to
nodulate P. alba, have genetic polymorphism and the strain behavior under osmotic
stress and IAA production is correlated with the hydric characteristics of the
environment where the strains come from.
MATHERIAL AND METHODS
Soil samples were collected from five locations in Parque Chaqueño region, Argentina:
1) San Miguel 31°45’59’’ S, 65°25’00’’ W, Córdoba province; Arid Chaco; 2) Colonia
Benítez 27°20’00” S, 58°55’60’’ W, Chaco province; Humid Chaco; 3) Padre Lozano
23°12’51’’ S, 63°50’39’’ W, NE of Salta province; Semi-arid Chaco; 4) Isla Cuba
24°17’31’’ S, 61°51’10’’ W, Formosa province; Semi-arid Chaco; 5) Bolsa Palomo 24°
13’15’’S, 61°57’42’’W, Formosa province; Semi-arid Chaco.
Rhizobial isolates were obtained from nodules developed in P. alba trap plants 120 days
after sowing. DNA of rhizobial isolates and reference strains was used to perform RepPCR fingerprinting with BOX-A1R primer. PCR electrophoretic pattern were analyzed,
for each location with (UPGMA) clustering method and Dice’s coefficient as similarity
index. These data were used to describe molecular variability by the Principal
Coordinate Analysis (PCO) (Balzarini et al., 2011). The selected rhizobial isolates were
assayed for their tolerance to water deficit treatment (0.6 Mpa, -2 Mpa) and indolic
compound production (Glickman and Dessaux, 1995) in free-living state. 16S rRNA of
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rhizobial isolates representing the main BOX-REP-PCR clusters was sequenced and
published in data base.
RESULTS AND DISCUSION
PCO analysis showed the highest molecular variability in rhizobial isolates from Semiarid Chaco (Figure 1).
Tolerance to water deficit treatment as well as higher levels of indolic compound
production showed that strains from San Miguel (Arid Chaco) and Padre Lozano (Semiarid Chaco), both locations with higher soil water shortage conditions, showed the
highest survival under water deficit and higher levels of indolic compound production.
16S rDNA from rhizobial strains that showed higher contribution to P alba growth were
analyzed. Three of the five strain from Bolsa Palomo (2 KC 759691, 3 KC 759692 and
54 KC 759694) were classified as Mesorhizobium genus; and the other two (12 KC
759693 and 63 KC 759695) as Sinorhizobium. Isolates from Padre Lozano (53 KC
759699 and 40 KC 759698) were species of Bradyrhizobium and Sinorhizobium genera
respectively. The isolates obtained from Colonia Benitez (66 KC 759696) and Isla Cuba
(36 KC 759697) were species of Mesorhizobium and Rhizobium genera, respectively
(Figure 2). To our knowledge, this is the first report of P. alba nodulation by strains
other than Mesorhizobium chacoense, which was already described for the Parque
Chaqueño.
0,40
PCO 2 (8.2%)
0,20
B. japonicum USDA138
0,00
R. etli SC15
M. loti
M. chacoense
-0,20
E. meliloti1021
ACKNOWLEGMENTS
-0,40
-0,40
-0,20
REFERENCES
Bolsa Palomo
Isla Cuba
0,00
0,20
0,40
PCO 1 (11%)
Padre Lozano
Colonia Benitez
San Miguel
Figure 1. PCO based on UPGMA similarity
matrix and Dice’s coefficient of the rep-PCR
(primer BOX-A1R) products of DNA from
rhizobial isolates from locations in Parque
Chaqueño in Argentina
Figure 2. 16S rDNA gene phylogeny of Parque Chaqueño
rhizobial isolates from P. alba and other. The tree was
constructed from 1163 bp aligned nucleotide sequence data by
using the UPGMA algorithm. The numbers at branch points
are the significant bootstrap values (1000 replicates).
Accession nº are written in parentheses after the species name.
ACKNOWLEDGEMENTS
This work was supported by INTA-PNFOR-044341 and Secyt-UNC 162/12 projects.
REFERENCES
Balzarini, M.G., et al. (2011) In: Phytopathology in the Omics Era. (Rodriguez Herrera, R. Aguilar, C.N.,
Simpson-Williamson, J.K., and Gutierrez Sanchez, G., eds). Research Signpost, Kerala, India, pp 120.
Glickman, E., and Dessaux, Y. (1995). Appl. Environ. Microbiol. 61: 793-796.
84
Session I
SI-CP-32
Condiciones de estrés hídrico o salino modifican la composición y
capacidad formadora de biofilm de comunidades bacterianas aisladas
de rizósfera de alfalfa.
Bogino, P., Abod, A., Nievas, F., Santoro, V., Vicario, J. *, Giordano, W.
Departamento de Biología Molecular, Facultad de Cs Exactas, Físico-Químicas y Naturales, Universidad
Nacional de Río Cuarto. Río Cuarto, Argentina.
* [email protected].
RESUMEN
Se aislaron comunidades bacterianas procedentes de suelo rizosférico de alfalfa
sometido a distintas condiciones: a) control, b) estrés hídrico y c) estrés salino. Las
mismas se caracterizaron en función de la capacidad formadora de biofilm total
(CFBT), la cual fue mayor en la comunidad procedente de la condición de estrés
hídrico. La composición fue estudiada mediante métodos moleculares y se determinó
mayor variabilidad para las comunidades obtenidas bajo condiciones de estrés.
INTRODUCCIÓN
En los suelos el potencial agua (ψ) incluye el potencial osmótico (por solutos disueltos)
y el potencial mátrico (fuerza de retención del agua por el suelo) (Papendick y
Campbell, 1980). Aunque tales potenciales son termodinámicamente equivalentes,
representan formas de deprivación del agua que posiblemente afecten a la fisiología
bacteriana de maneras distintas. Ha sido estimado que más del 99% de toda la actividad
bacteriana en los ecosistemas naturales está asociada con bacterias organizadas en
biofilms (Potera, 1996). Aunque la vida en biofilm presenta varias ventajas para las
bacterias del suelo, entre las que se pueden mencionar la protección contra la desecación
(Flemming, 1993), es escaso el conocimiento de los mecanismos que utilizan las
bacterias para desarrollar estructuras del tipo biofilm, como así también de su
composición y funcionalidad en los distintos micronichos edáficos (Chang et al., 2007).
El objetivo del presente trabajo fue estudiar las comunidades multibacterianas
establecidas en estructuras tipo biofilm asociadas a rizósfera de M. sativa bajo distintas
condiciones de disponibilidad de agua.
MATERIAL Y MÉTODOS
Suelo y tratamientos. Se tomaron porciones de 0-20 cm de diferentes sitios de un suelo
ubicado en la localidad de Bulnes, Córdoba, Argentina. Se homogeneizó, se dispuso en
macetas, se sembraron semillas de alfalfa variedad Pampeana Córdoba y se
establecieron tres condiciones: Control (regado regularmente), Estrés Hídrico (escaso
riego) y Estrés Salino (NaCl 2,5 g por Kg de suelo). Se mantuvieron 40 días en cámara
de crecimiento.
Aislamiento de las comunidades rizosféricas. Se obtuvieron los suelos rizosféricos (SR)
para cada condición (SRC: Control; SRM: Mátrico; SRS: Salino) y se realizaron
recuentos en medio rico (AN). De las placas de mayor dilución, se aislaron 95 cepas por
tratamiento.
Formación de biofilm. Se siguió la metodología descrita por O’Toole y Kolter (1998).
Caracterización molecular. Se realizó un análisis de restricción del gen ARNr 16S
(ARDRA) con la enzima HaeIII. La identidad se obtuvo mediante secuenciación del
gen ARNr 16S.
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SI-CP-32
Tamaño y CFBT de las comunidades aisladas de suelos rizosféricos de alfalfa.
Los recuentos obtenidos para los suelos rizosféricos (SRC, SRS y SRM) fueron
superiores al recuento obtenido en el suelo entero (SE), esto es debido a que la rizosfera
representa un micronicho nutricionalmente más rico. Las ufc ml-1 en la rizosfera de
alfalfa bajo condiciones de estrés (SRM y SRS) fueron menores en comparación con
SRC (sin estrés).
Interesantemente, la CFBT fue mayor para la comunidad obtenida del suelo rizosférico
sometido a estrés por desecación (SRM), mientras que la comunidad recuperada del
suelo rizosférico salino (SRS) fue la que presento la CFBT más reducida. Podría
especularse que la condición de estrés afecta la composición de los exudados radicales y
por lo tanto la comunidad bacteriana que se establece en las proximidades de las raíces.
Considerando la fisiología bacteriana, es probable que condiciones de desecación
conduzcan a la selección de cepas con mayor CFB para colonizar la superficie de las
raíces otorgando ventajas de supervivencia, mientras que otros mecanismos no
necesariamente relacionados a la CFB tendrían mayor relevancia en la supervivencia
bacteriana en condiciones de estrés salino.
Caracterización de las comunidades rizosféricas.
Las cepas aisladas de cada tratamiento fueron agrupadas en subpoblaciones de alta y
baja CFB (ACFB y BCFB respectivamente). La mayoría de los perfiles de ARDRA
obtenidos en los subgrupos de ACFB fueron diferentes a los de BCFB, demostrando
que los ribotipos que constituyen cada subpoblación son diferentes y la existencia de
cierta correspondencia entre fisiología (CFB) y caracterización genética. En general, las
subpoblaciones de ACFB y BCFB procedentes de suelos rizosféricos sometidos a
condiciones de estrés (SRM y SRS) presentaron mayores perfiles de restricción respecto
de la condición no sometida a estrés (SRC), indicando la posible existencia de procesos
de diversificación en tales situaciones. Podría especularse que ante la presencia de
estrés, un grupo más variado de bacterias se establece como consorcio mixto en las
zonas cercanas a las raíces de alfalfa, probablemente en respuesta a la búsqueda de
micronichos menos hostiles. Por el contario, en condiciones no estresantes, la
comunidad mixta se establecería con predominio de los grupos más efectivos para
colonizar la zona próxima a las raíces y a que en tal condición otros grupos no necesitan
del microambiente rizosférico para sobrevivir. En función de su pertenencia a perfiles
de ARDRA mayoritarios, se seleccionaron cepas para su identificación mediante
secuenciación del gen ARNr 16S. Se destaca la identidad de las cepas aisladas con cepas
típicas encontradas en los suelos, en particular resulta interesante la presencia de cepas
filogenéticamente vinculadas a los rizobios como parte del biofilm mixto que constituye
la comunidad asociada a rizósfera de alfalfa.
AGRADECIMIENTOS
Este trabajo ha sido financiado por medio del Programa PPI SECYT-UNRC 18/C408 y de los Proyectos
PICT FONCYT 2007-02228, 2011-0965 y PIP CONICET 112-201101-00086.
BIBLIOGRAFÍA
Flemming, H.C. (1993). Water Sci. Technol. 27: 1-10.
Chang, W.S., et al. (2007). J. Bacteriol. 189: 8290-8299.
Papendick, R.I., and Campbell, G.S. (1980). Water potential relations in soil microbiology. p. 1-22
Potera, C. (1996). 273: 1795-1797.
O'Toole, G.A., and Kolter, R. (1998). Mol. Microbiol. 28: 449-461.
86
Session I
SI-CP-33
Supervivencia y capacidad formadora de biofilms por rizobacterias.
Abod, A.*, Bogino, P., Vicario, J., Giordano, W.
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales,
Universidad Nacional de Río Cuarto. Río Cuarto, Argentina.
* [email protected]
RESUMEN
Las rizobacterias constituyen un amplio grupo de bacterias las cuales han desarrollado
estrategias como la agregación microbiana y la formación de biofilms para adaptarse a
diversas condiciones edafo-ambientales. En el presente trabajo se estudió la
supervivencia y capacidad de formación de biofilm de distintas rizobacterias bajo
condiciones de estrés hídrico y salino. También se evaluó el desarrollo de biofilms
mixtos formado entre rizobios y Pseudomonas. En general, las rizobacterias que
presentaron elevada capacidad para formar biofilms en condiciones de desecación
también mostraron mayores niveles de supervivencia en esa condición de estrés.
INTRODUCCIÓN
La rizósfera es definida como la zona del suelo inmediata al sistema radical de los
vegetales, donde tiene lugar una interacción dinámica con los microorganismos. En la
rizósfera existe un variado grupo de bacterias conocidas como PGPR (Plant Growth
Promoting Rhizobacteria) (Spaepen et al., 2009). La capacidad para desarrollar
estructuras del tipo biofilm representa una estrategia clave para responder a variaciones
en la disponibilidad nutricional, sobrevivir en el suelo y mejorar sus posibilidades para
colonizar la planta hospedadora. La mayoría de los biofilms en la naturaleza consisten
de consorcios microbianos de múltiples especies (Kawarai et al., 2007), cuya
funcionalidad y arquitectura es influida por diversas situaciones estresantes (Chang et
al., 2007).
MATERIAL Y MÉTODOS
Cepas bacterianas: se utilizaron las cepas silvestres de Sinorhizobium meliloti (Rm1021
y Rm8530) y la mutante no productora de EPS II, Rm8530 expA. Además se usó la cepa
WCS417r de P. fluorescens.
Medio de cultivo: Se utilizó el medio LB reducido (LB/2) para generar un potencial
osmótico de -0,25 MPa, al cual se le agregaron cantidades crecientes de NaCl o de PEG
8000 para generar condiciones de reducción del potencial agua (estrés salino o hídrico
respectivamente) del orden de -0,5 y -1,0 MPa.
Cuantificación de la formación de biofilm: La capacidad de las cepas de formar biofilm
creciendo individualmente y en co-cultivo fue examinada por el método basado en la
tinción y medición de la absorbancia del colorante cristal violeta (O'Toole y Kolter,
1998), adaptada en nuestro laboratorio para ensayos en rizobios (Rinaudi et al., 2006).
Ensayos de viabilidad bacteriana en condiciones de estrés hídrico y salino: Se utilizó
arena estéril como sustrato, la misma se dispuso en tubos de vidrio y se humedeció a
capacidad de campo con suspensiones conteniendo 106 UFC de cada cepa de S. meliloti.
Los tubos fueron incubados durante distintos períodos de tiempo a 30°C. Se incluyeron
controles sin deshidratar. Para el estrés salino, se procedió de la misma manera, pero la
arena fue humedecida con soluciones de 430 mM y 250 mM de NaCl conteniendo 10 6
UFC de cada cepa de S. meliloti. Los controles fueron humedecidos con solución
fisiológica (NaCl 155 mM). Se realizaron recuentos bacterianos a diferentes tiempos.
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Ensayos de viabilidad bacteriana en arena.
Estrés hídrico. La cepa Rm8530 presentó mayor capacidad de supervivencia que las
cepas Rm1021 y Rm8530 expA. Interesantemente, se observó que Rm8530 incrementó
el número de ufc por gramo de arena en condiciones de desecación. Probablemente la
producción de EPSII por esta cepa y la vinculación del mismo con la capacidad de
formar biofilms (Rinaudi y Giordano, 2010) permitan explicar estos resultados.
Estrés salino. La cepa Rm1021 tuvo mejor capacidad de supervivencia cuando la
concentración de NaCl fue de 155 mM mientras que la exposición a altas
concentraciones de NaCl (250 y 430 mM) determinó una marcada disminución de la
viabilidad celular, con reducciones de alrededor de 5 órdenes de magnitud. En los casos
de Rm8530 y Rm8530 expA la exposición al estrés salino no determinó grandes
cambios de la viabilidad celular. Especulamos que la presencia del elemento regulador
ExpR (Marketon y González, 2002) en Rm8530 y Rm8530 expA estaría vinculado a la
supervivencia de estas cepas en condiciones salinas.
Cuantificación de la formación de biofilm por consorcios mono y multiespecies.
Se determinó que frente a condiciones de estrés hídrico y salino, a medida que el
potencial agua del medio se redujo, la cepa WCS417r de P. fluorescens disminuyó el
crecimiento y aumentó la formación de biofilm, este efecto fue más marcado en
presencia del soluto PEG respecto de NaCl. En el caso de la cepa Rm1021, la formación
de biofilm fue reducida en todas las variables estudiadas excepto en el tratamiento con
PEG -1,0 MPa, en cuyo caso se registró un aumento de dicho parámetro. La cepa
Rm8530 no fue afectada frente a estrés salino en su capacidad para formar biofilm, por
el contrario, condiciones de estrés hídrico ocasionaron un gran aumento en la formación
de biofilm. Cuando se examinó el biofilm mixto entre Pseudomonas y Rm8530 se vio
que la presencia de NaCl (-0,5 y -1,0 MPa) produjo incrementos en el crecimiento y
formación de biofilm respecto de LB/2 (-0,25 MPa), mientras que la adición de PEG (1,0 MPa) determinó un crecimiento despreciable y una marcada formación de biofilm
por el co-cultivo. Llamativamente, los recuentos diferenciales indicaron que el biofilm
formado en medio conteniendo PEG -1,0 MPa estuvo formado casi exclusivamente por
la cepa de rizobio, mientras que en condiciones de estrés salino (NaCl -1,0 MPa) el
biofilm mixto estuvo constituído por ambas cepas en proporciones similares. Se puede
concluir que ante un estrés salino, la bacteria usa diferentes mecanismos (como la
acumulación de solutos compatibles) para afrontar y persistir en esta condición. Por el
contrario, en condiciones de estrés hídrico la estrategia de supervivencia más
conveniente sería el desarrollo de estructuras tipo biofilms.
AGRADECIMIENTOS
BIBLIOGRAFÍA
Chang, W.S., et al. (2007). J. Bacteriol. 189: 8290-8299.
Kawarai T., et al. (2007). Appl. Environ. Microbiol. 73: 4673-4676.
Marketon M.M., and González J.E. (2002). J. Bacteriol. 184: 3466-3475.
O'Toole G.A., and Kolter R. (1998). Mol. Microbiol. 28: 449-461.
Rinaudi L., et al. (2006). Res. Microbiol. 157: 867-875.
Rinaudi L., and Giordano W. (2010). FEMS Microbiol. Lett. 304: 1-11.
Spaepen S., et al. (2009) Adv. Bot. Res. 51: 283-320.
88
Session I
SI-CP-34
Caracterización morfológica de rizobios asociados a cultivos de arveja
(Pisum sativum L.), chocho (Lupinus mutabilis S.), fréjol (Phaseolus
vulgaris L.), haba (Vicia faba L.) y vicia (Vicia atropurpurea) en suelos
del Ecuador.
Carpio, M.J., Paucar, B.M.*, Alvarado, S.P.
Departamento de Manejo de Suelos y Aguas (DMSA), Estación Experimental Santa Catalina (EESC),
Instituto Nacional Autónomo de Investigaciones Agropecuarias (INIAP). Quito, Ecuador.
RESUMEN
Una de las alternativas para cubrir la deficiencia de nitrógeno (N) en los suelos es el uso
de microorganismos fijadores de este elemento. Rizobios asociados con arveja, chocho,
fréjol, haba y vicia en suelos ecuatorianos fueron aislados y caracterizados. Se
identificaron diferencias en los parámetros de elevación y producción de goma entre
aislados del mismo cultivo y entre cultivos.
INTRODUCCIÓN
Uno de los principales elementos que limitan la producción agrícola en suelos de la
sierra ecuatoriana es N, y para suplir este requerimiento se aplican fertilizantes
nitrogenados con una baja eficiencia; y consecuentemente, con problemas de tipo
económico y ambiental (Beman et al., 2005; Galloway et al., 2008; Schlesinger, 2009).
Existen varias alternativas para manejar adecuadamente la fertilidad de los suelos y la
nutrición de los cultivos, entre estas se encuentran el uso de abonos verdes dentro de un
sistema de rotación de cultivos y el uso de microorganismos eficientes. Los
microorganismos fijadores de N contribuyen con un aporte de N al suelo de forma
natural (Revelo et al., 2010; Bernal, 2012), sin dañar el medio ambiente y a la vez
favorecen la economía del agricultor (Campaña, 2003; González, 2001; Subía, 2001).
En el Ecuador se ha realizado investigación sobre rizobios en algunos cultivos (fréjol,
arveja, alfalfa, trébol, soya y maní), pero sólo se cuenta con caracterización fenotípica y
genotípica del rizobio de fréjol y maní, habiéndose evidenciado una alta heterogeneidad
en los suelos ecuatorianos (Bernal, 2004; Aguilar et al., 2004). Con respecto a los
rizobios asociados al cultivo de haba, chocho y vicia, no se han realizado estudios y en
lo referente a arveja hay poca información.
En el presente estudio se realizó el aislamiento, purificación y caracterización
morfológica de los rizobios asociados a los cultivos de arveja, chocho, fréjol (voluble),
haba y vicia como parte inicial de la evaluación de los mismos en cultivos con potencial
uso como abonos verdes.
MATERIAL Y MÉTODOS
Las muestras de rizobios colectadas en cultivos de arveja, chocho, fréjol, haba y vicia en
la provincia Imbabura-Ecuador, se tomaron en la época seca (octubre) y época lluviosa
(enero), con cultivos en estado de floración.
El aislamiento se realizó utilizando medio Levadura manitol agar (LMA) con rojo
congo, y luego se purificó para realizar la caracterización mediante las siguientes
pruebas: Tiempo de crecimiento, acidificación o alcalinización del medio LMA
(Levadura manitol agar) con ABT (Azul de bromotimol) y características morfológicas
(textura, cantidad de goma, aspecto, forma, elevación y borde) (CIAT, 1988; Jerez,
2004; Marquina et al., 2011).
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Session I
SI-CP-34
Los aislamientos de rizobios asociados a arveja, fréjol, haba y vicia, tienen
características similares en textura, aspecto y borde, diferenciándose entre ellas por la
cantidad de goma que producen. En tanto, los rizobios asociados a chocho fueron muy
diferentes con respecto a los antes mencionados por la abundante cantidad de goma y
por la elevación convexa. Esto indicaría que son diferentes tipos de géneros concordante
con lo manifestado por Marquina et al. (2011).
En cuanto a los aislamientos de rizobios asociados a arveja se observaron diferencias en
la elevación y en la cantidad de goma producida; mientras los aislados de haba se
diferenciaron por la cantidad de goma producida. Estos resultados posiblemente se
deben a que sean rizobios de diferente especie o biovar en cada caso, pues las muestras
efectivamente fueron recolectadas en diferentes localidades.
Los aislamientos de rizobio de fréjol tienen características distintas en elevación a los
reportados por Marquina et al. (2011), lo cual indicaría que podría tratarse de otra
especie y además estas fueron recolectadas en diferentes localidades. En cuanto al
tiempo de crecimiento, todos los rizobios aislados son de crecimiento rápido y
modifican el pH del medio a ácido, lo cual es consistente con lo manifestado por el
CIAT (1988).
En general, estos resultados corroboran con investigaciones previas, donde se menciona
la gran variabilidad de rizobios en suelos ecuatorianos (Bernal, 2004; Aguilar et al.,
2004).
AGRADECIMIENTOS
Esta investigación es parte del Proyecto PIC 12 INIAP 009 “Manejo adecuado de abonos verdes y
microorganismos fijadores de nitrógeno dentro de sistemas de producción agroecológicos” financiado por
el Gobierno Nacional del Ecuador a través de SENESCYT y ejecutado por el Departamento de Manejo de
Suelos y Aguas de la EESC del INIAP.
BIBLIOGRAFÍA
Aguilar, M., et al. (2004). Proc. Natl. Acad. Sci. USA 101: 13548-13553.
Beman, M.J., et al. (2005). Nature 434: 211-214.
Bernal, G. (2004). INIAP. Resumen Ejecutivo. 80 p.
Bernal, G. (2012). Memorias SECS. 9 p.
Campaña, D. (2003). Tesis de Ing. Agr. UCE. 117 p.
CIAT (1988). Simbiosis leguminosa rizobio -Man. met. eval., sel. manejo agr. 178 p.
Galloway, J., et al. (2008). Curr. Sci. 94: 1375-1381.
Jerez, M. (2004). Tesis Ing. Agr. UCE. 127 p.
González, P. (2001). Tesis Ing. Agr. UCE. 102 p.
Marquina, M., et al. (2011). Biol. Trop. 59: 1017-1036.
Revelo, J., et al. (2010). Informe Proyecto SENESCYT. 12 p.
Schlesinger, W.H. (2009). Proc. Natl. Acad. Sci. USA 106: 203-208.
Subía, C. (2001). Tesis Ing. Agrp. ESPE. 151 p.
90
Session I
SI-CP-35
Bacterias con actividad ACC desaminasa en huertos de aguacate en
Michoacán, México.
Chávez-Bárcenas, A.T.1*, Hernández-Valdés, E.F.1, Bárcenas-Ortega, A.E.1, LozunaLópez, F.1, García-Saucedo, P.A.1, Olalde-Portugal, V.2
1 Facultad de Agrobiología “Presidente Juárez” de la Universidad Michoacana de San Nicolás de
Hidalgo, México. 2 CINVESTAV Unidad Irapuato, México.
RESUMEN
Se determinó la cuenta total de bacterias viables con actividad ACC desaminasa en
suelo de huertos de aguacate ‘Hass’ bajo dos tipos de manejo del cultivo, orgánico y
convencional, en el municipio de Uruapan, Michoacán, México. En suelos bajo manejo
orgánico se encontró 40 % más UFC de bacterias que en los de manejo convencional.
Adicionalmente se seleccionaron 19 aislados con morfología colonial distinta que
mostraron un efecto positivo en el desarrollo radicular de sorgo.
INTRODUCCIÓN
El aguacate presenta raíces poco profundas y carentes de tricoblastos (Salazar, 2002),
por lo que las relaciones simbióticas con microorganismos de la rizosfera seguramente
juegan un papel muy importante en su desarrollo. El cultivo del aguacate en Michoacán
se lleva a cabo bajo dos esquemas de manejo contrastantes, el convencional con el uso
de fertilizantes y plaguicidas químicos sintéticos y el orgánico en el que se utilizan
insumos de origen orgánico como estiércol animal, compostas y control biológico de
plagas. Un grupo de bacterias promotoras del crecimiento vegetal (BPCV) disminuye
los niveles de etileno, que inhiben la elongación radical, por acción de la enzima 1amino-ciclopropano-1-carboxilato (ACC) desaminasa (Caballero, 2006). En este trabajo
se reporta la presencia de BPCV con actividad ACC desaminasa en suelo adyacente a
las raíces de aguacate con mayor abundancia en huertos bajo manejo orgánico respecto
al manejo convencional.
MATERIAL Y MÉTODOS
En cuatro huertos de aguacate ‘Hass’ establecidos por pares, uno de manejo orgánico
frente a otro de manejo convencional, se tomaron muestras compuestas de 200 g de
suelo cercano a raíces jóvenes de árboles de aguacate (cuatro árboles por huerto). Se
determinó la cuenta total microbiana en medio mínimo de sales con ACC como única
fuente de nitrógeno (Penrose y Glick, 2003). Colonias aisladas de rizosfera de aguacate
por cultivo de raíces en el mismo medio selectivo fueron clasificadas por su morfología
colonial y su efecto en el desarrollo de raíces se evaluó en co-cultivo in vitro con sorgo.
El número de bacterias con actividad ACC desaminasa es mayor en suelos de huertos
bajo manejo orgánico que en aquellos bajo manejo convencional.
Todos los huertos presentaron bacterias viables con actividad ACC desaminasa, sin
embargo en los huertos bajo manejo orgánico se encontró mayor número de bacterias
que en los de manejo convencional (Figura 1). Estos resultados sugieren que bajo las
condiciones de manejo orgánico en huertos de aguacatero se promueve un mayor
desarrollo de poblaciones de bacterias con capacidad para disminuir las concentraciones
de etileno en raíces de plantas. Estos resultados coinciden con los datos de biomasa y
actividad microbiana total, así como porcentaje de materia orgánica en los mismos
huertos (datos no mostrados) y con Sánchez et al. (2006), al comparar biomasa y
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actividad microbiana edáfica en manejo agroecológico y convencional de maracuyá. Se
sugiere que el esquema de manejo orgánico del aguacate en Uruapan, Michoacán,
promueve mejores condiciones ambientales para el desarrollo de bacterias benéficas,
respecto al manejo convencional.
.
Figura 1. Efecto del tipo de manejo del cultivo
del aguacate en Uruapan, Michoacán sobre la
abundancia de bacterais con actividad ACC
desaminasa. Letras distintas indican diferencias
estadísticas significativas.
Figura 2. Co-cultivo in vitro de semillas de sorgo
y aislado bacteriano. Semillas desinfectadas se
sembraron en medio MS al 10 % (flecha blanca),
al mismo tiempo se aplicó una azada de uno de
los aislados bacterianos (a) o agua estéril (b) en el
otro extremo de la caja de Petri (flecha negra).
Las bacterias con actividad ACC desaminasa aisladas de huertos de aguacatero
promueven el desarrollo radicular.
El desarrollo de raíces de sorgo co-cultivadas in vitro con 19 de los 20 morfotipos de
aislados de bacterias con actividad ACC desaminasa obtenidos, mostraron un mayor
crecimiento que el testigo 72 h después de la siembra, sobresaliendo el crecimiento de
las raíces de plantas con los aislados C18 y C22 (Figura 2). Adicionalmente se observó
que en las raíces co-inoculadas con los aislados C10 y C12 se promovió el desarrollo de
tricoblastos. Estos resultados muestran que la bacterias aisladas pueden considerarse
promotoras del crecimiento vegetal, lo cual se ha reportado para diversas especies de
bacterias de la rizobiota con estas propiedades bioquímicas en distintas especies
vegetales (Yim et al., 2013).
AGRADECIMIENTOS
A CONACYT y CIC-UMSNH, México, por el financiamiento del proyecto
BIBLIOGRAFÍA
Caballero, M.J. (2006). Rev. Lat Amer. Microbiol. 48: 154-161.
Penrose, D.M, and Glick, B.R. (2003). Physiol. Plant. 118: 10-15.
Salazar, G.S. (2002). Nutrición del aguacate, principios y aplicaciones. Primera Edición INIFAP,
INPOFOS. Querétaro, Qro. pp:165.
Sánchez, M., et al. (2006). Acta Agron. 55: 7-12.
Yim, W., et al. (2013). Plant Physiol. Biochem. 14: 95-104.
92
Session I
SI-CP-36
Caracterização genotípica de bactérias diazotróficas isoladas de
plantas de Brachiaria brizantha e B. humidicula no estado de Roraima,
Amazônia, Brasil.
Chalita, P.B.1, Cunha, E.N.1, Zilli, J.E.2, Silva, K.1*
1
Laboratório Microbiologia do Solo. Embrapa Roraima, Rodovia BR-174, Km 8, Distrito Industrial, CEP
69301-970, Boa Vista-RR, Brasil. 2 Embrapa Agrobiologia, Rodovia BR-465 Km 7, CEP 23890-000,
Seropédica-RJ, Brasil.
*
[email protected].
RESUMO
Através do sequenciamento parcial do gene 16S rDNA de 29 bactérias diazotróficas
isoladas de Brachiaraia brizantha e B. humidicula em Roraima na região Amazônica,
Brasil, foi verificada que a maioria das bactérias pertencem ao gênero Bacillus.
INTRODUÇÃO
Estudos com bactérias diazotróficas associadas a espécies de Brachiaria são
importantes para o fornecimento deste nutriente que é um dos mais limitantes nas
pastagens brasileiras. No Brasil, a bovinocultura de corte é fortemente sustentada por
diferentes espécies do gênero Brachiaria, tornando-as principal fonte de nutrientes para
animais em pastejo. Portanto o objetivo deste trabalho foi caracterizar genotipicamente
bactérias isoladas de plantas de Brachiaria no Estado de Roraima, Amazônia, Brasil.
MATERIAL E MÉTODOS
Vinte e nove bactérias isoladas de plantas de braquiária (Tabela 1) no Estado de
Roraima foram caracterizadas genotipicamente através da amplificação do gene 16S
rDNA utilizando os iniciadores Y1 (5’ TGG CTC AGA ACG AAC GCT GGC GGC
3’) e B3 (TAC CTT GTT ACG ACT TCA CCC CAG TC). O sequencimaneto parcial
foi realizado utilizando o inciador Y1 no sequenciador 3730xl.
Tabela 1. Local de coleta e planta de isolamento das 29 bactérias caracterizadas genotipicamente.
Planta de isolamento
Brachiaria humidicula
Local de coleta
B. brizanta
Parte aérea
Raiz
Parte aérea
Raiz
Fazenda São
Paulo – Roxinho*
ERR 535,
ERR 536
ERR 513, ERR 514,
ERR 515, ERR 516,
ERR 517
ERR 537,
ERR 538
ERR 518, ERR 519,
ERR 520, ERR 521,
ERR 522, ERR 523,
ERR 524
Campo
Experimental
Água Boa – Boa
Vista**
-
ERR 525, ERR 526,
ERR 527, ERR 528,
ERR 529, ERR 530
ERR 539,
ERR 540,
ERR 541
ERR 531, ERR 532,
ERR 533, ERR 534
* Área de transição cerrado-mata: B. brizanta (N 02º17’34,4”, W 61º15’09,8”); B. humidicula (N 02º18’05,5”, W
61º15’32,9”). ** Área de cerrado: B. brizanta (N 02º39’34,7”, W 60º50’00,8”); B. humidicula (N02o39’51,7”,
W60o50’60,5”).
RESULTADOS E DISCUSSÃO
Através do sequenciamento do gene 16S rDNA (Figura 1) foi possível observar que a
maioria dos isolados pertence aos gênero Bacillus. Além do gênero Bacillus, foram
encontrados isolados próximos a Brevibacillus e Lysinibacillus. Também foi encontrado
um isolado próximo de Agrobacterium tumefaciens e dois isolados próximos a
Klebsiella. Estes resultados demonstram há existência de outros gêneros diferentes dos
93
Session I
SI-CP-36
encontrados em outros trabalhos no Brasil, como Azospirillum (Magalhães and
Döbereiner, 1984; Reis Júnior et al., 2004), Burkholderia (Boddey, 2003) e
Herbaspirillum (Baldani et al., 1992).
54
(AJ831844) Bacillus aerophilus partial 28K
ERR520
ERR532
ERR541
(AJ831842) Bacillus altitudinis 41KF2b
ERR524
44
ERR526
ERR537
ERR521
ERR525
27
ERR523
ERR531
ERR516
26
(D16273) Bacillus megaterium IAM 13418
85
ERR518
(AB021192) Bacillus mycoides
(D16281) Bacillus thuringiensis IAM 12077
57
ERR535
59 ERR529
ERR538
28
ERR540
ERR513
ERR514
(AF234854) Bacillus safensis FO-036b
87
34
(AY876289) Bacillus pumilus ATCC 7061
ERR527
ERR539
ERR536
69
(FJ233848) Bacillus rhizospherae SC-N012
(AB112715) Brevibacillus reuszeri DSM 9887T
100 ERR515
100
(X60608) B.azotofixans
37
(D16276) Paenibacillus polymyxa IAM 13419
100
(AY677116) Lysinibacillus massiliensis
74 ERR528
(D14500) Agrobacterium tumefaciens
100 ERR530
(EF488759) Enterobacter oryzae Ola 51
(AF129440) Klebsiella oxytoca
ERR533
84 ERR534
0.005
Figura 1. Árvore filogenética do sequenciamento parcial do gene 16S rDNA das bactérias isoladas de
braquiária em Roraima.
Árvore inferida através do método de Neighbor-joining, utilizando o modelo de Jukes-Cantor, com 1000
repetições.
AGRADECIMENTOS
A Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) pelo financiamento, Projeto FBNPlus:
02.09.01.011.00.00.
REFERÊNCIAS
Baldani, V.L.D., et al. (1992). Symbiosis, 13: 65-73.
Boddey, L.H. (2003). Tese (Doutorado em agronomia). UFRRJ, Seropédica, Rio de Janeiro.
Magalhães, F.M.M., and Döbereiner, J. (1984). R. Microbiologia 15: 246-252.
Reis Júnior, F.B., et al. (2004). R. Bras. Ci. Solo, 28: 103-113.
94
Session II
Genetics and genomics of beneficial
microorganisms and associated plants
Session II
SII-P-1
Imperial, J.1, 2*, Laguerre, G.3, Brito, B.1, Jorrín, B.1
1
Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de
Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España. 2 Consejo Superior de Investigaciones
Científicas (CSIC). Madrid, España. 3 LSTM, CIRAD-IRD, Université de Montpellier 2, Supagro, USC
INRA, Montpellier.  Deceased, 17/1/2013.
*
[email protected]
ABSTRACT
Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate
plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus.
However, previous evidence suggests that some genotypes are more adapted to a given
plant host than others, and that the plant host can select specific genotypes among those
present in a given soil population. We have used a population genomics approach to
confirm that this is indeed the case, and to analyze the specific genotypic characteristics
that each plant host selects.
One of the key aspects of the Rhizobium-legume symbiosis is its well-known
specificity: specific rhizobia nodulate and fix nitrogen in specific legume hosts.
However, this specificity is not absolute. Some tropical legumes (such as Phaseolus or
siratro) are quite broad in their specificity requirements and are promiscuously
nodulated by a large number of different rhizobial species and genera, whereas some
rhizobia (such as Sinorhizobium sp. NGR234) are able to establish symbioses with very
different plants. Some rhizobia, such as Sinorhizobium meliloti (Ballard et al., 2005) or
Bradyrhizobium japonicum (Koch et al., 2010) can nodulate different hosts depending
on a specific genetic complement, often uncovered after mutant screening or isolation of
specific strains that are symbiotically active with just some of the hosts.
However, in some cases very subtle mechanisms of adaptation to a specific plant host
might be in operation. This, for example, seems to be the case of rhizobial species were
all (or most) of the isolates can effectively establish a diazotrophic symbiosis with
plants of several different genera, often from different habitats and with different
lifestyles, and is well exemplified by Rhizobium leguminosarum bv. viciae. Isolates
belonging to this biovar establish effective symbioses with legumes belonging to four
genera: Pisum, Lens, Lathyrus and Vicia. The last genus, in particular, includes species
as diverse as vetch (V. sativa) and broad bean (V. faba). One set of nodulation and
nitrogen fixation genes, harbored on a symbiotic plasmid, allows successful
establishment and development of symbiosis with the different hosts (Surin and
Downie, 1989) and, in cross-inoculation experiments, when challenged with any one of
the above legume hosts, any R. leguminosarum bv. viciae strain is able to establish an
efficient symbiosis. However, it has long been hypothesized that different rhizobial
strains may be more adapted to a specific plant host than others, which may result in
selection and enrichment of a specific strain or set of strains by the legume host from
those present in a particular soil. Molecular evidence for plant-mediated selection of
specific rhizobial genotypes from soil populations has been obtained by the research
groups of Gisèle Laguerre (Depret et al., 2004; Laguerre et al., 2003; Louvrier et al.,
1995) and J. Peter W. Young (Mutch and Young, 2004; Palmer and Young, 2000). They
used molecular markers and specific PCR amplification to obtain evidence that different
plant hosts enrich specific genotypic marker variants of R. leguminosarumbv. viciae
from those available in the soil.
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These molecular studies were limited by the number and nature of the markers selected,
and did not clarify the bases for enrichment of a given specific genotype. With the
development and availability of new generation sequencing technologies this problem
has been reappraised using genomic and population genomic approaches.
Genomics of Rhizobium.
After the original reports on genome sequencing of model rhizobia (Mesorhizobium loti
2000, Sinorhizobium meliloti 2001, Bradyrhizobium japonicum 2002, R. etli 2006 and
R. leguminosarum 2006, a very large number of rhizobial genomes have been
sequenced or are in the process of being sequenced, and among them about 100
Rhizobium isolates (as many as 96 complete or ongoing genome sequence projects in
GOLD, the Genomes OnLine Database –http://www.genomesonline.org, as of June 30,
2013). Although data are still quite recent, several general conclusions on the genomic
structure and organization of Rhizobium rhizobia emerge. In general, the Rhizobium
contain large genomes, of ca. 7 Mb, and even larger in the case of members of the genus
Bradyrhizobium (ca. 9 Mb).The occurrence of very large genomes in soil bacteria has
been interpreted as an adaptation to this habitat, a complex, hostile and changing
environment that demands the large metabolic and behavioral plasticity that can be
provided by a large gene-encoding capacity. Contrary to members of the genus
Bradyrhizobium, members of the genus Rhizobium (and both Sinorhizobium) present a
multi-partite genome, harboring several large plasmids, some of which resemble
chromosomes (“chromids”, Harrison et al., 2010). On average, 30-40% of the genome
in these bacteria is present in the form of plasmids (Galardini et al., 2013; Harrison et
al., 2010; Mazur et al., 2011). This characteristic is shared with the Roseobacter clade
(Petersen et al., 2013), and affords a large genomic plasticity, especially since many of
these plasmids incorporate conjugative systems (Crossman et al., 2008). This plasticity
liberates these bacteria from the constraints of long replication times associated to a
single, very large chromosome, the situation found with the bradyrhizobia.
Genomics and the rhizobia in the soil.
Soil microbial communities have the highest level of prokaryotic diversity (up to 109
microorganisms per gram, Knietsch et al., 2003). Metagenomic approaches would
appear to be the ideal approximation to such a complex system, allowing the study of
the nature, composition and function of microbial communities in soil. However, even
metagenomics is limitedby this complexity, in view of: a) the very large size of these
datasets limits our ability to analyze them; b) soil changes rapidly not only temporally
but also spatially, even at the micro level, and its physicochemical properties affect
microbial distribution within the soil matrix, imposing important technical and
methodological problems. Despite these caveats metagenomics constitutes a powerful
approach to obtain information about the nature, composition and function of microbial
communities in soil.
The specificity of the Rhizobium-legume symbiosis has classically allowed the use of
most probable number (MPN) techniques to enumerate soil Rhizobia that are able to
nodulate trap plants. For R. leguminosarum bv. viciae, representative abundances are on
the order of 104-105 viable cells per gram of soil). Louvrier and collaborators developed
a semi-selective medium to isolate R. leguminosarum directly from soils (Louvrier et
al., 1995). The numbers they obtained in soils from Eastern France were ca. 104 per
gram of soil. Overall, it can be concluded that, although cultivation of the plant host
results in an increase in rhizobial soil counts (modest in the case of R. leguminosarum),
established soil populations of R. leguminosarum are, at most, on the order of 104 to 105
per gram of soil. If typical soils contain ca. 10 9 bacteria per gram of soil, R.
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leguminosarum would amount to less than 0.01% of the total soil microbiota. In
practical terms, this implies that even in one of the largest metagenomic datasets (ca. 1
Tb), at most 100 Mb would be R. leguminosarum DNA. This, barring the crucial
problem of how to specifically identify these sequences, would represent at most a 15x
coverage of a single R. leguminosarum genome and would barely be representative of
the population diversity. Thus, the low natural abundance of rhizobia in soils precludes
the use of purely metagenomic methods to study their diversity and demands an
alternative approach.
In view of these difficulties, we decided to adopt a Pool-Seq approach to the study of
genotype selection by the host plant. Kofler and collaborators, working with Drosophila
melanogaster populations, proposed for the first time the Pool-Seq term in 2011 for the
next-generation sequencing and analysis of pooled DNA samples from natural
populations. It constitutes a feasible (and affordable) genome-wide approach for
comparison of population samples, thus allowing an easy scaling from the limitations of
single markers to population genomics (Kofler et al., 2011 and references therein).
We reasoned that sequencing pooled DNA samples from R.leguminosarum bv. viciae
nodule isolates obtained from different legume plant hosts used as rhizobial traps would
allow an experimental test of the hypothesis that different plant hosts select specific
subpopulations of rhizobia from the available population present in a given soil. We
compared four populations (P. sativum, L. culinaris, V, sativa and V. faba) originating
from the same agricultural soil and consisting each of one hundred nodulae isolates that
were grown independently and then pooled; the genomic DNA of the pools was
extracted and sequenced (Illumina Hi-Seq 2000, 180 bp PE libraries, 100 bp reads, 12
Mreads) at BGI (Hong Kong and Shenzhen, China).
For analysis of the rhizobial Pool-Seq data, two specific considerations were taken into
account.First, plant-specific subpopulations derive from the same unselected, resident
soil population, whose genomic composition is, by definition, unknown, since their low
numbers preclude any unselected genomic analysis. It is likely that this resident
population contains both major and minor genomic types, resulting both from the soil’s
edapho-climatic properties and from its agricultural history. Thus, specific selection by
the legume host will operate –if it does– on this original composition which, although
distorted by the plant effect, will still be present in the plant specific isolates. Second,
the large size and the multipartite composition of the R. leguminosarum genome favor
both an open pan-genome and a large non-conserved genome. With R. leguminosarum
bv. viciae we have estimated that 20-30% of the genes are strain specific. This suggests
that plant host selection of specific rhizobial genotypes may implicate specific genes or
groups of genes (eg. transport and metabolism of substrates). However, identification of
these genes from Pool-Seq data is technically complex, since any DNA assembly will
result, necessarily, in the formation of chimaeras with no biological meaning.
With these limitations in mind, we decided to restrict the Pool-Seq comparative data
analysis to conserved genes, and reads for those genes were identified following
recruitment by a reference genome, which in our case was that of R. leguminosarum bv.
viciae 3841. A data analysis pipeline was designed and implemented, where reads are
aligned to the reference genome, and both coverage and single nucleotide
polymorphism (SNP) analysis are performed and compared between subpopulations.
These analyses were carried out both for the complete genome and for relevant markers
(16S rDNA, nod genes, nif genes, recA and glnII housekeeping markers, etc.). The data
clearly show, both at the genome-wide and at the specific marker levels, that specific
genotypes are indeed selected by the plant host, thus confirming previous indications.
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A major outcome of this study is one of methodology for the study of natural rhizobial
populations in the soil. Given unlimited resources for sequencing and data analysis, it is
clear that individual genome sequencing of isolates, followed by assembly and multiple
genome comparisons represents a more powerful tool than the Pool-Seq approach.
However, such a situation is unlikely to occur, and the advantages and disadvantages of
Pool-Seq must be evaluated for each project. When this project was designed (2010), it
was not feasible to individually sequence and assemble 200 rhizobial strains. Even with
today’s higher capacity and lower costs, the pooled DNA approach allows for higher
sequencing depths, and thus for potentially better descriptions of the populations, and
for more facile analysis with our optimized pipeline. However, the Pool-Seq approach
suffers from important drawbacks for this type of analysis in rhizobia. First, since the
plant specific genotype enrichment will necessarily reflect the original structure of the
rhizobial population in the soil, and this can vary from soil to soil, the analysis should
be repeated with different types of soil. More importantly, the impossibility to assemble
reads without the generation of chimeras makes it very difficult to identify specific
genes that are not present in the reference genome but that may be specifically enriched
in plant-selected subpopulations. These genes are important because they can provide
not only specific host-linked markers but also evidence for the structural or functional
nature of the phenotypes selected by the plant. We are addressing these limitations by
means of two complementary approaches. First, the complexity of the plant-enriched
subpopulations can be reduced by any number of typing methods, for instance RAPD
analysis, making this reduced number of isolates more amenable to direct genome
sequencing and assembly. Second, the Pool-Seq pipeline for coverage and SNP data
analysis can be repeated with different R. leguminosarum reference genomes in order to
incorporate coverage and SNP analyses for genes that were absent from the original
reference genome. Results from both these strategies strengthen the power of the PoolSeq approach with a mínimum investment in sequencing and data analysis.
ACKNOWLEDGMENTS
This work was supported by the Spanish Consolider-Ingenio Program (Microgen Project, CSD200900006) to J.I. We thank Rosabel Prieto for help with isolation of strains from root nodules and DNA
preparations. We also thank Gonzalo Martín for IT.
REFERENCES
Ballard, R.A., et al. (2005). Aust. J. Exp. Agr. 45: 209-216.
Crossman, L.C., et al. (2008). PloS ONE 3: e2567.
Depret, G., et al. (2004). FEMS Microbiol. Ecol. 51: 87-97.
Galardini, M., et al. (2013). Genome Biol. Evol. 5: 542-558.
Harrison, P.W., et al. (2010). Trends Microbiol. 18: 141-148.
Knietsch, A., et al. (2003). J. Mol. Microbiol. Biotechnol. 5: 46-56.
Koch, M., et al. (2010). Mol. Plant-Microbe Interact. 23: 784-790.
Kofler, R., et al. (2011). Bioinformatics 27: 3435-3436.
Louvrier, P., et al. (1995). Soil Biol. Biochem. 27: 919-924.
Mazur, A., et al. (2011). BMC Microbiol. 11: 123.
Mutch, L.A., and Young, J.P.W. (2004). Mol. Ecol. 13: 2435-2444.
Palmer, K.M., and Young, J.P.W. (2000). Appl. Environ. Microbiol. 66: 2445-2450.
Petersen, J., et al. (2013). Appl. Microbiol. Biotechnol. 97: 2805-2815.
Surin, B.P., and Downie, J.A. (1989). Plant Mol. Biol. 12: 19-29.
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Ormeño-Orrillo, E.*, Martínez-Romero, E.
Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, CP
62251, Cuernavaca, Morelos, Mexico.
*
[email protected]
Rhizobia is a common name used to refer to members of several genera of α- and βProteobacteria which are able to induce the formation of root or stem nodules in legume
plants, invade the nodules and differentiate into nitrogen-fixing intracellular bacteroids.
These bacteria are used in agriculture as biofertilizers to reduce or replace the use of
nitrogen chemical fertilizers (Hungria et al., 2000). The term rhizosphere refers to the
root surface and adjacent soil that is influenced by root exudates. Despite being best
known as symbiotic partners of legumes, rhizobia are able to colonize the rhizospheres
of non-legume plants (Rosenblueth and Martínez-Romero, 2004) and act as plant
growth promoting bacteria (PGPB) in several crops (García-Fraile et al., 2012 and
references therein). Rhizobia have been safely used in agriculture for over a hundred
years, an advantage over other PGPB known to be potential human pathogens.
Extensive studies of many rhizobia-legume models have produced a detailed inventory
of molecular determinants and mechanisms used by rhizobia during nodulation and
nitrogen fixation with legumes. In contrast, little is known about the genetic repertoire
used by rhizobia during rhizosphere colonization. Here we report on genes that may
promote rhizosphere colonization in the genomes of two rhizobial strains used as
models in our research group, Rhizobium tropici CIAT 899 and Rhizobium phaseoli
Ch24-10, and report on the transcriptome of the latter strain during rhizosphere
colonization.
Rhizosphere colonization-related traits encoded in selected rhizobial genomes.
Strain Ch24-10 was isolated from the stem of a maize plant (Zea mays cv. Criollo
Cholula) growing intercropped with common bean (Phaseolus vulgaris) (Rosenblueth
and Martínez-Romero, 2004). Originally ascribed to Rhizobium etli, Ch24-10 has been
recently reclassified as R. phaseoli (López-Guerrero et al., 2012). This strain is both a
maize endophyte and common bean rhizobia as it possesses a symbiotic plasmid. R.
tropici CIAT 899 was isolated from a P. vulgaris nodule (Martínez-Romero et al.,
1991) but is also able to colonize the rhizosphere and interior of maize roots
(Rosenblueth and Martínez-Romero, 2004). Both strains are competitive for rhizosphere
colonization and behave as PGPB.
Recently we obtained the genome sequences of Ch24-10 (López-Guerrero et al., 2012)
and CIAT 899 (Ormeño-Orrillo et al., 2012). Analysis of both genomes revealed a wide
array of genes that may promote colonization and support a rhizospheric lifestyle. Like
other rhizosphere bacteria, both strains encode hundreds of transporters in their
genomes that may allow them to take up numerous nutrients available in root exudates.
Genes coding for enzymes like peptidades/proteases, glycosyl hydrolases, oxireductases
and oxygenases are also abundant in both genomes and many of them could be involved
in the catabolism of rhizosphere nutrients.
Traits known to promote rhizosphere colonization in other bacteria were found encoded
in both genomes. These included siderophore production, bacteriocin synthesis,
antimicrobial efflux pumps, vitamin prototrophy (except for biotin in CIAT 899
although it possesses redundant biotin uptake transporters), surface polysaccharides,
type IV pili, motility and chemotaxis. Both strains have genes involved in biosynthesis
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of the plant hormones auxins and gibberellins supporting their role as PGPB. In CIAT
899, and acdS gene coding for a 1-aminocyclopropane-1-carboxylate deaminase may
promote plant growth by reducing ethylene levels.
Genes for biosynthesis of cellulose microfibrils and adhesin-like proteins which may
contribute to root surface attachment were also found in both genomes. In CIAT 899,
filamentous hemagglutinin-like and YadA-like adhesins may be exported by several
type V secretion systems. Both strain genomes encoded type IV secretion systems
although they are more related to conjugation machineries than to effector-exporting
systems. In Ch24-10, two type VI secretion systems were found that may be related to
its superior competitive ability (Records, 2011).
Rhizobial genes expressed at the rhizosphere of maize and common bean.
We investigated the transcriptome of R. phaseoli Ch24-10 during its early interaction
with maize or common bean roots. Around 1250 genes were highly expressed in either
plant rhizosphere. Transporter genes comprised 15% of these transcripts. Genes coding
for uptake transporters of oligopeptides, sugars and amino acids were the most
abundant, but uptake transporters of other molecules like iron/siderophore, sulphate,
nitrate, and some metals were also expressed suggesting that Ch24-10 is able to use a
wide range of the nutrients present in root exudates. We have reported that rhizobia can
simultaneously utilize different carbon sources (Romanov and Martínez-Romero, 1994)
a feature that seems advantageous for rhizosphere colonization given the diversity of
nutrients present in root exudates (López-Guerrero et al., 2013; Ormeño-Orrillo and
Martínez-Romero, 2013). Several expressed genes like putA, hut, asn and rha for
proline, histidine, asparagine and rhamnose catabolism, respectively, as well as some
glycosidase and peptidase genes are surely involved in utilization of substrates imported
by the uptake transporters.
In rhizospheres, bacteria are exposed to plant antimicrobials and expression of genes
involved in resistance to these compounds seems to be advantageous. Several drug
efflux transporters were found among the Ch24-10 highly expressed genes, including
the Rmr extrusion pump involved in common bean nodulation proficiency (GonzalezPasayo and Martínez Romero, 2000) that was found expressed in both rhizospheres.
The lpsβ2 gene, required for biosynthesis of the O antigen moiety of lipopolysaccharide
(LPS) (Garcia-de los Santos and Brom, 1997), was highly expressed. This observation
is in agreement with our report that O antigen-defective mutants of R. tropici CIAT 899
are impaired in maize root colonization as LPS seems to protect rhizobia from plant
antimicrobials (Ormeño-Orrillo et al., 2008). Besides plant antimicrobials, bacteria
seem to face other adverse conditions in the rhizosphere (Matilla et al., 2007;
Ramachandran et al., 2011). Various genes involved in osmotic stress response were
highly expressed in our system including ndv for perisplasmic glucan biosynthesis, kup
and kdp for potassium transport, several genes for utilization of external
osmoprotectants like betaine, and treS and treY for biosynthesis of trehalose (another
osmoprotectant). Other stress-related genes included relA required for biosynthesis of
the alarmone ppGpp, katG coding for a catalase/peroxidase, and a cyclopropane fattyacyl-phospholipid synthase gene.
Synthesis of vitamins seems to be required for successful rhizosphere colonization
(Karunakaran et al., 2006). Accordingly, we found that thiamin biosynthesis genes were
highly expressed. Genes for the flagellum and type IV pilus, surface appendages that are
important in other bacteria for rhizosphere colonization, were expressed by R. phaseoli
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Ch24-10. Other expressed genes included those involved in the synthesis of some
known, poorly characterized or putative novel colonization factors like siderophores,
cellulose microfibrils (Ausmees et al., 1999), trifolitoxin (Robleto et al., 1998), two
different type VI secretion systems (Records, 2011) and an alpha-2-macroglobulin
(Budd et al., 2004). Interestingly, genes putatively involved in biosynthesis of the plant
hormone gibberellin were also expressed in t he rhizosphere by Ch24 -10.
Several orthologues to previously reported R. leguminosarum genes expressed in plant
rhizospheres (Ramachandran et al. 2011) were highly expressed in maize and common
bean roots by R. phaseoli Ch24-10. As in R. leguminosarum, many R. phaseoli Ch24-10
highly expressed genes were hypothetical. Expanding our understanding of rhizobial
genes expressed at the rhizosphere will surely aid in selecting or designing competitive
and proficient PGPB rhizobial strains.
REFERENCES
Ausmees, N., et al. (1999). Microbiology 145: 1253-1262.
Budd, A., et al. (2004). Genome Biol. 5: R38.
García-de los Santos. A., and Brom, S. (1997). Mol. Plant Microbe Interact. 10: 891-902.
García-Fraile, P., et al. (2012). PLoS ONE 7: e38122.
González-Pasayo, R., and Martínez-Romero, E. (2000). Mol. Plant Microbe Interact. 13: 572-577.
Hungria, M., et al. (2000). In: Nitrogen Fixation: From Molecules to Crop Productivity (Current Plant
Science and Biotechnology in agriculture Volume 38). Pedrosa, F.O., Hungria, M., Yates, G., and
Newton, W.E. (eds.). ISBN: 978-0-7923-6233-3. Springer. pp. 515-518.
Karunakaran, R. et al. (2006). J. Bacteriol. 188: 6661-6668.
López-Guerrero, M.G. et al. (2012). Syst. Appl. Microbiol. 35: 353-358.
López-Guerrero, M.G., et al. (2013). Front. Plant Sci. 4: 188.
Martínez-Romero, E., et al. (1991). Int. J. Syst. Bacteriol. 41: 417-426.
Matilla, M.A., et al. (2007). Genome Biol. 8: R179.
Ormeño-Orrillo, E., et al. (2008). Environ. Microbiol. 10: 1271-1284.
Ormeño-Orrillo, E., and Martínez-Romero, E. (2013). Syst. Appl. Microbiol. 36: 145-147.
Ormeño-Orrillo, E., et al. (2012). BMC Genomics 13: 735.
Ramachandran, V.K, et al. (2011). Genome Biol. 12: R106.
Records, A.R. (2011). Mol. Plant Microbe Interact. 24: 751-757.
Robleto, E.A., et al. (1998). Appl. Environ. Microbiol. 64: 2630-2633.
Romanov, V.I., and Martínez-Romero, E. (1994). Plant Soil 161: 91-96.
Rosenblueth, M., and Martínez-Romero, E. (2004). Arch. Microbiol. 181: 337-344.
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First analyses of the genomic sequence of the soybean symbiont
Sinorhizobium fredii HH103.
Vinardell, J.M.1*, Göttfert, M.2, Becker, A.3, Acosta-Jurado, S.1, Baena, I.4, Blom, J.5,
Bonilla, I.4, Buendía, A.M.1, Crespo-Rivas, J.C.1, Goesmann, A.5, Jaenicke, S.5, Krol,
E.3, Lloret, J.4, McIntosh, M.3, Margaret, I.1, Pérez-Montaño, F.1, Schneiker-Bekel, S.5,
Serranía, J.3, Szczepanowski, R.5, Zehner, S.2, Pühler, A.5, Ruiz-Sainz, J.E.1, Weidner,
S.5
1
Departamento de Microbiología, Universidad de Sevilla, Spain. 2 Institut für Genetik, Technische
Universität Dresden, Dresden, Germany. 3 LOEWE-Zentrum für Synthetische Mikrobiologie, Marburg,
Germany. 4 Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain; 5 Centrum für
Biotechnologie, Universität Bielefeld, Bielefeld, Germany.
* [email protected]
ABSTRACT
Sinorhizobium fredii HH103 is a fast-growing rhizobial strain with a broad host-range
including soybean. Here we present its genome, which consists of one chromosome and
six plasmids with a total size of 7239684bp.
INTRODUCTION
Sinorhizobium fredii shows an extremely broad host-range of nodulation, which
includes determinate (such as Glycine max and Vigna unguiculata) and indeterminate
(such as Cajanus cajan and Glycyrrhiza uralensis) nodule forming legumes. Among the
different S. fredii strains, HH103 is particularly interesting because it can effectively
nodulate both Asiatic and American (i.e., commercial) soybeans. S. fredii is closely
related to S. meliloti and Sinorhizobium sp. NGR234, although these bacteria do not
nodulate soybeans. S. fredii HH103 genes involved in the production of Nod factors, the
symbiotic T3SS, and surface polysaccharides have been already reported (for a review
see Margaret et al., 2011). The chemical structure of LCOs, cyclic glucans, capsular
polysaccharides, and exopolysaccharides has been determined. Field trials using strain
HH103 as soybean inoculant have also been carried out. This makes S. fredii HH103 to
one of the best studied rhizobial strains.
The complete genomic sequence of S. fredii HH103 has been established as described
by Weidner et al. (2012), leading to a circa 35-fold coverage. Manual annotation of the
genome and comparative analyses are being carried out by using the GenDB and
EDGAR software respectively (CeBiTec, University of Bielefeld, Germany). Molecular
biology techniques have been carried out as described by Vinardell et al. (2004).
Our first attempt to assemble the complete genome of S. fredii HH103 led to a genome
structure composed of 6 replicons: the chromosome and five plasmids (Margaret et al.,
2011, Weidner et al., 2012). New experiments indicate the presence of a putative sixth
plasmid (pSfHH103a2) that comigrates with the smallest plasmid in agarose gels. The
existence of this new plasmid has been confirmed by purification of the small plasmids
of HH103 and hybridisation experiments. The manual annotation of the genome (about
6919 genes) is close to completion. Table 1 summarizes the main characteristics of the
different replicons constituting the S. fredii HH103 genome. Figure 1 shows the number
of genes shared by S. fredii strains HH103 and USDA257 and by Sinorhizobium sp.
NGR234 (panel A). It also depicts a comparison of HH103, S.meliloti 1021, and the
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slow-growing soybean symbiont Bradyrhizobium japonicum USDA110 (panel B). As
expected, HH103 shares a higher number of genes with strains USDA257 and NGR234
than with S. meliloti 1021. Not surprisingly, the number of genes shared by HH103 and
B. japonicum USDA110 is very low. The analysis of the set of symbiotic genes shared
by S. fredii strains and B. japonicum might help to identify the essential bacterial traits
for nodulation of soybeans.
Table 1. Main characteristics of the seven replicons constituting the S. fredii HH103 genome.
GC
Gene
Other interesting
Replicon
Size (bp)
Symbiotic genes a
(%)
number
characteristics b
sinIsinR (QS)
CG, KPS, LPS,
Putative secretion systems: Sec4014
nolR, exoR, exoS,
Chromosome 4305723 62.61
independent, T1SS, T2SS,
mucR1, mucR2
T2/T4SS,
2096125 62.38
1973
EPS, KPS, LPS
minCDE;T2SS, T3SS, T4SS
pSfHH103e
Met tRNA (anticodon: CAT)
nod, noe, nol, nif,
582757
59.57
651
T3SS (sym), T4SS,
pSfHH103d
fix, nop, tts
trb (T4SS)- traItraR (QS)
T4SS
144082
58.68
167
nopM2, nopP2
Not essential for growth or
pSfHH103c
symbiosis c
Several unsuccessful attempts
61880
58.47
58
pSfHH103b
have been carried out to cure it
Not essential for growth or
24036
58.21
18
pSfHH103a1
symbiosis c
Several
genes
encoding
25081
58.02
38
pSfHH103a2
chaperonines
7239684 62.14
6919
Total
a
CG, cyclic glucans. KPS, K-antigen capsular polysaccharide. LPS, lipopolysaccharide. EPS,
exopolysaccharide; b QS, quorum sensing. TXSS, type X secretion system; c Elimination of this plasmid
neither affected growth in TY or YMA media nor symbiotic ability with soybean, cowpea, pigeonpea.
Figure 1. Number of shared genes between HH103 and different rhizobial strains (EDGAR software).
ACKNOWLEDGEMENTS
This work was supported by grants BIO2008-05736-C02-01/02 and BIO2011-30229-C02-01 from the
Spanish Ministry of Science and Innovation and grant 0313805A from the Bundesministerium für
Forschung und Technologie, Germany
REFERENCES
Margaret, I., et al. (2011). J. Biotechnol. 155: 11-19.
Vinardell, J.M., et al. (2004). Mol. Plant-Microbe Interact. 17: 676-685.
Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618.
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Identificación y caracterización del regulador maestro del flagelo
lateral de Bradyrhizobium japonicum USDA 110.
Mongiardini, E.J. *, Quelas, J.I., Lodeiro, A.R.
IBBM-Facultad de Ciencias Exactas, UNLP-CONICET, La Plata, Argentina
* [email protected]
RESUMEN
En este trabajo se identificó el regulador maestro del flagelo lateral de Bradyrhizobium
japonicum USDA 110 mediante mutagénesis insercional. Además se muestran
resultados preliminares relacionados con el mecanismo de acción de dicha proteína
mediante mutagénesis puntual de un residuo clave relacionado con su funcionalidad.
INTRODUCCIÓN
Bradyrhizobium japonicum es una bacteria Gram negativa que se utiliza en la industria
agrícola como fertilizante biológico para soja, dada su capacidad para nodular raíces de
soja y una vez allí, fijar nitrógeno atmosférico en simbiosis con esta leguminosa.
En los suelos donde se cultiva soja a menudo existen poblaciones de rizobios capaces de
nodular sus raíces compitiendo de esta manera con el fertilizante biológico. En este
sentido, trabajos previos de nuestro laboratorio demostraron que la movilidad bacteriana
puede jugar un papel en la competitividad del fertilizante biológico para nodular. Así,
cuando en el campo se inocularon bacterias seleccionadas en el laboratorio por su
mayor movilidad, éstas fueron capaces de competir mejor que las bacterias establecidas
en el suelo y concomitantemente, condujeron a aumentos de rendimiento en grano
(Althabegoiti et al., 2008; López-García et al., 2009).
La capacidad de movimiento de estas bacterias está dada por una estructura denominada
flagelo. B. japonicum es el único rizobio que cuenta con dos sistemas flagelares, uno
lateral e inducible y otro subpolar y constitutivo. Remarcablemente, encontramos que
las bacterias seleccionadas por mayor movilidad tienen desreprimida la expresión del
flagelo lateral (Althabegoiti et al., 2008). Dada la importancia de la regulación de este
flagelo en la competitividad para nodular y la escasez de información al respecto,
decidimos profundizar nuestro conocimiento sobre este tema. En general, la regulación
de la expresión de los flagelos bacterianos se da en diferentes etapas y está controlada a
modo de cascada por varios reguladores transcripcionales. El regulador clave que
desencadena la síntesis de los flagelos se denomina regulador maestro. (Smith and
Hoover, 2009). En este trabajo se identificó y se comenzó con la caracterización del
regulador maestro que controla la expresión del flagelo lateral en B. japonicum.
MATERIAL Y MÉTODOS
Se construyó un mutante insercional en el ORF blr6846 introduciendo un cassette de
resistencia a kanamicina entre los nucleótidos 7542881 y 7543631, y en paralelo se
construyeron plásmidos replicativos llevando el gen completo, para todo lo cual se
emplearon las metodologías ya descriptas (Quelas et al., 2010). Con una modificación
de estos métodos se realizaron los reemplazos de aminoácidos en la secuencia
codificada. La extracción de flagelina de cultivos líquidos y su visualización en SDSPAGE se realizaron como se describió (Althabegoiti et al., 2008).
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Mediante estudios bioinformáticos se identificó el probable regulador maestro que
controla la expresión del flagelo inducible, cuyo gen estaría codificado en el locus
blr6846. Esta proteína fue previamente caracterizada en Ensifer meliloti y Brucella
melitensis (Léonard et al., 2007, Rotter et al., 2006). Este regulador pertenece a la
familia de proteínas de reguladores de respuesta de sistemas de dos componentes
presentando un dominio sensor capaz de ser fosforilado y otro de unión a ADN que le
permite reconocer las regiones donde ejerce su acción. En E. meliloti y B. melitensis se
probó que este regulador de respuesta presenta una mutación puntual en el residuo que
debería sufrir la fosforilación, de manera que su funcionalidad no dependería de esta
modificación post-traduccional y por lo tanto sería independiente de su par quinasa. Sin
embargo, un análisis de la secuencia aminoacídica de esta proteína en B. japonicum
muestra que este residuo no presenta dicha mutación sino que se encuentra conservado
como en los típicos reguladores de respuesta de sistemas de dos componentes. Así, se
esperaría que para ejercer su función dependa de una fosforilación previa.
El mutante insercional construído en blr6846 resultó incapaz de expresar el flagelo
lateral en las condiciones de inducción. La mutación se corroboró complementando en
trans con el gen salvaje completo en un vector replicativo, lo cual restituyó el fenotipo
salvaje.
Para probar si el regulador de respuesta de B. japonicum depende de una modificación
por fosforilación para ejercer su función, se construyó un mutante puntual en el residuo
Asp58 por Gly58, residuo que no puede sufrir este tipo de modificaciones. De esta
manera se determinó que el mutante puntual con el reemplazo Asp58Gly sigue siendo
capaz de expresar el flagelo inducible, lo cual indica que la presencia del residuo Asp58,
y por lo tanto la fosforilación, no sería necesaria para cumplir su función. Este hecho
particular está siendo investigado con mayor detalle en el laboratorio.
AGRADECIMIENTOS
Este trabajo fue financiado por la ANPCyT de Argentina. EJM, JIQ y ARL son miembros de la
carrera de investigador científico de CONICET, Argentina.
BIBLIOGRAFÍA
Althabegoiti, M.J., et al. (2008). FEMS Microbiol. Lett. 282: 115-123.
Léonard, S., et al. (2007). J. Bacteriol. 189: 131-141.
López-García, S.L., et al. (2009). Agronomy J. 101: 357-363.
Quelas, J.I., et al. (2010). Mol. Plant-Microbe Interact. 23: 1592-1604.
Rotter, C., et al. (2006). J. Bacteriol. 188:6932-6942.
Smith, T.G., and Hoover, T.R. (2009). Adv. Appl. Microbiol. 67: 257-295.
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Pyrosequencing reveals the presence of diverse bacterial genera which
have not previously described to soil and rhizosphere.
Lagos, L.1*, Jorquera, M.1, Maruyama, F.2, Mora, M.L.1
1
Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Tokyo, Japan.
* [email protected]
2
ABSTRACT
Rhizosphere is a complex environment that harbors diverse microorganisms which
carry out essential functions for plants. Multitude of biotic and abiotic factors are
assumed to influence the structure and activity of microbial communities in the
rhizosphere. In this work the diversity of bacterial communities in ryegrass (Lollium
perenne var. Nui) rhizosphere grown in two Andisols was analyzed by pyrosequencing
technique. Our results reveal the presence of 65 bacterial genera which have no records
in the GenBank as groups present in plant rhizospheres and soils. However, the
distribution and functions of these 65 bacterial groups in soil or rhizosphere remain
unknown.
INTRODUCTION
The rhizosphere, defined as soil zone affected by plant roots, is a complex environment.
The diversity of microbial communities in the rhizosphere differs in their composition,
activity and abundance according to diverse biotic and abiotic factors. Bacterial
diversity in these environments is much greater than previous estimated by using
conventional molecular techniques (T-RFLP and DGGE). In traditional molecular
studies, dominant populations have masked the detection of low abundance OTUs,
genetic diversity, and their individual distribution in soil environments. The study of
soil metagenome (all the genetic material present in soil), particularly of the
rhizosphere, could be crucial to a better understanding how structure, diversity and
activity are distributed along the roots of plants. In this context, the objective of this
study was to explore by using pyrosequencing the diversity of bacterial communities in
rhizosphere of ryegrass grown in two Andisols under greenhouse conditions.
Sterile seeds of Lollium perenne var. Nui were sown in rhizoboxes containing unsterile
volcanic soils (Andisols) and maintained for 30 days under greenhouse conditions.
Rhizosphere soil samples were aseptically collected from adhering soil to roots and
immediately processed in laboratory. Total bacterial DNA was extracted from
rhizosphere samples by using Power Soil® DNA Isolation Kit (MoBio Inc., USA)
according to manufacturer instructions. Then, the DNA extracts were analyzed by
pyrosequencing at Macrogen, Inc. (Seoul, Korea). Pyrosequencing was performed by
using Genome Sequencer-FLX system (Roche). The genera identified were compared
with those present in the National Center for Biotechnology Information’s GenBank
database (NCBI; www.ncbi.nlm.nih.gov/).
The sequencing data revealed a high diversity of low-abundance bacterial groups and
the occurrence of 65 genera which have not been previously described for soil or
rhizosphere according to GenBank database. The genera included: Dethiosulfovibrio
(Synergistetes), Asteroleplasma (Tenericutes), Aspromonas, Desulfopila, Sulfurimonas,
Albidiferax (Proteobacteria), Vitellibacter (Bacteroidetes), Anaerofustis (Firmicutes),
107
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Bryobacter (Acidobacteria) isolated from diverse habitats, such as: sediments and
waters from sea and lakes, gastrointestinal tract and gut of human and animals, slurry
and industrial sludges, sedimentary rock, cave, glacial, etc. Other genera present have
been related with functions such as biopolymer-degrader (Proteobacteria; Rugamonas),
sulfate-reducer
(Proteobacteria; Desulfopila), thermophilic alkane-degrader
(Firmicutes;
Coprothermobacter),
succinate-utilizing
bacteria
(Firmicutes;
Succiniclasticum) oil-degrader, (Actinobacteria; Aestuariimicrobium), glycogenaccumulator (Actinobacteria; Micropruina). In traditional molecular studies, dominant
populations have masked the detection of low-abundance OTUs, genetic diversity, and
their individual distribution in soil environments. Pyrosequencing studies, has recently
uncovered relatively “rare” (Dini-Andreote and Elsas, 2013) species in soil
communities. The distribution of these 65 bacterial groups and their role in rhizosphere
in nutrient cycling and beneficial effects on plant growth remain unknown.
ACKNOWLEGMENTS
This work was supported by the CONICYT Doctoral Scholarships 21120698 and FONDECYT Project
no. 1120505.
REFERENCES
Dini-Andreote, F., and Elsas, J.D. (2013). Plant and Soil DOI 10.1007/s11104-013-1687-z.
108
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Temporal profile of nif gene expression in Azotobacter vinelandii: effect
of nifA mutation.
Navarro-Rodríguez, M.*, Poza-Carrión, C., Jiménez-Vicente, E., Rubio, L.M.
* [email protected]
ABSTRACT
Azotobacter vinelandii, a free-living bacterium, is considered a model organism for the
study of biological nitrogen fixation. Expression of nitrogen fixation (nif) genes is
regulated in response to environmental conditions by the NifA activator and the NifL
anti-activator. An A. vinelandii mutant strain, UW356, carrying a chromosomal inframe deletion of nifA was generated and its nif gene expression profile compared to
that of the wild-type strain. Quantitative real-time PCR was performed normalizing
absolute mRNA values. In addition, quantitative immunoblot analysis of Nif protein
accumulation was carried out at different derepression times. Our results show that
nifU, nifS and nifV gene expression is not exclusively dependent on nifA.
INTRODUCTION
Azotobacter vinelandii is one of the model organisms to study the biological process of
nitrogen fixation. This complex process carried out by the nitrogenase enzyme(s) plays
a key role in the biogeochemical nitrogen cycle. The majority of biological nitrogen
fixation is carried out by the molybdenum nitrogenase, although alternative vanadium
and iron-only nitrogenases exists. Molybdenum-dependent nitrogen fixation depends on
nif genes, which are grouped into operons and expressed from NifA-dependent
promoters (Setubal J.C. et al., 2009). NifA transcriptional activating ability is
modulated by association/dissociation with the nifL protein that depends on the nitrogen
and environmental conditions such as the presence of oxygen or the C/N balance of the
cell (Dixon R., 1998). Therefore, when nifA does not interact with nifL, it activates nif
gene transcription.
A mutant strain of Azotobacter vinelandii that carried a chromosomal in-frame deletion
of the nifA gene was generated by replacing it with an spectinomicin resistant cassette,
UW356 (∆nifA::Spc). Cultures of wild-type and UW356 strains were grown in modified
Burk media supplemented with ammonium at 30ºC and 200 rpm. Cells were collected
by centrifugation and resuspended in media with or without ammonia. Twenty three-ml
aliquots were collected at different times after resuspension (0', 10', 30', 60', 120', 180',
240' where time 0' indicates 0 minutes after ammonium removal from the culture
medium) and divided into three samples: 20 ml for RNA extraction and RT-qPCR
analysis, 1ml for immunoblot assays, 1ml for acetylene reduction assays and 1ml for
OD measure. Total RNA was isolated using RiboPureTM-Bacteria Kit (Ambion, life
technologies), treated with Turbo DNA-free™ (Ambion, life technologies) and the
cDNA synthesis was carried out using High Capacity cDNA Reverse Transcription Kit
(Ambion, life technologies). Each reaction used 2 µg of total RNA and an aliquot of 1
µl of a 1:10 dilution (1:10,000 for reference gene 16SrDNA) of the cDNA was used as
template for RT-qPCR amplification. Immunoblot analysis (Brandner et al., 1989) has
been described. Protein samples were prepared by mixing pelleted cells with Laemmli
sample buffer 2X supplemented with DTT and 2-mercaptoethanol to a final OD600 of
109
Session II
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4. In vivo nitrogenase activity was determined by the acetylene reduction assay at 30ºC
for 15 minutes as described (Stewart et al., 1967).
Our results show a clear and fast pulse of nifA expression peaking at 10-30 minutes after
ammonium removal and descending rapidly afterwards. This peak is followed by
expression of structural and biosynthetic nif genes, which peaks have variable times
between 30 min and 120 min and exhibit smoother profiles. In all cases, nif genes
within each of the operons followed similar expression patterns. Surprisingly, the nifA
mutant showed significant expression on nifU, nifS and nifV genes, indicating that they
were not strickly dependent on nifA.
t=
4h
0m
t=
30
m
t=
1h
t=
2h
t=
0
t=
1
t=
4h
t=
3h
t=
2h
m
m
t=
1h
t=
30
t=
0
t=
10
t=
3h
ΔnifA
DJ
nifH
nifD
nifK
0
50
0
50
0
5
0
5
0
0.5
0
0.5
nifY
nifE
nifN
nifX
nifU
nifS
nifV
nifA
nifB
fdxN
nifQ
nafY
nifL
Figure 1. Different responses of nif gene expression under diazotrophic conditions in the mutant UW356
and. wild-type strains of A. vinelandii. Gene expression was quantified by q-RTPCR. mRNA expression
data is visualized with the Multi Experimental Viewer program (http:://www.tm4.org/). Color bar shows
normalized absolute signal. Each data point represents the mean of three biological replicates.
REFERENCES
Dixon, R. (1998). Arch. Microbiol. 169: 371-380.
Brandner, J.P., et al. (1989). J. Bacteriol. 171: 360-368.
Stewart W.D.P., et al. (1967). Proc. Natl. Acad. Sci. USA. 58: 2071-2078.
Setubal J.C., et al. (2009). J. Bacteriol. 191: 4534-4545.
Figure 3
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Singularidades adaptativas del genoma de Pseudomonas fluorescens
F113. Comparación con otras cepas del género Pseudomonas.
Redondo-Nieto, M.*, Martínez-Granero, F., Muriel, C., Martín, M., Rivilla, R.
Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. c/ Darwin, 2. 28029.
Madrid.
* [email protected]
RESUMEN
Pseudomonas fluorescens F113 es una rizobacteria promotora del crecimiento vegetal
(PGPR) que presenta actividad protectora ante patógenos fúngicos y además sirve de
modelo para la colonización de la rizosfera. La secuenciación completa de su genoma y
su posterior comparación con otras Pseudomonas ha puesto de manifiesto una alta
conservación del genoma central. A pesar del alto grado de conservación, P. fluorescens
F113 muestra un gran número de rutas propias que le conceden una alta capacidad
competitiva y adaptativa en la rizosfera.
INTRODUCCIÓN
Pseudomonas fluorescens F113 es una PGPR aislada de la rizosfera de remolacha que
posee un elevado potencial como cepa biocontroladora dada su capacidad para la
producción de compuestos antibióticos y antifúngicos. Además, su capacidad adaptativa
y competitiva en rizosfera conferida por la plasticidad genómica debida a fenómenos de
variación de fase hace de F113 un interesante candidato para aplicaciones
biotecnológicas. Se ha completado la secuenciación y el borrador de la anotación de esta
cepa y se ha comparado con los genomas de otras Pseudomonas con el fin de establecer
por un lado las relaciones filogenómicas y por otro los genes compartidos y genes
propios de F113 que le confieren ventajas adaptativas.
MATERIAL Y MÉTODOS
El genoma de F113 fue secuenciado por las plataformas 454 FLX Titanium (Roche) y
Solexa (actualmente Illumina). Para el ensamblaje se recurrió a varios algoritmos,
Velvet, MIRA y NewBler. Los contigs obtenidos con cada software fueron comparados
mediante Blast y analizados con scripts propios con el fin de fusionarlos. Las regiones
que no pudieron ser calculadas se cerraron amplificándolas por PCR y secuenciándolas
por Sanger. La anotación se llevo a cabo utilizando el protocolo RAST y Blast2GO. La
búsqueda de ortólogos se realizó con el paquete de software OrthoMCL. Para el estudio
filogenómico, se descargaron todos los genomas completos y borradores del género
Pseudomonas presentes en la base de datos PATRIC y para calcular las distancias se
utilizó una aproximación del tipo “Composition Vector” usando el algoritmo CVtree
con una ventana de 6 aminoácidos por péptido. Los árboles se construyeron mediante el
método de Neighbor-Joining.
El genoma de P. fluorescens F113 está compuesto por un cromosoma circular con un
tamaño de 6,8 Mpb, un contenido G+C medio del 60,8% y una densidad codificante del
86,7%. La anotación actual muestra que F113 presenta 5862 secuencias codificantes,
nueve ncRNAs, cinco operones de rRNA y 66 loci de tRNA (Redondo-Nieto et al.,
2012). El genoma de F113 presenta una variedad de genes codificantes que le confieren
ventaja en el ambiente rizosférico. Estos incluyen adaptaciones metabólicas inusuales
dentro de la especie P. fluorescens, como puede ser la capacidad denitrificadora y
111
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metabolismo de sustancias terpénicas cíclicas; nuevos sistemas de movilidad como son
un segundo aparato flagelar no descrito hasta la fecha en esta especie y sistemas
quimiotácticos adicionales; genes codificantes de posibles toxinas para distintos
organismos y un vasto número de genes implicados en el ensamblaje de distintos
sistemas de secreción de tipo II, III y VI.
Las características fenotípicas de F113 en el momento de ser aislada hicieron que fuera
incluida en el grupo de las P. fluorescens, pero como ya ha sido indicado por Silby et al.
(2009), Loper et al. (2012), y más recientemente por Redondo-Nieto et al. (2013), la
clasificación de este grupo requiere un estudio en mayor profundidad. El análisis
filogenómico del genoma y/o borradores de 162 cepas del género Pseudomonas
disponibles en las bases de datos, han mostrado una distribución prácticamente
concordante con la obtenida por métodos filogenéticos como MLSA. El denominado
grupo P. fluorescens está compuesto por 50 cepas de las especies P. fluorescens, P.
brassicacearum, P. protegens, P. mandelii, P. chlororaphis, P. tolaasii, P.
extremaustralis y Pseudomonas spp. Según el árbol filogenómico, este grupo se forma a
una profundidad mayor que otros grupos más compactos como P. aeruginosa o P.
syringae. Por ello se realizó un análisis más detallado de esas 50 cepas y se concluyó
que el grupo P. fluorescens puede clasificarse en 5 subgrupos, estando F113 en el
subgrupo I con otras dos cepas de P. fluorescens y dos de P. brassicacearum. El análisis
de ortólogos muestra que F113 comparte un 35% con todas las cepas asignadas al grupo
de P. fluorescens pero ese valor se eleva al 76% al compararlo con las cepas de su
subgrupo. Estos datos junto con que las cepas del subgrupo I comparten habilidades
inusuales como la denitrificación, variación de fase durante la colonización de la
rizosfera y sistemas propios de quimiotaxis altamente conservados, conducen a la
conclusión de que estas cepas corresponden a la misma especie.
Adicionalmente F113 presenta 344 genes que no presentan el resto de las cepas del
subgrupo I entre los que podemos destacar los dirigidos al ensamblaje de un segundo
aparato flagelar y a la producción de un posible antibiótico de tipo poliquétido. Estos
grupos de genes podrían explicar la excelente capacidad competitiva de F113 de
rizosfera y están actualmente en estudio.
AGRADECIMIENTOS
Esta investigación ha sido financiada por los proyectos BIO2009 08254, BIO2012 316034,
Microambiente CM.
BIBLIOGRAFÍA
Loper, J.E., et al. (2012). Plos Genet. 8(7):e1002784.
Redondo-Nieto, M., et al. (2012). J. Bacteriol. 194: 1273-1274.
Redondo-Nieto, M., et al. (2013). BMC Genomics 14: 54.
Silby, M., et al. (2009). Genome Biol. 10: R51.
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AmrZ es un regulador transcripcional global en Pseudomonas
fluorescens F113.
Martínez-Granero, F., Redondo-Nieto, M., Vesga, P., Martín, M., Rivilla, R. *
* [email protected]
RESUMEN
Mediante análisis de ChIP-seq se ha determinado que la proteína AmrZ es un regulador
transcripcional de múltiples genes implicados en movimiento, producción de
exopolisacáridos y homeostasis de hierro, entre otros. Se ha determinado un motivo
conservado para la unión de AmrZ al ADN.
INTRODUCCIÓN
AmrZ (originalmente denominada AlgZ) es una proteína reguladora de la familia ArcC
que contiene un dominio de unión a ADN Ribbon-Helix-Helix (RHH). Esta proteína fue
descrita originalmente en Pseudomonas aeruginosa como un activador transcripcional
que regulaba positivamente la producción del exopolisacárido alginato. A su vez, AmrZ
se auto-regula de forma negativa y reprime la expresión de genes implicados en la
síntesis de otro exopolisacárido. AmrZ también regula el movimiento a nivel
transcripcional mediante el control de la producción de pili tipo IV y del flagelo. AmrZ
requiere para su transcripción el factor sigma alternativo AlgU y está conservada en los
genomas de todas las pseudomonas secuenciadas hasta la fecha.
Pseudomonas fluorescens F113 fue aislada de la rizosfera de remolacha y su capacidad
para producir compuestos con actividad antibiótica y antifúngica la convierte en una
estirpe de interés en biocontrol. Los usos de F113 en sistemas integrados
planta/microorganismo dependen de la eficacia de la colonización de la rizosfera. Uno
de los caracteres de interés en la colonización competitiva de la rizosfera es la capacidad
de movimiento (Capdevila et al., 2004; Martínez-Granero et al., 2006). En esta cepa,
AmrZ regula la movilidad a través de la represión de fleQ, que a su vez codifica el
principal regulador de la síntesis del flagelo (Martínez-Granero et al., 2012). Debido al
importante papel que AmrZ puede tener en la ecología de las pseudomonas y
aprovechando la secuenciación completa del genoma de F113 (Redondo-Nieto et al.,
2013), hemos realizado un análisis genómico por inmunoprecipitación de cromatina
(ChIP-seq) para determinar los sitios de unión de AmrZ al ADN y analizar la
importancia de esta proteína en la adaptación ambiental de las pseudomonas.
MATERIAL Y MÉTODOS
Se inmunoprecipitó ADN de un derivado de F113 que contenía una fusión traduccional
HA-AmrZ, utilizando un anticuerpo anti-HA. Se construyó una librería que se
secuenció a través de Illumina. Las lecturas obtenidas se alinearon frente al genoma de
F113 usando el programa BowTie 2. Los picos de lecturas acumuladas fueron
detectados mediante el programa MACS 1.4. Los picos observados se validaron por
PCR cuantitativa de un experimento de ChIP independiente. Se estudió la expresión
génica de genes seleccionados mediante PCR cuantitativa en fondos genéticos wt y
amrZ-. Se utilizó el programa MEME para determinar los motivos de unión de AmrZ al
ADN.
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El análisis del ChIP-seq mostró la presencia de 154 picos que podrían corresponder a
sitios de unión de AmrZ. El 76% de estos picos se localizaban en regiones intergénicas
indicando que AmrZ se une fundamentalmente a regiones promotoras. Se validaron 20
de estos picos mediante PCR en tiempo real de un experimento independiente de ChIP.
Los picos se asignaron a genes implicados en múltiples funciones, destacando genes
relacionados con homeostasis de hierro, movilidad, quimiotaxis y producción de
polisacáridos. Algunos de los picos se asociaron a genes que codifican proteínas
reguladoras y de transducción de señal, incluidas proteínas implicadas en la síntesis y/o
degradación del c-di-GMP. El análisis de las secuencias contenidas en los picos,
permitió determinar un motivo de unión de AmrZ, que contenía la secuencia
identificada previamente por cristalografía en los promotores de algD y amrZ en P.
aeruginosa.
Se analizó la expresión de 20 de estos genes, pertenecientes a distintas clases
funcionales en la estirpe silvestre y en un mutante amrZ, mostrando que en las
condiciones estudiadas, AmrZ participaba en la expresión de algunos de ellos. Además,
se seleccionaron 12 genes implicados en la homeostasis de hierro y que estaban
precedidos por un pico. El análisis de expresión mostró que AmrZ reprimía a todos
estos genes en condiciones de deficiencia de hierro.
El alto número de sitios de unión de AmrZ al ADN, así como la identidad de los genes
regulados por esta proteína, muestran que AmrZ es un importante regulador global de la
transcripción en P. fluorescens. Además, al regular a otras proteínas reguladoras y
señalizadoras, es de esperar que el regulón de AmrZ sea muy amplio, convirtiendo a
AmrZ en uno de los reguladores más importantes para la adaptación de las
pseudomonas a condiciones ambientales cambiantes.
AGRADECIMIENTOS
Esta investigación ha sido financiada por los proyectos BIO2009 08254, BIO2012 316034,
Microambiente CM.
BIBLIOGRAFÍA
Capdevila, S., et al. (2004). Microbiology SGM 150: 3889-3897
Martínez-Granero, F., et al. (2006). Appl. Environ. Microbiol. 72: 3429-3434
Martínez-Granero, F., et al. (2012). PLoS ONE Vol 7: e31765
Redondo-Nieto, M., et al. (2013). BMC Genomics 14:54
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The rkp-2 region of Sinorhizobium fredii HH103 is involved in LPS and
EPS production.
Acosta-Jurado, S.1*, Crespo-Rivas, J.C.1, Murdoch, P.S.2, Rodríguez-Carvajal, M.A.3,
Ruiz-Sainz, J.E.1, Vinardell, J.M.1
1
Departamento de Microbiología and 2 Departamento de Bioquímica Vegetal y Biología Molecular,
Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012-Sevilla, Spain; 3
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. C/ Profesor García
González 1, 41012-Sevilla, Spain
* [email protected]
ABSTRACT
Bacterial surface polysaccharides play a crucial role in the rhizobia-legume symbiotic
interaction. In this work we characterize the Sinorhizobium fredii HH103 rkp-2 genetic
region and demonstrate its involvement in exopolysaccharide (EPS) and
lipopolysaccharide (LPS) production. This region is composed of two genes, lpsL and
rkpK. Inactivation of any of these genes results in mucus reduction, LPS electrophoretic
profile alterations, reduced capacity to form biofilms, and enhanced autoaggregation in
liquid cultures. In addition, rkp-2 mutants, in contrast to other S. fredii HH103 mutants
affected in surface polysaccharide production, bind Congo red and calcofluor.
INTRODUCTION
In the rhizobia-legume symbiotic interaction, bacterial surface polysaccharides appear
to play a crucial role either acting as signals required for the progression of the
interaction and/or preventing host defence mechanisms (Fraysse et al., 2003). We have
previously described the rkp-1 and rkp-3 regions, which are required for KPS (Kantigen polysaccharide) production (Margaret et al., 2012a,b) in S. fredii HH103. Here
we present our first studies of the HH103 rkp-2 region, which comprises two genes:
lpsL, that encodes a UDP-glucuronate 4-epimerase, and rkpK, that encodes a UDPglucose 6-dehydrogenase. Here we report that, in contrast to that described for S.
meliloti, the HH103 rkp-2 region is not involved in KPS synthesis. However, HH103
rkp-2 mutants are affected in LPS and EPS production.
Mutagenesis of the lpsL and rkpK genes, LPS extraction, separation on SDS
polyacrylamide gel electrophoresis, silver staining and immunostaining procedures were
performed as described by Margaret et al. (2012a,b). To observe EPS production on
solid YM medium, rhizobial strains were grown for 120 h at 28°C and then incubated
48 h at room temperature. Congo red adsorption was observed on plates of YM medium
supplemented with 25mg/L of this dye. Calcofluor binding was tested on TY plates
containing calcofluor 0.02% m/v. Biofilm formation on plastic surfaces was studied by
using the method of O'Toole and Kolter (1998), with some modifications. The
autoaggregation assays was carried out as described by Sorroche et al. (2012).
S. fredii HH103 mutant derivatives SVQ701 (lpsL::lacZ-GmR), SVQ703 (rkpK::lacZGmR), and SVQ704 (rkpK::lacZ-GmR) were unable to produce EPS as observed on
YMA plates (Figure 1 Panel A) and after precipitation with 3 volumes of cold ethanol.
This defect could be due to the fact that glucuronic acid (whose production is dependent
on RkpK) is present in the HH103 EPS structure. All the mutants tested were able to
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absorb Congo red and calcofluor (Figure 1, panels B and C). Moreover, HH103 rkp-2
mutants produced a LPS which showed an altered profile in SDS-PAGE experiments
(Figure 1, panel D) and was not recognized by the monoclonal antibody NB6-228.22 (It
recognizes the wild type LPS) (Figure 1, panel E). All S. fredii HH103 rkp-2 mutants
were unable to form biofilms and showed increased autoaggregation ability in MGM
liquid medium (Figure 1, panel F and G) when compared with the parental strain
HH103. The fact that HH103 does not contain glucuronic acid in its KPS justifies that
HH103 rkp-2 mutants were not affected in KPS production (data not shown) in contrast
to Sinorhizobium meliloti Rm41 (whose KPS contains glucuronic acid).
We have also obtained a mutant in the rkpZ gene, SVQ702 (rkpZ::lacZ-GmR). Strain
SVQ702 did not show differences with regard to HH103 in any of the characteristics
studied above.
A
B
C
SVQ269
SVQ701
SVQ701
SVQ269
SVQ269
SVQ702
SVQ703
SVQ701
SVQ703
SVQ702
bluB
SVQ704
SVQ704
D
1
2
SVQ703
SVQ704
E
3
1
2
3
G
F
Figure 1. (A,B,C) Growth of HH103 RifR (= SVQ269) and different rkp derivatives on YMA (A), and
YMA supplemented with calcofluor (B) or Congo red (C). S.meliloti bluB was used as a positive control for
calcofluor binding. (D) SDS-PAGE and silver-staining or (E) immuno-staining (monoclonal antibody NB6228.22) of lipopolysaccharides (LPS) crude extracts from HH103 and its rkp-2 derivatives. (F) Biofilm and
(G) autoaggregation of HH103 and its rkp-2 derivatives. 1. SVQ269, 2. SVQ701, and 3. SVQ703.
ACKNOWLEDGEMENTS
This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO201130229-C02-01) and from the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de
Excelencia CVI2506). Authors also thank Modesto Carballo (CITIUS, Biology Service, University of
Sevilla) for helping with biofilm measurement experiments.
REFERENCES
Fraysse, A.L., et al. (2003). Eur. J. Biochem. 270: 1365-1380.
Margaret, I., et al. (2012a). Arch. Microbiol 194: 87-102.
Margaret, I., et al. (2012b). Mol. Plant-Microbe Interact. 25: 825-838.
O'Toole, G.A., and Kolter, R. (1998). Mol Microbiol 28: 449-461.
Sorroche, F.G., et al. (2012). Appl Environ Microbiol.78: 4092-101.
116
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Study of the quorum sensing Sin system of Sinorhizobium fredii HH103.
Crespo-Rivas, J.C.1*, Pérez-Montaño, F.1, Acosta-Jurado, S.1, Payán-Bravo, L.1,
McIntosh, M.2, Meyer, S.2, Becker, A.2, Ruiz-Sainz, J.E.1, Vinardell, J.M.1
1
Departamento de Microbiología Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6.
41012, Sevilla, Spain. 2 LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of
Biology, Philipps-Universität Marburg, Marburg, Germany.
* [email protected]
ABSTRACT
Quorum sensing (QS) is a genetic regulation system depending on population density.
In rhizobia the small autoinducer molecules involved in QS regulation are N-AcylHomoserine-Lactones (AHLs). Two kinds of AHLs produced by HH103 were detected
in chromatoplate assays using the biosensor strain Agrobacterium tumefaciens NT1
(pZRL4). The kinetic productions of both AHLs are different and vary according to the
different points of the growing curve. An in silico search in the S. fredii HH103
genomic sequence revealed the presence of two genes putatively responsible for AHL
synthesis: sinI and traI. Inactivation of these genes removed the production of AHLs.
Chromatoplate assays and expression experiments revealed that the production of these
AHLs is repressed by genistein, a nod gene inducer flavonoid.
INTRODUCTION
QS is a regulatory process by which bacteria perceive their population density (Fuqua et
al., 1994). Bacteria synthesize small molecules called autoinducers that are able to
move across the membrane. The concentration of these molecules in the extracellular
medium increases according to the number of bacteria. When the concentration of these
molecules reaches a threshold level, they can activate or suppress the expression of
genes involved in a collective behavior. In rhizobia, as in other Gram negative bacteria,
the QS system autoinducer molecules known to date are AHLs. Some recognised
features of QS responses observed in rhizobia are the strain-specificity and the high
diversity of genes involved in this mechanism (Sánchez-Contreras et al., 2011). In this
work we have verified that S. fredii HH103 produces at least two different AHLs in a
population density-depending manner, and we have initiated the study of the complex
genetic regulation by AHLs in this strain.
The biosensor strain Agrobaterium tumefaciens NT1 (pZRL4) was used to detect AHLs
produced by HH103 in culture supernatants by Thin Layer Chromatography (TLC)
assay experiments (Holden et al., 1999). Surface polysaccharide analyses and rt-RTPCR experiments were done according to Crespo-Rivas et al. (2009). Fluorescence
measurement experiments were done according to McIntosh et al. (2009).
In order to find genes involved in AHL synthesis in S. fredii HH103, we analysed its
genomic sequence to find orthologues of Vibrio fischeri luxI. Thus, the sinI and traI
genes were identified. In order to verify whether these genes are responsible for AHL
synthesis, two HH103 mutants were generated: SVQ693 (sinI::lacZ-GmR) and SVQ697
(traI::Ω SpcR). By using the TLC approach described above, two different AHLs were
found to be produced by HH103: a long acyl chain AHL and a short acyl chain AHL.
The analysis of mutants SVQ693 and SVQ697 demonstrated that these AHLs are
synthesized by SinI and TraI respectively (Figure 1, panels A, B, and C). It was also
117
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observed that the rate of production of each of these AHLs is not dependent on the
presence of the other AHL. The absence of long acyl chain AHL did apparently not
affect the production of LPS, EPS, KPS neither its swimming mobility (data not
shown), which could be due to the absence of a functional expR gene.
We have also studied whether AHL production in HH103 could be affected by the
presence of flavonoids, as it has been described for S. fredii SMH12 (Perez-Montaño et
al., 2011). TLC assays (Figure 1D) revealed that the production of the two types of
AHLs decreased when HH103 was grown in the presence of genistein, although this
reduction was clearly more evident for the long acyl chain AHL (dependent on sinI). rtRT-PCR and fluorescence measurement experiments (using a transcriptional fusion
between the sinI promoter region and the gfp gene) showed that the presence of
genistein led to a reduction of sinI expression (Figure 1 E and F), which is in agreement
with the results obtained in TLC assays.
E
A
B
C
D
F
Figure 1. (A, B, C, D) TLCs from supernatant culture extracts at different O.D. of Sinorhizobium fredii
HH103 (A) and its sinI (B) and traI (C) derivatives; D: HH103 cultured in the presence or absence of
genistein. E and F: rt-RT-PCR relative expression data of nodA and sinI genes in HH103 (= 269) in the
presence (+) or absence of genistein. St= AHL standards, Gen= genistein.
CONCLUSIONS
S. fredii HH103 produces at least two kinds of AHLs whose synthases are encoded by
sinI and traI. The production of these AHLs is somehow repressed by genistein, which
clearly suggests a connection between QS and the nod regulon in S. fredii HH103.
ACKNOWLEDGEMENTS
This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO201130229-C02-01) and from the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de
Excelencia CVI2506). Authors also thank Modesto Carballo (CITIUS, Biology Service, University of
Sevilla) for helping with fluorescence measurement experiments.
REFERENCES
Crespo-Rivas, J.C., et al. (2009). Mol. Plant-Microbe Interact. 22: 575-588.
Fuqua, W.C., et al., (1994). J. Bact. 176: 269–275.
Holden, M.T., et al. (1999). Mol. Microbiol. 33, 1254-1266.
McIntosh, M., et al. (2009). Mol. Microbiol. 74: 1238–1256.
Pérez-Montaño, F., et al. (2011). Res. Microbiol. 162: 715-23.
Sánchez-Contreras, M., et al. (2011). Fundamentos y aplicaciones agroambientales de las interacciones
beneficiosas plantas-microorganismos. Edita: Sociedad Española de Fijación de Nitrógeno (SEFIN),
España pp.227-240 ISBN: 978-84-614-7364-9.
118
Session II
SII-CP-05
Lotus japonicus Gifu and L. burttii responses to inoculation with a
collection of S. fredii HH103 mutants affected in symbiotic signals.
Rodríguez-Navarro, D.N.1 Jin, H.2, Kawaharada, Y.2, Sandal, N.2, Andersen, S.U.2,
Stougaard, J.2, Ruiz-Sainz, J.E.3*
1
IFAPA, Centro Las Torres-Tomejil. Apartado Oficial 41200, Alcalá del Río, Sevilla, Spain. 2
Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK-8000
Aarhus C, Denmark. 3 Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla,
Avda. Reina Mercedes 6, C.P. 41012. Sevilla, Spain.
*
[email protected]
ABSTRACT
A collection of S. fredii HH103 mutants affected in genes that are known to be
symbiotically relevant has been tested with the model legume Lotus japonicus Gifu and
also with L. burttii. S. fredii HH103 and most of its mutant derivatives analysed did not
form nitrogen fixing (Fix+) nodules with L. japonicus. However, S. fredii HH103 nodD2
or nolR mutants showed improved nodulation capacity with L. japonicus. Most of the S.
fredii HH103 mutants were still able to form Fix+ nodules with L. burttii. Mutations
affecting S. fredii HH103 surface polysaccharide (SP) production [exopolysaccharides
(EPS), lipopolysaccharides (LPS), capsular polysaccharides (KPS) or cyclic glucans
(CG)] did not prevent the formation of Fix+ nodules with L. burttii. Fix+ nodules formed
by inoculation of L. burttii with S. fredii HH103 appear to be the result of crack entry.
These results indicate that when S. f. HH103 invades L. burttii roots through cracks, the
bacterial SP are not strictly required for successful nodulation.
INTRODUCTION
Sinorhizobium fredii HH103 is a fast-growing strain able to nodulate American and
Asiatic soybean (Glycine max) cultivars as well as many other legumes. The S. fredii
HH103 genome has been sequenced (Margaret et al., 2011; Weidner et al., 2012). For
details about the HH103 genome see the abstract/poster Vinardell et al.
S. fredii HH103 effectively nodulates with L. burttii but it only induces the formation of
ineffective pseudonodules on L. japonicus roots (Sandal et al. 2012). L. japonicus is
being used as a model in genetic studies of determinate nodule forming legumes. L.
burttii is becoming attractive for genetic studies because it represents a valid alternative
to L. japonicus MG-20 as a L. japonicus Gifu mapping partner. In fact, we have
demonstrated the utility of the L. japonicus Gifu x L. burttii RILs in QTL mapping by
identifying an Nfr1-linked QTL for S. fredii nodulation (Sandal et al. 2012).
Numerous S. fredii HH103 mutants have been tested for their symbiotic capacity with
soybean. We have now investigated the symbiotic capacity of these mutants with Lotus
burttii and L. japonicus. Plant tests on L. burttii should reveal which mutations impair
the symbiosis of S. fredii HH103 with L. burttii. Plant tests on L. japonicus might lead
to the identification of HH103 genes that block nodulation on this plant.
The infection of L. japonicus by Mesorhizobium loti is through the formation of
infection threads but in the background of particular symbiotic mutants, examples of
crack entry were also found (Madsen et al., 2010). Here we are investigating the mode
of infection in the symbiosis between L. burttii and S. fredii HH103.
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Surface sterilization of L. japonicus Gifu and L. burttii seeds and nodulation tests in
square plastic boxes were carried our as described by Sandal et al. (2012). In other
cases, miniaturized Leonard jars were used to grow the plants.
Mutants affected in genes that are transcriptionally activated by flavonoids.
1. Mutants affected in genes that are transcriptionally activated by flavonoids.
Mutated in nod genes (nodA and noeI) or in genes involved in the secretion of Nops
(nodulation outer proteins) through the Type Three Secretion System (T3SS). Only the
nodA mutant, unable to produce Nod factors, failed to nodulate with L. burttii. None of
the mutants gained the capacity to form Fix+ nodules with L. japonicus.
2. Mutants affected in surface polysaccharide production.
None of the HH103 mutants affected in EPS biosynthesis, in KPS production, in LPS
biosynthesis, or in CG production (cgs, also called ndvB) totally lost the capacity to
induce pink (Fix+) nodules on L. burttii roots. To our knowledge, this is the first report
in which a rhizobial mutant unable to produce CG is still capable of inducing pink
nodules on any of its host legumes. Three pink nodules were also found in L. japonicus
plants inoculated with an HH103 lpsB mutant.
3. Mutants affected in regulatory genes.
Mutated in transcriptional regulators of nodulation genes (nodD2 and nolR), in a
regulator of the T3SS (ttsI), or in a transcription elongation factor (greA). All these
mutants formed pink nodules with L. burttii. L. japonicus roots inoculated with mutants
in nodD2 or nolR formed pseudonodules and pink nodules, indicating that these
mutations enhance the symbiotic capacity of S. fredii HH103 with L. japonicus.
The symbiosis of L. burttii with S. fredii HH103 is established through crack entry.
The symbiosis between L. burttii and Mesorhizobium loti is established through
infection threads. In contrast to this microscopic analysis of L. burttii plants inoculated
with S. fredii HH103 DsRED, including microtome and vibratome sections, revealed
microcolonies at the root hair tips but infection threads formed very rarely and they did
not reach any further than the middle of the root hair. Infection threads were not
detected in the nitrogen fixing nodules. Therefore the infection most likely is formed
through crack entry. The rate of crack entry infections is clearly higher in symbiosis
between S. fredii HH103 and L. burttii than in L. burttii and L. japonicus symbioses
with M. loti where it is only noticed as a rare event in particular plant mutant
backgrounds. The analysis of S. fredii HH103 mutants on L. burttii can therefore show
which bacterial genes are needed for crack entry. For instance, it seems that cyclic
glucans are not needed for crack entry in L. burttii but Nod factors are needed.
ACNOWLEDGEMENTS
This work was supported by grants from the Andalusia Government P07-CVI-02506 and P11-CVI-7500.
REFERENCES
Madsen, L.H., et al. (2010). Nat. Commun. 1: 1-12.
Sandal, N., et al. (2012). DNA Res. 19: 317-323.
120
Session II
SII-CP-06
Analyses of the Sinorhizobium fredii HH103 and USDA257 secretomes
in the presence and absence of the flavonoid genistein.
Yao, W.1, Thomas, J.R.1, Jiménez-Guerrero, I.2, López-Baena, F.J.2*, Ruiz-Sainz, J.E.2,
Vinardell, J.M.2
1
Department of Biology, University of York. Y010 5DD, York, United Kingdom. 2 Departamento de
Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla,
España.
* [email protected]
ABSTRACT
The genome of the fast-growing soybean-rhizobia S. fredii HH103 has recently been
sequenced. This bacterium possesses many protein secretion systems to interact with the
environment and also with its eukaryotic counterparts. The identification of proteins
secreted by HH103 could provide valuable information for identifying particular genes
that could play relevant roles in the symbiosis.
INTRODUCTION
Plant rhizosphere bacteria must overcome different environmental and biotic stresses to
establish a successful relationship with their host plants. These bacteria use different
protein secretion systems (Type 1 to Type 7) to deliver effectors and toxins that modify
the host physiology to promote colonization.
S. fredii HH103 is a broad host-range bacterium that possesses twelve putative protein
secretion systems (Margaret et al., 2011; Weidner et al., 2012). Interestingly, cultivar
specificity, which is the impairment to nodulate agronomically improved American
soybean varieties shown by some S. fredii strains (such as S. fredii USDA257), is
mediated by proteins secreted through the Type 3 secretion system (Yang et al., 2010).
In this work, we have identified the extracellular proteins (the secretome) that are
present in S. fredii HH103 and USDA257 liquid cultures grown with and without
genistein, a flavonoid that is present in the soybean rhizosfere and activates the
transcription of rhizobial nodulation genes.
Protein samples were briefly subjected to SDS-PAGE such that proteins entered only 1
cm into the gel. In-gel tryptic digestion was followed by LC-MS/MS using a Waters
nanoAcquity coupled to a Bruker maXis. Tandem mass spectral data were matched to
proteins in the NCBInr database using Mascot (Matrix Science). Mascot-calculated
emPAI values and direct comparison of Mascot ions scores were used to asses protein
relative abundance in different samples. The S. fredii HH103 genome sequence was
analyzed by using the GenDB software (CeBiTec, University of Bielefeld, Germany).
The analyses of the S. fredii HH103 and USDA257 secretomes have shown that these
bacteria secrete a high number of proteins to the extracellular milieu. Thus, whereas 119
proteins were detected in USDA257 uninduced cultures, 126 were found in the presence
of genistein. In the case of HH103 cultures, 37 proteins were identified in the absence of
genistein and 119 in the presence of the flavonoid. The analyses of these data showed
some differences between S. fredii strains USDA257 and HH103. For instance, five
effector proteins (NopA,B,L,P,X) secreted through the USDA257 T3SS were detected
in t he presence of genist ein. In contrast, seven HH103 putat ive effectors
(NopA,B,L,P,X plus NopC and NopM) were identified. These results are in agreement
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with previous reports suggesting that S. fredii USDA257 might not be particularly
efficient in Nop secretion (Krishnan et al., 2011). The secretome analyses of strains
HH103 and USDA257 showed other interesting differences. For instance, 8 putative
components of the flagellum were identified in the secretome of USDA257 but only
three of them were detected in HH103 cultures. This difference in the number of
putative flagella components identified in USDA257 and HH103 cultures occurs in the
presence and absence of genistein. S. fredii strains USDA257 and USDA191 also differ
in the number of putative flagella proteins (Krishnan et al., 2011). Five proteins that
might play symbiotic roles were chosen for further work (Table 1).
Table 1. Proteins of the S. fredii HH103 and USDA257 secretomes that were chosen for further analyses.
Predicted functions
of some HH103 and
USDA257
orthologues.
Putative metal
binding protein
Phosphoserine
aminotransferase
Peptidyl-prolyl cistrans isomerase,
cyclophilin type
Peptidyl-prolyl-cistrans isomerise,
cyclophilin type
Peptidase M29,
aminopeptidase II
USDA257
genes
Presence of
protein in
Gen Gen +
HH103
genes
Presence of
protein
Gen - Gen +
c53490
-
+
02943
-
+
serC_c51910
-
+
serC_02741
-
+
ppi2_c40150
-
+
ppiA_01477
-
+
ppi1_c40140
D
-
-
ppiB_01476
-
+
c61510
-
-
03876
-
+
Gen-, absence of genistein; Gen+, presence of genistein; USDA257 and HH103 genes occupying the sale
line are orthologues.
Cyclophilins proteins belong to a group of proteins that have peptidyl-prolyl cis-trans
isomerase activity and are found in all prokaryotes and eukaryotes investigated. Recent
studies have implicated cyclophilins in a diverse array of cellular functions, including
roles as chaperones and in cell signaling. Genes coding for cyclophilins will be tagged
with the commercial oligopeptide HA to confirm extracellular secretion and induction
with genistein by western blot analyses. In addition, a mutant in the ppiB gene will be
constructed to study the role of these cyclophilins in the symbiosis with soybean.
The knowledge of the bacterial capacity to use secretion systems to be adapted to
environmental conditions, such as the plant lifestyle, is growing fast. However, the role
of secretion systems in beneficial plant-bacteria interactions is poorly understood.
ACKNOWLEDGEMENTS
This work has been supported by project BIO2011-30229-C02-01. Irene Jiménez-Guerrero work was
financed with a predoctoral contract from the program IV Plan Propio of the University of Seville.
REFERENCES
Krishnan, H.B., et al. (2011). Appl. Environ. Microbiol. 77: 6240-6248.
Weidner, S., et al. (2012). J. Bacteriol. 194: 1617-1618
Yang, S., et al. (2010). Proc. Natl. Acad. Sci. USA 107: 18735-18740.
122
Session II
SII-CP-07
Ocorrência e caracterização de bactérias isoladas de nódulos de
amendoinzeiro (Arachis hypogaea L.) em solos paranaenses, Brasil.
Andrade, D.S.1*, Cardoso, J.D.1, Pertinhez, G.N.1, Saturno, D.F., Lovato, G.M. 1,
Nomura, R.B.G.1, Hungria, M.2
1
*
Instituto Agronômico do Paraná, Londrina, PR, Brasil. 2 Embrapa Soja, Londrina, PR, Brasil.
[email protected]
RESUMO
O objetivo deste trabalho foi avaliar a ocorrência e caracterizar morfofisiologicamente e
geneticamente bactérias isoladas de nódulos de amendoinzeiro (Arachis hypogaea L.),
em solos paranaenses, Brasil. Em amostras de 36 municípios representativos do estado
Paraná foi observada nodulação do amendoim em 84,4%, independente se em áreas
cultivadas, ou em florestas. Nos testes de caracterização morfofisiológica de 44 estirpes
autenticadas, 75% das estirpes apresentaram taxa de crescimento rápido em meio de
cultura com manitol; 25 acidificaram o meio, cinco alcalinizaram e 14 não resultaram
em modificação do pH. A capacidade de produzir sideróforos foi observada em 43% das
estirpes e 16% foram capazes de solubilizar fosfato de cálcio em meio de cultura. O
sequenciamento do gene 16S rRNA mostrou a formação de três principais grupos,
correspondentes a três filos: Alfaproteobacteria, Betaproteobacteria e Firmicutes. O
gênero com maior ocorrência foi o Bacillus.
INTRODUÇÃO
O amendoinzeiro (Arachis hypogaea L.) é uma leguminosa oleaginosa originárioa da
América do Sul, ocorrendo em regiões tropicais e subtropicais. Na maioria dos sistemas
de cultivo o amendoinzeiro consegue realizar a fixação biológica de nitrogênio em
quantidades adequadas para suprir suas necessidades (Thies et al., 1991). No Paraná,
não se recomenda a aplicação de fertilizantes nitrogenados para a cultura. A avaliação
da ocorrência de rizóbios em áreas cultivadas e sob floresta, bem como a caracterização
destas estirpes pode fornecer informações sobre a filogenia de microrganismos
simbióticos nativos simbiontes do amendoinzeiro. Sendo assim, o objetivo deste
trabalho foi avaliar a ocorrência de rizóbios nodulantes do amendoinzeiro e analisar as
características morfofisiológicas e genéticas destas bactérias simbiônticas.
MATERIAL E MÉTODOS
A ocorrência de rizóbio simbionte do amendoinzeiro (Arachis hypogaea L.) foi avaliada
pela presença de nódulos em plantas semeadas em amostras de solos coletadas de 36
municípios do Paraná, em áreas sob cultivos de milho (Zea mays L.), pastagens e solos
de mata, do Estado do Paraná, na camada 0–10 cm. Para o estudo da caracterização
mofofisiológica e genética, também foram obtidas estirpes bacterianas de nódulos de
plantas de amendoinzeiro de um experimento de campo, onde foram coletados nódulos
de maneira aleatória e realizado o isolamento. Os isolados obtidos foram autenticados
duas vezes para certificar a capacidade simbiótica no amendoinzeiro. As estirpes foram
autenticadas em vasos Leonard com areia e vermiculita contendo solução nutritiva sem
N mineral e a cultivar de amendoim IAC-Tatu ST. As estirpes foram avaliadas quanto à
produção de sideróforos e à capacidade de solubilização de fosfato de cálcio. O DNA
genômico total foi submetido à amplificação para análise do BOX-A1R e procedeu-se
ao sequenciamento do gene 16S rRNA. A estirpe de Bradyrhizobium sp, SEMIA 6144
(Coleção de Cultura do laboratório da FEPAGRO, RS), autorizada para a produção de
inoculante para o amendoinzeiro no Brasil, foi incluída como controle.
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De um total de mais de 100 isolados, apenas 44 foram comprovados quanto à formação
de nódulos efetivos, com coloração interna avermelhada e foram codificadas (IPR-Ah) e
depositadas na Coleção de microrganismos do Instituto Agronômico do Paraná
(IAPAR). A capacidade de produzir sideróforos foi observada em 43% das 44 estirpes.
A capacidade de solubilizar fosfato de cálcio em meio de cultura foi observada em 16%
das estirpes. A estirpe recomendada SEMIA 6144 não apresentou capacidade para
produzir sideróforos nem para solubilizar fosfato de cálcio. O sequenciamento do gene
16S rRNA das 41 estirpes mostrou a presença de oito gêneros, sendo Bacillus com 63%
de ocorrência (Figura 1).
2
2
2
2
2
2
Bacillus
2 2
Acinetobacter
Paenibacillus
Pectobacterium
7
2
63
10
Cohnella
2
Mesorhizobium
2
Stenotrophomonas
Rhizobium
Lysinibacillus
Figura 1. Porcentagem de ocorrência dos gêneros de bactérias do total de 41 estirpes (IPR Ah)
identificadas pelo sequenciamento do gene 16S rRNA.
Bactérias de crescimento rápido nodulando leguminosas tropicais têm sido relatadas, o
que pode ser atribuído à dupla ocupação nos nódulos, sendo sugerido o uso de
antibióticos como uma estratégia para isolar apenas as de crescimento lento (Trinick,
1982). Neste estudo, todas as estirpes foram autenticadas duas vezes, então, nossa
hipótese é que ocorreu simbiose entre duas bactérias no isolamento, o que significa a
formação de uma colônia a partir de duas células com crescimento rápido e lento. Estes
resultados mostram a necessidade de buscar estratégias para aumentar a eficiência da
inoculação, evitando a competição desta alta população nativa.
REFERÊNCIAS
Thies, J.E., et al. (1991). Appl. Environ. Microbiol. 57:1540-1545.
Trinick, M.J. (1982). In: Vincent, J.M. (Ed.), Nitrogen Fixation in Legume. Academic Press Australia,
Sydney, pp. 229-238.
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The Type 3 secretion system effector NopP from Ensifer
(=Sinorhizobium) fredii HH103 is phosphorylated by a soybean kinase.
Calero, B.1*, Jiménez-Guerrero, I.1, Pérez-Montaño, F.1, Monreal, J.A.2, Ollero, F.J.1,
López-Baena, F.J.1
1
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. Reina Mercedes,
6. 41012-Sevilla, Spain. 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología,
Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012-Sevilla, Spain.
* [email protected]
ABSTRACT
NopP from E. fredii NGR234 is a Type 3 secreted effetor that is phosphorylated by
plant kinases (Skorpil et al., 2005). E. fredii HH103 also secretes NopP and its presence
is detrimental for nodulation with soybeans (López-Baena et al., 2009). However, the
function of this protein in the symbiotic interaction with soybean is still unknown.
The nopP gene was cloned in plasmid pGEX for in vitro expression and purification of
NopP. The purified protein was used for in vitro phosphorylation and in-gel kinase
assays. Results showed that the E. fredii HH103 NopP is phosphorylated by a soybean
kinase that seems to be calcium dependent.
INTRODUCTION
Some rhizobial strains possess a specialized apparatus for protein secretion called Type
3 secretion system (T3SS), which is also present in pathogenic bacteria. This apparatus
is used to deliver effectors directly into the cytoplasm of the host cells. Nodulation outer
protein P (NopP) is a Rhizobium-specific type 3 effector. Phosphorylation of NopP in E.
fredii NGR234 by kinases of legume roots is blocked by general inhibitors of tyrosine
and serine/threonine kinases. However, this phosphorylation is not MAP kinase
dependent as described for NopL (Skorpil et al., 2005). Inactivation of nopP led to an
increase in the symbiotic capacity of E. fredii HH103 to nodulate Williams soybean
(López-Baena et al., 2009).
Results shown in this work indicate that the HH103 NopP protein was phosphorylated
by a kinase obtained from soybean root extracts. This kinase is currently being
characterized to try to elucidate its role in the symbiosis.
Strains E. fredii HH103 RifR, E. fredii HH103 RifR ΩnopP, and E. coli BL21 with the
nopP gene cloned in plasmid pGEX were used in this study.
Production of the recombinant NopP protein and in vitro phosphorylation assays were
performed as described by Monreal et al. (2013).
In-gel kinase assay was performed as described by Monks et al. (2001) with some
modifications.
In vitro phosphorylation assay showed that NopP was phosphorylated by a soybean root
kinase (Figure 1). This kinase was calcium dependent. In-gel kinase assays were
performed to identify the kinase responsible for this phosphorylation and two different
bands corresponding to two possible kinases were detected.
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Figure 1. In vitro phosphorylation assay. Soybean root extracts obtained from uninoculated plants or
inoculated with E. fredii HH103 RifR were mixed with the recombinant protein NopP. Lane 1, Control
with recombinant NopP and GST; lane 2, molecular weight marker; lane 3, recombinant NopP with
soybean root extract from plants inoculated with E. fredii HH103 RifR; lane 4, control with soybean root
extract from plants inoculated with E. fredii HH103 RifR; lane 5, recombinant NopP with uninoculated
soybean root extract; lane 6, Control with uninoculated soybean root extract.
The analysis of the genome of E. fredii HH103 confirmed the presence of another
possible effector, similar to NopP, which was named NopP2. Regulation of the
expression of this gene depended on NodD, TtsI and flavonoids, as confirmed by qPCR.
To determine the role of all the Rhizobium-specific phosphorylated effectors in the
symbiosis with soybean and elucidate whether their functions could be redundant, the
double mutants ΩnopL LacZ-GmRnopP, LacZ-GmnopP ΔnopP2, and ΩnopL ΔnopP2,
and a triple mutant ΩnopL LacZ-GmRnopP ΔnopP2 were constructed. These mutant
derivatives will be tested in nodulation assays in soybean to study their effect in
symbiosis.
ACKNOWLEDGEMENTS
This work was financed by projects AGL2009-13487-C04 from the Spanish MEC and P11-CVI-7050
from the Junta de Andalucía.
REFERENCES
López-Baena, F.J., et al. (2009). Mol. Plant Microbe Interact. 22: 1445-1454.
Monks, D.E., et al. (2001). Plant Cell. 13: 1205-1219.
Monreal, J.A., et al. (2013). Planta. In press.
Skorpil, P., et al. (2005). Mol. Microbiol. 57: 1304-1317.
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A yeast-based array to study the function of Ensifer (=Sinorhizobium)
fredii HH103 Type 3 secretion system effectors in symbiosis.
Ollero, F.J.1*, Jiménez-Guerrero, I.1, Pérez-Montaño, F.1, Mesa, B.2, Medina, C.2,
López-Baena, F.J.1
1
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. de Reina
Mercedes, 6. 41012 - Sevilla, España. 2 Área de Microbiología. Centro Andaluz de Biología del
Desarrollo. Universidad Pablo de Olavide/CSIC. Ctra. Utrera, Km 1. 41013-Sevilla, España.
* [email protected]
ABSTRACT
Little is known about the functions of rhizobial Type 3 secretion system effectors in the
symbiosis with their host legumes. Expression of bacterial effectors in yeast has been
extensively used to study effector functions within the host cell. A yeast-based array
was used to identify potential function of rhizobial effectors in the context of plantrhizobia interactions.
INTRODUCTION
Many pathogenic Gram-negative bacteria encode a type 3 secretion system (T3SS), a
molecular syringe evolved to deliver proteins into the host cell during infection. These
virulence proteins, called type 3 effectors (T3Es), modulate several host cellular
processes to promote disease. A current challenge is the determination of their virulence
functions and host targets.
The yeast Saccharomyces cerevisiae is currently being used as a tool to investigate
bacterial T3Es due to the observation that these proteins often target cellular processes
that are conserved among all eukaryotes.
Kramer et al. (2007) described an approach to study bacterial effectors in yeast, which
uses yeast synthetic lethal (SL) interaction data. SL is defined as the situation in which
two genes that are non-essential when individually mutated cause lethality when they
are combined as a double mutant. Their analysis is based on the assumption that
phenotypes resulting from the activity of the T3E would resemble phenotypes of a
mutation in the target gene of the effector. Accordingly, genes are defined as congruent
to an effector if their sets of SL interactions overlapped with the deletion strains
hypersensitive to that effector and therefore represent putative cellular targets. The
major disadvantage of this approach is that it requires the screening of all 4,750 deletion
strains, which limits its wide application to laboratories.
Bosis et al. (2011) have developed a simple method based on the finding that it is
possible to cover the majority of the interacting genes (i.e. genes having at least one
known SL interaction) with 90 deletion strains. An array of yeast deletion strains fitted
into a single 96-well plate covers 69% of the interacting genes with less than 2% of the
deletion strains in the yeast collection. The small number of deletion strains in the array
simplifies the analysis, reduces costs and facilitates the screening of a large number of
bacterial T3Es in a short period of time.
The E. fredii HH103 effector genes nopP, nopP2, nopL, nopM and nopC were cloned
into the pGML10 vector of expression in yeast and individually transferred to the 90
deletion strains of the array. Yeast strains carrying the empty pGML10 vector were used
as controls. The method described by Bosis et al. (2011) was used to identify SL
interactions with some modifications. Briefly, transformed yeast strains were grown in
liquid cultures in 96-well plates in inducing medium with galactose and in growing
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medium with glucose. The plate with glucose was used as a control to normalize the
number of cells, and hence growth, in the inducing medium. Significant differences in
the OD600nm 48h after inoculation indicated possible target genes.
There were no differences in growth of the yeast strains expressing nopP2 and nopC. It
could be possible that NopC was not a real effector but a component of the secretion
machinery, as suggested by some authors (personal communication). Therefore, no
metabolic route in the eukaryotic host would be affected. In the case of nopP2, despite
this gene seems to be an effector because its transcription is regulated as other effectors
such as NopP, this protein could not have a clear effect in the metabolism of the host or
the target gene would not be included in the array.
With respect to NopM, NopP, and NopL, some differences in yeast growth could be
observed and possible target genes could be assigned. The effector NopM, previously
described as an ubiquitin ligase (Xin et al., 2012), was used as a control to know if this
method could be used with rhizobial effectors. Results obtained (Table 1) showed that
the E. fredii HH103 NopM effector could be a protein involved in ubiquitination of
proteins. This was in agreement with previous published results in other rhizobial
strains and in animal and plant pathogens.
Table 1. FuncAssociate 2.0 results assigning GO attributes for the E. fredii HH103
NopM effector.
Rank N
1
3
2
3
X
83
104
LOD
1.779
1.676
p-value
0.00007419
0.0001456
GO attribute
Protein ubiquitination
Protein modification
conjugation
by
small
protein
N = number of congruent genes that have the GO attribute.
X = total number of interacting genes covered by our array that have the GO attribute.
LOD = Logarithm (base 10) of the odds ratio.
ACKNOWLEDGEMENTS
from the Junta de Andalucía. We would like to thank Dr. Guido Sessa for providing the yeast array. Irene
Jiménez-Guerrero work was supported by a predoctoral fellowship of the University of Seville.
REFERENCES
Bosis, E., et al. (2011). PloS One 11: e27698
Kramer, R.W., et al. (2007). PLoS Pathog. 3: e21.
Xin, D.W., et al. (2012). PLoS Pathog. 5: e1002707.
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Implicación del gen bgvA en la formación de biofilm por Rhizobium
tropici CIAT899.
Del Cerro, P.1*, Ollero, F.J., Megías, M.2, Bellogín, R.A.1, Guasch-Vidal, B.1, PérezMontaño, F.1, Espuny, M.R.1
1
41012-Sevilla. España. 2 Departamento de Microbiología y Parasitología, Facultad de Farmacia,
Universidad de Sevilla. Profesor García González, s/n. Sevilla.
* [email protected]
RESUMEN
Rhizobium tropici CIAT899, bacteria altamente tolerante a diversos tipos de estrés,
sintetiza factores de nodulación cuando es crecida a concentraciones de 300 mM de
NaCl en ausencia del flavonoide inductor apigenina. El gen bgvA de esta bacteria, que
parece estar implicado en esta actividad, se encuentra en un operón de síntesis y
secreción de polisacáridos y hasta la fecha sólo se ha detectado en el genoma de
CIAT899 y como una proteína hipotética en el de Agrobacterium radiobacter K84.
Nuestros resultados muestran que este gen parece estar implicado en el desarrollo de
biofilm de CIAT899 puesto que dos mutantes por inserción en el gen bgvA de esta
bacteria, uno con Tn5-Mob y otro con el casete Ω, desarrollan menor biofilm bajo
condiciones normales y especialmente cuando crece bajo estrés salino.
INTRODUCCIÓN
Rhizobium tropici CIAT899 es un simbionte de judía catalogado como altamente
tolerante a diversos tipos de estrés abiótico (Hungría et al.., 2003). De hecho, el análisis
de su genoma ha revelado una gran variedad de rasgos que hacen que esta bacteria sea
una gran colonizadora de la rizosfera (Ormeño-Orrillo et al.., 2012). Además, se ha
visto que concentraciones de 300 mM de NaCl inducen la expresión de los genes nod en
ausencia de flavonoides (B. Guasch-Vidal and J. Estévez et al., 2013). Se ha
seleccionado y caracterizado, por inserción de un trasposón Tn5-mob, un mutante de
CIAT899 denominado C9, que está afectado en la producción de factores Nod en
condiciones de estrés salino (NaCl 300 mM) y sin apigenina. El gen mutado, que se
registró como bgvA (GenBank HM768892) no ha sido detectado hasta la fecha en
ninguna otra bacteria, con excepción de Agrobacterium radiobacter K84, donde se
desconoce su función (Tesis doctoral de Guasch-Vidal, B., 2011). Este gen forma parte
de un operón que se caracteriza por contener mayoritariamente genes implicados en la
biosíntesis y transporte de polisacáridos superficiales bacterianos. En los rizobios, la
matriz del biofilm bacteriano está compuesta principalmente por exopolisacáridos,
factores de nodulación y agua (Rinaudi and Giordano, 2010). Con estos antecedentes,
nos propusimos determinar la implicación de este gen en el desarrollo de biofilm, e
intentar relacionarlo con posibles defectos en la producción de EPS y movilidad
bacteriana.
MATERIAL Y MÉTODOS
Las estirpes empleadas se detallan en la Tabla 1. El medio de cultivo en todos los
ensayos fue B-, al que se añadió cuando fue necesario apigenina (1 μg/ml), o NaCl
(300mM) y los antibióticos adecuados. Los ensayos de biofilm se realizaron en placas
de microtítulo de poliestireno, según el método de Mueller and Gonzalez (2011) y
empleando los mismos medios.
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Tabla 1. Estirpes utilizadas.
Estirpes
CIAT899
SVQ689
SVQ689
(pMUS1066)
C9
C9 (pMUS1066)
Características relevantes
Rhizobium tropici. Estirpe silvestre. Produce factores Nod con NaCl
Mutante de CIAT899 en el gen bgvA por inserción de Ω. Produce factores Nod
con NaCl
SVQ689 complementado con el gen bgvA silvestre en el plásmido pBBR1MCS-5.
Mutante de CIAT899 por inserción de Tn5-Mob del gen bgvA. No produce
factores Nod con NaCl
Mutante C9 complementado con el gen bgvA silvestre en el plásmido
pBBR1MCS-5.
Los resultados obtenidos demuestran que existen diferencias en la formación de biofilm
entre la estirpe silvestre y los mutantes SVQ689 y C9, de tal forma que los mutantes
desarrollan menor biofilm en todas las condiciones (B-, B- suplementado con NaCl 300
mM y B- suplementado con apigenina). En el mutante C9 en condiciones salinas se
produce un descenso aún mayor en la formación de biofilm, quizá debido a que los
factores Nod forman parte de la matriz del biofilm. En el futuro se realizarán estudios de
cuantificación de la producción de polisacáridos y movilidad bacteriana que pueden
relacionarse con estos resultados.
AGRADECIMIENTOS
Este trabajo ha sido financiado por los proyectos AGL2009-13487-C04-03/AGR y AGL2012-38831del
Ministerio de Economía y Competitividad de España.
BIBLIOGRAFÍA
Guasch-Vidal, B. (2011). Tesis doctoral. Departamento de Microbiología. Universidad de Sevilla.
Guasch-Vidal, B., et al. (2013). Mol Plant Microbe Interact. 26: 451-460.
Hungría, M., et al. (2003). Biol. Fertil. Soils. 39: 88-93.
Lloret, L., and Martínez-Romero, E. (2005). Rev. Lat. Microbiol. 47: 43-60.
Mueller K., and González, E. (2011). J. Bacteriol. 193: 485-496.
Ormeño-Orrillo, E., et al. (2012). BMC Genomics 13: 735.
Rinaudi L.V., and Giordano W. 2010. FEMS. Microbiol. Lett. 304: 1-11.
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Identification and characterization and of a soybean kinase that
phosphorylates the Type 3 secretion system effector NopL from Ensifer
(=Sinorhizobium) fredii HH103.
Jiménez-Guerrero, I.1*, Pérez-Montaño, F.1, Ollero, F.J.1, Monreal, J.A.2, Cubo, M.T.1,
López-Baena, F.J.1
1
6. 41012 - Sevilla, Spain. 2 Departamento de Biología Vegetal y Ecología, Facultad de Biología,
Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 - Sevilla, Spain.
* [email protected]
ABSTRACT
Little is known about the exact role of the nodulation outer proteins (Nops) secreted by
the Type 3 secretion system (T3SS) of E. fredii HH103 in the symbiosis with soybeans.
One of these proteins, NopL, has been described as a phosphorylated effector that could
be involved in the modulation of plant defence responses by interfering with signal
transduction cascades associated to MAP kinases.
This work is the first approach to identify possible soybean targets for the E. fredii
HH103 NopL protein. A nopL mutant was constructed to study the symbiotic phenotype
in soybean and Vigna unguiculata. The absence of NopL significantly affected
symbiosis with both legumes. The nopL gene was also cloned in plasmid pGEX for in
vitro expression and purification of NopL. The purified protein was used for in vitro
phosphorylation and in-gel kinase assays. Results showed that the E. fredii HH103
NopL is phosphorylated by a soybean kinase that is calcium dependent.
INTRODUCTION
Some Gram-negative bacteria possess a specialized apparatus for protein secretion
named T3SS. Symbiotic and pathogenic bacteria use the T3SS to translocate effectors,
which are involved in the modulation of host defense responses, directly into the host
cytoplasm. Nodulation outer protein L is a Rhizobium-specific Type 3 effector. NopL
from E. fredii NGR234 is phosphorylated by plant kinases and seems to modulate host
defense responses (Bartsev et al., 2004). Previous results suggest that NopL of NGR234
mimics a MAPK substrate and suppresses premature nodule senescense by impairing
MAPK signaling in host cells (Zhang et al., 2011). However, the role of E. fredii
HH103 NopL in the symbiotic interaction with soybean is currently unknown.
Strains E. fredii HH103 RifR, E. fredii HH103 RifR ΩnopL mutant, and E. coli BL21
with the nopL gene cloned in plasmid pGEX were used in this study.
Nodulation assays on Glycine max (L.) and Vigna unguiculata (L.) were performed as
described by de Lyra et al. (2006).
Production of recombinant NopL protein and in vitro phosphorilation assays were
performed as described by Monreal et al. (2013).
In-gel kinase assay was performed as described by Monks et al. (2001) with some
modifications.
The symbiotic phenotype of the mutant strain HH103 ΩnopL in Williams soybean
showed significant differences when compared to the parental strain. Thus, nodules
fresh mass and plant-top dry mass from plants inoculated with the mutant strain were
higher than in plants inoculated with the parental strain. By contrast, the symbiotic
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phenotype of the mutant strain in Vigna unguiculata showed opposite results. In this
plant the number of nodules and plant-top dry mass of plants inoculated with HH103
RifR ΩnopL were lower than in plants inoculated with HH103 RifR. Therefore, NopL
seems to play an important role in the symbiosis with soybean and V. unguiculata.
In vitro phosphorylation assays showed that NopL was phosphorilated by a kinase
present in soybean root extracts. Different kinase inhibitors are currently being used to
try to characterize this plant kinase. In this sense, preliminary studies showed that NopL
was phosphorylated by a calcium dependent not inducible kinase (Figure 1).
On the other hand, in-gel kinase assays allowed the determination of the approximate
size of the kinase involved in the phosphorylation of NopL.
Figure 1. In vitro phosphorylation assay. Soybean root extracts obtained from uninoculated plants or
inoculated with E. fredii HH103 RifR were mixed with the recombinant protein NopL. Lane 1, Control
with recombinant NopL and GST; lane 2, molecular weight marker; lane 3, recombinant NopL with
soybean root extract from plants inoculated with E. fredii HH103 RifR; lane 4, control with soybean root
extract from plants inoculated with E. fredii HH103 RifR; lane 5, recombinant NopL with uninoculated
soybean root extract; lane 6, Control with uninoculated soybean root extract.
ACKNOWLEDGEMENTS
from the Junta de Andalucía. Irene Jiménez-Guerrero work was supported by a predoctoral fellowship of
the University of Seville.
REFERENCES
Bartsev, A.V., et al. (2004). Plant Physiol. 134: 871-879.
de Lyra, M.C.C.P., et al. (2006). Int. Microbiol. 9: 125-133.
Monks D.E., et al. (2001). Plant Cell. 13: 1205-1219.
Monreal, J.A., et al. (2013). Planta. In press.
Zhang, L., et al. (2011). J. Biol. Chem. 37: 32178-32187.
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Control de movilidad y producción de EPS I en Sinorhizobium meliloti:
nuevas funciones asignadas al sistema NtrY/X.
Calatrava, N.*, Nogales, J., Ameztoy, K., van Steenbergen, B., Soto, M.J.
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del Zaidín CSIC, 18008 Granada.
* [email protected]
RESUMEN
Bacterial surface motility can impact colonization and infection of eukaryotic hosts and
it is subject to strict control. To identify genetic determinants involved in S. meliloti
surface motility, GR4-derivative transposants with an altered ability to translocate over
surfaces relative to the wild type were selected. Three of these mutants contained
independent Tn5 insertions that interrupted the ntrY gene coding for the putative sensor
histidine kinase of the two-component system NtrY/X which has been involved in
nitrogen metabolism regulation in several bacteria. Characterization of the ntrY::Tn5
mutant GNS577 revealed a pleiotropic phenotype when compared to the parental strain:
i) reduced swimming and surface motilities, ii) altered cell morphology with low
number of flagella, iii) mucoid phenotype after growth in minimal medium, iv)
increased biofilm formation ability on abiotic surfaces, and v) impaired growth under
high salt conditions. Our results indicate that the defect in motility shown by GNS577 is
mainly due to reduced expression of the flagellar master regulator genes visNR whereas
the mucoid phenotype is caused by EPS I over-production. GNS577 is able to develop
nitrogen-fixing nodules on alfalfa plants but it is highly impaired in competitive
nodulation.
INTRODUCCIÓN
Diversos estudios indican que la capacidad de una bacteria de desplazarse sobre una
superficie puede afectar el establecimiento de interacciones con un hospedador
eucariota. No obstante, el conocimiento existente sobre motilidad en superficie de los
rizobios, y de cómo ésta puede afectar la colonización y establecimiento de
endosimbiosis mutualista con las leguminosas es muy escaso. Nuestro grupo fue el
primero en describir motilidad tipo swarming en un mutante fadD de la cepa GR4 de S.
meliloti (Soto et al. 2002), mutante que resultó estar afectado en su capacidad infectiva
y competitiva por la nodulación de alfalfa. Posteriormente, demostramos que la cepa
Rm1021 es capaz de desplazarse sobre la superficie de medios semisólidos utilizando
mecanismos dependientes e independientes de la acción flagelar, en los que la
producción del sideróforo rizobactina 1021 cumple un papel esencial (Nogales et al.
2012). Con el objetivo de identificar nuevos componentes implicados en motilidad en
superficie de S. meliloti, hemos llevado a cabo el aislamiento de transposantes derivados
de la cepa GR4 alterados en este tipo de motilidad. En este trabajo se presenta la
caracterización de uno de ellos: GNS577.
MATERIAL Y MÉTODOS
La cepa GR4 de S. meliloti se sometió a mutagénesis generalizada con Tn5 usando el
plásmido pSUP2021. La translocación en superficie se ensayó en medio mínimo (MM)
semisólido (0,6% agar Noble) y la motilidad swimming en medio Bromfield (0,3% agar
bacteriológico) tal y como se describe en Nogales et al. (2012). La secuencia de ADN
afectada por la inserción del Tn5 se identificó mediante PCR arbitraria y posterior
secuenciación. La súper-producción de EPS I se analizó iluminando con luz UV
colonias crecidas en MM conteniendo Calcoflúor (0,2 mg/mL). El fenotipo simbiótico
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(infectividad y competitividad) se analizó en plantas de alfalfa crecidas en cultivo
hidropónico siguiendo la metodología descrita en Soto et al. (2002).
El análisis de 5800 transposantes derivados de la cepa GR4 de S. meliloti nos permitió
identificar 14 clones con deficiencias en translocación sobre superficie de MM
semisólido. La caracterización genética de estos mutantes reveló que 3 de ellos
contenían inserciones independientes de Tn5 en el gen ntrY que potencialmente codifica
el sensor histidín quinasa del sistema regulador de dos componentes NtrY/X implicado
en el control de genes relacionados con metabolismo nitrogenado en bacterias
simbióticas fijadoras de N2. La caracterización fenotípica del mutante GNS577 reveló
estar afectado no sólo en motilidad en superficie (Figure 1A) sino también en motilidad
tipo swimming (Figure 1B), defectos que podrían ser debidos a la menor expresión de
los genes visNR detectada en el mutante ntrY con respecto a GR4. Observaciones
realizadas en TEM revelaron que GNS577 presentaba una morfología alterada (menor
tamaño y más redondeada) con respecto a la cepa parental, así como un reducido
número de flagelos (Figure 1C). Además, la mayor mucosidad exhibida por GNS577 en
placas de MM (Figure 1D), así como la fluorescencia emitida tras iluminar con UV
placas de MM conteniendo calcoflúor (Figure 1E), sugerían que este mutante producía
mayores cantidades de EPS I que GR4, hipótesis que ha sido confirmada al revertir
estos fenotipos en un doble mutante ntrYexoY. GNS577 presenta mayor sensibilidad a
estrés salino (MM adicionado de 300 mM NaCl) (Figure 1F), y mayor capacidad de
formar biofilm en superficie de vidrio que GR4 (Figure 1G). Aunque capaz de formar
nódulos fijadores de N en alfalfa, el mutante ntrY muestra claros defectos en capacidad
competitiva por la nodulación (Figure 1H) que pueden ser recuperados tras la sobreexpresión de VisNR. Nuestros resultados sugieren que el sistema NtrY/X de S. meliloti
participa en el control coordinado de motilidad y producción de EPS I.
Figura 1. Fenotipos mostrados por GNS577 (ntrY::Tn5) en comparación con la cepa parental GR4. (Ver
detalles en texto)
AGRADECIMIENTOS
Este trabajo ha sido financiado por MICINN (BIO2010-18005), Junta de Andalucía (Proyecto de
Excelencia CVI-03541) y fondos FEDER.
BIBLIOGRAFÍA
Soto, M.J., et al. (2002). Mol. Microbiol. 43: 371-382.
Nogales, J., et al. (2012). J. Bacteriol. 194: 2027-2035.
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Auxotrophy accounts for nodulation impairment in a Sinorhizobium
meliloti mutant defective for meso-diaminopimelate biosynthesis.
García-Rodríguez, F.M., Ortigosa, A., Millán V., Toro, N., Martínez-Abarca, F. *
Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo
Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.
* [email protected]
ABSTRACT
Sinorhizobium meliloti establishes an effective nitrogen-fixing symbiosis with its
leguminous host plant, alfalfa (Medicago sativa). A S. meliloti dapB (encoding 2,3dihydrodipicolinate reductase) mutant has defective nodulation that is restored by the
addition of diaminopimelate (DAP) to the plant growth medium.
INTRODUCTION
The soil bacterium Sinorhizobium meliloti establishes a nitrogen-fixing symbiosis with
its legume plant partner (Medicago sativa), through a series of signal exchanges (van
Rhijn and Vanderleyden 1995). Auxotrophic mutations affecting a number of aminoacid biosynthetic pathways do not affect the ability of S. meliloti to initiate nodule
formation (Randhawa and Hassani 2002). However, several mutants with defects in the
biosynthesis of branched-chain amino acids have been shown to display impaired
initiation of nodule formation (Aguilar and Grasso 1991; Hassani et al., 2002; SanjuánPinilla 2002; de las Nieves Peltzer, et al., 2008).
The dapB gene, encoding L-2,3-dihydrodipicolinate reductase, is required for the
biosynthesis of meso-diaminopimelate (DAP; Born and Blanchard 1999). DAP is an
essential component of peptidoglycan and also serves as a direct precursor of lysine.
Mutations of the dapB gene impair the growth of Pseudomonas stutzeri in the absence
of DAP and lysine (Rediers et al., 2003). We studied the free-living and symbiotic
phenotypes of a dapB mutant of S. meliloti.
dapB mutant construction. 1021-ΔdapΩ was constructed by insertion of the Sm/SpR
cassette (Prentki and Krisch 1984) into the chromosome (between positions 186859 and
187585).
Growth and Symbiotic phenotype. TY and MM growth media assays and plant assays
were performed as described in (Soto et al., 2004).
The S. meliloti deleted dapB (1021-ΔdapΩ)mutant displayed impaired growth in both
TY and MM media, but complementation of this defect was observed when 2.5 mM
DAP was added to the medium. Complementation of the mutant with the wild-type
dapB ORF expressed in trans fully restored wild-type growth in media lacking DAP.
We then investigated the effect of this mutation on symbiosis (Figure 1). Plants
inoculated with the dapB mutant had yellow or pale green leaves, indicating a lack of
nitrogen fixation. By contrast, the plants inoculated with the dapB+ parental strain or
the mutant strain complemented with pKmDAP had healthy green leaves and the
presence of elongated pink nodules (Figure 1A and B). Plants inoculated with the dapB
mutant had white nodules, which developed on the roots later than wild-type nodules
and were generally not elongated (less than 10% were elongated). Histochemical
analyses of longitudinal sections of elicited nodules revealed that the nodule produced
by the parental strain had a clear zonal distribution, whereas the mutant induced
135
Session II
SII-CP-13
structures essentially resembling non invaded pseudonodules and displaying no nitrogen
fixation. In these conditions, the nodules induced by the dapB mutant appeared at the
same time as those elicited by the wild-type strain, and dry weight did not differ
significantly between the two sets of inoculated plants.
These results suggest that the concentration of DAP in alfalfa root exudates is limiting;
however, the supplementation of this compound restored nitrogen fixation and normal
infectious processes. These properties define the dapB gene as a good selective marker
for screening promoters involved in early stages in the alfalfa-S. meliloti symbiosis
Figure 1. Symbiosis defects of the
dapB mutant. (A) Alfalfa plants 25
days after inoculation with the wt
1021 strain (panel 1), the dapB mutant
(panel 2), the complemented dapB
mutant (carrying pKmDAP; panel 3)
and the dapB mutant in the presence
of 2.5 mM DAP added to the plant
growth medium (panel 4). (B)
Enlarged image of the nodules elicited
on alfalfa. Panel identification
numbers are as described above.
ACKNOWLEDGMENTS
This work was supported by research grants AGR 252 (Proyecto de Excelencia de la Junta de Andalucía),
and CSD 2009-0006 of the Consolider-Ingenio Program, including ERDF (European Regional
Development Funds).
REFERENCES
Aguilar, O.M., and Grasso, D.H. (1991). J. Bacteriol. 173: 7756-7764.
Born, T.L,, and Blanchard, J.S. (1999). Curr. Opin. Chem. Biol. 3: 607-613.
de las Nieves Peltzer, M., et al. (2008). Mol. Plant Microbe Interact. 21: 1232-1241
Hassani, R., et al. (2002). Indian J. Exp. Biol. 40: 1110-1120.
Prentki, P., and Krisch, H,M. (1984). Gene. 29: 303-313.
Randhawa, G.S. and Hassani, R. (2002). Indian J. Exp .Biol. 40: 755-764.
Rediers, H., et al. (2003). Appl. Environ. Microbiol. 69: 6864-6874.
Sanjuán-Pinilla, J., et al. (2002). Arch. Microbiol. 178: 36-44.
Soto, M,J., et al. (2004) FEMS Microbiol. Ecol. 48: 71-77.
van Rhijn, P., and Vanderleyden, J. (1995). Microbiol. Rev. 59: 124-142.
136
Session II
SII-CP-14
Genomic analysis of Azospirillum brasilense Az39, the most extensively
used strain for inoculant production in Argentina.
Cassán, F.1*, Rivera Bottia, D.1, Molina, R.1, Revale, S.2, Vazquez, M.2, Spaepen, S.3,
Vanderleyden, J.3, Perticari, A.4
1
Universidad Nacional de Río Cuarto. Córdoba. Argentina. 2 Instituto de Agrobiotecnología de Rosario
[INDEAR]. Rosario. Argentina. 3 Centre of Microbial and Plant Genetics. KU Leuven. Belgium. 4
Instituto de Microbiología y Zoología Agrícola INTA Castelar [IMYZA-INTA]. Argentina.
* [email protected]
ABSTRACT
We sequenced and analyzed the genome of A. brasilense Az39, the most extensively
used strain for inoculants production in Argentina. The genome sequence was obtained
using a 454 GS FLX Titanium pyrosequencer. Genome annotation was done using the
Standard Operating Procedures for Prokaryotic annotation from ISGA and from RAST
annotation server. The putative plant growth promotion and related mechanisms were
analyzed in A. brasilense Az39 and compared with other Azospirillum sp. strains
previously sequenced.
INTRODUCTION
In the 1980s, an intensive program to select and identify Azospirillum sp. strains able to
improve productivity of wheat and corn was started by INTA-IMYZA in Argentina. A.
brasilense Az39 was selected as one of the most effective PGPR and recommended for
wheat and maize inoculants formulation. After 40 years of agronomic use, numerous
laboratory, greenhouse and field experiments since then have demonstrated the
effectiveness of Az39 to increase growth and productivity in increasing number of crops
species. In this work, we present a comprehensive analysis of the: (1) genomic features
and (2) plant growth promotion and related mechanisms of Az39 in comparison with
sequenced strains belonging to this genus.
The genome sequence was obtained using a combined Whole Genome Shotgun and 8kb
Pair-Ends strategy with a 454 GS FLX Titanium pyrosequencer at INDEAR. Replicons
were identified via a PFGE analysis. Assembly was done using 454 Newbler v2.6 with
a 16X genome coverage. Genome annotation was done using the Standard Operating
Procedures for Prokaryotic annotation from ISGA (Hemmerich et al., 2010) and from
RAST annotation server (Aziz et al. 2008). The putative plant growth promotion
mechanisms of Azospirillum brasilense Az39 (16S ribosomal RNA gene accesion
number JQ844453.1) were analyzed and compared with those previously obtained for
Azospirillum brasilense Sp245, Azospirillum lipoferum 4B (Wisniewski-Dyé et al.,
2011) and Azospirillum sp. B510 (Kaneko et al., 2010). Comparative analysis was
performed by the use of KEGG (Kanehisa et al., 2012) and RAST (Aziz et al., 2012)
web services.
137
Session II
SII-CP-14
Genomic features.
Table 1. General features of the A. brasilense Az39 genome, database analyzed and compared with those
obtained for Azospirillum brasilense Sp245, Azospirillum lipoferum 4B and Azospirillum sp. strain B510.
Features
A.brasilense Az39
A. brasilense Sp245
A. lipoferum 4B
Azospirillum sp. B510
Size
GC Content [%]
Number of features
Number of RNAs
tRNAs
rRNAs
Number of Coding Sequences
in Subsystems
non-hypothetical
hypothetical
not in Subsystems
non-hypothetical
hypothetical
Number of Subsystems
COG Assignement
Not in COG
Replicons [plasmids or chromids]
7.421.756
68,6
6760
79
68
11
6681
2717
2570
147
3964
1605
2359
467
4703
1939
5*
7.530.241
68,5
7027
105
80
25
6922
2589
2460
129
4333
1737
2596
472
4716
2206
6
6.846.400
67,7
6263
108
79
29
6155
2473
2356
117
3682
1482
2200
486
4302
1853
6
7.599.738
67,6
6886
105
79
26
6781
2683
2543
140
4098
1811
2287
484
4761
2020
6
*PFGE analysis: P1: 1.6; P2: 0.95; P3: 0.7; P4: 0.68 and P5: 0.15 Mpb
Plant growth promotion and survival mechanisms.
The plant growth promotion mechanisms previously described for PGPR were
evaluated in genome sequence of Az39 and compared with other Azospirillum sp.
genomes. Here, we included the indole-3-acetic acid biosynthesis pathways, while other
mechanisms as nitrogen fixation or nitrate reduction; phytohormones and growth
regulators (i.e. cytokinins, gibberellins, ethylene, abscisic acid, polyamines and nitric
oxide) biosynthesis; siderophores and biofilms production; secretion system;
widespread colonization island and other related mechanisms will be presented in full
version of this work during the Congress.
Table 2. Summary of the IAA pathways according to putative gene sequences identified in the genome of
A. brasilense Sp245, Az39 and CBG497; A. lipoferum 4B, A. amazonense Y2 and Azospirillum sp. B510.
IAA biosynthesis
Enzyme name
A. brasilense Sp245
A. brasilsense Az39
A. brasilense CBG497
A. lipoferum 4B
Azospirillum sp. B510
A. amazonense Y2
IPyA
indole piruvate
decarboxylase
aromatic amino
aldehyde
transferase
deshydrogenase
ipdC/ppdC
+
+
+
-
*hisC1 from A. brasilense Sp7;
†
IAN
hisC1 *
+
+
+
+
+
-
+
+
-
nitrilase
NIT1 †
+
+
+
NIT2 †
+
+
+
IAM
tryptophan
monooxygenase
iaaM
+
-
indole
acetamide
hydrolase
iaaH
+
-
NIT1 and NIT2 from Arabidopsis thaliana
ACKNOWLEDGEMENTS
This work was supported by projects granted by FONCyT, CONICET, UNRC, INTA and FWO.
REFERENCES
Aziz, R.K., et al. (2008). BMC Genomics 9: 75
Hemmerich, E., et al. (2010). Bioinformatics 26: 1122-1124.
Kanehisa, M., et al. (2012). Nucleic Acids Res. 40: 109-114.
Kaneko, T., et al. (2010). DNA Res., 17: 37-50.
Wisniewski-Dye, F., et al. (2011). PLoS Genet. 7 (12).
138
Session II
SII-CP-15
Two-dimensional proteomic reference map
diazoefficiens strain CPAC 7 (=SEMIA 5080).
of
Bradyrhizobium
Gomes, D.F.1, Batista, J.S.S.3*, Hungria, M.2
1
Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brasil; Fellowship from CNPq. 2
Embrapa Soja, Londrina, Brasil. 3 Departamento de Biologia Estrutural, Molecular e Genética,
Universidade Estadual de Ponta Grossa, Ponta Grossa, Brasil.
* [email protected]
ABSTRACT
A two-dimensional gel electrophoresis profile was generated for Bradyrhizobium
diazoefficiens CPAC 7 (=SEMIA 5080), a highly competitive strain against naturalized
soil rhizobia and efficient in fixing nitrogen in symbiosis with soybean. We selected
150 spots and 124 proteins were effectively identified. The majority of the identified
proteins were related to metabolic functions.
INTRODUCTION
Strain CPAC 7 was recently reclassified into a novel species named Bradyrhizobium
diazoefficiens (Delamuta et al., 2013). Due to its outstanding efficiency in fixing
nitrogen, the strain is employed in soybean (Glycine max L.) commercial inoculants in
Brazil, since 1992 (Hungria et al., 2006). The genome sequencing of CPAC 7 is now in
progress; so, the establishment of a proteomic reference map can add important proteinexpression information into the genomic annotation process. In this study we present the
two-dimensional proteomic reference map of CPAC 7 that will allow a comparative
analysis with the published proteomic reference map of B. diazoefficiens, strain USDA
110 (Delmotte et al., 2010).
B. diazoefficiens CPAC 7 was grown in AG medium until exponential phase. Wholecell proteins were extracted and separated by two-dimensional gel electrophoresis, using
IPG-strips with pH range 4-7 (Gomes et al., 2012). The experiment was performed in
triplicate and 150 well-defined spots, present in all three gels were randomly selected
for MALDI-TOF/TOF identification. Spectra generated were searched against the
public database NCBInr, Proteobacteria, using the Mascot software v. 2.3 (Matrix
Science).
Well defined and reproducible 2D gel profiles were generated (Figure 1). Among 150
protein spots randomly selected to be analyzed by mass spectrometry, 124 proteins were
successfully identified. In addition to Mascot identification, we also made a prediction
of the subcellular location using the software PLSpread and PsortB.
Functional classification in clusters of orthologous groups (COG) was also performed,
and proteins were distributed in 17 COG categories, belonging to four functional
groups. Proteins related to metabolic functions were the majority, representing 39% of
identified proteins (Figure 2). The first hit of most proteins was with USDA 110,
defined as the type strain of B. diazoefficiens. In relation to the cellular location, 105 of
the proteins are located in the cytoplasm.
139
Session II
SII-CP-15
Figure 1. 2D gel profile of B. diazoefficiens CPAC 7
Figure 2. Distribution of the identified proteins into COG categories.
Among the proteins identified in our study, it is worth mentioning that ATP-dependent
Clp protease, GTP-binding tyrosin phosphorylated protein and electron transfer
flavoprotein, besides others, were up-regulated when B. japonicum strain CPAC 15 was
grown in the presence of genistein (Batista and Hungria, 2012). This reference map will
be useful to add an expression point-of-view in the oncoming genome sequencing of
strain CPAC 7.
ACKNOWLEGMENTS
Project CNPq-Repensa 562008/2010-1
REFERENCES
Batista, J.S.S., and Hungria, M. J. (2012). J. Proteomics, 75: 1211-1219.
Delamuta, J.R.M., et al. (2013). Int J Syst Evol Microbiol, ijs.0.049130-0v1-ijs.0.049130-0.
Delmotte, N., et al. (2010). Proteomics, 10: 1391-1400.
Gomes, D.F., et al. (2012). Proteomics, 12: 859-863.
Hungria, M., et al. (2006). In: Nitrogen Nutrition and Sustainable Plant Productivity. 43-93.
140
Session II
SII-CP-16
Emprego da metodologia de MLSA em avaliações de filogenia e
taxonomia de rizóbios: estudo com Rhizobium spp. microssimbiontes
de feijoeiro (Phaseolus vulgaris L.).
Ribeiro, R.A.1, 2*, Delamuta, J.R.M.1, 2, Hungria, M.1, 2
1
Laboratório de Biotecnologia do Solo; Embrapa Soja. 2 Programa de Pós-Graduação em Microbiologia
(Universidade Estadual de Londrina).
* [email protected]
RESUMO
Estudos taxonômicos desempenham um papel relevante na valoração da diversidade
microbiana do solo. A metodologia de MLSA (Multilocus Sequence Analysis) tem
possibilitado incrementar o conhecimento da biodiversidade bacteriana do solo, bem
como a descrição de novas espécies. Neste estudo, quatro genes housekeeping foram
utilizados para estabelecer as relações filogenéticas entre 19 estirpes de Rhizobium spp.
microssimbiontes do feijoeiro. Além de obter maior definição das relações filogenéticas,
o MLSA indicou possíveis novas espécies de rizóbios ainda não descritas.
INTRODUÇÃO
A diversidade procariótica presente no solo é elevada, particularmente nos trópicos, mas
um grande desafio consiste em classificar os microrganismos, identificando o seu
potencial biotecnológico em favor de maiores rendimentos das culturas e
sustentabilidade ambiental. Nesse contexto, estudos taxonômicos são fundamentais e a
metodologia de MLSA aponta como uma ferramenta de grande utilidade. O objetivo
deste trabalho foi o de elucidar as relações filogenéticas e a taxonomia de Rhizobium
spp. microssimbiontes do feijoeiro (Phaseolus vulgaris L.), através da análise do gene
16S rRNA e de quatro genes housekeeping.
MATERIAL E MÉTODOS
Foram analisadas 19 estirpes de rizóbios microssimbiontes do feijoeiro, isoladas de
regiões andinas, mesoamericanas e do Brasil. A extração do DNA, os primers, as
condições de amplificação dos genes 16S rRNA, recA, glnII, gyrB e rpoA, bem como a
purificação dos produtos de PCR, o sequenciamento e a construção das árvores
filogenéticas foram realizadas conforme descrito por Ribeiro et al. (2009).
Com base na árvore do 16S rRNA, das 19 estirpes analisadas, 12 foram posicionadas no
grupo de R. etli, três se posicionaram no de R. tropici e as demais foram alocadas no
grupo de R. radiobacter. O baixo poder de resolução do gene 16S rRNA não permitiu
boa definição da posição taxonômica das estirpes deste estudo, principalmente daquelas
do grupo R. etli (Figura 1A). As sequências concatenadas e alinhadas resultaram em um
fragmento comum de 1.846 nucleotídeos e foram utilizadas para a construção da árvore
filogenética. Através da combinação das informações filogenéticas dos quatro genes
housekeeping concatenados, obteve-se uma melhor resolução na definição das espécies,
em comparação com a análise baseada em um único gene (Figura 1B). Novas linhagens,
representando possíveis espécies novas foram evidenciadas na análise de MLSA. A
estirpe CNPSo 669, do México, foi agrupada com a estirpe IE4771, previamente
designada como R. etli; contudo, houve indicação de representar uma espécie
independente de R. etli e R. phaseoli, recebendo a denominação de PEL1. A estirpe
equatoriana CNPSo 679 foi posicionada independentemente como nova linhagem e
denominada PEL2, enquanto as estirpes CNPSo 661, 666 e 668 formaram o grupo
PEL3. As estirpes CNPSo 676, 683, 670, 671 e 672 do Equador e 659 do México
141
Session II
SII-CP-16
agruparam com um bootstrap de 100% e formaram outra nova linhagem, PEL4. As
estirpes WSM2304 e CCGE510, embora tenham sido identificadas como R.
leguminosarum na análise do 16S rRNA, formaram dois grupos por MLSA, PEL 5 e
PEL6, respectivamente (Figura 1B.). A posição taxonômica de outras estirpes foi
melhor definida, por exemplo, as estirpes brasileiras CNPSo 657 e 660 agruparam
estreitamente com R. leucaenae (CFN 299T) e apresentaram a inserção de 72
nucleotídeos no gene 16S rRNA, o qual é característico da espécie (Ribeiro et al.,
2012).
Rhizobium sp. IE 4771 (KC261566, ABRC00000000, ABRC00000000, KC261567)
100
Rhizobium sp. CNPSo 669 (KC293530, KC293516, KC293519, KC293533)
A
B
Rhizobium sp. CNPSo 679 (JN129355, JN129310, JN129340, JN129370)
99
PEL1
PEL2
R. phaseoli CNPAF512 (AEYZ00000000, AEYZ00000000, AEYZ00000000, AEYZ00000000)
R. phaseoli Ch24-10 (AHJU00000000, AHJU00000000, AHJU00000000, AHJU00000000)
100
100
R. phaseoli CIAT 652 (CP001074, CP001074, CP001074, CP001074)
100 Rhizobium sp. CNPSo 661 (JN129346, JN129301, JN129331, JN129361)
80
PEL3
100
R. etli CFN42T (CP000133, CP000133, CP000133, CP000133)
100
R. etli CNPSo 664 (JN129348, JN129303, JN129333, JN129363)
100
99
100
99
PEL4
Rhizobium sp. WSM 2304 (CP001191, CP001191, CP001191, CP001191)
96
Rhizobium sp. CCGE 510 (AEYF00000000, AEYF00000000, AEYF00000000, AEYF00000000)
85
PEL5
PEL6
R. leguminosarum USDA 2671 (EU488811, EU488784, KC293526, EU488837)
100
R. leguminosarum 3841 (AM236080, AM236080, AM236080, AM236080)
R. leguminosarum WSM 1325 (CP001622, CP001622, CP001622, CP001622)
96
R. pisi DSM30132T (DQ431676, JN580715, KC293522, KC293535)
R. gallicum R602spT (AY907357, AF529015, AM418828, EU488840)
73 R. leucaenae CFN 299T (AJ294372, AF169583, KC293524, EU488845)
100 Rhizobium sp. CNPSo 657 (JN129343, JN129298, JN129328, JN129358)
R. multihospitium CCBAU 83401T (EF490029, EF490040, KC293528, JF318207)
99
R. tropici CIAT 899T (AJ294373, AF169584, HQ438238, EU488833)
85
R. miluonense CCBAU 41251T (HM047131, HM047120, KC293527, JF318206)
78
R. lusitanum P1-7T (DQ431674, EF639841, KC293525, JF318205)
R. rhizogenes K84 (CP000628, CP000628, CP000628, CP000628)
R. grahamii CCGE 502T (JF424622, JF424618, AEYE00000000, AEYE00000000)
100
R. mesoamericanum CCGE 501T (JF424620, JF424617, PRJNA63213 , PRJNA63213)
R. endophyticum CCGE 2052T (HM142767, JF424619, PRJNA63221, PRJNA63221)
R. giardinii H52T (AM182123, EU488778, HQ438240, EU488829)
R. radiobacter ATCC 19358T (FM164311, JN580718, FR695243, KC293536)
0.02
Figura 1. Ávores filogenéticas entre as estirpes de Rhizobium spp. e estirpes tipo, com base no gene 16S
rRNA (A) e nos genes housekeeping concatenados (recA, glnII, gyrB e rpoA) (B).
CONCLUSÃO
Quando comparada com a análise do gene 16S rRNA, a metodologia de MLSA
permitiu melhor definição taxonômica de estirpes de Rhizobium microssimbiontes de
feijoeiro provenientes de regiões andinas, mesoamericanas e do Brasil. Houve indicação
de novas espécies, mostrando o poder de resolução do MLSA e confirmando o potencial
elevado dessa técnica para o conhecimento da biodiversidade e para a definição
taxonômica de estirpes com potencial biotecnológico.
AGRADECIMENTOS
Projeto CNPq-Repensa 562008/2010-1.
REFERÊNCIAS
Ribeiro, R.A., et al. (2009). Res. Microbiol. 160: 297-306.
Ribeiro, R.A., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 1179-1184.
142
Session II
SII-CP-17
Relative expression of hypothetical
Bradyrhizobium diazoefficiens CPAC 7.
protein-related
genes
for
Gomes, D.F.1, 2, Rolla-Santos, A.A.P. 1*, Batista, J.S.S.3, Hungria, M.1
1
Embrapa Soja, Londrina, Brazil, 2 Departamento de Genética, Universidade Federal do Paraná, Curitiba,
Brasil, 3 Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta
Grossa, Ponta Grossa, Brazil.
* [email protected]
ABSTRACT
From the proteomic map of Bradyrhizobium diazoefficiens strain CPAC 7 we selected
nine hypothetical protein-related genes and evaluated their relative expression by Realtime quantitative PCR (RT-qPCR). The experiment was performed with two treatments
(induced or not with 5 µM genistein). Six out of nine protein-related genes were upregulated in the presence genistein.
INTRODUCTION
Bradyrhizobium diazoefficiens strain CPAC 7 (Delamuta et al., 2013) is a
microsymbiont that fixes nitrogen in association with soybean (Glycine max L.) and is
used in commercial inoculants in Brazil; therefore it has high ecological and economical
importance. It is estimated that more than 30% of the genome of B. diazoefficiens type
strain USDA 110 encodes hypothetical proteins. The hypothetical protein nomenclature
is used when the existence of a gene is supported only by the prediction of gene-finding
softwares, and when they do not show significant homology with any characterized
gene (Batista et al., 2010). However, these proteins can bring interesting information to
gene bioprospection. In these sense, our goal was to study the relative expression of
some hypothetical protein-related genes from strain CPAC 7 in response to genistein.
Strain CPAC 7 was grown in AG medium until exponential phase in two treatments
(Induced or not with genistein 5 µM). Total RNA was isolated using Trizol and
employed for cDNA synthesis using Superscript III TM reverse transcriptase
(InvitrogenTM). Platinum® SYBR Green®qPCRSuperMix-UDG was used according to
the manufacturer’s instructions. Amplification reactions were conducted in triplicate
using a 7500 Real Time System thermocycler (Applied Biosystems). We used 16S
rRNA (GeneID: 1055154) as reference gene for normalization. Relative gene
expression was determined by the 2-ΔΔCT method (Livak and Schmittgen, 2001).
Statistical analysis of the data was performed using the REST 2009 v.2 software, which
enables the calculation of P values for each sample group and 95% confidence intervals.
Based on an unpublished B. diazoefficiens CPAC 7 proteomic reference map of our
group, we selected 18 hypothetical proteins identified and performed a preliminary
functional inference using bioinformatics tools and based on the presence of function
domains/motifs (Table 1).
Nine genes classified as hypothetical proteins that did not fit into any functional
category of COGnitor (http://www.ncbi.nlm.nih.gov/COG/old/xognitor.html), assigned
as “NO related KOG”, were selected and analyzed by RT-qPCR. The analysis was
performed to monitor the relative expression of these genes in response to genistein. Of
the nine genes, six were up-regulated (Figure 1). Curiously, all genes coding for
143
Session II
SII-CP-17
periplasmic proteins (bll0565, blr7534, bll5307 and blr0227) were differentially
expressed in response to the flavonoid.
Table 1. Identified hypothetical proteins of B. diazoefficiens CPAC 7.
Hypothetical
Cellular
Functional inference
protein
location
BJ6T_08050
Cytoplasmic Thioredoxin-like protein
Bll7551
Periplasmic
Blr5678
Cytoplasmic
L-aminopeptidase, probably related to arginine biosynthesis
Bll5131
Extracellular
ATP/GTP binding site, small transmembrane domain
Blr2961
Cytoplasmic
Fumarylacetoacetase
Blr3798
Cytoplasmic
Demethylmenaquinone methyltransferase
Blr5067
Cytoplasmic
Blr0227
Periplasmic
Blr2191
Periplasmic
Histidine phosphotransferase
Bll4565
Cytoplasmic
Ribonuclease, high similarity with Blr3798
Bll5307
Periplasmic
Blr2761
Cytoplasmic
Universal stress protein UspA
Blr7436
Cytoplasmic
Predicted transcriptional regulator containing the HTH
Bll4752
Cytoplasmic
domain
Bll0565
Periplasmic
Bll5663
Cytoplasmic
ATPase
Blr3064
Cytoplasmic
Succinyl-diaminopimelate desuccinylase
Blr7534
Periplasmic
2
*
Relative expression
*
1,6
*
1,2
*
*
*
0,8
0,4
0
Figure 1. Gene expression analysis by
RT-qPCR of nine hypothetical proteins.
The differential expression of these genes in response to genistein suggests that their
respective proteins are probably involved in symbiosis establishment process.
ACKNOWLEGMENTS
REFERENCES
Batista, J.S., et al., (2010). Proteomics. 10: 3176-3189.
Delamuta, J.R.M., et al. (2013). Int J Syst Evol Microbiol, ijs.0.049130-0v1-ijs.0.049130-0.
Gomes, D.F., et al. (2012). Proteomics. 12:859-863.
Livak K.J., and Schmittgen, T.D. (2001). Methods 25: 402-408.
144
Session II
SII-CP-18
PoolSeq analysis of the selection of the Rhizobium genotypes by the
legume host plant.
Jorrín, B.1*, Imperial, J.1, 2
1
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Madrid.
Investigaciones Científicas (CSIC).
* [email protected]
2
Consejo Superior de
ABSTRACT
Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We
have applied a Pool-Seq approach to study plant host selection of genotypes. Our results
confirm, at the genomic level, previous observations regarding plant selection of
specific genotypes
INTRODUCTION
Rhizobium leguminosarum bv. viciae establishes highly specific nitrogen-fixing
symbioses with different legume genera (Pisum, Lens, Lathyrus and Vicia). The
molecular bases of specificity, establishment and functioning of the symbioses have
been described from the bacterial (Murray et al., 2011) and plant (Yokota and Hayashi,
2011) points of view.
Thanks to the advent next generation sequencing techniques and bioinformatics tools,
the genome of a large number of rhizobial mycrosimbiont strains have been sequenced.
Nevertheless, there are aspects of the Rhizobium-legume interaction that are yet to be
explained, and they could be relevant for efficient inoculant design.
Classic studies using trap plants provided evidence that, given a choice, specific hosts
select specific genotypes of rhizobia which are, apparently, particularly adapted to that
host (Mutch and Young, 2004; Louvrier et al., 1996).
In the Pool-Seq approach (Kofler et al., 2011), population genomics insights are gained
through high-throughput DNA sequencing of pools of bacterial isolates.
We have applied a Pool-Seq approach to study plant host selection of genotypes from
the available genomic diversity in a well-characterized soil.
A well-characterized agricultural soil (INRA Bretennieres) was used as source of
rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were
employed as traps. We pooled 100 nodules from each host, and the pooled DNAs were
sequenced (BGI-Hong Kong; Illumina Hi-seq 2000, 180 bp PE libraries, 100 bp reads,
12 Mreads). Reads were quality filtered with FastQC and Trimmomatic. Filtered reads
were mapped with Bowtie2 using Rhizobium leguminosarum bv. viciae 3841 as
reference genome. Single Nucleotide Polymorphisms (SNPs) were called with VarScan
(minimum coverage 20 reads, minimum frequency 0.1). Results were visualized with
SeqMonk and IGV.
An important fraction of the filtered reads were not recruited by the reference genome
(Table 1), suggesting that plant soil isolates contain a large number of genes that are not
present in the reference genome.
Single Nucleotide Polymorphism (SNP) analysis was carried out with Pool-Seqs, and a
plant-specific SNP distribution was observed within the nodDABC cluster.
145
Session II
SII-CP-18
Population dissimilarities were obtained from the complete SNP genome analysis.
Pairwise Euclidean distances were calculated from SNP frequencies with SPSS
software (Table 2).
Table 1. Reads mapped and unmapped against R. leguminosarum bv. viciae 3841.
Total reads
% Mapped reads
% Unmapped reads
PEA
LENTIL
FAVA
VETCH
11,697,508
84.17
16.24
11,893,439
77.79
23.27
11,905,328
77.24
23.29
11,862,580
81.88
18.22
Table 2. Population dissimilarities.
Pea
Lentil
Fava
Vetch
Rlv 3841
Pea
Lentil
Fava
Vetch
Rlv 3841
0
76
90
54
157
0
56
58
187
0
65
195
0
170
0
Our results confirm, at the genomic level, previous observations regarding plant
selection of specific genotypes. We expect that further, ongoing comparative studies on
differential Pool-Seq sequences will identify specific gene components of the plantselected genotypes.
ACKNOWLEDGMENTS
Supported by the Microgen Project (Consolider-Ingenio 2010, CSB2009-00006, MCINN, Spain) to JI.
REFERENCES
Kofler, R., et al. (2011). Bioinformatics, 27: 343-3436.
Louvrier P., et al. (1996). Appl. Environ. Microbiol. 62: 4202.
Murray, J.D. (2011). Mol. Plant Microbe Interact. 24: 631.
Mutch, L.A., and Young, J.P. (2004). Mol. Ecol. 13: 2435.
Yokota, K., and Hayashi, M. (2011). Cell Mol. Life Sci. 68: 1341.
146
Session II
SII-CP-19
Incorporación estable y expresión del gen de la diguanilato ciclasa
PleD* en el genoma de bacterias que interaccionan con plantas.
Romero-Jiménez, L. *, Rodríguez, D., Prada-Ramírez, H., Gallegos, M.T., Sanjuán, J.,
Pérez-Mendoza, D.
Dpto. Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín, Consejo
Superior de Investigaciones Científicas (CSIC), Profesor Albareda 1, 18008 Granada, España.
* [email protected]
RESUMEN
En este trabajo hemos desarrollado una serie de herramientas genéticas para la manipulación de
los niveles intracelulares de c-di-GMP en bacterias. Los nuevos vectores, derivados de miniTn7, permiten la integración estable del gen de la diguanilato ciclasa pleD* de Caulobacter
crescentus en una localización específica del genoma de bacterias Gram-negativas. Los nuevos
mini-Tn7 han sido utilizados con éxito en bacterias de los géneros Rhizobium, Sinorhizobium y
Pseudomonas.
INTRODUCCIÓN
El c-di-GMP es un segundo mensajero implicado en la regulación de funciones bacterianas
como la motilidad, virulencia, adhesión y formación de biopelículas. Los niveles intracelulares
de este segundo mensajero son controlados por dos tipos de enzimas antagónicas: diguanilato
ciclasas que sintetizan c-di-GMP y fosfodiesterasas que lo degradan. La sobreexpresión del gen
de la diguanilato ciclasa pleD* (Aldridge et al., 2003) en el vector de expresión pJBpleD*
(Pérez-Mendoza, sin publicar), provoca aumentos significativos de los niveles intracelulares de
c-di-GMP
y, en consecuencia, cambios fenotípicos asociados a la producción de
exopolisacáridos, la formación de biopeliculas y la motilidad de bacterias que interaccionan con
plantas. Sin embargo, esta construcción presenta una baja estabilidad en ausencia de presión
selectiva de antibiótico, particularmente in planta. Para superar esta inestabilidad hemos
construido derivados del transposón mini-Tn7 portadores de pleD*, capaces de insertarse
establemente, en una sola copia y en una localización específica del genoma, sin afectar la
viabilidad de las bacterias.
MATERIAL Y MÉTODOS
La introducción de los vectores mini-Tn7pleD* en las cepas receptoras se realizó siguiendo el
protocolo utilizado por (Bao et al., 1991). Para la extracción y cuantificación de c-di-GMP se
siguió una modificación del protocolo descrito por (Amikam et al., 1995). La producción de
exopolisacáridos por las bacterias se puso de manifiesto utilizando los colorantes rojo congo y
calcoflúor (Teather y Wood, 1982).
El cultivo axénico de plantas de alfalfa se realizó según la técnica descrita por (Olivares et al.,
1980) y para el cultivo de veza y judía se siguió la técnica de (Leonard, 1943).
RESULTADOS Y DISCUSION
Construcción e introducción de los vectores mini-Tn7 en el genoma de bacterias.
En el vector pUC18T-miniTn7T se clonó el gen pleD* en solitario o acompañado de
genes de resistencia a antibióticos (kanamicina o tetraciclina). Además, se construyeron
versiones de los mini-Tn7 carentes de pleD*, mediante una deleción interna de dicho
gen. Se verificó la capacidad de inserción en el genoma y la estabilidad de los minitransposones en varias especies de rizobios y en Pseudomonas syringae pv. tomato. Las
frecuencias de inserción de los nuevos mini-Tn7 variaron dependiendo de la bacteria,
mientras que su estabilidad en ausencia de presión selectiva llegó a ser del 100%.
Determinación de los niveles de c-di-GMP e impacto del c-di-GMP en vida libre.
Mediante HPLC se cuantificaron los niveles de c-di-GMP en todas las cepas portadoras
pleD* en multicopia (pJBpleD*), en monocopia (mini-Tn7pleD*Km), así como de sus
respectivos controles (pJB3Tc19 y Tn7::Km). Los resultados mostraron aumentos muy
147
Session II
SII-CP-19
significativos de los niveles de c-di-GMP en las cepas portadoras de pleD* con
independencia de su localización (vector en multicopia o insertado en el genoma). Estos
elevados niveles de c-di-GMP provocaron en todos los casos un aumento en la
producción de exopolisacáridos extracelulares, que pudieron ser revelados al crecer las
bacterias sobre medio adicionado con rojo congo (CR) o calcoflúor (CF). Los
transposantes con pleD* en monocopia tuvieron un comportamiento similar que las
cepas con pleD* en multicopia, produciendo colonias rugosas rojas o fluorescentes
(bajo luz ultravioleta) en presencia de CR y CF, respectivamente, en contraposición a lo
observado en las cepas silvestres. Además, todas las cepas portadoras de pleD*
(multicopia y monocopia), mostraron un aumento significativo de la capacidad de
formar biopelículas con respecto a las cepas control. También se analizaron la
motilidad "swimming" y la motilidad en superficie de las cepas portadoras del gen
pleD* (pJBpleD* y transposantes Tn7::pleD*). Los resultados mostraron un descenso
de ambos tipos de motilidad en las cepas portadoras de pleD* tanto en mono como en
multicopia, en comparación con las cepas control.
Impacto del c-di-GMP en la interacción rizobio-leguminosa.
La expresión de pleD* tanto en mono como en multicopia en R. etli y R.
leguminosarum provocó un aumento de hasta 160 veces en la adhesión a las raíces de
sus plantas hospedadoras, Phaseolus vulgaris y Vicia sativa, respectivamente. Además
se realizaron ensayos de infectividad en los sistemas S. meliloti-Medicago sativa, R.
leguminosarum-V. sativa y R. etli-P. vulgaris. Solo en el caso de la interacción R. etliP. vulgaris, la simbiosis se vio claramente afectada en aquellas plantas inoculadas con
cepas portadoras de pleD* tanto en mono como multicopia, observándose un tamaño
reducido de la parte aérea y un menor número de nódulos que las plantas inoculadas
con las cepas control.
CONCLUSIONES
Se han construido una serie de vectores mini-Tn7 que permiten la integración
localizada y estable del gen de la diguanilato ciclasa pleD* en el genoma de bacterias
Gram-negativas. La expresión de pleD* provoca una serie de cambios fenotípicos
compatibles con los significativos aumentos de c-di-GMP intracelular observados. En
algunos casos, estos aumentos del c-di-GMP intracelular provocaron alteraciones
importantes de las interacciones de las bacterias con sus respectivas plantas
hospedadoras, lo que sugiere la importancia de este segundo mensajero durante el
establecimiento de asociaciones planta-bacteria.
AGRADECIMIENTOS
Este trabajo ha sido financiado por los proyectos BIO2011-23032 (MICIIN) y CVI-5800 (J. Andalucía).
L.R-J es perceptora de una beca predoctoral JAE-CSIC. H.P-R es perceptor de una beca predoctoral FPI.
D.P-M fue perceptor de contratos postdoctorales JAE-CSIC y de la J. Andalucía.
BIBLIOGRAFÍA
Aldridge, P., et al. (2003). Mol. Microbiol. 47: 1695-1708.
Amikam, D., et al. (1995). Biochem. J 311: 921-927.
Bao, Y., et al. (1991). Gene 109: 167-168.
Choi, K.H., and Schweizer, H.P. (2006). Nat. Protoc. 1: 153-161.
Leonard, L.T. (1943). J. Bacteriol. 45: 523-527.
Olivares, J., et al. (1980). Appl. Environ. Microbiol. 39: 967-970.
Teather, R.M., and Wood, P.J. (1982). Appl. Environ. Microbiol. 43: 777-780.
148
Session II
SII-CP-20
Identification of the gene responsible for a non-nodulating phenotype
in chickpea.
Ali, L.1, Madrid, E.2, Sánchez, R.1, Temprano, F.3, Gil, J.1, Rubio, J.4, Millan, T.1*
1
Dpto Genética, Univ Córdoba, Campus de Excelencia Internacional CeiA3, Córdoba, Spain. 2 Institute
for Sustainable Agriculture, CSIC, Córdoba, Spain. 3 IFAPA Centro “Las Torres-Tomejil”, Alcalá del
Río, Sevilla, Spain. 4 Área de Mejora y Biotecnología, IFAPA Centro “Alameda del Obispo”, Córdoba,
Spain.
* [email protected]
ABSTRACT
A non-nodulating chickpea mutant (PM233) was crossed by a wild type (CA2139).
Segregant populations were advanced until F7 generation. Taking profit of residual
heterocigositiy a pair of NILs (Near Isogenic Lines) was obtained. The polymorphic
region between the NILs was localized in chickpea linkage group 5 (LG5),
corresponding to Medicago truncatula chromosome 3 (Chr3). . A gene located Chr3,
NSP2, was selected as a candidate gene for nodulation in chickpea. The gene (CaNSP2)
consists of a 1,503 bp exon with no introns. Partial amplification in the mutant will be
used to start genomic walking or inverse PCR in order to characterize this mutation.
INTRODUCTION
Chickpea (Cicer arietinum L.) is the second most cultivated grain legume in the world.
In this crop, there have been no reports of molecular studies of the nodulation pathway.
Development and study of nodulation (Nod) mutants is a very useful tool to dissect this
transduction pathway. Seven nodulation mutants have been described in chickpea:
PM233, PM665, PM679, PM405, PM796 and ICC435M (Davis 1988; Singh et al.
1992). The mutant PM233 carries the recessive gene rn1 (Paruvangada and Davis
1999). The aim of this study was the molecular characterization of the gene related with
the non-nodulating mutation in PM233.
A cross between PM233, a non-nodulating mutant Desi type, and CA2139, a nodulating
Spanish Kabuli type landrace, was used for the development of a pair of NILs following
Rajesh et al. (2002) scheme. The pair of NILs developed was designated as NIL7-2A
(nod)/NIL7-2B (non-nod). An F3 population derived from a cross between this pair of
NILs was inoculated with a suspension of Mesorhizobium sp. (isolates ISC7 and ISC11)
to establish the nod/non-nod inheritance ratio and perform linkage analysis.
Molecular characterization: DNA was isolated using the DNAzol method (Invitrogen).
STMS markers distributed through different linkage groups (LGs) of the chickpea
genetic map were chosen to detect the polymorphic region between the NILs. Using
genetic information available for Medicago truncatula the nodulation signaling pathway
2 gene (NSP2)was selected to design primers to be amplified in chickpea. Six different
primer combinations were tested in genomic DNA of each parental line (CA2139 and
PM233). The clearest amplicon was excised from acrylamide gel, cloned and
sequenced.
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Pairs of NILs for a given trait differ for a target region and are almost identical in the
rest of the genome facilitating the identification of the gene responsible of the trait of
interest. The molecular characterization of NIL7-2A (nod) and NIL7-2B (non-nod)
showed polymorphisms for markers located in LG5 that corresponds to M. truncatula
Chr3. The transcription factor NSP2 (MTR_3g072710) (Oldroyd and Long 2003) was
selected as a candidate gene responsible for the non-nodulating phenotype. Six different
primer combinations designed in M. truncatula gene were tested in PM233, CA2139
and NILs in order to amplify the gene sequence in chickpea. Only one combination
gave a clear amplicon, present in CA2139 and NIL7-2A (nod) and absent in PM233 and
NIL7-2B (non-nod). The full-length sequence obtained in CA2139 after assembling the
reads was 1,067 bp, covering the gene except 353 bp at the 5’ end. The recently released
chickpea genome sequence (Varshney et al. 2013) permitted us to obtain the complete
NSP2 gene sequence. The sequence has been deposited in the GenBank
(www.ncbi.nlm.nih.gov) database and assigned the accession number KC534503 for
chickpea genotype CA2139 (CaNSP2). The deduced amino acid sequence of CaNSP2
contains 501 residues, with a molecular weight of 55.270 kDa. BLASTX analysis
showed the highest similarity with Pisum sativum (89%) and M. truncatula (87%)
(Figure 1). Characterization of the gene in PM233 mutants is necessary to be able to
know the nature of this mutation and to confirm whether CaNSP2 function is similar to
that reported other species.
M.truncatula
Pisum sativum
Cicer arietinum
Figure 1. Aligment of NSP2 protein among Medicago truncatula, Pisum sativum and Cicer arietinum,
showing high similarity in the conserved regions of this family protein.
ACKNOWLEDGEMENT
This work has been supported by the project INIA contract RTA2010-00059, co-financed by EU funds
(FEDER).
REFERENCES
Davis, T.M. (1988). J. Hered. 79: 476-478.
Paruvangada, V., and Davis, T.M. (1999). J. Hered. 90: 297-299.
Rajesh, P., et al. (2002). Theor. Appl. Genet. 105: 604-607.
Oldroyd, G.E.D., and Long, S.R. (2003) J. Plant Physiol. 131: 1027-1032.
Singh, O., et al. (1992). Crop Sci. 32: 41-43.
Varshney, R.K., et al. (2013). Nat. Biotech. 31: 240-246.
150
Session II
SII-CP-21
Functional characterization of Ensifer meliloti denitrification genes.
Torres, M.J.1*, Rubia, M.I.1, Coba de la Peña, T.2, Pueyo, J.J.2, Bedmar, E.J.1, Delgado,
M.J.1
1
Estación Experimental del Zaidin, Consejo Superior de Investigaciones Científicas (CSIC), P. O. Box
419, 18080-Granada, Spain. 2 Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones
Científicas (CSIC), Madrid, Spain.
* [email protected]
ABSTRACT
Despite possessing the complete set of denitrification genes, Ensifer meliloti 1021 is
considered a partial denitrifier due to its inability to grow under anoxic conditions with
nitrate or nitrite as terminal electron acceptors. In this work, we have demonstrated that,
under microoxic conditions, E. meliloti is able to use nitrate or nitrite as respiratory
substrates to support bacterial growth and that the nap, nir, nor and nos genes are
involved in microoxically nitrate respiration and denitrification.
INTRODUCTION
Ensifer (formerly Sinorhizobium) meliloti is the endosymbiont of alfalfa plants that also
possess the genes for the complete denitrification pathway (Barnett et al., 2001).
Several studies have been carried out on the regulation and symbiotic characterization
of E. meliloti nap, nir, nor and nos genes (Becker et al., 2004, Bobik et al., 2006,
Meilhoc et al., 2010, Horchani et al., 2011). However, up to date the role of E. meliloti
denitrification genes in free-living conditions remains unknown.
Bacterial strains. E. meliloti wild type (WT) strain 1021, and napA, napC, nirK, norC
and nosZ mutants (RhizoGATE; Becker et al. 2009) were used in this study. Cells were
grown in TY medium and further incubated in minimal medium under microoxic (2%
O2) or anoxic (filled tubes) conditions.
MV+-dependent nitrate reductase (MV+-NR) and nitrite reductase (MV+-Nir) activity,
and Haem-staining analysis were performed as described by Bueno et al. (2010).
Nitric oxide (NO) and nitrous oxide (N2O) determination. NO was measured
amperometrically with an NO electrode APOLO 4000, and N 2O with a gas
chromatograph type HP 4890D equipped with an electron capture detector (ECD).
After RNA isolation Real-time PCR reactions were run in a 7300 Real Time PCR
System.
Nitrate-dependent growth and Nap, Nir, Nor and Nos activity under microoxic
conditions.
WT cells incubated under microoxic conditions without nitrate showed an OD 600 up to
0.6 (Figure 1A). Addition of nitrate to the growth medium provoked an increase in the
OD 600 up to 1 (Figure 1A). Under these conditions, nitrite was produced and
accumulated in the medium being consumed further by the cells (Figure 1A). Cells of
the napA and nirK mutants showed a growth defect after incubation under microoxic
conditions with nitrate compared to WT cells (Figure 1A). Nitrite was not observed in
the napA medium, however the nirK mutant accumulated nitrite in the medium, but it
was not further reduced (Figure 1B). Similarly, the norC mutant had a significant
growth defect under microoxic conditions with nitrate, however the nosZ mutant
showed WT growth rates (data not shown). Levels of MV +-NR, MV+-NiR, and Nor
activities of the cells incubated under microoxic conditions with nitrate were strongly
151
Session II
SII-CP-21
reduced in the napA, nirK and norC mutants, respectively (data not shown). Under these
conditions, a nosZ mutant accumulated higher N2O levels than the WT (data not
shown).
B
[NO2-] (mM)
A
A
9
8
7
6
5
4
3
2
1
0
0
12
24
36
48
60
72
84
96
108
120
Time (hours)
Figure 1. (A) Nitrate-dependent growth and (B) extracellular nitrite concentration of wild type E. meliloti
1021 (▲, ), and napA (■, □), and nirK (●, ) mutant strains. Cells were incubated under microoxic
conditions in minimum medium supplemented (closed symbols) or not (open symbols) with KNO3.
Expression of E. meliloti denitrification genes under anoxic conditions.
Figure 2. Expression of E. meliloti dentrification
genes under microoxic and anoxic conditions.
By performing qRT-PCR analyses we have
found that napA, nirK, norC and nosZ genes
were induced not only under microoxic but
also under anoxic conditions with nitrate
relative to microoxic conditions without
nitrate (Figure 2). In fact, these results
demonstrated that anoxia and the presence
of nitrate are required for maximal
expression of E. meliloti napA, nirK, norC and nosZ denitrification genes (Figure 2).
Furthermore, anoxically incubated cells also expressed MV+-NR, MV+-Nir, Nor and
Nos activities (data not shown). However, haem staining analyses of WT cells
incubated under anoxic conditions with nitrate showed a strong defect of FixP, FixO as
well as NorC expression compared to microoxic conditions (data not shown). Taken
together, these results suggest that the inability of E. meliloti to growth anoxically with
nitrate is not due to a defect in the expression of the denitrification genes or in the
activity of the denitrification enzymes. It might be possible that the low protein level
could be one of the reasons of the limited growth of E. meliloti under anoxic conditions
with nitrate.
ACKNOWLEDGMENTS
This work was supported by Fondo Europeo de Desarrollo Regional (FEDER)-cofinanced grant
AGL2010-18607 and grant AGL2009-10371 from Ministerio de Economía y Competitividad.
REFERENCES
Barnett, M.J., et al. (2001). Proc. Natl. Acad. Sci. U.S.A. 98:9883-9888.
Becker, A., et al. (2004). Mol Plant Microbe Interact 17:292-303.
Becker, A., et al., (2009). J. Biotechnol. 140: 45-50.
Bobik, C., et al. (2006). J. Bacteriol. 188:4890-4902.
Bueno, E., et al. (2010). Environ. Microbiol. 12: 393-400.
Horchani, F., et al. (2011). Plant Physiol. 155:1023-1036.
Meilhoc, E., et al. (2010). Mol. Plant. Microbe Interact. 23:748-759.
Torres, M.J., et al. (2013). J. Appl. Microbiol. doi: 10.1111/jam.12168.
152
Session II
SII-CP-22
Genome sequence of Herbaspirillum rubrisubalbicans M1, an
endophytic diazotroph and mild phytopathogen of sugarcane.
Souza, E. M.*, Chubatsu, L., Cardoso, R.A., Raittz, R.T., Weiss, V.A., Monteiro, R.A.,
Faoro, H., Wassem, R., Baura, V.A., Balsanelli, E., Huergo, L., Muller-Santos, M.,
Tadra-Sfeir, M., Cruz, L.M., Pedrosa, F.O.
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, C. Postal 19046,
81531-980, Curitiba, PR, Brasil.
*
[email protected]
INTRODUCTION
H. rubrisubalbicans strain M1 is capable of developing mild disease symptoms in
susceptible some varieties of sugarcane such as B4362. It is also capable of endophytic
colonization of non-susceptible sugarcane, maize and sorghum without producing
disease. Despite the clear symptoms observed upon inoculation of sugarcane B-4362, H.
rubrisubalbicans is used as a component of a bacterial consortium recommended by the
Brazilian Agricultural Research Company (EMBRAPA) as a commercial inoculant for
sugarcane. This bacterial mixture can supply part of the nitrogen required by sugarcane
and increase productivity, representing a positive impact on the sugarcane business by
saving chemical fertilizers
The H. rubrisubalbicans genome was sequenced using mate-paired reads from 454 FLX
Titanium Roche Pyrosequencer and from Sanger automatic sequencing. De novo
genome sequence assembly was performed using GS de novo Assembler v2.0
(Newbler; Roche). A total of 472 contigs were obtained, with 79 contigs ordered in 9
scaffolds. The scaffolds were further ordered using H. seropedicae SmR1 complete
genome sequence as a reference.
Potential protein-coding regions were identified by RAST. Probable functions of
translation products of potential CDS were inferred using the Blast software by
searching public databases
The reads were assembled to produce a genome sequence of 5,611,430 bp encoding
4673 CDS. Among the traits potentially involved in both beneficial and pathogenic
interaction of this microorganism and graminaceous plants we found genes for auxin
biosynthesis, ACC deaminase, hydrolytic enzymes, adhesins and protein secretion
systems.
Comparison of proteins encoded by H. rubrisubalbicans M1 genome against the CAZy
database revealed 1putative glycoside hydrolase of the GH8 family involved in plant
cell wall degradation. Adhesins are involved in bacterial adhesion to host cells and
biofilm formation. Genes encoding proteins of the T1, T3 and T4 pili were found in H.
rubrisubalbicans. Furthermore, H. rubrisubalbicans has 21 genes encoding adhesins of
the nonfimbrial group. Genes encoding proteins from the general secretory pathway
(Sec pathway), Tat pathway and bacterial secretion systems type I, type II, type III, type
V and type VI (two T6SS clusters) were found in the H. rubrisubalbicans genome. In
addition, at least 10 genes encoding putative T3SS effector proteins were identified.
Among these, the Hrop4 effector protein shares homology with the type III effector
protein AvrPto1 from Pseudomonas syringae pv. Lachrymans. In addition, H.
rubrisubalbicans M1 has at least six genes encoding proteins secreted by T6SS.
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Session II
SII-CP-22
Homologous proteins in other organisms have been shown to be involved in
pathogenicity.
Comparison of M1 genome sequence with that of 8 Herbapirillum spp. revealed unique
regions spanning several genes, indicating putative horizontally transferred genes. The
nif and T3SS gene clusters were in these variable regions suggesting that these genes
are in a genomic island. The genome comparison allowed identifying the core- and pangenome of the genus, which consist of 1,526 and 13,874 genes, respectively. Functional
distribution of the core-genome showed a high number of genes involved in the
following COG categories: amino acid transport and metabolism; energy production
and conversion; translation, ribosomal structure and biogenesis; and general function
prediction only. On the other hand, the pan-genome contains a relatively low frequency
of genes distributed in the COG functional categories. Conversely, the proportion of
non-COG genes (genes with no homologous in COG database) is substantially higher in
the pan-genome (72.6 %) than in the core-genome (15.9 %). The result suggests that
genes of the pan-genome are responsible for the main the differences among the
Herbaspirillum species and/or sub-groups of the genus.
ACKNOWLEDGEMENTS
We thank the Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de
Nitrogênio/CNPq.
154
Session II
SII-CP-23
Sinorhizobium meliloti differentially expressed non-coding RNAs
modulating nitrogen fixation and cell cycle progression.
Robledo M.*, Frage B., Schlüter J.P., Becker A.
Center for Synthetic Microbiology (SYNMIKRO), University of Marburg, Germany
* [email protected]
ABSTRACT
In order to investigate the role of ncRNAs in post-transcriptional regulation of
symbiosis-related genes, in this work we first focus on ncRNAs candidates orientated in
antisense or overlapping the 5’- or 3’-untranslated regions (UTRs) of protein encoding
genes located in pSymA that are required for nodule formation and nitrogen fixation.
Interestingly, some transcripts accumulate differentially in response to different
environmental factors, and seem to repress nif genes under conditions in which
functional nitrogenase is dispensable. In addition, we study a broad conserved
chromosomal trans-encoded RNA whose overexpression leads to elongated polyploid
bacteroid-like cells due to interaction with mRNA derived from cell-cycle related genes
at transcriptional or translational level . Therefore, this work contributes to unravel
sRNA-mediated modulation of nitrogen fixation and cell cycle progression in rhizobia.
INTRODUCTION
Transcripts that do not encode proteins (ncRNAs) have emerged as a widespread type of
regulators in all domains of life. They usually modulate translation and stability of
mRNAs through base pairing. Antisense ncRNAs normally are encoded in the region
that they regulate. In contrast, trans-ncRNA genes map separately from their target
genes and show more limited complementarity. It is tempting to speculate that processes
relevant to symbiosis are modulated by ncRNAs, especially those related to bacterial
adaptation during the transition from free-living to host conditions, e.g. induction of
root nodules, invasion and completion of the morphological differentiation process to
become bacteroids capable of fixing nitrogen. However, nothing is hitherto known
about the biological relevance of ncRNAs in symbiosis. Recently, a RNA-sequence
approach delivered ~ 17,000 experimentally mapped transcriptional start sites (TSS) in
S. meliloti that were assigned to protein-encoding genes or non-coding transcripts
(Schlüter et al., 2013) showing an over-representation of trans- and cis-encoded
ncRNAs on pSymA, specially in the region including the symbiotic genes. Besides this,
sequence conservation analyses suggest strong similarities of several chromosomal
trans-encoded RNAs to genomic regions in related α-proteobacteria (Reinkensmeier et
al., (2011), but the functions of the vast majority remain to be assigned.
The following assays were basically performed as described: Northern analysis (Wilms
et al., 2012), nodulation assays (Robledo et al., 2008), double plasmid assay (Sharma
and Vogel, 2009) and microarray hybridizations (Bahlawane et al., 2008).
The screening of antisense sRNA candidates that overlap partially or completely with
annotated ORFs related with symbiosis lead to a great abundance of nod, fix and nif
genes that harbour a sRNA encoded on the reverse strand. Between them, we chose
sRNAs overlapping genes encoding essential proteins either for nodulation (NodD2) or
nitrogen fixation (NifK and NifE). We confirmed seven of these candidates by Northern
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Session II
SII-CP-23
blot analysis and monitored its expression in different growth phases and media, stress
shifts, luteolin and plant root exposure, nodule and low O 2, micro-oxic and conditions,
inducing the expression of fix and nif genes, respectively. Hybridization signals
corresponding to small RNA transcripts (≤120 nt), were reliably detected. The two
longer sRNA candidates showed more than one signal, suggesting that they are
processed. Three of the identified nif-related sRNAs are differentially expressed under
different conditions including O2 and N2 availability, and in nodules. These asRNAs are
repressed under conditions in which functional nitrogenase is required, suggesting a role
in N2-fixation regulatory pathways. Furthermore, plasmid-based expression of one of
this asRNA resulted in a slight decrease in nitrogen fixation. These experiments
provided the first validation of rhizobial cis-acting riboregulators in response to diverse
abiotic and symbiotic conditions.
On the other hand, the single copy trans-ncRNA EcpR1 (elongated cell phenotype
RNA1) is upregulated at late stationary phase and under several stress conditions. Flow
cytometry analysis showed that after overexpression of EcpR1 the majority of cells
present two or more chromosomes, suggesting that DNA replication uncoupled from
cell division resulting in filamentous cells. This elongated phenotype is conserved
among other rhizobia, and seems to be independent of the RNA chaperone Hfq. Using
an eGFP-based reporter system we observed EcpR1-dependent altered mRNA
translation rates for cell-cycle related genes. However, overexpression of an EcpR1
mutant version carrying nucleotide exchanges in the conserved loop motif predicted to
bind mRNAs led to wild type-like cells and alleviated the altered translation rates.
Concomitantly, 3nt-changes in the predicted target mRNA region also restored the
reporter fluorescence, showing that this motif is responsible for the sRNA-mRNA
interaction. Transcriptome studies revealed known and new cell cycle homologs in S.
meliloti among the differentially expressed genes. Interestingly, 3´translational
chromosomal integrated fusions of cell cycle related genes (Greif et al., 2010) revealed
similar protein accumulation patterns in elongated EcpR1-overexpressing cells and in
bacteroids. Our results suggest that EcpR1 is part of a regulatory network connecting
stress adaptation and cell cycle. To our knowledge, this is the first sRNA that mediates
regulation of cell cycle progression related genes in bacteria.
ACKNOWLEDGES
This work was supported by Humboldt and German Research Foundation (SPP 1258).
REFERENCES
Schlüter, J.P., et al. (2013). BMC Gen 14: 156.
Reinkensmeier, J., et al. (2011). Genes 4: 925-952.
Wilms, I., et al. (2012). Mol. Microbiol. 19: 5209-5217.
Robledo, M., et al. (2008) PNAS 105: 7064-7069.
Sharma, C.M., and Vogel, J. (2009). Curr. Opin. Microbiol. 12: 536-546.
Bahlawane, C., et al. (2008). Mol. Plant Microbe Interact. 21: 1498-1509.
Greif, D., et al. (2010). J. Biotechnol. 149: 280-288.
156
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Symbiotic plant/microbe Interactions
Session III
SIII-P-1
Priming plant defences by beneficial soil microorganisms
Pozo, M.J.*, López-Ráez, J.A., Barea, J.M., Azcón-Aguilar, C.
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín. Consejo
Superior de Investigaciones Científicas, Profesor Albareda 1, 18008 Granada, Spain.
*
[email protected]
ABSTRACT
Beneficial soil microorganisms are frequent in nature and confer major benefits to plant
fitness and ecosystems dynamics. They are able to promote plant growth and to increase
the plants’ ability to cope with biotic and abiotic stresses. In particular, some
microorganisms are able to reduce damages caused by potential deleterious organisms
including microbial pathogens and phytophagous insects and even parasitic plants.
Several mechanisms may operate during this bioprotection, including direct interactions
in the rhizosphere and plant mediated effects, in which alterations in plant nutrition and
activation of plant defence mechanisms play major roles. Generally, during the
interaction of the plant with the beneficial microbe a mild, but effective activation of the
plant immune responses occur. This activation can lead to a primed state of the plant
that allows a more efficient activation of defence mechanisms in response to attack by
potential enemies. As a consequence, it is common that beneficial microorganisms
increase the plant resistance to pathogens and pests, a phenomenon known as induced
systemic resistance (ISR). Multiple examples illustrate that ISR relies in priming of
jasmonate regulated plant defense mechanisms, as in the case of ISR triggered by
arbuscular mycorrhizal fungi (AMF) and Trichoderma, and by beneficial bacteria from
different genera as Pseudomonas, Bacillus and Micromonospora.
INTRODUCTION
Rhizosphere microbes have different trophic/living habits and establish a variety of
saprophytic or symbiotic relationships with the plant, either detrimental or beneficial
(Barea et al., 2013). The plant has to constantly fine-tune its immune responses to
promote interactions with beneficial microbes while restricting the access to deleterious
ones. Indeed, beneficial organisms interacting with plant roots, like pathogens, are
confronted with the plant innate immune system and colonization success essentially
depends on the evolution of strategies for mutual recognition and/or immune evasion.
The modulation of plant defence responses by microbial symbionts aids establishing the
delicate balance between the two partners (Pozo and Azcón-Aguilar 2007; Soto et al.,
2009; Zamioudis and Pieterse 2012). As a consequence of a successful interaction,
alterations in the plant primary and secondary metabolism are common and they may
affect plant responses to the environment, usually increasing the plant ability to cope
with stresses. The activation of the surveillance system and priming of defences is
manifested not only in roots but also in aboveground plant tissues, thereby providing the
plant with an ISR effective against a broad spectrum of plant pathogens and herbivorous
insects below and aboveground (Pozo and Azcón-Aguilar 2007; van Wees et al. 2008,
Pineda et al., 2010). Considerable research efforts have been devoted in the last decades
to unravel the signalling mechanisms regulating ISR, and key regulatory elements have
been identified. The role of defense signalling in the interaction between environmental
conditions and the success of ISR is also a challenge for research (Pineda et al., 2013).
Session III
SIII-P-1
PROTECTION AGAINST PATHOGENS AND PESTS
Most of the early studies on biocontrol of plant diseases by beneficial soil organisms
focused on their direct ability to interact with soil borne pathogens, mainly through
direct mechanisms as production of antibiotics, mycoparasitism, and/or competition for
nutrients in the rhizosphere. In most cases, several of these mechanisms operate
simultaneously, and in addition, changes triggered by the beneficials in the host plant
also contribute to the alleviation of the damage: changes in root morphology and
exudations may alter the dynamics of pathogen infection, and the activation of the plant
defense mechanisms also contributes to resistance. But the enhanced resistance is not
only restricted to root pathogens. Beneficial soil organisms can also impact plant
interactions aboveground. The pathogens’ lifestyles have been shown to be crucial for
the outcome of the interaction, especially in the biocontrol of shoot diseases by
mycorrhizal symbioses (Pozo and Azcón-Aguilar 2007, Jung et al. 2012). As direct
interaction between the beneficial and the pathogen is excluded in these systems, two
main mechanisms drive the effect on aboveground interactions: i) the improvement of
the nutritional status of the host plants and/or alterations of the source-sink relation that
may help a plant overcome an herbivore attack or quickly recover after the attacker has
been fought off and ii) the potentiation of plant defence mechanisms. These
mechanisms may also impact herbivorous insects (Pineda et al., 2010). The final impact
on insect performance will depend on the interplay between a positive effect derived
from the enhanced plant growth and a negative effect derived from the induced
resistance in the plant, and is largely variable depending on the type of the attacking
insect. Generalist insects, able to feed on diverse plants and sensible to the plant defence
mechanisms, are usually negatively affected by the presence of resistance inducers.
However, specialist insects, which feed from one or only a small number of host species
and show a high degree of adaptation to their hosts’ defence responses, may perform
better on the symbiotic plants, probably because of the improved nutritional quality of
the host (Pineda et al., 2010; Jung et al., 2012).
MODULATION OF THE PLANT’S IMMUNE SYSTEM
Both symbiotic and pathogenic fungi are initially recognized as alien organisms through
conserved microbe associated molecular patterns (MAMPs), and the plant reacts with
the activation of an immune response. This defensive response is not completely
abolished but maintained to a minimum level to keep colonization under an uncritical
threshold. Hence, active interference with the plant immune system is fundamental for
the establishment of intimate mutualistic relationships (Soto et al., 2009, Zamidious and
Pieterse, 2012). A prime example of plant immune modulation by microbes can be
found in the intimate symbiotic relationship between plants and AMF, where successful
recognition between both partners leads to a massive colonization of the plant’s root
cortex by the fungus. AMF behave as biotrophic microorganisms and thus a quick but
transient increase of endogenous salicylic acid (SA) occurs in the roots with a
concurrent accumulation of defensive compounds. These initial reactions are temporally
and spatially limited compared to the reaction during plant-pathogen-interactions,
suggesting a role in the establishment or control of the symbiosis (García-Garrido and
Ocampo 2002). In later stages AM fungi evade and manipulate the host innate immune
system to promote successful colonization. Not only SA, but also the levels of other
phytohormones related to defence, such as abscisic acid (ABA), ethylene (ET) and
particularly jasmonic acid (JA) are altered during the plant interaction with the AM
fungus (López-Ráez et al., 2010). The modulation of plant defense signalling system by
AMF seems to play major roles in mycorrhiza-induced resistance. The dependence of a
successful mycorrhiza on JA and SA signalling would explain the range of protection
Session III
SIII-P-1
conferred by this symbiosis (Pozo and Azcón-Aguilar 2007; Jung et al. 2012):
mycorrhizal plants are more resistant to necrotrophs and chewing insects, which are
targeted by JA-dependent defence responses, while the resistance to biotrophs is not
improved, targeted by SA-regulated defences. This pattern correlates with an activation
of JA-dependent defences and repression of SA-dependent ones in a well-established
mycorrhiza. Similarly, JA signalling is likely to be manipulated by Piriformospora
indica to suppress both early (reactive oxygen species production) and late (SAmediated responses and indole glucosinolate production) defence responses. This
suggests that activation of the JA signalling pathway may be key in the control of plant
interactions with beneficial endophytic fungi (Van Wees et al. 2008).
The induction of resistance by beneficial microbes does not necessarily require direct
activation of defence mechanisms but can result from a sensitization of the tissue to
express basal defence mechanisms more efficiently after subsequent pathogen attack.
This priming of the plant’s innate immune system has important fitness benefits
compared to direct activation of defences (Van Hulten et al. 2006; Van Wees et al.
2008; Conrath 2009) and is common in beneficial interactions. Induction of the primed
state is usually associated with a moderate accumulation of defence-related regulatory
molecules, such as transcription factors or MAP kinases (Van der Ent et al. 2009; Pastor
et al. 2012). For example, rhizobacteria-induced systemic resistance in Arabidopsis is
related to priming of JA-dependent responses through the accumulation of MYC2, a
transcription factor with a key role in the regulation of JA responses (Pozo et al. 2008).
Examples of primed defence responses in mycorrhizal plants were first observed in root
tissues. In tomato AMF colonization systemically protected roots against Phytophthora
parasitica infection. Only mycorrhizal plants formed papilla-like structures around the
sites of pathogen infection, preventing the pathogen from spreading further, and they
accumulated significantly more PR proteins than non-mycorrhizal plants upon
Phytophthora attack. Priming has been also involved in mycorrhizal induction of
resistance against other pathogens and nematodes (reviewed in Jung et al., 2012). But
the primed response is not restricted to the root system. AM symbiosis and Trichoderma
colonization induce systemic resistance in tomato plants against the necrotrophic foliar
pathogen Botrytis cinerea. While the amount of pathogen in leaves is significantly
lower, the expression of some jasmonate-regulated, defence-related genes is higher in
these plants (Jung et al., 2013, Martinez Medina et al., 2013). The use of tomato
mutants impaired in JA signalling confirmed that JA is required for the induced
resistance against Botrytis, confirming that ISR by beneficial fungi is similar to the
well-studied rhizobacteria-induced systemic resistance (ISR) in Arabidopsis and
requires a functional JA signalling pathway for the efficient induction of resistance
(Pieterse et al. 1998). Recently, priming of JA dependent responses has been shown to
mediate also Micromonospora induced resistance against Botrytis cinerea in tomato
(Martinez-Hidalgo et al., unpublished). Thus, activation of the JA signalling pathway
appears as a common mechanism in the induction of resistance by beneficial soil
microorganisms.
CONCLUSIONS
While beneficial soil microbes may vary in their lifestyle, host range and the benefits
they confer to the plant, experimental evidences point to important common points in
the mechanisms mediating improved stress resistance. Interaction with the plant
immune system and modulation of the jasmonate signalling pathway have been shown
to be key elements in the induction of resistance. The identification of defense
regulatory elements coordinating ISR is a major challenge for research and a crucial
Session III
SIII-P-1
step for the development of sustainable strategies for the integrated management of
pests and diseases.
Figure 1. Impact of beneficial soil microorganisms on plant biotic interactions. Interaction with beneficial
soil microorganisms often leads to a primed state of the host plant that allows a more efficient activation
of defense mechanisms upon attack. As a consequence, the plant can be more resistant to herbivorous
insects and microbial pathogens below and aboveground. This priming of plant defenses relies on the
jasmonic acid (JA) signaling pathway.
ACKNOWLEGMENTS
This work was supported by projects AGL-2006-08029 and AGL2009-07691 of the Spanish Ministry of
Science and Technology.
REFERENCES
Barea, J.M., et al. (2013) In: Molecular Microbial Ecology of the Rhizosphere (de Bruijn, F. Ed.) WileyBlackwell USA. pp. 29-44.
Conrath, U., et al. (2006). Mol. Plant-Microbe Interact. 19: 1062-1071
Garcia-Garrido, J.M., et al. (2002). J. Exp. Bot. 53: 1377-1386.
Jung, S.C., et al. (2012). J. Chem. Ecol. 38: 651-664.
López-Ráez, J.A., et al. (2010). J Exp Bot 61: 2589-2601.
Martinez Medina A., et al. (2013). Front. Plant-Microbe Interact. doi: 10.3389/fpls.2013.00206
Pineda, A., et al. (2010). Trends Plant Sci. 15: 507-514.
Pineda, A., et al. (2013). Funct. Ecol. 27: 574-586.
Pozo, M.J. et al. (2005). J. Plant Growth Regul. 23: 211-222.
Pozo, M.J., and Azcón-Aguilar C. (2007). Curr. Op. Plant Biol. 10: 393-398.
Pozo, M.J., et al. (2008). New Phyt. 180: 511-523.
Soto, M.J., et al. (2009). Cell. Microbiol. 11: 381-388
Van der Ent, S., et al. (2009). Phytochem. 70: 1581-1588.
van Hulten, M,. et al. (2006). PNAS 103: 5602-5607.
Van Wees, S., et al. (2008). Curr. Op. Plant Biol. 11: 443-448.
Zamioudis, C., and Pieterse C.M.J. (2012). Mol. Plant-Microbe Interact. 25: 139-150.
Session III
SIII-P-2
Una visión general del papel de los polisacáridos superficiales de
Sinorhizobium fredii HH103 en su simbiosis con la soja y otras
leguminosas.
Ruiz-Sainz, J. E.*, Crespo-Rivas, J. C., Acosta-Jurado, S., Margaret, I., Buendía, A.,
Vinardell, J. M.
Departamento de Microbiología, Universidad de Sevilla, España.
*
[email protected]
INTRODUCCIÓN
Las bacterias que hoy llamamos Sinorhizobium fredii fueron descritas por primera vez
en 1982 (Keyser et al., 1982). Su descubrimiento causó una gran sorpresa en “el mundo
de los rizobios” porque se demostraba que había bacterias de crecimiento rápido (1,5 a
3,0 horas de tiempo de generación) que nodulaban con la soja (Glycine max). Hasta ese
momento, todas las bacterias que se aislaban de los nódulos de soja eran de crecimiento
lento (6 o más horas de tiempo de generación) y se agrupaban en la especie
Bradyrhizobium japonicum. Este primer trabajo, que se publicó en Science, tenía sin
embargo un segundo mensaje que era poco alentador de cara a que, en un futuro, las
estirpes de S. fredii fueran usadas como inoculantes comerciales para la soja: estas
estirpes formaban nódulos fijadores de nitrógeno con las variedades asiáticas de soja,
pero sólo inducían la formación de pseudonódulos en las variedades americanas de soja,
que son las comúnmente utilizadas en muchas partes del mundo. Muchos laboratorios
de investigación que iniciaron estudios sobre S. fredii dejaron de hacerlo.
Esta situación cambió en 1985 cuando se describen nuevos aislamientos de S. fredii que
sí son capaces de fijar nitrógeno tanto con las sojas asiáticas como con las americanas
(Dowdle y Bohlool, 1985). La estirpe S. fredii HH103 pertenece a este nuevo lote de
estires aisladas. S. fredii HH103 es plenamente efectivo con las sojas asiáticas, pero fija
menos nitrógeno que B. japonicum USDA110 con las sojas americanas.
Los estudios sobre S. fredii han seguido su curso en los últimos 30 años y, a día de hoy,
podemos decir que es la especie más utilizada en los estudios de rizobios con amplio
rango de nodulación. Tres son las estirpes que más se han estudiado: 1) S. fredii
NGR234, aislada de Lablab y con el mayor rango de nodulación descrito (Pueppke y
Broughton, 1999). Por razones desconocidas no nodula con la soja. 2) S. fredii
USDA257, perteneciente al grupo de estirpes aisladas por Keyser et al. en 1982 y, por
tanto, inefectiva con las variedades americanas de soja. Su rango de nodulación es muy
amplio y aparece como un subconjunto del mostrado por S. fredii NGR234 (Pueppke y
Broughton, 1999). 3) S. fredii HH103, capaz de formar nódulos fijadores de nitrógeno
con todas las sojas probadas, aunque los niveles de fijación son mayores en las asiáticas
que en las americanas hasta ahora probadas. Aparte de las sojas, el rango de nodulación
de HH103 no parece diferir del de S. fredii USDA257 (Margaret et al., 2011).
Se ha secuenciado el genomio completo de la estirpe S. fredii HH103 [ver póster
presentado por J. M. Vinardell et al.; Weidner et al. (2012); Margaret et al. (2011)]. S.
fredii HH103 es, por tanto, la bacteria mejor conocida entre aquellas que muestran un
crecimiento rápido, un amplio rango de nodulación y son capaces de hacer simbiosis
fijadoras de nitrógeno con todos los cultivares de soja ensayados.
Session III
SIII-P-2
LOS POLISACÁRIDOS SUPERFICIALES DE S. fredii HH103.
Tres grupos de señales químicas bacterianas son los más estudiados en cuanto a su
implicación en las simbiosis Sino/Meso/BradyRhizobium-leguminosa:
I, El sistema compuesto por flavonoides (de la planta) y factores de nodulación (de la
bacteria). Se puede encontrar información sobre estas señales químicas de S. fredii
HH103 en los artículos Vinardell et al. (2004a) y Gil-Serrano et al. (1997).
II, Las proteínas Nop (outer nodulation proteins) secretadas por el Sistema de Secreción
de Tipo Tres (T3SS). Se puede encontrar información sobre el T3SS de S. fredii HH103
y sobre las Nops producidas en los artículos de Lyra et al. (2006) y López-Baena et al.
(2009).
III, Cuatro polisacáridos superficiales diferentes: glucanos cíclicos (GC),
exopolisacáridos (EPS), polisacáridos capsulares tipo antígeno K (KPS) y el
lipopolisacárido (LPS). Es a este grupo de señales de S. fredii HH103 al que
dedicaremos el resto de este capítulo.
Los glucanos cíclicos (GC) de S. fredii HH103.
S. fredii HH103 produce beta-glucanos cíclicos (GC) no ramificados que constan de 18
a 24 residuos de glucosa, con o sin sustituciones de fosfoglicerol. Hay dos genes
cromosómicos principalmente implicados en la producción de GC: ndvA y ndvB.
La proteína NdvA está implicada en el transporte del GC al espacio periplásmico. La
proteína NdvB, también llamada Cgs (Cyclic glucan synthase), es muy grande y estaría
implicada en la todas las fases de la biosíntesis del GC. La mutación en ndvB mediante
la inserción del cassette lacZΔp-GenR produce mutantes de S. fredii HH103 que son
pleiotrópicos (Crespo-Rivas et al., 2009). Este mutante (SVQ562) no se mueve en los
medios YMA (yeast-mannitol agar) o GYM (hipoosmótico), ni en este último
suplementado con 100 mM de NaCl. Los tiempos de generación (tg) de HH103 y de
SVQ562 en YMB (yeast-mannitol broth) y en GYM suplementado con 100 mM NaCl
fueron iguales (aprox. 228 min). SVQ562 en medio GYM creció más lentamente (tg de
324 min, aprox.). Por tanto, como sucede con los mutantes ndvB de S. meliloti, los GC
aparecen implicados en la tolerancia de la bacteria al estrés hipoosmótico. Sin embargo,
el mutante SVQ562 es capaz de sobrevivir 3 días en agua destilada, cosa que no sucede
con los mutantes ndvB de S. meliloti. Este hecho indica que, además de los GC, S. fredii
debe tener otros mecanismos adicionales de protección frente a la hipoosmosis.
SVQ562 no solamente produce más EPS que la estirpe silvestre HH103 sino que,
además, este EPS contiene un mayor grado de sustituciones acetato y piruvato. El EPS
producido por SVQ562 se distribuye en dos picos cromatográficos (size-exclusion
chromatography) con respecto a su peso molecular, uno de unos 50-60 kDa (como el
EPS de HH103) y otro de altísimo peso molecular que supera los 2.000 kDa. El gen
exoA [glucosil transferasa que en S. meliloti coloca la primera glucosa en la cadena
naciente (lípido-galactosa) del EPS] muestra mayores niveles de transcripción en el
mutante SVQ562 respecto a HH103.
El mutante SVQ562 sólo forma pseudonódulos en soja (nódulos de tipo determinado) y
en Glycyrrhiza uralensis (nódulos de tipo indeterminado). Las raíces de Vigna
unguiculata inoculadas con SVQ562 no muestran ninguna respuesta a nivel
macroscópico. Por tanto, las mutaciones que eliminan la producción de glucanos
cíclicos afectan tanto a las simbiosis formadoras de nódulos determinados como a las de
Session III
SIII-P-2
nódulos indeterminados. Es posible que, entre otras actividades, los GC actúen de
señales para la planta. Así parece suceder con el fitopatógeno Xanthomonas campestris,
cuyos GC suprimen diferentes reacciones de defensa en Nicotiana benthamiana.
Los exopolisacáridos (EPS) de S. fredii HH103.
La subunidad de repetición del EPS producido por S. fredii HH103 está ramificada y
tiene D-glucopiranosa, D-galactopiranosa y ácido glucurónico (el poster presentado por
Rodriguez-Carvajal et al. muestra la estructura química de este polisacárido). Esta
estructura es igual a la descrita para Sinohizobium fredii NGR234.
La producción de EPS por S. fredii HH103 se ve reducida si al medio de cultivo se le
adicionan flavonoides (como la genisteína) que activen la transcripción de los genes de
nodulación. Esta inhibición de la producción de EPS por flavonoides es dependiente de
NodD porque los mutantes en nodD1 no muestran este fenómeno. Por otro lado, el
incremento del número de copias de nolR (por clonación en un plásmido incP) provoca
un aumento de la producción del EPS de S. fredii HH103. Así pues, en presencia de
flavonoides, el gen nolR de HH103 actúa como un represor de la transcripción de los
genes de nodulación y de la producción de proteínas Nop por el Sistema de Secreción
de Tipo Tres (T3SS) y, a su vez, como un activador de la producción de EPS. Estos
resultados casan bien con el hecho de que un mutante en el gen exoA de S. fredii HH103
no produzca EPS y, aparentemente, no muestre deficiencia simbiótica con la soja
(Parada et al., 2006). Es más, este mutante (SVQ530) es más competitivo que su estirpe
parental HH103 (productora de EPS) para nodular en la variedad Williams de soja y
menos competitivo con Vigna unguiculata (cowpea). Además, el incremento del
número de copias de nolR en HH103 (que entre otras cosas produce una sobreexpresión del EPS) reduce la formación de nódulos con la soja y la aumenta con cowpea
(Vinardell et al., 2004b). Estos hechos ilustran una vez más que, aunque los
Brady/Sinorizobios que nodulan con la soja también nodulan siempre con cowpea, los
mecanismos de nodulación, o los sistemas de percepción de señales en estas dos
leguminosas, deben tener diferencias notables.
Los polisacáridos capsulares tipo antígeno-K (KPS) de S. fredii HH103.
Estos polisacáridos ricos en Kdo, o derivado de Kdo, sólo se han descrito y estudiado en
S. meliloti y S. fredii. En S. meliloti AK631 se han definido tres regiones de genes
involucrados en la producción del KPS (síntesis de la subunidad de repetición,
polimerización y/o transporte). Estas regiones (llamadas rkp-1, rkp-2 y rkp-3) también
se encuentran en S. fredii HH013. Las regiones rkp-1 y rkp-2 se localizan en el
cromosoma y la rkp-3 en el mayor plásmido (pSfHH103e, de 2.096.125 pares de bases).
La estructura química del KPS de S. fredii HH103 consiste en un homopolisacárido de
un derivado del ácido pseudoamínico (Gil-Serrano et al., 1998). Esta estructura es única
entre los KPS de las estirpes de S. meliloti y S. fredii analizados, que suele consistir en
un disacárido en el que uno de los componentes puede ser el Kdo, el Kdn (derivados de
los ácidos octulosónico y nonulosónico, respectivamente) o un derivado de este último.
En S. meliloti es suficiente que la bacteria produzca EPS o un KPS simbióticamente
activo para que la bacteria pueda nodular. Si no produce ninguno de los dos sólo se
forman pseudonódulos en las raíces de Medicago sativa. Esta situación es diferente en
S. fredii HH103 porque la no producción de KPS conlleva un deterioro de la capacidad
simbiótica con la soja independientemente de que se produzca, o no, el EPS. Las raíces
Session III
SIII-P-2
de soja Williams inoculadas con mutantes en genes de la región rkp-1 forman muchos
pseudonódulos y algunos nódulos. Estas plantas muestran claros síntomas de hambre de
nitrógeno (Parada et al., 2006; Hidalgo et al., 2010; Margaret et al., 2012a). Los genes
rkp-1 están involucrados en la biosíntesis de un lípido que debe intervenir en la
biosíntesis y transporte del KPS. Los mutantes en la región rkp-3 (genes de la
biosíntesis de la subunidad de repetición del KPS) solo inducen pseudonódulos en la
soja (Margaret el al. 2012b). Estos mutantes, a diferencia de la mayoría de los genes de
la región rkp-1, también tienen alterado el LPS. Los mutantes en la región rkp-2 (lpsL y
rkpK) no están involucrados en la síntesis de KPS. Los mutantes de S. fredii HH103 en
estos genes están afectados negativamente en la producción de EPS y biopelículas, el
perfil electroforético de su LPS está alterado y tienen un retraso en la nodulación con la
soja (ver póster presentado por Acosta-Jurado et al.). Aunque el KPS es importante para
la capacidad simbiótica de S. fredii HH103 con la soja, los mutantes rkp no parecen
estar afectados en su simbiosis con V. unguiculata.
El lipopolisacárido (LPS) de S. fredii HH103.
Se desconoce la estructura del LPS de S. fredii HH103. Se han mutado los genes lpsB y
lpsE (glicosil transferasas que actúan en la biosíntesis del core del LPS. Los mutantes
en lpsB y lpsE tienen alterado el perfil electroforético del LPS y forman muchos
pseudonódulos y algunos nódulos de morfología externa normal en las raíces de soja.
Las células infectadas en estos nódulos muestran claros signos de senescencia
prematura, posiblemente asociada a reacciones de defensa de la planta (paredes de las
células vegetales muy engrosadas y acumulación de compuestos fenólicos). Todos estos
resultados indican que el LPS de S. fredii HH103 no solamente es importante en las
primeras fases de la nodulación con la soja sino también en la estabilidad de los nódulos
maduros (Margaret et al., 2013, aceptado con “minor revision” en PLOS ONE).
AGRADECIMIENTOS
Los trabajos aquí presentados han sido parcialmente subvencionados por los proyectos de Excelencia de
la Junta de Andalucía P07-CVI-02506 y P11-CVI-7500 y por los proyectos BIO2008-05736-C02 y
BIO2011-30229-C02-01 del Ministerio de Ciencia e Innovación.
BIBLIOGRAFÍA
Crespo-Rivas, J.C., et al. (2009). Mol. Plant-Microbe Interact. 22: 575-588.
Dowdle, S.F., and Bohlool, B.B. (1985). Appl. Environ. Microbiol. 50: 1171-1176.
Gil-Serrano, A.M., et al. (1997). Carbohydr. Res. 303: 435-443.
Gil- Serrano, A.M., et al. (1998). Biochem J. 334: 585-594.
Hidalgo, A., et al. (2010), Microbiology 156: 3398-3411.
Keyser, H.H., et al. (1982). Science 215: 1631-1632.
López Baena, F.J., et al. (2009). Mol. Plant-Microbe Interact. 22: 1445-1454.
Margaret, I., et al. (2012a). Arch. Microbiol. 194: 87-102.
Margaret, I., et al. (2012b). Mol. Plant-Microbe Interact. 25: 825-838.
Parada, M., et al. (2006). Mol. Plant-Microbe Interact. 19: 43-52.
Pueppke, S.G., and Broughton, W.J. (1999). Mol. Plant-Microbe Interact. 4: 293-318.
Vinardell, J.M., et al. (2004a). Arch. Microbiol. 181: 144-154.
Vinardell, J.M., et al. (2004b). Mol. Plant-Microbe Interact. 17: 676-685.
Session III
SIII-CO-1
Quijada, N.M., Cerda, M.E., Abreu, I. *, Pérez de Nanclares, M., Bonilla, I., Bolaños, L.
* [email protected]
ABSTRACT
Boron (B) deficiency negatively affects the legume–rhizobia symbiotic interaction and
the development of N2-fixing nodules. Many described alterations are related to plantderived carbohydrates involved in plant–microbe interactions; however, the effects of B
on the bacterial polysaccharides that are crucial for correct symbiosis are unknown.
Growth of rhizobial strains was slightly affected by B deprivation; however
exopolysaccharide (EPS) production and lipopolysaccharide electrophoretic profiles
were altered in several strains grown in B-free media. These results provide evidence of
a role of B in bacteria that can diminish the infection capacity of rhizobia. We are
already analysing whether boron deficiency is affecting the expression of genes
involved on bacterial cell surface polysaccharide synthesis or it is stabilizing bacteria
cell surface by interacting with synthesized polysaccharides.
INTRODUCTION
Boron (B), a micronutrient required for plants and other organisms in micromolar
concentrations plays an essential role in rhizobia-legume symbioses. As in other plant
tissues, B is needed for the maintenance of nodule cell wall structure, and the
involvement of B in each step of the symbiosis, from early preinfection and infection
events until symbiosome development and nodule organogenesis, has now been
established. The strong effect of B on legume–rhizobia symbiosis has always been
related with the structure and stability of plant-derived components crucial for nodule
development. As a major role, B has been involved in the modulation of cell surface
interactions among legume glycoconjugates and bacterial cell surface polysaccharides
important for the development of a symbiotic rather than a pathogenic interaction
(Bolaños et al., 2004). Actually, induction of pathogenesis-related proteins of the PR-10
family during development of B-deficient nodules has been reported. Moreover, we
recently reported that B starved Rhizobium leguminosarum apparently lacks a capsule of
surface polysaccharides, leading to abortion of infection (Reguera et al., 2010), similar
to the phenotypes described for mutants affected in cell surface polysaccharide
production. To test this hypothesis, the effects of B deficiency on the expression of key
genes related with cell surface polysaccharides, on exopolysaccharide (EPS) production
and on lipopolysaccharide (LPS) structure, as well as the sensitivity of B-deprived
rhizobia to several stresses is being analysed.
Bacterial growth.
R. leguminosarum 3841, Sinorhizobium meliloti 1021 and S. meliloti 8530 grew in
defined liquid or solid (1% B-free agarose) minimal media (MM). Boron deficient
media were prepared eliminating boron traces with the resin Amberlite IRA743 (Asad et
al., 1997).
EPS quantification.
EPS were extracted from supernatant using acetone, lyophilized and weighed.
Alternatively, EPS I synthesis was assayed by fluorescence in solid nutrient plates
supplied with Calcofluor.
Session III
SIII-CO-1
LPS isolation and electrophoresis.
LPS were isolated in SDS buffer, and after treated with proteinase K. Then LPS were
separated by Tricine-SDS-PAGE and silver stained (Shindu et al., 1990).
Boron deficiency affects EPS production and LPS electrophoretic profile.
In a previous report (Reguera et al., 2010), we observed that rhizobia inside the
infection thread apparently lacked a polysaccharide capsule, and that R. leguminosarum
3841 grew in boron-free media developed non mucoid colonies. In the present work we
analyse the effect of boron deficiency on growth and in capsule formation of strains S.
meliloti 1021 and 8530, because the role of B is mainly related with its interaction with
carbohydrates, and the main components of the bacterial capsule and bacterial outer
membrane, EPS and LPS, are enriched in sugars. Supporting that B is not essential for
rhizobia we did not detected differences of growth in B-sufficient or B-deficient media.
However, B-deficiency led to a 65–80% reduction in the amount of total EPS extracted
from liquid cultures. Interestingly, when bacteria were platted in media supplied with
Calcofluor to test EPSI production, the fluorescence was increased in the boron free
plates. These results seem to be controversial, but because Calcofluor staining is not
specific to EPSI, we suggest that the lack of B resulted in the increase of β1-4 bonds
able to interact with Calcofluor. An analysis of sugar composition will be performed to
clarify such observation. In order to deeply study the causes of the decrease of
extractable EPS, we are now analysing whether boron is affecting expression of genes
regulating the synthesis of both EPSI y EPSII. Concerning LPS, and similarly to what
was reported as responses to different environmental changes, bacteria grown on boron
deficient media show an alteration in the electrophoretic silver-stained LPS profile.
Those alterations resembled some non-infective LPS mutants. As described by the EPS,
we are trying to analyze if boron deficiency is altering gene expression or it is
stabilizing these compounds. Moreover, we are trying to discover, if some components
of the LPS are boron ligands, using an affinity chromatography with Amberlite IRA743.
The results provide evidence that B is important for production of rhizobia cell surface
polysaccharides essential to establish a symbiotic rather than a pathogenic-like
interaction. Finally, we are studying whether alterations of bacterial cell surface in low
B conditions increase sensitivity to stresses like salinity, high osmolarity o detergent
pollution. In case of positive results, we could postulate that B is not essential for lab
growing rhizobia, but indeed it is for environmental adaptation of the tested strains.
ACKNOWLEGMENTS
This work was supported by projects MINECO BIO2012-32796 and MICROAMBIENTECM Program
from Comunidad de Madrid. N.M. Quijada is recipient of an Ayuda de Inicio de Estudios de Postgrado
from UAM, and I. Abreu is recipient of a FPU fellowship from Ministerio de Educación (AP2010-4786).
REFERENCES
Asad, A., et al. (1997). Ann. Bot. 80: 65-73.
Bolaños, L., et al. (2004). Mol. Plant-Microbe Interact. 17: 216-223.
Reguera, M., et al. (2010). Plant Cell Environ. 33: 2112-2120.
Shindu, S.S., et al. (1990). J. Bacteriol. 172: 1804-1813.
Session III
SIII-CO-2
Trehalose is a key carbon regulatory player in the Legume-Rhizobium
symbiosis.
Sanchez, F.1*, Barraza, A.1, Estrada-Navarrete, G.1, Quinto, C.1, Merino, E.2
1
Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca,
México. 2 Departamento de Microbiologia Molecular, Instituto de Biotecnología, Universidad
Autónoma de México. Av. Universidad # 2001, Col. Chamilpa. C.P.: 62210, Cuernavaca,
México.
*
[email protected]
Nacional
Morelos,
Nacional
Morelos,
ABSTRACT
Trehalose metabolism during common bean (Phaseolus vulgaris L.) nodule ontology
has been characterized by down-regulation of trehalase (PvTRE1) using RNAi. In
addition, structural and phylogenetic analysis of trehalases suggested a high degree of
evolutionary conservation of trehalose metabolism regulation.
INTRODUCTION
Trehalose is a ubiquitous non-reducing disaccharide that has been found accumulated in
several plant-microorganism interactions (Paul et al., 2008). The nodule specific
expression of PvTRE1 strongly suggests a functional role for trehalose in the symbiotic
nodule (Barraza et al., 2013). The phylogenetic analysis unveiled three major branches
that suggest a prokaryotic origin, a high degree for structural conservation and a marked
trend for the conservation of the catalytic amino acids among trehalases from several
phylogenetic origins (Barraza and Sanchez, 2013). In combination with experimental
and bioinformatics approaches we were able to start unraveling the remarkable role for
trehalase and trehalose in plant-microorganisms interactions and cell physiology.
Rhizobium re-isolation viability assay. Bacteroid re-isolation from transgenic nodules
and viability was assessed according to Müller et al. (2001).
Acetylene reduction assay. Nitrogenase activity in trangenic nodules was determined by
measuring acetylene reduction according to Vessey (1994).
qRT-PCR measurements. Relative expression for the genes analyzed was determined
using six biological replicates and data were normalized to the elongation factor 1α
(EF1α).
Phylogenetic and structural analysis. Phylogenetic analysis were carried out with the
neighbor-joining methodology coupled with 1000 bootstrap replicates. Structural
analysis were performed with PyMol (DeLano, 2002).
Rhizobium survival inside symbiotic nodules, nitrogen fixation, and trehalose content
In common bean symbiotic nodules, PvTRE1 down-regulation by RNAi induced 78%
increase of trehalose content. Interestingly, CFUs of re-isolated bacteria from these
nodules increased by almost one order of magnitude, as well as nitrogenase activity
(71%), without whatsoever negative side-effect on the aerial non-transgenic organs. In
addition, the relative expression by qRT-PCR (Livak and Schmittgen, 2001), for genes
directly involved in carbon metabolism and growth (SUS1, HXK1, TOR, SnRK1),
autophagy (ATG3, Beclin), and nitrogen assimilation (GOGAT, GS) were assessed. In
PvTRE1 silenced nodules transcript accumulation increase for SUS1 (203%), HXK1
(134%), TOR (43%), SnRK1 (81%), ATG3 (26%), GOGAT (80%) also suggests an
Session III
SIII-CO-2
increase in nitrogen assimilation (Barraza et al., 2013). Contrarily, for Beclin nosignificant fold-changes were found (Barraza et al., 2013). Therefore, a positive effect
on Rhizobium cell number and nitrogen fixation and assimilation; accumulation of
transcripts of genes directly involved in carbon metabolism and growth and autophagy
(ATG3), without the induction of Beclin 1 suggest autophagy activation towards cell
recycling instead of programmed cell death.
Trehalose phylogeny and structural conservation.
The sequences analysis performed for trehalases from bacteria, animals, fungi and
plants unveiled three major phylogenetic branches that suggest a prokaryotic origin for
trehalases, and grouping plant and animal trehalases in the same branch, bacterial
trehalases, and fungi trehalases (fungi acid trehalases in a discrete sub-clade). Structural
analysis for all the trehalases deduced structures compared with the trehalase
crystallographic structure (PDB code: 2WYN) gave RMSD values less than 0.1 Å, these
results strongly suggest a structural conservation to maintain the function to regulate the
trehalose content in the cell. In order to support this data, we carried a maximum
likelihood analysis of natural selection codon-by-codon to determine a marked trend for
synonimous substitutions mostly in the catalytic residues (Barraza and Sanchez, 2013).
Altogether, the modification of trehalose metabolism in the symbiotic nodules shed
light onto the mechanism that regulates the bacteroid survival, nitrogen fixation and the
genes that directly orchestrate nodule physiology, and the marked trend for the
structural conservation across taxonomic kingdoms to maintain the activity for trehalose
content regulation.
ACKNOWLEDGEMENTS
This work was supported by Consejo Nacional de Ciencia y Tecnología (CB-2007-83324 and CB-2012177744 to F. S.) with a postdoctoral fellowship (18134) to A. B.
REFERENCES
Barraza, A., et al. (2013). New Phytol. 197: 194-206.
Barraza, A., and Sanchez, F. (2013). Plant Signal. Behav. 8 e24778.
DeLano, W.L. (2002). DeLano Scientific, San Carlos, USA.
Livak, K.J, and Schmittgen, T.D. (2001). Methods 25: 402-408.
Müller, J., et al. (2001). J. Exp. Botany 52: 943-947.
Paul, M.J, et al. (2008). Ann. Rev. Plant Biol. 59: 417-441.
Vessey, J.K. (1994). Plant and soil. 158:151-162.
Session III
SIII-CO-3
RbohB-dependent reactive oxygen species regulates rhizobial infection,
bacteroid development and nitrogen fixation in common bean.
Quinto, C.*
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, México.
*
[email protected]
ABSTRACT
The role of RbohB during the symbiotic interaction between P. vulgaris (common bean)
and Rhizobium tropici was assessed by RNAi and over-expression approaches using a
hairy root system. The results obtained show that RbohB regulates the establishment and
progression of the infection threads and also the number of nodules formed. Analysis of
the nodule ultrastructure revealed the participation of RbohB in rhizobial colonization
and in determining the number and size of bacteroids in infected cells of the bean
nodules.
INTRODUCTION
Plant homologs of membrane-associated NADPH oxidases (Rbohs; respiratory burst
oxidase homologs) have been associated with plant defense against pathogens (Torres et
al. 2002). Recently, it has been reported that during the legume-rhizobia symbiosis,
NADPH oxidases regulate the early steps of the bacterial infection in P. vulgaris
(Montiel et al., 2012) and impair nodule functioning in M. truncatula (Marino et al.
2011). The PvRbohB transcript is the most highly expressed of the NAPH oxidases
found in P. vulgaris roots and nodules. Its promoter becomes active in response to
Rhizobium stimuli and was found associated with growing infection threads (Montiel et
al,. 2012). In this study we present data on the effect of silencing and over-expression of
PvRbohB in transgenic roots.
Cloning and characterization of PvRbohB
PvRbohB was cloned into pTdT-Dc-RNAi and pH7WG2D.1 binary vector respectively
and expressed in P. vulgaris hairy roots. Transcripts were measured by RT-qPCR.
ROS detection assay
Accumulation of superoxide (O2ˉ) radicals was measured according to Ramel et al.
2009.
Acetylene reduction assay
Nitrogenase activity in transgenic nodules (at 28 dpi) was determined by measuring
acetylene reduction as described by Ramírez et al. (1999).
Electron microscopy of transgenic nodules
Tissues were processed according to Sánchez-López et al. (2011) and examined with a
Zeiss EM900 transmission electron microscope (Zeiss) at 50 kV.
PvRbohB modulates ROS accumulation and rhizobia infection process in transgenic
roots of the common bean.
Herein, we cloned 3´ UTR of PvRbohB into pTdT-DC-RNAi and the open reading frame
of PvRbohB into pH7WG2D.1 binary vectors for silencing and over-expression,
respectively. The transgenic roots expressing the above constructs were analysed by
RT-qPCR. Our results confirmed that PvRbohB-RNAi or PvRbohB-OE roots
Session III
SIII-CO-3
significantly down-regulated and over-expressed the transcript over the empty vector
(control). The quantitative assay of NBT-formazan precipitation in transgenic roots
showed a positive correlation with the PvRbohB transcript levels indicating an altered
O2ˉ accumulation (ROS) in silenced and over-expressed roots compared to controls.
After Rhizobium tropici inoculation, the PvRbohB promoter was active at growing
infection threads (ITs). At 7 dpi, the ITs prematurely aborted in the roots hairs of
PvRbohB-RNAi and no such defect was seen in PvRbohB-OE roots; instead, the number
of ITs significantly increased over controls. At four weeks, the total number of nodules
reduced by ~80% in silenced roots and increased in over-expressed roots by ~13%
compared to controls. Furthermore, nodules in silenced roots were unable to fix
nitrogen (5.6±5.6 µmolC2H2/g NDW/h-1) whereas, in PvRbohB-OE nodules the
efficiency increased 54% (88.6±7.1 µmolC2H2/g NDW/h-1) compared to control nodules
(41.6±4.1 µmolC2H2/g NDW/h-1). Together, these results suggest that RbohB-dependant
ROS production is not only required for infection thread progression but also for
bacteroid development and nitrogen fixation in common bean.
Analysis of the ultrastructure of transgenic nodules suggests that PvRbohB regulates
the number and size of the bacteroids.
To gain further insight into the architecture of PvRbohB-RNAi and PvRbohB-OE
nodules, a transmission electron microscopy analysis was performed. The PvRbohB
sparse silenced nodules show poorly colonized rhizobia in infected cells; in contrast, an
enhanced number of bacteria per cell were found in PvRbohB over-expressed nodules.
Tightly packed symbiosomes were very common in PvRbohB-OE nodules and no such
phenotype was seen either in PvRbohB-RNAi or control nodules. Interestingly, the
bacteroid size increased one order of magnitude, and also underwent additional volume
change. Furthermore, accumulation of polyhydroxybutyrate granules increased
noticeably in PvRbohB-OE nodules as compared in PvRbohB-RNAi or control nodules.
Thus, we propose that PvRbohB functions in regulating size and number of bacteriods.
The presence of a high number of polyhydroxybutyrate granules indicates the
involvement of PvRbohB in directing carbon source to rhizobia.
ACKNOWLEDGEMENTS
This work was supported by Consejo Nacional de Ciencia y Tecnologìa (CB-2010-153718 to C.Q. and
132155 to L.C) with a post-doctoral fellowship (17656) to A.M.K.
REFERENCES
Marino, D., et al. (2011). New Phytol. 189: 580-592.
Montiel, J., et al. (2012). Plant Cell Physiol. 53: 1751-1767.
Ramel, F., et al. (2009). BMC Plant Biol. 9:28.
Ramírez, M., et al. (1999). Mol. Plant-Microbe Interact. 12: 1008-1015.
Sánchez-López R., et al. (2011). Plant Cell Environ. 34: 2109-2121.
Torres, M.A., et al. (2002). Proc. Natl. Acad. Sci. USA. 99: 517-522.
Session III
SIII-CO-4
Quorum Sensing systems and flavonoids via NodD1 coordinate and
regulate the biofilm transition in Sinorhizobium fredii SMH12, which is
necessary for a successful root colonization of Glycine max cv Osumi.
Pérez-Montaño, F.1*, Jiménez-Guerrero, I.1, Del Cerro, P.1, López-Baena, F.J.1, Ollero,
F.J.1, Bellogín, R.A.1, Lloret, J.2, Espuny, M.R.1
1
6. 41012 - Sevilla, Spain. 2 Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de
Madrid. 28049. Madrid. Spain.
* [email protected]
ABSTRACT
The role and regulation of biofilm formation in S. fredii SMH12 during the symbiosis
with Glycine max cv. Osumi has been studied. Results suggest that flavonoids, besides
inducing the synthesis of Nod factors and other symbiotic molecules, potentiate the QS
systems, and are necessary for the formation of biofilms in S. fredii SMH12.
INTRODUCTION
In all species studied until now, bacterial surface components, especially
exopolysaccharides, flagella, and lipopolysaccharides are crucial for the formation of
biofilms, in combination with the presence of bacterial QS signals. Biofilm formation
allows soil bacteria to colonize their surrounding habitat and to survive to common
environmental stresses such as desiccation and nutrient limitation. The biofilm mode of
life is often crucial for survival in bacteria of the genera Mesorhizobium, Sinorhizobium,
Bradyrhizobium, and Rhizobium. However, no direct evidence has been already found
that biofilm formation significantly promotes effective symbiosis with the legume host
(Rinaudi and Giordano, 2010).
Strains: wild type S. fredii SMH12, a nodD1 mutant, and a lactonase derivative strain
were constructed.
Nodulation assays on Glycine max (L.) Merrill cv. Osumi were performed as described
by de Lyra et al. (2006).
Colonization assays: rhizosphere attachment, epifluorescence microscopy and electronic
barrier microscopy were carried out as described by
Bacterial biofilm formation was studied by two complementary assays: measure of
bacterial attachment to polystyrene surfaces (Pérez-Montaño et al., 2013) and
observation of biofilm structure by confocal microscopy in crystal surfaces.
Thin layer chromatography and quantitative RT-PCR from biofilm cultures were
performed using a modified protocol described by Pérez-Montaño et al. (2011).
In this work, the role and regulation of biofilm formation in S. fredii SMH12 during the
symbiosis with Glycine max cv. Osumi has been studied. For this purpose and taking
into consideration previous reports, the wild-type strain SMH12, a nodD1 mutant, and a
lactonase derivative strain were constructed and tested in nodulation assays with the
legume. Both strains negatively affect nodulation with respect to the wild-type strain.
Three different colonization assays showed lower root colonization in both derivative
strains, especially in the case of the strain that carries the lactonase gene. Furthermore,
by means of adhesion assays and confocal microscopy, it was determined that in the
presence of inducer flavonoids, SMH12 changed the type of biofilm formed from
Session III
SIII-CO-4
monolayer to microcolony (Figure 1). This process requires the presence of flavonoids
and NodD1, and is coordinated by QS systems. Interestingly, these systems are
activated only by inducer flavonoids.
without flavonoid
with nod gene inducer flavonoid
WT
nodD1-
lactonase+
Figure 1. Biofilm formation in SMH12 and two derivative strains in the presence of nod gene inducer
flavonoids. Bacterial cultures were inoculated in 8-well chambers Lab-Tex Chamber Slide System
(Thermo Fischer Scientific Inc., USA) and grown 4 days without shaking. Confocal microscope images
were captured using a Leica TCS SP2 microscope (Leica Microsystems, Germany). In silico 3D
reconstruction analysis was performed using the software Bitplane (Imaris software, Switzerland).
All these results suggest that flavonoids, besides inducing the synthesis of Nod factors
and other symbiotic molecules, potentiate the QS systems, and are necessary for the
formation of biofilms in S. fredii SMH12. To our knowledge, this is the first report that
unequivocally connect biofilm formation, root colonization and a successfully
symbiosis between a rhizobial strain and its host legume.
ACKNOWLEDGEMENTS
This work was supported by grants AGL2009-13487-C04 from the Spanish Ministerio de Educación y
Ciencia and AGR-5821 from the Junta de Andalucía, Consejería de Innovación, Ciencia y Empresa.
REFERENCES
Pérez-Montaño, F., et al. (2011). Res. Microbiol. 162: 715-723.
Pérez-Montaño, F., et al. (2013). Res. Microbiol. In press.
Rinaudi, L.V., and Giordano, W. (2010). FEMS Microbiol. Lett. 304: 1-11.
Session III
SIII-CP-01
Proteolytic control of the Bradyrhizobium japonicum transcriptional
regulator FixK2.
Fernández, N.1*, Bonnet, M.2, Stegmann, M.2, Maglica, Z.3, Weber-Ban, E.3, Hennecke,
H.2, Bedmar, E.J.1, Mesa, S.1
1
Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo
Superior de Investigaciones Científicas, Granada, Spain. 2 Institute of Microbiology, ETH Zurich, Zürich,
Switzerland. 3 Institute of Molecular Biology and Biophysics, ETH Zurich, Zürich, Switzerland.
*
[email protected]
ABSTRACT
Here, we describe different approaches to study the posttranslational control of the
Bradyrhizobium japonicum regulatory protein FixK2 by specific proteolysis, between
valine 220 and leucine 221, and by general degradation mediated by ClpAP 1. Our
results showed that, for both proteolytic processes, a C-terminal stretch of twelve amino
acids within FixK2 protein sequence plays a crucial role.
INTRODUCTION
FixK2 is an important regulator involved in the control of different lifestyles of the
facultative soybean endosymbiont B. japonicum. This transcription factor regulates
several hundreds of genes required for microoxic, anoxic and symbiotic growth (Mesa
et a1., 2008). FixK2 is an unusual member of the CRP/FNR superfamily, because it is
active in vitro without an additional effector molecule (Mesa et a1., 2005) and is
regulated posttranslationally by the oxidation of its singular cysteine residue (Mesa et
a1., 2009). In addition to its oxidation-mediated control, FixK2 seems to be regulated by
proteolysis. Despite the induction of fixK2 gene expression at low-oxygen
concentrations, the steady-state levels of the FixK 2 protein remains constant regardless
of the growth conditions (Mesa et a1., 2009). Further, a truncated variant is always copurified together with the full-length His6-FixK2 protein from FixK2-overproducing
Escherichia coli cells. Moreover, this shorter FixK2 species is also present in cells of B.
japonicum. In this context, it is worth mentioning that the C-terminal sequence of FNR,
a FixK2-homologue in E. coli, plays a crucial role in the degradation of the inactive
form of this protein which is mediated by the proteolytic system ClpXP, both in vivo
and in vitro (Mettert and Kiley, 2005).
DNA work and enzymatic activities were performed according to standard protocols.
Protein expression and purification, and degradation assays were carried out as
previously described (Bonnet et a1., 2013). In vitro transcription (IVT) assays were
done according to Mesa et al. (2005). Markerless mutant strains were constructed as
described elsewhere (Mesa et a1., 2009).
FixK2 is C-terminally cleaved between residues V220 and L221
Mass spectrometric analysis revealed that the secondary form co-purified with the fulllength FixK2 is a C-terminally cleaved derivative (between V220 and L221), which
lacks the last twelve amino acids. Next, we constructed a series of protein variants
around the cleavage site. To limit the overall change in structure upon mutation, V220
and L221 were individually substituted to asparragine or threonine. In particular, the
L221T FixK2 derivative was more prone to cleavage and showed reduced capacity to
bind DNA and to activate transcription from a FixK2-dependent promoter in an IVT
Session III
SIII-CP-01
activation assay. Thus, the C-terminal part of FixK2 and particularly L221, have a role
in FixK2 protein cleavage and activity. In order to rule out the putative function of the
carboxy-terminal cleavage of FixK2 in its in vivo activity, different proteins variants are
currently being tested with regard to the ability to complement a fixK2 strain.
Degradation of FixK2 by ClpXP1 and ClpAP1 chaperone-proteases.
In analogy to E. coli FNR, FixK2 might be proteolytically regulated by the B. japonicum
Clp system. Therefore, the Clp proteins from B. japonicum, specifically ClpAP1 and
ClpXP1, were purified and tested for their ability to degrade FixK2. Contrary to FNR,
FixK2 is not a substrate for ClpXP1 but it was found to be a substrate for ClpAP1 in vitro
(Figure 1A). This degradation was specific and was inhibited by the ClpS 1 adaptor
protein (Figure 1A), indicating that FixK2 is a novel direct substrate for ClpAP1. By
fusing C-terminal sequence stretches of FixK2 to green fluorescent protein (GFP), we
showed that the last 12 amino acids of FixK2 play the main role in the recognition by
ClpA (Figure 1B).
A
B
Figure 1. (A) FixK2 is a direct substrate for ClpAP1. FixK2 degradation assay by ClpAP1 in the absence
or presence of ClpS1. (B) The C-terminus of FixK2 is the main recognition site for ClpAP1. Degradation
of different GFP derivatives was followed by fluorescence.
Taken together, our results suggest the possibility that the ClpAP system is involved in
the turnover of FixK2 in vivo. To confirm this hypothesis, clpA, clpP1, and clpP2
strains are currently being constructed to analyze the steady-state levels of FixK2 in
different growth conditions and to elucidate whether FixK 2 degradation occurs
continuously in the cells or at a particular scenario. Further, limited information is
available about the role of Clp-like proteins in the symbiotic interaction of rhizobial
species with leguminous plants. Hence, the putative function of Clp proteases in the
symbiotic interaction B. japonicum-soybean is presently under investigation.
ACKNOWLEDGEMENTS
This work was supported by grants from ETH Zurich, the Swiss National Science Foundation, and the
ERDF-cofinanced grant AGL2011-23383 from Ministerio de Economía y Competitividad of Spain.
REFERENCES
Bonnet, S., et al. (2013). FEBS Lett. 587: 88-93.
Mesa, S., et al. (2005). J. Bacteriol. 187: 3329-3338.
Mesa, S., et al. (2008). J. Bacteriol. 190: 6568-6579.
Mesa, S., et al. (2009). Proc. Natl. Acad. Sci. USA. 106: 21860-21865.
Mettert, E., and Kiley, P.J. (2005). J. Mol. Biol. 354: 220-232.
Session III
SIII-CP-02
Effect of temperature on ectomycorrhizal fungi associated with Pinus
sylvestris L. in organic vs mineral soils.
Gómez-Gallego, T.1, García-Rabasa, S.2, Flores-Rentería, D.2, Rincón, A. *
1
Group of Beneficial Plant-Microbial-Interactions, Instituto de Ciencias Agrarias, Consejo Superior de
Investigaciones Científicas ICA-CSIC. 2 Museo Nacional de Ciencias Naturales MNCN-CSIC.
* [email protected]
ABSTRACT
Ectomycorrhizal fungi of Pinus sylvestris L. were characterized in two contrasted soils.
The effect of temperature on mycorrhization and fungal diversity was also tested. Eight
fungi were identified by morphotyping and a total of thirteen by sequencing. Both soils
had a similar number of fungi, although completely differed in the identity of the
dominant ones. The number of fungi and ectomycorrhizas per seedling, together with
fungal diversity significantly decreased at high temperature. Nearly half of fungi were
not found forming mycorrhizas at the highest temperature and contrarily, only one
fungus significantly increased in frequency with this factor. Our results emphasize the
strength of environmental conditions affecting host mycorrhizal rates and structuring
EM communities, which might have profound implications in forest dynamics.
INTRODUCTION
Pinus sylvestris is a representative ectomycorrhizal (EM) tree species highly spread in
Europe. Ectomycorrhizas are the symbiotic association between roots and fungi in
which basically, the fungus improves plant water and nutrient uptake in exchange of
carbohydrates (Smith and Read, 1997). The EM symbiosis plays an essential role in
plant nutrition and forest dynamics, which can be crucial especially under unfavourable
environmental conditions such as those related to global change (i.e. drought, increased
temperature) (Smith and Read, 1997). The objectives of this work were to characterize
the EM fungi associated with P. sylvestris growing in two contrasted soils, and to check
how different temperatures could affect the mycorrhizal status of seedlings and the
diversity of their fungal associates.
Soils were collected in a P. sylvestris forest (Valsaín, Segovia), in an altitudinal
gradient: a) mineral soil at 1.250 m, and b) organic soil at 1.850 m. Seedlings were
grown in containers filled with each soil, at three different temperatures (day/night): a)
low 10/15 ºC, b) medium 15/20 ºC and c) high 20/25 ºC. A total of six treatments were
established with four replicates in each. Four months after, EM percentages were
assessed, and ectomycorrhizas classified by morphotypes (Agerer, 1995) and collected
for DNA extraction (Rincón et al. 2007). The internal transcribed spacer region (ITS) of
the rDNA was PCR-amplified with the primers ITS1F/ITS4 (Gardes and Bruns, 1993),
the PCR-products digested by endonucleases (Hinf I, Msp I), and restriction patterns
(RFLPs) analysed. Samples sharing identical RFLP profile were classed as unique
Operational Taxonomic Units (OTU). Each OTU was sequenced and tentatively
identified by comparison with sequences in the GenBank database. The relative
frequencies of each fungal OTU were calculated and analysed by t-Student test (P ≤
0.05). The EM percentages, the number of OTUs per seedling and the fungal diversity
(Shannon index) were analyzed by one-way analysis of variance (ANOVA) and
differences among treatments separated by the Tukey test (P ≤ 0.05). All analyses were
done with the SPSS 19.0 software.
Session III
SIII-CP-02
Characterization of ectomycorrhizal fungi of organic and mineral soils
Eight EM morphotypes were classified attending to morphological features, whereas
sequencing allowed identifying a total of 13 different fungal OTUs. This highlighted the
convenience of using a combined morphological and molecular approach for identifying
EM fungi, which might have highly similar and/or cryptic characteristics leading to
confusing identification (i.e. Pezizales) (Tedersoo et al., 2006).
A total of 12 and 10 OTUs were respectively identified in the organic and mineral soil,
with 69 % of OTUs found in both soils. Thelephora terrestris Ehrh., S. luteus and
Unkown-5 were only found in the organic soil, and Unkown-9 only in the mineral one.
In both soils, the fungal community structure was similar with few dominant fungi and
many less frequent ones, although the specific composition clearly differed between
soils. In the organic one, the most frequent fungi were: Unkown-3
(Archaeorhizomycetes), Suillus bovinus (Pers.) Roussel and T. terrestris, whereas in the
mineral soil were Unkown-2 (Atheliaceae), Unknown-4, Cenococcum geophilum Fr.
and Wilcoxinia sp. Fungi Unk-2 and Unk-4 significantly decreased in frequency in the
organic soil compared with the mineral one (P = 0,00; P = 0,01), whereas the opposite
happened to T. terrestris and S. bovinus (P = 0,04; P = 0,04). The results showed that
both soils had a high potential of active EM fungal inoculum, but a totally different
cohort of fungi distinctively adapted to mineral or organic conditions.
Effect of temperature on seedling mycorrhization and fungal diversity
The EM percentages (P=0,00), the number of fungi per sample (P=0,03), and fungal
diversity (P=0,04) significantly decreased at high temperature. Reduced mycorrhization
rates could indicate a lower symbiotic-dependence for nutrient uptake by the host,
probably due to a enhanced photosynthetic activity at high temperature (Van derHeijden
and Sanders, 2002). Nearly half of the fungi (46%) were not found forming mycorrhizas
at the highest temperature, and three among them (Unk-5, Tomentella sp. and Unk-9)
were only found at low temperature. The Unk-3 (Archaeorhizomycetes) was the only
fungus that significantly increased in frequency in response to high temperature (P
=0,04), even disappearing at the lowest one.
Replacements of species and changes in the relative dominance of particular fungi
denoted a variable inter-specific response pointing out to temperature as an important
driver structuring EM fungal communities, which might have important consequences
for the functioning of forest ecosystems (Van derHeijden and Sanders, 2002).
ACKNOWLEGMENTS
This work was supported by the projects S2009/AMB-1511 of the Comunidad de Madrid and CGL201129585 of the Spanish Ministry of Innovation and Science.
REFERENCES
Agerer, R. (1995). In: Varma A, Hock B (eds). Springer-Verlag. Berlin, Heidelberg. pp: 687-734.
Gardes, M., and Bruns, T. (1993). Molecular Ecology 2: 113-118.
Rincón, A., et al. (2007). Mycorrhiza 18: 23-32.
Smith, S., and Read, D. (1997). Mycorrhizal Simbiosis. Academic Press. New York. pp: 605.
Tedersoo, L., et al. (2006). New Phytologist 170:581-596.
Van der Heijden, M., and Sanders, I., eds. (2002). Mycorrhizal Ecology. Springer-Verlag. Berlin, pp: 469.
Session III
SIII-CP-03
Ensifer meliloti is the preferred symbiont of Medicago arborea in
eastern Morocco soils.
Missbah El Idrissi, M.1, Guerrouj, K.2, Pérez-Valera, E.3, Abdelmoumen, H.2*, Bedmar,
E.J.3
1
Laboratoire d’Amélioration des sols et Environnement, Ecole Normale Supérieure-Av. Mohamed
Belhassan El Ouazzani-Takaddoum. BP: 5118. Rabat, Moroccco. 2 Laboratoire de Biologie des Plantes et
des Microorganismes, Faculté des Sciences, Université Mohamed Premier, Oujda 6000. Morocco. 3
Department of Soil Microbiology and Symbiotic Systems. Estación Experimental del Zaidín, Consejo
Superior de Investigaciones Científicas (CSIC). E-419, 18080-Granada, Spain.
* [email protected]
ABSTRACT
Medicago arborea (Tree Medic) is the oldest species in the genus Medicago and the
only one with a shrub habit. We carried out a polyphasic approach to identify its
symbionts in different soils of the eastern Morocco. Although the high phenotypic and
genetic diversity observed all the isolates belong to the species Ensifer meliloti.
INTRODUCTION
The main features in M. arborea are drought resistance, cold and salt tolerance, absence
of summer and winter dormancy, evergreen plant, resistance to pathogens and it can
reduce soil erosion (Elgin and Ostazeski, 1982; Andreu et al., 1994). De Mita et al.
(2007) reported that Medicago species are nodulated by two symbiotic bacterial species
of the genus Ensifer (Sinorhizobium); Ensifer medicae and/or E. meliloti but there are
no data about the main symbionts of Medicago arborea species. In this work, we
carried out an analysis of the phenotypic and genetic diversity of 61 bacteria isolated
from this plant root nodules in different soils in Eastern Morocco identify the main
symbionts of this medic tree in these areas.
Sixty one bacteria were isolated from the root nodules of Medicago arborea plants
grown for 3 month in four soils of eastern Morocco using the method of Vincent (1970).
The isolates were first fingerprinted by ERIC PCR (Versalovic et al., 1991) to check for
their genetic diversity and to avoid strains duplication. The phenotypic characterization
consisted in the determination of 116 properties including physiological and cultural
characteristics.
PCR amplifications of 16S rRNA gene fragments were done using the two opposing
primers fD1 and rD1 previously described (Weisburg et al., 2001). The pair primers
nodCFn/nodCI was used for amplification of the nodC genes (Laguerre et al., 2001).
Purified amplification products were subjected to cycle sequencing using the same
primers as for PCR amplification, with ABI Prism Dye Chemistry, and analyzed with a
3130xl automatic sequencer at the sequencing facilities of Estación Experimental del
Zaidín, CSIC, in Granada, Spain.
The phenotypic, symbiotic and cultural characteristics analyzed allowed the description
of a wide physiological diversity among the isolates. The results obtained suggest that the
phenotype of these rhizobia might have evolved to adapt the local conditions. The genetic
characterization consisted in an analysis of the rep-PCR fingerprints and the PCR-based
RFLP of the 16S rDNA patterns. The isolates diversity was investigated by rep-PCR
giving in similarity 62%, 3 clusters, 4 groups and 6 sub-clusters. The results show
Session III
SIII-CP-03
relationship between rep-PCR fingerprinting and sugar assimilation which are
complementary in diversity investigation.
The nearly complete 16S rRNA gene sequence from representative strains of each soil
showed they are closely related to members of the genus Ensifer of the family
Rhizobiaceae within the Alphaproteobacteria and shows the highest similitude values
(99,93/100%) with Ensifer meliloti LMG 6133T (X67222). Sequencing of the symbiotic
nodC gene from seven representative strains revealed they had 94.89% identity with the
nodC sequence of the type strain Ensifer meliloti LMG 6133T (EF428922). Therefore,
the 61 M. arborea isolates from the 4 different soils have the same phylogenetic
affiliation, which proves the restricted host specificity among M. arborea species.
B
MI4 (JX524728)
A
ME5 (JX524735)
MB9 (JX524731)
E. meliloti LMG 6133T (X67222)
MG2 (JX524730)
ME21 (JX524736)
ME1 (JX524733)
70
ME22 (JX524737)
ME2 (JX524734)
MI5 (JX524729)
MB12 (JX524732)
80
E. numidicus ORS 1407T (AY500254)
E. arboris LMG 14919T (AM181744)
E. medicae WSM419T (CP000738)
100
E. garamanticus ORS 1400T (AY500255)
93
S. americanum CFNEI 156T (AF506513)
E. fredii ATCC 35423T (D14516)
E. kummerowiae CCBAU 71714T (AY034028)
E. kostiensis LMG 19227T (AM181748)
E. saheli LMG 7837T (X68390)
95
100
E. sojae CCBAU 05684T (GU593061)
S. chiapanecum ITTG S70T (EU286550)
91
T
E. mexicanus ITTG R7 (DQ411930)
E. terangae LMG 7834T (X68388)
ME21 (JX524727)
E. meliloti LMG 6133T (EF428922)
MG2 (JX524723)
100 MB9 (JX524724)
MI5 (JX524722)
MB12 (JX524725)
MI4 (JX524721)
ME5 (JX524726)
E. kummerowiae CCBAU 71714T (GU994071)
E. medicae USDA 1037T (EF209422)
E. arboris LMG 14919T (EU123539)
E. saheli LMG 7837T (GU994073)
E. fredii ATCC 35423T (GU994072)
E. sojae CCBAU 05684T (GU994069)
R. leguminosarum bv. trifolii USDA 2071 (AF217271)
0.05
E. adhaerens LMG 20216T (AM181733)
R. giardinii H152T (U86344)
0.005
Figure A. Neighbor-joining phylogenetic tree based on partial 16S rRNA sequences of strains from
nodules of Medicago arborea and phylogenetically related species within the genus Ensifer. The tree is
rooted on Rhizobium giardinii H152T (U86344). T = type strain.
Figure B. Neighbor-joining phylogenetic tree based on nodC sequences of strains from nodules of M.
arborea and phylogenetically related species within the genus Ensifer. The tree is rooted on Rhizobium
leguminosarum bv. trifolii USDA 2071 (AF217271). T = type strain.
REFERENCES
Altschul, S.F., et al. (1990). J. Mol. Biol. 215: 403-410.
Andreu, V., et al. (1994). Soil Use Manag. 10: 95-99
Brenner, D.J., et al. (1982). J. Clin. Microbiol. 15: 1133-1140.
De Mita, S., et al. (2007). BMC Evol. Biol. 7: 210-218.
Elgin, J.H., and Ostazeski, S.A. (1982). Crop Sci. 22: 39-42.
Laguerre, G., et al. (2001). Microbiology 147: 981- 993.
Versalovic, J., et al. (1991). Nucleic Acids Res. 19: 6823-6831.
Vincent J.M. (1970). A manual for the practical study of root nodule bacteria. Blackwell Scientific
Publications, Oxford.
Session III
SIII-CP-04
Interaction between Arbuscular Mycorrhizal Fungi and rhizobia on
the growth of subclover under Mn toxicity: The role of Extraradical
Mycelium.
Alho, L.1*, Carvalho, M.1, Goss, M.J.2, Brito, I.1
1
ICAAM, University of Évora, Apartado 94, 7002-554 Évora, Portugal. 2 Kemptville Campus, University
of Guelph, Kemptville, Ontario K0G 1J0, Canada.
* [email protected]
ABSTRACT
When Arbuscular Mycorrhizal (AM) colonization started from an intact extraradical
mycelium (ERM) its bioprotective effect on subclover was enhanced in comparison
with other sources of inoculum. The presence in the soil of an intact ERM, developed
previously on mycotrophic plants tolerant to Mn toxicity, resulted in the earlier
colonization of subclover, reduced Mn concentration in the roots, improved
development and activity of root nodules, and enhanced N acquisition.
INTRODUCTION
Arbuscular Mycorrhizal Fungi (AMF) can protect plants against several different abiotic
stresses, including Al and Mn (Yano and Takaki, 2005; Nogueira et. al., 2007).
However, a well-established AM infection is crucial for an adequate degree of
protection (Garg and Chandel, 2010). When compared with other sources of AM
propagules, colonization initiated from an intact ERM starts earlier and develops faster
(Fairchild and Miller, 1988; Martins and Read, 1997). We hypothesized that the
presence in the soil of an intact ERM developed on Mn tolerant plants, at the time of
subclover seeding, could enhance the bioprotection of subclover, and its associated
rhizobia, against Mn toxicity.
A two-stage experiment was conducted in pots containing a sandy loam Cambisoil,
where toxic levels of Mn were detected previously. In Stage 1, Mn tolerant weed
species, Silene gallica L., Rumex bucephalophorus L., Lolium rigidum L. and
Ornithopus compressus L. (developer plants), with different levels of mycotrophy, grew
for 6 weeks. At the end of Stage 1, half of the pots were disturbed (ERM of
mycotrophic developers disrupted), while glyphosate was applied to the weed shoots in
the other half, thereby keeping the ERM of mycotrophic developers intact. For Stage 2
of the experiment, 6 seedlings of clover were introduced into the pots inoculated with an
appropriate and effective strain of rhizobia. Three of these plants were sampled after 3
weeks to determine the arbuscular colonization of the clover, while the three remaining
plants were allowed to grow for a total of 6 weeks.
By the end Stage 1, the arbuscular colonization rate (AC) of ERM developer plants
indicates different levels of mycotrophy and, therefore, different amounts of ERM were
present in the soil. Developer plants did not change the availability of Mn in the soil.
The AM colonization of subclover after 3 weeks was significantly greater when an
intact ERM was present at the time of planting, that is, after Lolium and Ornithopus in
the undisturbed treatment (Table 1). The dry matter and N content of subclover after 6
weeks was also significantly increased after the mycotrophic, than non-mycotrophic
plants. AC of the clover at 3 weeks (AC3) was positively and significantly related to
shoot weight at 6 weeks but inversely proportional to the Mn concentration in the roots
Session III
SIII-CP-04
(Figure 1A). The AC3 was significantly and positively related with N content (NS) of
subclover shoots and nodule dry weight at 6 weeks (NDW) (Figure 1B), whereas Mn
concentration in the roots was inversely related to NS and NDW (Figure 1C). An intact
ERM, at the seeding of the subclover enhanced earlier AM colonization and with Nfixing rhizobia, was the basis of a more effective tripartite symbiosis. This resulted from
better N acquisition associated with larger and more active root nodules, the
consequence of the smaller concentration of Mn in the roots. We conclude that AM
colonization starting from an intact ERM greatly enhances the bioprotection granted by
AMF against Mn toxicity.
Table 1. Arbuscular colonization rate (AC) and Mn in soil solution of developers at the end of stage 1;
and AC at 3 weeks, Shoot dry weigh (SDW), Nodule dry weight (NDW), Shoot N and Mn concentration,
Root Mn concentration and Shoot N content of clover at 6 weeks of growth.
ERM Developer Plants
AC
Mn in
soil
solution
(mg/L)
Silene
0.01 C
0.76
Rumex
0.01 C
0.22
Lolium
0.54 B
0.24
Ornithopus 0.73 A
0.83
Subclover
Disturbance
AC
3 wks
Undisturbed 0.355
Disturbed 0.145
Undisturbed 0.329
Disturbed 0.219
Undisturbed 0.615
Disturbed 0.404
Undisturbed 0.682
Disturbed 0.240
SDW
6 wks
(g/pot)
BC 1.372
E 0.720
BD 1.246
DE 0.836
A 3.670
B 1.341
A 4.373
CE 0.707
CD
DE
CE
BE
B
CE
A
DE
NDW
6 wks
(µg/nod)
24 B B
42 B B
28 B B
61 B B
418 A A
105 B B
160 B B
25 B B
Concentration
Content
Shoot Shoot
Root
Shoot
N
Mn
Mn
N
(g/Kg) (mg/Kg) (mg/Kg) (mg/pot)
4.9
5.0
4.9
4.8
3.2
4.6
3.6
4.6
A
A
A
A
B
A
B
A
141
118
132
135
125
130
129
140
312
231
171
247
134
191
108
261
66 B
36 B
61 B
41 B
120 AB
59 B
155 A
33 B
Figure 1. (A) Relationship between arbuscular colonization rate (AC) at 3 weeks and shoot dry weight
(open circles) and Mn concentration in the roots (closed triangles) of subclover at 6 weeks; (B)
Relationship between AC at 3 weeks and N content in the shoots of subclover at 6 weeks (crosses ) and
the dry weight of nodules at 6 weeks (open circles); (C) Relationship between Mn concentration in the
roots and dry weight of nodules (crosses) and N content in the shoots (open circles) at 6 weeks of clover
growth.
ACKNOWLEDGMENTS
This work was financed by National Funds through FCT – Foundation for Science and Technology, in the
frame of the project PTDC/AGR-PRO/111896/2009. The authors wish to thank to Filipa Santos and
Manuel Figo for their technical assistance and to Fertiprado for providing the subclover seeds.
REFERENCES
Fairchild, G.L., and Miller, M.H. (1988). New Phytologist 110: 75-84.
Garg, N., and Chandel, S. (2010). Agron. Sustain. Dev. 30: 581-599.
Martins, M.A., and Read, D. J. (1997). Pesqui. Agropecu. Bras. 32: 1183-1189.
Nogueira M.A., et. al. (2007). Plant Soil 298: 273–284.
Yano K., and Takaki, M. (2005). Soil Biol. Biochem. 37: 1569-1572.
Session III
SIII-CP-05
Autochthonous drought-tolerant arbuscular mycorrhizal fungi and
bacteria can increase nutrients acquisition and alleviate drought stress
in lavandula plants.
Armada, E.1, Roldán, A.2, Azcón, R.1*
1
CSIC, Prof. Albareda 1, 18008 Granada, Spain. 2 CSIC-Centro de Edafología y Biología Aplicada del
Segura, Department of Soil and Water Conservation, P.O. Box 164, Campus de Espinardo, 30100
Murcia, Spain.
* [email protected]
ABSTRACT
In a natural arid Mediterranean soil we determined that Lavandula dentata plants
increased growth, nutrition and drought tolerance when inoculated with autochthonous
bacteria or arbuscular mycorrhizal (AM) fungi and particularly with both. The highest P
and K acquisition by the dual inoculated plants protected against the drought stress
imposed as the decreasement of antioxidant GR, APX and CAT enzymatic activities
show. The role of microbial inoculants is important in the restoration of arid
ecosystems.
INTRODUCTION
The establishment of a plant cover based on autochthonous plant species as Lavandula
is an effective strategy in the restoration of the arid degraded Mediterranean zones.
Some microorganisms can promote the plant establishment in these arid soils. The
arbuscular mycorrhizal (AM) symbiosis improves plant growth under extreme
environments but different AM fungi differ in drought tolerance and in their ability to
protect plants against water deficiency (Alguacil et al., 2003). Autochthonous
microorganisms are more resistant to this environmental stress. Thus, the use of
autochthonous AM fungi alone or in combination with native PGPR bacteria could
decrease plant drought stress (Marulanda et al., 2006). In this study we determine the
effectiveness of drought-tolerant autochthonous microorganisms in improving plant
nutrition and stress tolerance tested as plant antioxidant activities.
For the isolation of autochthonous bacteria or AM fungi and respective inocula
production the conventional procedures were carried out using the natural
Mediterranean arid soil from Murcia province (Spain).
In a microcosm experiment, Lavandula was grown in this arid soil for 1 year under
drought conditions (50% whc) inoculated or not with the autochthonous bacteria
identified as Enterobacter sp., B. thuringiensis or Bacillus sp. A consortium of AM
autochthonous fungi were also assayed associated or not to each one of the
autochthonous bacteria. After one year of growth, plant biomass, nutrition and
antioxidant (GR, APX, CAT and SOD) activities were evaluated following the methods
proposed by Aebi (1984); Carlberg and Mannervik (1985); Amako et al. (1994); Burd
et al. (2000), respectively.
Plant parameters analyzed resulted affected by the microbial treatments applied being
important the particular AM-fungi and bacteria interactions found. The plant growth
increased by 123% when AM inoculated but this value was increased by the dual AM
plus Bacillus sp. inoculation. These both treatments also highly increased P, K and Ca
nutrition. Comparatively, K was the nutrients particularly increased by these
Session III
SIII-CP-05
microorganisms. The effectiveness of both Bacillus strains on K acquisition was as
relevant as this of AM fungi and such K enhancement was maximized by the
inoculation of both microorganisms. This is an important mechanism to increase plant
drought tolerance. Concomitantly, the antioxidants APX, GR and CAT activities
resulted highly reduced in these plants. These results indicate the relation between K
nutrition, antioxidant responses and Lavandula adaptation to drought. The reduced
APX, GR and CAT activities as affected by the microbial associations is index of
drought resistance. The lower values in such enzymatic activities in response to drought
represent a better drought adaptation of dually inoculated plants evidencing the lowest
detrimental effect caused by drought in the inoculated plants (Benabdellah et al., 2011).
Thus, inocula applied are important to promote plant growth and nutrition and also to
alleviate drought stress.
Effect of autochthonous bacterial strains (Enterobacter sp.; B. thuringiensis and Bacillus sp.) and
autochthonous arbuscular mycorrhizal (AM) single or dually inoculated, on shoot dry weight P, K and Ca
content (mg/plant) and APX, CAT, GR and SOD antioxidant activity in shoot, of Lavandula dentata
growing in natural arid Mediterranean soil under drought.
Lavandula dentata Shoot dry weight (mg)
Control
Enterobacter sp.
B.thuringiensis
Bacillus sp.
(-)
655a
678a
1086c
864b
APX nmol/mg
prot·min
(-)
AM
13112d 10222c
11582cd 9938c
Control
Enterobacter
sp.
B.thuringiensis 7068a 13203d
Bacillus sp.
8263ab 6913a
AM
1459d
1838e
1646de
1947e
P
mg/plant
(-)
0,62b
0,50a
0,64b
0,64b
K
mg/plant
AM
0,78c
1,07d
1,04d
1,11d
(-)
13,51a
15,03a
21,96b
19,47b
AM
(-)
AM
19,73b 13,29b 32,71c
28,74c 9,83a 35,96c
30,56cd 16,83b 33,46c
35,22d 15,45b 44,05cd
CAT nmol/mg
prot·min
(-)
AM
1275c
1171b
1214c
1090b
GR nmol/mg
prot·min
(-)
AM
1882e
89a
920c
141a
1678d
1320c
1074d
894c
1635d
814a
Ca
mg/plant
370b
106a
USOD nmol/ mg
prot·min
(-)
AM
2,7a
4,2c
2,7a
4,1c
2,9ab
2,4a
ACKNOWLEDGMENTS
This study has been carried out in the framework of the Project AGL 2009-12530-CO2-02.
REFERENCES
Aebi, H. (1984). Methods Enzymol. 105: 121-126.
Alguacil, M.M., et al. (2003). Physiol. Plant. 118: 562-570.
Amako, K., et al. (1994). Plant Cell Physiol. 35: 497-504.
Benabdellah, K., et al. (2011). Eur. J. Soil Biol. 47: 303-309.
Burd, G.I., el al. (2000). Can. J. Microbiol. 46: 237-245.
Carlberg, I., and Mannervik, B. (1985). Methods Enzymol. 113: 484-489.
Marulanda, A., et al. (2006). Microb. Ecol. 52: 670-678.
6,6d
3,1b
Session III
SIII-CP-06
Análisis filogenético de la diversidad de hongos formadores de
micorrizas arbusculares presentes en los distintos tipos de propágulos
asociados a especies de plantas características del Parque Natural
Sierra de Baza (Granada, España).
Varela-Cervero, S., López-García, A., Berrio, E., Barea, J. M., Azcón-Aguilar, C.*
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín
(CSIC), Granada, España.
*
[email protected]
RESUMEN
En este estudio se analiza la diversidad que albergan los distintos tipos de propágulos de
hongos formadores de micorrizas arbusculares (raíces micorrizadas, esporas e hifas)
asociados a especies de plantas representativas de ecosistemas semiáridos
mediterráneos. La diversidad se evaluó mediante la técnica de T-RFLP. Los resultados
evidencian diferencias en la diversidad de filotipos de hongos micorrícicos en los
distintos tipos de propágulos. También sugieren que las estrategias de colonización y
supervivencia de estos hongos difieren considerablemente y que estas diferencias tienen
una base taxonómica a nivel de familia.
INTRODUCCIÓN
Los hongos formadores de micorrizas arbusculares (MA) son simbiontes obligados que
forman asociaciones mutualistas con la mayoría de las plantas terrestres. Las hifas del
hongo que se desarrollan en el suelo (micelio extrarradical) absorben y transfieren
nutrientes y agua a las plantas con las que se asocian (Barea et al., 2013). La
colonización micorrícica se puede iniciar a partir de tres tipos de progágulos distintos:
esporas, raíces previamente micorrizadas y micelio extrarradical o hifas. Estudios
recientes han mostrado una diferencia notable en la composición de hongos MA entre
las esporas presentes en el suelo y las raíces micorrizadas (Hempel et al., 2007;
Sanchez-Castro et al., 2012). El objetivo del presente estudio fue el análisis de la
diversidad que albergan los distintos compartimentos de hongos MA (hifas, esporas y
raíces micorrizadas) asociados a especies de plantas representativas de ecosistemas
mediterráneos semiáridos.
MATERIAL Y MÉTODOS
Se muestrearon las raíces y el suelo adyacente de cinco especies representativas
(Rosmarinus officinalis, Thymus zygis, Thymus mastichina, Genista cinerea, Lavandula
latifolia) de un ecosistema mediterráneo del Parque Natural Sierra de Baza (Granada).
Se separaron los tres tipos de propágulos mediante tamizado húmedo y decantación y se
analizó la diversidad de hongos MA presente en cada uno de ellos mediante T-RFLP
(Terminal Restriction Fragment Length Polymorphism). Esta técnica permite detectar
presencia o ausencia de filotipos de hongos MA en las muestras analizadas (LópezGarcía et al., 2013). Para el análisis mediante T-RFLP se utilizaron cebadores
específicos para amplificar una región del gen del ARN ribosómico 18S.
Se detectaron un total de 16 filotipos distintos de hongos MA, pertenecientes a 6
familias diferentes (Glomeraceae, Diversisporaceae, Pacisporaceae, Paraglomeraceae,
Claroideoglomeraceae y Scutellosporaceae). No se detectaron diferencias significativas
en la diversidad de filotipos encontrados en las raíces de las distintas especies botánicas
Session III
SIII-CP-06
estudiadas, ni en la de los propágulos presentes en sus correspondientes rizosferas. Sin
embargo, sí que se observaron diferencias importantes en la diversidad de hongos MA
presente en los distintos tipos de propágulos micorrícicos.
En todos los propágulos micorrícicos predominaban los filotipos pertenecientes a la
familia Glomeraceae. Sin embargo, mientras que estos representaban más del 80 % del
total de filotipos detectados en raíces, solo representaban el 36 % de los encontrados en
esporas y algo más del 50 % de los detectados en hifas (Tabla 1). Por el contrario, los
filotipos pertenecientes a otras familias de hongos MA se encontraron preferentemente
en hifas o esporas. Así, los de Pacisporaceae y Paraglomeraceae eran más abundantes
en esporas, mientras que los de Diversisporaceae, Claroideoglomeraceae y
Scutellosporaceae se detectaban con mayor frecuencia en esporas e hifas.
Estas tendencias generales se observaron en todos los filotipos pertenecientes a la
misma familia, lo que permite concluir que los hongos MA presentan estrategias vitales
y de colonización diferentes y que estas diferencias tienen una base filogenética.
Sugieren, además, que la taxonomía de los hongos MA tiene una base funcional. Los
resultados obtenidos confirman la existencia de distintas estrategias vitales en hongos
MA (Hart et al., 2001) y que estas son independientes de la planta hospedadora
implicada en la simbiosis. Ello tiene implicaciones importantes en el diseño de
inoculantes con una composición y estructura optimizada para maximizar su eficacia.
Tabla 1. Frecuencia de detección de diferentes familias de hongos
MA en los distintos tipos de propágulos de hongos micorrícicos.
Glomeraceae
Diversisporaceae
Pacisporaceae
Paraglomeraceae
Claroideoglomeraceae
Scutellosporaceae
Raíces
81,3 a
9,2 b
0,5 b
2,7 b
1,9 b
4,4 b
Esporas
36,0 a
20,9 ab
10,2 b
16,0 b
7,1 b
9,8 b
Hifas
50,9 a
25,1 b
3,4 c
6,4 c
6,1 c
8,1 c
Datos en la misma columna que no comparten una letra en común difieren
significativamente de acuerdo a la prueba del Χ2.
AGRADECIMIENTOS
Este trabajo ha sido subvencionado por el proyecto CGL2009-08825 (Ministerio de Ciencia e
Innovación).
BIBLIOGRAFÍA
Barea, J.M. et al. (2013). In: Beneficial Plant-Microbial Interactions: Ecology and Applications (Rodelas,
B., and Gonzalez-Lopez, J., eds.). CRC Press. ISBN: 9781466587175.
Hart, M.M., et al. (2001). Mycologia 93: 1186-1194.
Hempel, S., et al. (2007). Environ. Microbiol. 9: 1930-1938.
López-García, A., et al. (2013). Plant Soil DOI 10.1007/s11104-013-1625-0.
Sanchez-Castro, I., et al. (2012). J. Arid Environ. 80: 1-9.
Session III
SIII-CP-07
Influencia de la composición de la cubierta vegetal sobre los hongos
micorrícico-arbusculares y la estabilización de un suelo degradado en
un ecosistema mediterráneo.
Cornejo, P.1, 2, Ferrol, N.1, Barea, J.M.1*, Azcón-Aguilar, C.1
1
CSIC, Profesor Albareda 1, 18008 Granada, España. 2 Departamento de Ciencias Químicas y Recursos
Naturales, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile.
* [email protected]
RESUMEN
En un sistema de mesocosmos se analizó la influencia de la composición de la cubierta
vegetal sobre las densidades de propágulos de hongos formadores de micorrizas
arbusculares (HMA) y la estabilización del suelo durante 3 años. Se observaron
combinaciones de plantas que produjeron una mayor cobertura vegetal, una mayor
densidad de propágulos fúngicos y mayores índices de estabilización de suelos, que
sugieren la utilización de combinaciones sinérgicas de plantas en procesos de
estabilización de suelos degradados.
INTRODUCCIÓN
Numerosos estudios muestran que una colonización MA efectiva favorece procesos de
estabilización de suelos degradados, debido a diversas causas, como el crecimiento de
micelio fúngico y producción de glomalina, que favorecen la agregación de las
partículas del suelo y, por ende, evitando procesos erosivos (Curaqueo et al., 2011). En
ambientes mediterráneos predominan plantas arbustivas, muchas de las cuales dependen
en gran medida de la formación de MA para establecerse y crecer (Barea et al., 2011).
Sin embargo, poco se conoce acerca del efecto en la estabilización de suelos cuando
estas plantas crecen en combinación, como una forma de restituir una adecuada
diversidad de la cubierta vegetal en estos ecosistemas degradados. Este estudio reporta
resultados de tres años de estudio en diversas asociaciones vegetales usando plantas del
matorral mesomediterráneo, inoculadas con una mezcla de HMA, sobre diversos
parámetros del crecimiento vegetal, fúngico, y la estabilización de un suelo degradado.
MATERIAL Y MÉTODOS
Se establecieron mesocosmos utilizando un suelo degradado de la Sierra de Baza
(Granada, España) inoculados con una mezcla de seis especies de HMA del género
Glomus, en los que se hicieron crecer cuatro plantas de distintas especies del matorral
mediterráneo (lavanda-Lavandula latifolia, retama-Retama sphaerocarpa, romeroRosmarinus officinale y mejorana-Thymus mastichina) de forma monoespecífica o en
combinación de dos especies, por un período de tres años. Cada tres meses se evaluó la
superficie de cobertura en cada mesocosmos, las densidades de propágulos (esporas por
100 g de suelo, micelio y raíz colonizada) y diversos parámetros físico-químicos del
suelo (pH, P disponible, glomalina fácilmente extraíble, formación de agregados
estables al agua -AEA-), y se analizó la relación existente entre las diversas variables
analizadas en función de las asociaciones vegetales establecidas.
Los resultados mostraron una marcada influencia del aumento de densidad de los
propágulos de HMA sobre el crecimiento vegetal (datos no mostrados) y sobre la
mejora de las características del suelo (Figura 1). En particular, la formación de AEA
correlacionó fuertemente con los aumentos de la densidad de micelio a través del
Session III
SIII-CP-07
tiempo, aumentando en algunos casos desde cerca de un 40% inicial a valores
comprendidos entre 60 y 80% al final del estudio (Figura 1A y B). Por su parte, el
crecimiento vegetal correlacionó fuertemente con el aumento de estructuras fúngicas
(Figura 1 C), pero también fue posible observar efectos sinérgicos en algunas
asociaciones de especies vegetales (lavanda-mejorana, lavanda-retama, mejoranaretama), así como otras combinaciones que resultaron menos compatibles (en especial
con romero), probablemente por efectos alelopáticos.
Th
Th-Ret
3,2
Lav
Th-Ros
Ros
Lav-Ros
Th-Lav
Ret-Ros
3
C
A
2
2,4
1,6
0,8
0,0
100
B
80
AEA (%)
Ret
Lav-Ret
PC AM fungal propagules (64.5%)
Hifas HMA (m g-1)
4,0
60
40
Ret-Ros
Lav-Ros
1
Lav-Ret
Th-Ros
0
Th-Ret
Th-Lav
-1
Ros
Ret
-2
Lav
-3
Th
-3
20
-2
-1
0
1
2
PC soil properties (59.7%)
0
Año 1
Año 2
Año 3
Figura 1. Influencia de la composición de la comunidad vegetal sobre la evolución del crecimiento
fúngico y estabilización de las propiedades de un suelo degradado. A) Evolución de la densidad de hifas
de HMA; B) Evolución de la formación de agregados estables al agua (AEA); C) Relación entre las
componentes principales obtenidas para las variables de crecimiento de HMA (esporas, hifas,
colonización) y mejora de las características del suelo (pH, P disponible, Glomalina, AEA). Abreviaturas:
Thymus mastichina (Th); Lavandula latifolia (Lav); Retama sphaerocarpa (Ret); Rosmarinus officinale
(Ros).
Las combinaciones que presentaron los mejores incrementos globales, tanto en el
aumento de los propágulos de HMA, crecimiento vegetal, como en la mejora de las
características del suelo fueron lavanda y mejorana, tanto solas como en combinación,
lo que sugiere que una alternativa para mejorar los procesos de estabilización de suelos
degradados, además de la utilización de plantas inoculadas con HMA, es la elección de
especies vegetales compatibles, que puedan actuar sinérgicamente al ser utilziadas en
combinación.
AGRADECIMIENTOS
Estudio financiado por CICYT-EU (Proyectos REN2000-1506/GLO y REN2003-00968/GLO. Pablo
Cornejo agradece las becas MAE-AECI (España) y grupo Santander-Universia (Banco Santander, Chile).
BIBLIOGRAFÍA
Barea, J.M., et al. (2011). J. Arid Environ. 75: 1292-1301.
Curaqueo, G., et al. (2011). Soil Till. Res. 113:11-18.
Session III
SIII-CP-08
Salt tolerance evaluation in Casuarina glauca: impact on the
photosynthetic apparatus.
Batista-Santos, P.1*, Graça I.1, Semedo J.2, Lidon, F.3, Alves, P.1, Scotti, P.2, Pais, I.2,
Ribeiro, A.I.1, Ramalho, J.C.1
1
Centro de Ambiente, Agricultura e Desenvolvimento (BioTrop), Instituto de Investigação Científica
Tropical (IICT), Qta Marquês, Av. República, 2784-505 Oeiras, Portugal. 2 Instituto Nacional de
Investigação Agrária e Veterinária, I.P. (INIAV), Qta Marquês, Av. República, 2784-505 Oeiras,
Portugal. 3 Dept. Ciências e Tecnologia da Biomassa, Faculdade de Ciências e Tecnologia / Univ. Nova
de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.
*
[email protected]
ABSTRACT
Salt stress is one of the major abiotic stresses that imposes negative effects on plants
and consequently on plant productivity. Casuarina glauca belongs to the group of
actinorhizal plants that establish N2-fixing root-nodule symbiosis with actinomycetes of
the genus Frankia. It is commonly found in the saline soils of the coastal zones,
presenting an outstanding ability to cope with environmental constraints. In this context,
we are presently evaluating to which extent is C. glauca able to cope with salt stress and
what is the relationship of such ability with the symbiotic capacity. In this work stage,
we are analysing the impact of salt on the photosynthetic pathway by means of
morphological and physiological methodologies. In general, C. glauca tolerates high
levels of salt (up to 600 mM). The stressed plants present reductions in relative water
content (RWC) suggesting a salinity induced water stress, associated with a decrease in
the plant vigor (decreased growth). Similarly, leaf gas exchanges and fluorescence of
chlorophyll a parameters declined with salt stress imposition. The results pointed to the
impairment of photosynthesis with the salt imposition, probably related to stomatal and
non-stomatal limitations.
INTRODUCTION
Actinorhizal plants, a group of woody plants belonging to eight different families, are
capable of high rates of N2 fixation due to their capacity to establish root-nodule
symbiosis with N2-fixing actinomycetes of the genus Frankia. These plants are able to
grow in impoverished and disturbed soils and are important elements in plant
communities worldwide. They have a great capability for adaptation and can be used for
fuel, wood production, agro-forestry, and land reclamation (Diem and Dommergues,
1990).
According to (Wand et al., 2003), drought and salinity are becoming particularly
widespread in many regions, and may cause serious salinization of more than 50% of all
arable lands by the year 2050. Thus, salt tolerance is becoming increasingly more
relevant in agro-forestry systems. Besides being considered the model of actinorhizal
plants, C. glauca is commonly found in saline soils of the coastal zones and is widely
used to recover marginal soils and to prevent desertification. Under this context, we are
presently evaluating the impact of salinity in C. glauca and its association, if any, with
the symbiotic capacity.
Plants were grown in hydroponic culture and nodulated by Frankia Thr according to
standard procedures available in our lab. Six month-old plants were transferred to walkin growth chambers (10000 EHHF, ARALAB, Portugal) where they were kept at 26/25
ºC (day/night), 12 h photoperiod, 65-70% RH, ca 380 µL L-1 of external CO2 and ca.
Session III
SIII-CP-08
500 µmol m-2 s-1 of irradiance. Nodulated and non-nodulated plants were subjected to
salt stress (0 mM, 200 mM, 400 mM and 600 mM NaCl).
Relative water content (RWC) and membrane permeability were determined in
branchlets samples of 9 pieces (1.5-2 cm long each), respectively, according to
(Albouchi et al., 2003) and (Campos et al., 2003). Gas exchange measurements and
Chlorophyll a fluorescence parameters were determined as referred in (Batista-Campos
et al., 2011). Each evaluation was performed in 4-5 replicates.
One of the common symptoms of salinity stress in many plants is the decrease of the
relative water content (RWC). In C. glauca, the exposure to increased NaCl levels
decreased the RWC of both nodulated and non-nodulated plants. This reduction
reflected the water stress induced by salinity and was probably associated with a
decrease in plant vigor (Greenway and Munns, 1980). Concomitantly, leaf net
photosynthetic rate (Pn) and stomatal conductance to water vapour (gs) also declined
with the gradual salt imposition in both nodulated and non-nodulated plants. The results
suggest that the plants responded to increased NaCl concentrations by decreasing gs, in
order to avoid further dehydration, thus limiting Pn (and therefore growth) as the CO2
supply (Ci) to carboxylation sites was reduced. However, the impairment of
photosynthesis would also be related to non-stomatal limitations, linked to limitations
on PSII photochemical efficiency, thylakoid electron transport and membrane
selectivity.
ACKNOWLEGEMENTS
This work was supported by Fundação para a Ciência e a Tecnologia, through the project PTDC/AGRFOR/4218/2012 and by the grants SFRH/BPD/78619/2011 (P. Batista-Santos) and SFRH/BD/
41589/2007 (I. Graça), both co-financed by the Portuguese PIDDAC program and European Social Fund,
under the 3rd framework program.
REFERENCES
Albouchi, A., et al. (2003) Science et changements planétaires/Sécheresse 14: 137-42.
Batista-Santos, P,. et al. (2011). J. Plant Physiol. 168: 792-806.
Campos, P.S., et al. (2003) J. Plant Physiol. 160: 283-92.
Diem, H.G., and Dommergues, Y.R. (1990). In C. R. Schwintzer and J. D. Tjepkema (ed.), The biology
of Frankia and actinorhizal plants. Academic Press, San Diego. pp. 317-342.
Greenway, H. and Munns, R. (1980). Annu. Rev. Plant Physiol. 31: 149-190.
Wang, W., et al. (2003). Planta 218: 1-14.
Session III
SIII-CP-09
Rhizosphere symbiots valorisation: Common bean-rhizobia symbiosis
adaptation to P deficiency in Ain Temouchent agro-ecosystem in
Algeria.
Benadis, Ch.1*, Bekki, A.1, Lazali, M.2, Drevon, J.J.2
1
Laboratoire de biotechnologie des rhizobia et amélioration des plantes LBRAP. BP n°16 Faculté des
sciences d’Oran. ALGÉRIE. 2 INRA, UMR Eco & Sols, Place Viala F-34060, Montpellier, France.
* [email protected]
ABSTRACT
Leguminous have a high environmental, dietary and socio-economic importance
especially for the African countries. Despite of their interest, during these last years,
their culture is decreasing caused by biotic and abiotic stresses: Temperature variations,
impairment of Mediterranean soils in minerales especially phosphorus. The ability of a
symbiotic association with rhizobia allows the biological nitrogen fixation resulting can
be exploited to improve plant growth and fertility soil. The inoculation by rhizobia
plays an important role in improving and increasing the potential of fixing the
atmospheric nitrogen through increasing number and weight of nodules. In this context
that is our study whose objective is to select an efficient rhizobia and bean genotypes to
improve growth and production of this very important beans and adapt it to address
constraints in particular soil phosphorus deficiency.
INTRODUCTION
The biological nitrogen fixation in symbiosis established with bean-rhizobia and nodule
formation provides annually to the plant and soil an amount of nitrogen equivalent to
chemically synthesized in the fertilizer industry (Oliveira et al., 1998). Inside Common
beans are often considered as a poor nitrogen fixer (ISOI and Yoshida, 1991). A study
conducted by CIAT has also shown that 60% of cultivated soils beans are deficient in P
(Vadez, 1996).Witch are our challenges in this work: Improve symbiotic potential of
Phaseolus vulgaris in adverse conditions and Increase its adaptation to infertile soils
with Reasoned Inoculation that is study of natives rhizobia from selected agroecosystem
to preservate of biodiversity and agroecosystem functions.
Six recombinant inbred lines, namely RILs: (CIAT) and one common bean variety
widely cultivated in Algeria. Lines 115, 104 and 75 have been characterized as Pefficient whereas 147, 83 and 29 have been categorized as P-inefficient based on plant
growth and seed yield in relation to the availability of P. This RIls was sowing both in
vitro trapping and in natura multilocus test for 20 plots from Ain Temouchent
agroecosystem chose in northwest of Algeria. After 45 days after transplanting the
nodules are collected, according to macroscopically aspect 40 strains was selected to
PCR-RFLP analysis amplified 16S rDNA genes. Total DNA was extracted as Laguerre
(1992) described. Aliquots of PCR products were digested with restriction
endonucleases. The following enzymes were used: Msp I and Nde II.
All strains were tested in glass house at hydroponic cultural condition. The preparation
begins before sowing, by sterilization of the seeds (Tajini et al., 2009; Bargaz et al.,
2010). After, seeds were cultivated in bottles 1 -1 warped with aluminum foil to maintain
the rooting darkness condition, and contain the same nutriment solution changed
weekly, (Vadez, 1996; Adelson et al., 2008).
Session III
SIII-CP-09
Small collection of isolates revealed an interesting diversity. For 40 strains studied five
were identified as Rhizobium etli, 3 R. leguminosarum, 11 R. gallicum, 1 R. loti, 1 R.
ciceri and 6 Agrobacterium and 10 strains remaining will be sequenced to be identified.
For this strain studied, 29 can establish a nodule and 17 and 2 strains were more
efficiency that was identified as R. etli.
Figure 1. Data are means of five replicates of nodules number after 45 days transplanting (A) in natura and (B) in vitro
for 20 plots from Ain Temouchent agroecosystem.
Bo îte à M o u s ta c h e s p a r Gro u p e s
M oy enne
M o y e n n e ± Erre u r-Ty p e
M o y e n n e ± Ec a rt-Ty p e
Va ri a b l e :No d u l e s n u m b er
450
400
350
No d u l e s nu m b e r
300
250
200
150
100
50
0
-5 0
2
4
6
8
11
13
16
18
20
22
24
26
29
1a
10G 15a 28a 30a 33a 33c
s tra i n
Figure 2. Data are means of five replicates of nodules number after 45
days transplanting in vitro in hydroaeroponic cultural system.
AKNOWLEDGEMENT
This work was supported by Oran University for the stay of BENADIS Chahinez in Borj cedria (Tunisia)
and Montpellier (France), the Grand Federative Project Fabatropimed of Agropolis Montpellier France
and PNR of Algeria. The authors thank Adnane Bargaz for his help and Sabrine Saidi for her technical
assistance.
REFERENCES
Adelson, P.A., et al. (2008). Plant Soil. 312: 129-138.
Bargaz, A., et al. (2010). Plant Soil. 61: 602-611.
Isoi, T,. et al. (1991). Sci. Plant Nutr. 37: 559-563.
Laguerre, G., et al. (1992). FEMS. Microbiol. Ecol. 101: 17-26.
Olivera, M., et al. (2004). Physiol plant. 121: 79-84.
Tajini, F., et al. (2009). BMC Plant Biol. 9: 73-81.
Vadez, V., et al. (1996). Plant Physiol. Biochem. 34: 871-878.
Session III
SIII-CP-10
The genotypic variability of beans affects the growth parameters, the
nodulation and the microorganisms which initiate nodulation.
Benadis, Ch.1, Bekki, A.1*, Abed, N.H.2, Ouzane, H.2, Irekti, H.2
1
Laboratoire de biotechnologie des rhizobia et amélioration des plantes LBRAP. BP n°16 Faculté des
sciences d’Oran. Algérie. 2 Division Biologie du sol. Institut National de la Recherche Agronomique
d'Algérie 2, Rue Frères Ouadek, El Harrach, Alger. Algérie.
*
[email protected]
ABSTRACT
The exploitation of rhizobia-legume symbiosis, the choice of suitable varieties of
legumes and reasoned inoculation will contribute to a better management of ecosystems
and ecological preservation of the environment and biodiversity. The practice of these
symbioses in rotation with cereal crops will reduce the need of chemical fertilizers and
lead to a good functional agroecosyteme complex for a sustainable agriculture. In our
study the economic outputs of this strategy in a targeted agroecosystems mass
production of pulses will be tested. In natura, six contrasting lines of bean (provided by
the International Centre for Tropical Agriculture. Colombia) and a local varietal, the
most planted by farmers, will be tested on 20 plots located in four different regions of
Ain Temouchent agroecosystem.
INTRODUCTION
Food legumes are of great importance due to their high nutritional value, making them
an essential component in the food and feed in the world. On the other hand, they are a
way to improve soil fertility and associated crops yields and thus are of interest in
establishing a sustainable agriculture. Despite their agronomic benefit, in Algeria they
are poorly cultivated and represent only 1% of the total UAA and 2-3% of the cultivated
grains, and the efforts given to culture surface intensification have often failed to
achieve the desired results. In the western region of Algeria, Ain Témouchent is
considered as the first legumes production granary in Algeria. One of the less cultivated
legumes, bean remains in fourth position after chickpea, faba bean and lens; farmers are
reluctant to this culture because of its low yields. It is in this context that our Bean
project is focused, in order to use different bean genotypes inoculated with rhizobia and
then to choose the most effective Rhizobium-legume symbiotic couples adapting to a
specific settlement for sustainable and organic agriculture.
Stations of Observation.
20 agroecosystem stations selected in Ain Témouchent, northwest of Algeria, are
targeted in four areas: Sidi Ben Adda, Hammam Bouhjar, Ain Tolba and Terga. In each
station, a plot is sown in multisite with testing contrasting 6147 lines: 115, 104, 83, 34,
29 and a local line (Loc).
Growth nodulation and yield Measuring.
12 plants are harvested by digging up roots in a volume of 20 cm square at the
flowering stage. Sampling is carried out in five different randomly chosen points in the
plot. On each plant, the aerial part is separated from the roots at the cotyledonary node.
The number of nodules, nodule dry and aerial part weight was measured after 48 hours
of drying in an oven at 70 ° C for each plant.
Analysis of soil and irrigation water.
At each station, soils are sampled at planting.
Session III
SIII-CP-10
The results (Figures 1 and 2) show that genotypic variability affects the growth
parameters, the nodulation and the microorganisms which initiate nodulation with
seeded lines at the targetted parcels (Vadez et al., 1996; Bargaz et al., 2010).
Nodulation is limited in In natura conditions (Figure 2) and plots where high dry matter
yield was observed also had a significant nodulation (Tajini et al., 2009). The
hierarchical classification similarity index grouped the physicochemical properties of
soils plots into two main groups (Figure 3). A large variability between plots shows that
even if the plots are in the same microclimate, other factors had an influence on the
physico-chemical characteristics such as T and previous crops.
AKNOWLEDGEMENTS
This work was supported by Oran University for the stay of BENADIS Chahinez in Borj cedria (Tunisia)
and Montpellier (France), the PNR of Algeria and financial support of LBRAP.
REFERENCES
Bargaz, A., et al. (2010). Plant Soil 61: 602-611.
Tajini, F., et al. (2009). BMC Plant Biol. 9: 73-81.
Vadez, V., et al. (1996). Plant Physiol. Biochem. 34: 871-878.
Session III
SIII-CP-11
Phosphoenol pyruvate phosphatase transcript in nodule cortex of
Phaseolus vulgaris.
Bargaz, A.1*, Lazali, M.2, Ghoulam, C.3, Drevon, J.J.2
1
Swedish University of Agricultural Sciences, Department of Biosystems and Technology, PO Box 103,
2
3
SE-230 53 Alnarp, Sweden. INRA, UMR Eco&Sols, 1 Place Viala, F34060, Montpellier, France.
Equipe de Biotechnologie Végétale et Agrophysiologie des Symbioses, Faculté des Sciences et
Techniques Guéliz, BP 549, 40000, Marrakech, Maroc.
*
[email protected]
ABSTRACT
Increases of acid phosphatases (APases) enzymes are among mechanisms which lead to
increase efficiencies both of N2 fixation and nodule respiration under P deficiency. Our
findings have revealed that activities and differential expression of phosphoenol
pyruvate phosphatase (PEPase) transcripts were positively correlated with increases
both of the rhizobial symbiosis efficiency (EURS) and nodule O 2 permeability. The
induced enzyme activity and the marked transcript localization of this APase in nodule
cortex would control nodule respiration and contribute to adaption of nodulated legumes
to low P availability.
INTRODUCTION
In nodules, expression of large number of Glycine max APase “GmPAP” genes was
mainly detected under P-deficiency (Li et al., 2011). This organ was reported to be
strongly enriched in highly tissue-specific genes (Libault et al., 2010; Severin et al.,
2010). Overall, most APases are nonspecific hydrolyzing Pi from a broad spectrum of
Pi mono-esters and may have different functions as described for a soybean APase gene
GmPAP3 whose expression alleviates oxidative stress caused by salinity and osmotic
constraint (Li et al., 2008). The involvement of APases in tolerance to various abiotic
and biotic constraints, led us to assume that PEPase may have a multiplicity of functions
as well as those related to phosphate metabolism and should provide new insights into
adaptation to P-deficiency. Thus, the present work hypothesizes that differential
expression in the nodule cortex, of PEPase may contribute to low P adaptation and may
be involved in the regulation of nodule respiration.
Nodules of about 3 mm diameter of each RIL corresponding to 50 ± 05 mg of nodule
fresh weight (FW) were carefully detached at 42 days after transplanting (DAT) and
immediately frozen in liquid nitrogen and stored at -80 °C until use for PEPase activity
assay. Sample preparation and fixation for in situ reverse transcription polymerase chain
reaction (RT-PCR) were set according to the protocol described by Molina et al. (2011).
The method involves in situ amplification of specific nucleic acid sequences on nodule
sections, followed by fluorescence detection of the localized PCR product via epifluorescence microscopy.
To our knowledge, this study is the first to reveal that the PEPase transcription was
induced by P-deficiency with differential expression among nodule tissues. The
differential localization of transcripts encoded for PEPase in the outer cortex and
infected zone under P-deficiency (Figure 1) opens new insights into understanding the
physiology of N2-fixing legumes as well as requirements for N2 fixation and regulation
of nodule permeability to O2 diffusion. Under P-deficiency, the correlation between
Session III
SIII-CP-11
PEPase enzyme activity or transcript localization in infected cells and EURS or N 2
fixation (Figure 2) suggest that this APase is involved in nodule metabolism that is
linked to N2 fixation and the overall N2-dependent growth of the legume in
hydroaeroponics. The marked increase in PEPase transcripts in the nodule cortex of Pdeficient nodules not only suggests an increase in intracellular Pi scavenging but also
opens up a challenge to understand whether such sub-localization is involved in the
scavenging of Pi from extracellular organophosphates.
Figure 1. In situ localization of PEPase transcripts
(green spots) in nodules of common bean RIL115
and RIL147 grown under a sufficient (250 P)
versus a deficient (75 P) P supply. InC, infected
cell; IC, inner cortex; IZ, infected zone; OC, outer
cortex; UC, uninfected cell; VT, vascular trace
parenchyma.
Figure 2. Efficiency of use of rhizobial symbiosis
of common bean RIL115 and RIL147 inoculated
with R. tropici CIAT899 and grown under a
sufficient (open circles) versus a deficient (filled
circles) P supply. Data represent individual values
of 14 replicates harvested at 42 DAT. Ndw,
Nodule dry weight.
ACKNOWLEGMENTS.
This work was supported by the FABATROPIMED project financed by Agropolis Fondation under the
reference ID 1001-009.
REFERENCES
Li, W.Y.F., et al. (2008). New Phytol. 178: 80-91.
Li, C., et al. (2011). Ann Bot. 109: 275-285.
Libault, M., et al. (2010). Plant J. 63: 86-99.
Severin, A.G., et al. (2010). BMC Plant Biol. 10: 160.
Session III
SIII-CP-12
P-deficiency increases phytase activity and O2 uptake per N2 reduced
in Phaseolus vulgaris L.
Lazali, M.1, 2, 3*, Abadi, J.1, Ounane, S.M.2, Bargaz, A.4, Pernot, C.1, Drevon, J.J.1
1
Institut National de la Recherche Agronomique (INRA), UMR 1222 Ecologie Fonctionnelle &
Biogéochimie des Sols et Agroécosystèmes, INRA-IRD-CIRAD-SupAgro. Place Pierre Viala, 34060
Montpellier, France. 2 Ecole Nationale Supérieure Agronomique (ENSA), Département de phytotechnie.
Avenue Hassan Badi, El Harrach 16200 Alger, Algérie. 3 Université de Khemis Miliana, Faculté des
Sciences de la Nature et de la Vie & des Sciences de la Terre. Route Theniet El Had, Soufay, 44225 Ain
Defla, Algérie. 4 Swedish University of Agricultural Sciences (SLU), Department of Biosystems and
Technology, Box 103, SE-23053 Alnarp, Sweden.
* [email protected]
ABSTRACT
To understand the mechanisms used by legumes to improve their PUE for SNF under Pdeficiency, six RILs of P. vulgaris were inoculated with Rhizobium tropici CIAT899,
and grown at two levels of P supply. Our results have revealed that the increases of
APases and phytases activities under P-deficiency varied among RILs of P. vulgaris and
were positively correlated with increases both of the nodule conductance to O2 diffusion
and the EURS. This may represent an adaptive mechanism for N 2-fixing legumes to
respond to low P availability, by increasing the utilization and the uptake of P for SNF .
INTRODUCTION
Phosphorous (P) is one of the most important macronutrients involved in many
physiological and biochemical processes in plants (Tran et al., 2010). However, P
concentration in soil solution is often low because of a strong P fixation by organic
compounds, free Fe or Al oxides. Therefore, low P availability is a major constraint to
plant growth and development. While the induction of APase is a universal response to
P-deficiency in plants (Duff et al., 1994), the physiological role of these enzymes in
nodule O2 permeability of N2 fixing legume is crucial but still not fully understood. In
this context, recent studies have reported that high expression of large number of PAP
genes in the nodules of Glycine max is considered as an adaptive mechanism to tolerate
P-deficient conditions (Li et al., 2012). The present work hypothesizes that the
differences among common bean RILs for phytase activity may be involved in the
regulation of nodule respiration and can contribute to low P soils adaptation.
This study was carried out using six recombinant inbred lines, namely RILs 147, 115,
104, 83, 34 and 29 obtained from a cross of two parental lines, namely BAT477 and
DOR364 originated from CIAT. Root nodules were induced by Rhizobium tropici
CIAT899, and grown in hydroaeroponic culture under P-sufficiency (250 μmol P
plant−1 week−1) versus P-deficiency (75 μmol P plant −1 week−1) supply. At 42 days after
transplantion, nodules of about 3 mm diameter of each RIL corresponding to 60 mg of
fresh weight were carefully detached to roots after measuring O2 uptake by nodulated
roots (Conr) and nodule conductance to O2 diffusion. Plants were harvested at the stage
of pod setting and separated in shoots, roots and nodules. After drying for 3 days at 70°
C, plant and nodule dry weight was measured and used to determine the efficiency in
use of the rhizobial symbiosis (Drevon et al., 2011).
Session III
SIII-CP-12
The increases of phytase activity (Figure 1A) in nodules under P-deficiency were
accompanied with an increase in their conductance to O2 diffusion (Figure 1B) and have
shown several genotypic variations among the tested common bean RILs. The positive
correlation between phytase enzyme activity and nodule O 2 permeability (data not
shown) substantiates a physiological role of phytase in regulating nodule O 2 diffusion.
Although, the induced of APase activity in plants during P-deficiency has been widely
recognized. Phytases might play a major role for internal plant metabolism rather than
for obtaining P from the soil phytate.
Furthermore, phytase expression in nodule cortex (data not shown) and the regulation of
the nodule respiration since this zone, more particularly the inner cortex, which is
postulated to be a physical barrier for the regulation of nodule permeability to gas
diffusion (Witty and Minchin 1998) and osmotic conditions (Schulze and Drevon
2005).
Figure 1. Phytase activity (A) and conductance to O2 diffusion (B) in nodules of six common bean RILs
inoculated with R. tropici CIAT899 and grown under sufficient (white) versus deficient (grey) P supply.
Data are mean and standard deviation of seven replicates harvested at 42 DAT.
In summary, the present study suggests that the higher increased of phytases activity in
the nodules of all RILs under P-deficiency, indicating that N2-fixing legumes can
enhance P utilization within the nodules to tolerate low P conditions.
ACKNOWLEGMENTS
This work was supported by the Great Federative Project FABATROPIMED of Agropolis Fondation
under the reference ID 1001-009.
REFERENCES
Drevon, J.J., et al. (2011). Proc. Environ. Sci.9: 40–46.
Duff, S.M.G., et al. (1994). Physiol. Plant. 90: 791–800.
Li, C., et al. (2012). Ann. Bot. 109: 275–285.
Schulze, J., and Drevon, J.J. (2005). J. Exp. Bot. 56:1779–1784.
Tran, H.T., et al. (2010). Plant Sci. 179: 14–27.
Witty, J.F., and Minchin, F.R. (1998). J. Exp. Bot. 49:1015–1020.
Session III
SIII-CP-13
Effect of inoculation with selected rhizobia on symbiotic nitrogen
fixation and Faba bean grains yield in north Tunisia agro-ecosystems.
Sifi, B.1, 2, 3*, Maazaoui, H.1, Drevon, J.J.2
1
Laboratore des Sciences et Techniques Agronomique, Institut National Reverche Agronomique de
Tunisie, rue Hédi Karray, 2080 Ariana, Tunisie. 2 Faculté des sciences de Bizerte, Tunisie. 3 INRA, UMR
Eco&Sols - Ecologie Fonctionnelle & Biogéochimie des Sols & des Agroécosystèmes, 2 Place Pierre
Viala, F34060, Montpellier, France.
* [email protected]
ABSTRACT
In order to improve of agro-ecosystem productivity based on cereal and legumes
rotation, fourteen Rhizobium strains were tested in hydroponic culture with faba bean
″Bachar variety″ in glasshouse. The selected rhizobia were used to inoculate faba bean
in thirteen farmers’ fields during two years. Inoculation with the different isolates
enhanced nodulation and improved production parameters. Furthermore, Mat strain was
the most preferment compared to the tested rhizobia. Results were confirmed by those
obtained experiments carried out in farmer’s fields. Inoculation of faba bean with ″Mat″
Rhizobium strain increased significantly nodulation and grains yield. The variability of
faba bean nodulation and yield was related to soils fertilities.
INTRODUCTION
North Tunisia agro-ecosystems were based on cereal monoculture. Introduction of
legumes and inoculation with specific Rhizobium strain can improve soil fertility and
productivity. In recent decades there has been a great deal of interest in enhancing
symbiotic nitrogen fixation during the production of legumes for sustainable agriculture.
Reduction of nitrogen fixation was due to soil fertility will often be related to either an
excess of soil nitrates or a deficiency of some essential plant growth nutrient. Moreover,
the majority of soils were found to have very small concentrations of soluble
phosphorus and this was related to poor nodulation and (Amijee and Giller, 1998).
Investigations conducted in Mediterranean areas revealed the absence of Rhizobium
leguminosarum in many soils (Dhamane et al., 1994). In such cases, the improvement
of nodulation and symbiotic nitrogen fixation requires not only the supply of fertilizers
but also the application of efficient rhizobial inoculants for legumes crops.
The experiment was designed for the evaluation of nodulation potential of a collection
of local Rhizobium strains modulating faba bean under hydroponic culture in glasshouse
conditions. The experiment was designed to assess the effect inoculation of faba bean
with preferment selected rhizobia in farmer’s fields in Mateur, north of Tunisia.
Inoculation has ameliorated nodulation, but the number and biomass of nodule was
variable a cording field and significantly correlated with soil fertility (Figure 1). Faba
bean nodulation number and quality was significantly variable in farmer’s fields.
Session III
SIII-CP-13
Nod.pl-1
Dry W g.pl-1
Figure 1: Effect of inoculation with rhizobia
on faba bean nodules number.
on faba bean root dry weight (g.pl-1)
Faba bean plant root dray weight was significantly variable a cording the farmer’s field
trials (Figure 2). The root dry weight was correlated with nodules number and variable
throw the farmer field’s zone.
Dray weight shoot of inoculated faba bean plant was significantly variable a cording the
farmer’s field trials. The shoot dry weight was correlated with nodules number and
variable throw the farmer field’s zone (Figure 3).
Dry W.g.pl-1
on faba bean shoot dry weight (g.pl-1).
g.m-2
on faba bean grains yield (g.m-2)
Faba bean average yield was significantly variable (200 to 420 g.m-2) from field to
other. Grains yield was not significantly correlated with plant nodulation root and shoot
biomass production.
REFERENCES
Amijee, F., and Giller, K.E. (1998). African Crop Sci. J. 6: 159-169.
Dahmane, A., et al. (1994) In: Facteurs limitant la fixation symbiotique de l’azote dans le bassin
méditerranéen. Montpellier, 6-8 avril 1994. Ed. INRA, Paris 1995 (Les colloques, No 77). p175-182.
Session III
SIII-CP-14
Integrating soil microbial community context in plant response to
mycorrhizal symbionts.
Carvalho, L. *, Correia, P., Meleiro, A.I., Carolino, M., Dionisio, F., Cruz, C.
Centre for Environmental Biology, Faculty of Sciences, University of Lisbon. Campo Grande, Bloco C2,
1749-016 Lisbon, Portugal.
*
[email protected]
ABSTRACT
We found that more beneficial mycorrhizal species to a host plant, in terms of
phosphorus and nitrogen nutrition, did not coincide when in isolation and when
interacting with a bacterial community, showing that plant growth response to
mycorrhizal fungi is rhizospheric-associated bacterial community dependent.
INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) are likely the most abundant plant mutualists,
improving phosphorus and nitrogen plant nutrition in exchange of carbon. Each plant
simultaneously associates with several species of AMF that vary in symbiont quality,
that is, in the delivery of benefit they provide to the host plant (Klironomos et al.,
2003), which could threaten the stability of mycorrhizal mutualisms. Recent research
indicate that plants can favor more-beneficial against less-beneficial AMF symbionts
(Bever et al., 2009; Kiers et al., 2011). Studies testing mycorrhizal symbiont quality
have been conducted using plants and AMF in isolation, but plant response to
mycorrhizal fungi may be biotic community context dependent (Hoeksema et al., 2010).
In nature, plants interact with other symbiotic and associative soil microbial symbionts
involved in plant nutrition, and these plant-AMF-microbe interactions may influence
plant response to each AMF species. Here, we investigated whether the nutritional
effects of different AMF species to the host plant (symbiont quality) are dependent on
soil biotic community context, particularly on the presence of plant-growth promoting
bacteria.
Maize plants were inoculated with one of four AMF species, alone or together with a
community of plant-growth promoting rhizobacteria involved in phosphorus and
nitrogen plant nutrition; non-mycorrhizal control treatments were included. Plant
growth, phosphorus and nitrogen content, nitrogen isotope rations (15N), and
mycorrhizal colonization were assessed after two months of growth under greenhouse
conditions.
Mycorrhizal benefits in providing phosphorus and nitrogen to maize plants vary with
AMF taxa, but variation in AMF identity effect was influenced by the presence of a
rhizospheric bacterial community (Figure 1). For instance, AMF species conferring
more nutritional benefits when inoculated alone, provided similar or lower benefits than
other species when AMF were inoculated together with a bacterial community.
Plant benefits from AMF relatively to non-mycorrhizal controls were generally highest
in nitrogen rather than in phosphorus contents, and synergistic effects between AMF
and rhizospheric bacteria depended on mycorrhizal species. Overall, shoot 15N
signature of maize plants was influenced by AMF taxa.
Session III
SIII-CP-14
Figure 1. Phosphorus and nitrogen content of maize plants inoculated with one from four AMF species,
alone or together with a bacterial community of six PGPR.
This study indicates that the presence of a soil microbial community differentially
influences symbiotic function of mycorrhizal fungal species to host plants, which can
have important evolutionary and ecological implications for the stability of the
mutualism between plants, and for the design of effective microbial consortia to be
applied in agriculture.
ACKNOWLEDGEMENTS
This work was supported by the project PTDC/AGR-PRO/115888/2009 and
SFRH/BPD/33633/2009 (to LC) of the Fundação para a Ciência e a Tecnologia of Portugal.
REFERENCES
Bever, J.D., et al. (2009). Ecol. Lett. 12: 13-21.
Hoeksema, J.D., et al. (2010). Ecol. Lett. 13: 394-407.
Kiers, E.T., et al. (2011). Science 333: 880-882.
Klironomos, J.N. (2003). Ecology 84: 2292-2301.
grant
Session III
SIII-CP-15
Identificación y caracterización de sistemas de transporte de hierro en
el hongo micorrícico arbuscular Rhizophagus irregularis.
Tamayo, E., Ferrol, N.*
CSIC, Profesor Albareda 1, 18008, Granada.
* [email protected]
RESUMEN
Se han identificado en el genoma del hongo micorrícico arbuscular Rhizophagus
irregularis genes que codifican proteínas implicadas en la síntesis de sideróforos y en la
vía reductiva de captación de hierro (Fe). La caracterización del gen RiFTR1 indica que
codifica una permeasa de Fe de alta afinidad cuya expresión se inhibe en presencia de
niveles tóxicos de Fe y que se expresa mayoritariamente en el micelio intrarradical.
INTRODUCCIÓN
Los hongos disponen de dos vías principales de captación de hierro, una dependiente de
sideróforos y otra reductiva, consistente en la reducción extracelular de Fe con la
subsecuente absorción del ion Fe2+ mediante un complejo de transporte de alta afinidad
formado por una oxidasa multicobre (FET3) y una permeasa de Fe (FTR1) (Johnson,
2008). Los hongos micorrícicos arbusculares (hongos MA), pertenecientes al phylum
Glomeromycota, establecen simbiosis mutualistas con las raíces de la mayoría de las
plantas. El hongo ayuda a la planta a adquirir nutrientes minerales del suelo y a cambio
la planta le cede al hongo compuestos carbonados. En condiciones de deficiencia en Fe,
las micorrizas arbusculares (MA) contribuyen a la adquisición de este metal por la
planta y en condiciones de toxicidad las plantas micorrizadas son más tolerantes al
exceso de Fe (Smith and Read, 2008). Sin embargo, los mecanismos de homeostasis de
Fe en MA se desconocen actualmente. El objetivo del presente trabajo es identificar y
caracterizar los sistemas de absorción de Fe en el hongo MA Rhizophaugus irregularis.
MATERIAL Y MÉTODOS
Material Biológico
Se utilizaron cultivos monoxénicos de R. irregularis desarrollados en placas de Petri
compartimentadas en presencia de diferentes dosis de Fe.
Identificación de genes
Se realizó mediante búsquedas en las bases de datos del transcriptoma y del genoma de
R. irregularis (Tisserant et al., 2012). Las secuencias génicas completas se obtuvieron
mediante RT-PCR y/o RACE.
Caracterización de RiFTR1
El análisis funcional se realizó en la cepa Δftr1 de Saccharomyces cerevisiae. Los
análisis de expresión génica se realizaron mediante técnicas de qRT-PCR.
Identificación de genes candidatos implicados en transporte de hierro
En el genoma de R. irregularis se han identificado genes que codifican proteínas
implicadas en la biosíntesis de sideróforos y en la vía reductiva de absorción de hierro.
En concreto, se han identificado dos genes, RiNRPS1 y RiNRSP2, que presentan
homología (>40%) a los genes SidC de varios hongos que codifican sintetasas de
péptidos no ribosomales, RiFRE1 que presenta la mayor homología con la reductasa
férrica cloroplastídica FRO7 de Arabidospis, RiFTR1 y RiFTR2 que codifican posibles
permeasas de Fe y los genes RiFET1-3 que presentan la mayor homología (>50 %) con
Session III
SIII-CP-15
oxidasas multicobre de varios hongos. Estos resultados sugieren que los hongos MA
podrían usar tanto la vía reductiva como la de producción y secreción de sideróforos
para la adquisición del hierro que necesitan para su desarrollo. Con el fin de caracterizar
ambas vías actualmente estamos caracterizando varios de los genes identificados y más
concretamente, los implicados en la vía reductiva.
Caracterización de la permeasa de Fe RiFTR1
La expresión del gen RiFTR1 en la cepa Δftr1 de S. cerevisiae, incapaz de crecer en
condiciones de deficiencia de Fe, revirtió el fenotipo mutante (Figura 1). Estos
resultados permiten concluir que RiFTR1 codifica una permeasa de Fe de alta afinidad.
Figura 1. Análisis funcional de RiFTR1 en S. cerevisiae.
El análisis de la regulación de la expresión de RiFTR1 en el micelio extrarradical de R.
irregularis por Fe mostró que la expresión de este gen se inhibía en presencia de altas
concentraciones de este metal. La observación de que la estructura fúngica en la que
este gen se expresa mayoritariamente es el micelio intrarradical sugiere que FTR1 está
implicada en la adquisición de hierro en la interfase simbiótica y, por tanto, una
participación del hierro en el desarrollo de la simbiosis MA.
AGRADECIMIENTOS
Este trabajo ha sido financiado por el Programa Nacional de Proyectos de Investigación del Ministerio de
Economía y Competitividad, Proyecto AGL2012-35611.
BIBLIOGRAFÍA
Johnson, L. (2008). Mycol. Res. 112: 170-183.
Tisserant, E., et al. (2012). New. Phytol. 193: 755-769.
Smith, S.E., and Read, D.J. (2008). Mycorrhizal Symbiosis. Academic Press. London, UK.
Session III
SIII-CP-16
Structure of the exopolysaccharide isolated from Sinorhizobium fredii
HH103.
Rodríguez-Carvajal, M.A.1*, Acosta-Jurado, S.2, Rodriguez-Navarro, D.N.3, CrespoRivas, J.C.2, Margaret, I.2, Sanjuán, J.4, Soto, M.J.4, Vinardell, J.M.2, Ruiz-Sainz, J.E.2,
Gil-Serrano, A.1
1
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. Sevilla, Spain. 2
Departamento de Microbiología Vegetal y Biología Molecular, Facultad de Biología, Universidad de
Sevilla. Sevilla, Spain. 3 Centro Las Torres-Tomejil (IFAPA). Alcalá del Río (Sevilla), Spain. 4 Estación
Experimental del Zaidín, CSIC, Departamento de Microbiología del Suelo y Sistemas Simbióticos.
Granada, Spain.
* [email protected]
ABSTRACT
The structure of the exopolysaccharide isolated from Sinorhizobium fredii HH103 has
been determined on the basis of mass spectrometry and NMR studies. Its repeating unit
consists of a branched nonasaccharide with the following structure:
This structure is identical to that found in the exopolysaccharide isolated from
Rhizobium sp. strain NGR234.
INTRODUCTION
Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate
legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules
of the indeterminate type. Different bacterial surface polysaccharides, such as K-antigen
capsular polysaccharides (KPS) and cyclic glucans (CGs) are relevant for these
symbiotic interactions. In this work, we present the determination of the structure of the
S. fredii HH103 exopolysaccharide (EPS).
Isolation and purification of EPS: Culture medium was treated with proteinase K,
concentrated up to 20% and three volumes of cold ethanol were added. The resulting
precipitate was separated by centrifugation, re-dissolved in water and purified by
dialysis against distilled water at 4 °C.
Monosaccharide composition (Chaplin, 1982) and their absolute configurations (Gerwig
et al., 1978) were identified on GLC-MS analysis. Samples of polysaccharide having
carboxyl groups previously reduced (Rodriguez-Carvajal et al., 2008) were methylated
by using the method of Ciucanu and Costello (2003). Hydrolysis, reduction with
NaBD4, and acetylation were performed according to Kim et al. (2006), yielding the
corresponding partially methylated and acetylated alditols (PMAAs), which were
analysed by GLC-MS.
Partial hydrolysis: A solution of the exopolysaccharide in 0.5M TFA was heated for 1 h
at 100 °C and then dialysed against water. The diffusate was concentrated and the
residue was chromatographed on Bio-Gel P-2 using water as eluent. Fractions
Session III
SIII-CP-16
containing low molecular-weight oligosaccharides were chromatographed on Silica-gel
with a gradient of acetonitrile/water to elute the oligosaccharides (Fauré et al., 2006).
Lithium degradation: The polysaccharide was stirred in ethylenediamine with lithium
metal as described in Fernández de Córdoba et al. (2008), and then purified by size
exclusion chromatography on Biogel P-2.
NMR: Samples were deuterium-exchanged several times by freeze-drying from D2O
and then examined in solution (1-5 mg/750 μL) in 99.98% D2O. Spectra were recorded
at 303 K and 333 K on a Bruker AV500 spectrometer operating at 500.20 MHz ( 1H) and
125.8 MHz (13C). Standard Bruker sequences (DQFCOSY, TOCSY, ROESY, HSQC,
HMBC, and HMQC-TOCSY) were used for 2D experiments (Parella et al., 2004).
Although standard monosaccharide analysis shows that the polysaccharide is composed
by D-Glc and D-Gal (5:2), the study of the oligosaccharides obtained by partial
hydrolysis indicates that it also is composed by D-GlcA. Methylation analysis allow
identifying the units: →3)-D-Galp, →3)-D-6-d2-Glcp (from a glucuronic acid unit),
→4)-D-Glcp (coeluting with →4)-D-6-d2-Glcp), →6)-D-Glcp, →4,6)-D-Glcp, and
→4,6)-D-Galp in a ratio close to 1:1:4:1:1:1. Furthermore, the occurrence of →3) and
→4)-linked uronic acids makes applicable the degradation of EPS with lithium in
ethylenediamine [8], yielding a polysaccharide (EPS-Li) that was studied by NMR.
Moreover, the study by NMR of the polysaccharide recovered after partial hydrolysis
allowed the identification of, besides the units previously identified in EPS-Li, a
trisaccharidic branch composed of two units of α-D-GlcA and a unit of α-D-Gal bearing
a pyruvate group as ketal. Finally, NMR of the undegraded exopolysaccharide also
shows signals from acetyl groups located on ~50% of non-reducing terminal galactose
residues (at O-2 and O-3).
This structure has the same carbohydrate backbone of the exopolysaccharide isolated
from Rhizobium sp. strain NGR234 (Djordjevic et al., 1986; Staehelin et al., 2006).
ACKNOWLEDGEMENTS
We thank the Andalusian Consejería de Innovación, Ciencia y Empresa (Proyecto de Excelencia
CVI2506) and the Spanish Ministerio de Educación y Ciencia (MEC) (AGL2009-13487-C04-02) for
financial support. We also thank the Centro de Investigación, Tecnología e Innovación (CITIUS) of the
University of Seville for NMR and MS facilities.
REFERENCES
Chaplin, M.F. (1982). Anal. Biochem. 123: 336-341.
Ciucanu, I., and Costello C.E. (2003). J. Am. Chem. Soc. 125: 16213-13219.
Djordjevic, S.P., et al. (1986). Carbohydr. Res. 148: 87-99.
Fauré R., et al. (2006). J. Org. Chem. 71: 5151-5161.
Fernandez de Cordoba, F.J, et al. (2008). Biomacromolecules 9: 678-685.
Gerwig, G.J., et al. (1978). Carbohydr. Res. 62: 349-357.
Kim, J.S., et al. (2006). Carbohydr. Res. 341: 1061-1064.
Lau, J.M., et al. (1987). Carbohydr. Res. 168: 219-243.
Parella, T. (2004). Pulse Program Catalogue. NMR Guide v4.0. Bruker Biospin.
Rodriguez-Carvajal, M.A., et al. (2008). Carbohydr Res 343: 3066.
Staehelin, C., et al. (2006). J. Bacteriol. 188: 6168-6178.
Session III
SIII-CP-17
First evidence for interlinked control of surface motility and biofilm
formation in Sinorhizobium meliloti.
Amaya-Gómez, C.V.1, Hirsch, A.H.2, Soto, M.J.1*
1
Dpto. Microbiología del Suelo y Sistemas Simbióticos. Estación Experimental del Zaidín (CSIC), 18008
Granada, Spain. 2 Department of Molecular, Cell, and Developmental Biology and Molecular Biology
Institute, University of California-Los Angeles, CA 90095-1606, USA.
*
[email protected]
ABSTRACT
Swarming motility and biofilm formation are surface-associated behaviors, which in
some bacteria are subjected to inverse co-regulation. In Rhizobium, these two opposite
lifestyles are largely unexplored. In the current study, we investigated whether factors
known to influence swarming motility in Sinorhizobium meliloti have an impact on its
capability to form biofilms. We show that siderophore rhizobactin 1021 (Rhb1021),
together with flagella, are required for both surface motility and biofilm formation. In
addition, our results support the existence of control mechanisms that inversely coregulate these two different lifestyles in S. meliloti to allow for optimal plant root
colonization and in which iron, and the fadD and rirA genes are involved.
INTRODUCTION
The majority of bacteria spend most of their lifecycle associated to surfaces behaving
either as motile cells or sessile communities named biofilms. Swarming is a mode of
surface translocation dependent on rotating flagella and is characterized by the rapid and
coordinated movement of multicellular groups of bacteria. Several studies have revealed
the existence of a link between biofilm formation and swarming motility in pathogenic
bacteria (Verstraeten et al., 2008). However, little information is available about these
opposite surface-associated processes in rhizobia. Our previous work revealed that S.
meliloti Rm1021 and GR4 strains show different swarming motility phenotypes.
Whereas Rm1021 spreads over semisolid surfaces using flagella-dependent andindependent mechanisms, GR4 is non-motile under the same conditions (Nogales et al.,
2010, 2012). In both strains, a mutation in the fadD gene promotes surface translocation
by yet unidentified mechanisms (Nogales et al., 2010). A transcriptomic analysis of S.
meliloti under swarming-inducing conditions led to the discovery that biosynthesis of
the siderophore rhizobactin 1021 (Rhb1021) is essential for surface translocation of
Rm1021 (Nogales et al., 2010, 2012). Moreover, the global iron response regulator
RirA and levels of iron in the media, were shown to participate in the control of
swarming in Rm1021. The aim of this work was to investigate the existence of a
possible connection between swarming motility and biofilm formation in S. meliloti by
comparing the biofilm formation ability of GR4 and Rm1021 and analyzing the impact
of iron and genes such as fadD, rhb, and rirA on the development of biofilms.
S. meliloti wild-type strains GR4 and Rm1021, fadD mutants of GR4 and Rm1021
(GfadD and RmfadD, respectively) and rhbA and rirA mutant derivatives from Rm1021
were analyzed for biofilm formation on two different abiotic surfaces (PVC and glass)
and on alfalfa roots. Biofilm formation on abiotic surfaces was assessed by growing
cells in Minimal Media (MM). The biofilm formed on PVC was stained with crystal
violet and quantified by measuring the optical density. Biofilm formation on glass and
alfalfa roots was studied by confocal laser scanning microscopy (CLSM) using green
fluorescence protein (GFP)-labeled strains.
Session III
SIII-CP-17
The defects in biofilm development on glass shown by the rhbA mutant regardless of
iron concentration (Figure 1A), demonstrate that siderophore Rhb1021, which is
essential for Rm1021 surface motility, plays an important role in biofilm formation and
it is not exclusively related to iron acquisition. The surfactant properties inherent to
Rhb1021 might be responsible for its function in the two surface-associated phenotypes.
In addition, several results obtained during this study provide the first evidence in
Rhizobium of an inverse co-regulation of surface motility and biofilm formation: i)
Strain GR4, which exhibits stricter control over surface motility under laboratory
conditions, is more efficient in biofilm establishment on abiotic and root surfaces than
strain Rm1021, which exhibits greater surface motility (Figure 1A, and C); ii) fadD and
rirA mutations, which are known to promote S. meliloti surface translocation, interfere
with the development of mature biofilms on glass surfaces, regardless of iron levels
(Figure 1A); and iii) high-iron conditions that inhibit swarming motility in S. meliloti
promote biofilm formation by inducing the development of thicker and sponge-like
structured biofilms (Figure 1A, and B). S. meliloti fadD, rhb and rirA mutants were
affected in plant root colonization (Figure 1C). These results indicate that components
essential for biofilm formation and control mechanisms that inversely co-regulate
swarming motility and biofilm development in S. meliloti participate to allow for
optimal plant root colonization.
Figure 1. A) CLSM images showing the thickness of 3- and 10-day-old biofilms developed by GR4 or
Rm1021 and their derivative mutant strains on chambered cover glass slides in response to low (22 µM
FeCl3) and high (220 µM FeCl3) iron availability. The biofilm thickness is represented by the xz planes.
Structured biofilms developed in MM containing 220 µM of FeCl 3 are indicated with an asterisk (*). B)
CLSM images showing the architecture of 3-day-old biofilms developed by GFP-labelled GR4 cells on
glass after growth in MM containing different concentrations of FeCl3. Bars, 15 µm. C) CLSM images of
biofilms established on alfalfa root surfaces 3 dpi. Bars 70 µm.
ACKNOWLEDGMENTS
This work was supported by projects BIO2007-62988 and BIO2010-18005 of the Spanish MICINN.
REFERENCES
Verstraeten, N., et al. (2008). Trends Microbiol. 16: 496-506.
Nogales, J., et al. (2010). BMC Genomics 11: 157
Nogales, J., et al. (2012). J. Bacteriol. 194: 2027-2035.
Session III
SIII-CP-18
Symbiotic phenotype of different rhizobial species on Lotus japonicus
Gifu and Lotus burttii.
Rodríguez-Navarro, D.N.1*, Temprano, F.1, Velázquez, E.2, Ruiz-Sainz, J.E.3
1
IFAPA, Centro Las Torres-Tomejil. 41200-Alcalá del Río, Sevilla (España). 2 Departamento
Microbiología y Genética, Universidad de Salamanca, 37007 Salamanca (España). 3 Departamento de
Microbiología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla (España).
*
[email protected]
ABSTRACT
We have study the symbiotic behavior of strains belonging to Rhizobium,
Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium on Lotus japonicus Gifu
and L. burttii species. Extra-slow-growing bradyrhizobia were also included. In
comparison with L. japonicus plants, L. burttii nodulated with a wider range of rhizobial
species. These differences in nodulation range might be related to the fact that bacterial
entry through root-epidermis cracks might be more common in L. burttii than in L.
japonicus (see poster Rodríguez-Navarro et al.: “Lotus japonicus Gifu and L. burttii
responses to inoculation with a collection of S. fredii HH103 mutants affected in
symbiotic signals”).
INTRODUCTION
Lotus japonicus is the model legume currently used for studying the formation of
determinate (spherical) nodules. S. fredii HH103 is a fast-grower microsymbiont of
soybean that also forms nitrogen fixing nodules with Lotus burttii (Sandal et al., 2012).
This strain, however, only forms pseudonodules and ineffective white nodules with L.
japonicus Gifu, indicating that L. burttii and L. japonicus show clear differences in
symbiotic partner preference. In addition, Mesorhizobium loti appears to be an
infrequent microsymbiont of Lotus spp. and rhizobial strains belonging to
Mesorhizobium spp. and to Rhizobium spp. have been isolated from Lotus spp. nodules
(Estrella et al., 2009). All these facts prompted us to investigate a set of Rhizobium,
Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium strains for their
symbiotic capacity with L. japonicus and L. burttii.
Surface sterilization of L. japonicus Gifu and L. burttii seeds and nodulation tests in
square plastic boxes were carried our as described by Sandal et al. (2012).
Results of this work are presented in Table 1. Although there are previous works
describing rhizobial strains isolated from nodules of Lotus spp. plants growing in
different soils of various countries (Lorite et al., 2010) to our knowledge this is the first
attempt to evaluate the symbiotic capacity of a range of rhizobia on the model legume L.
japonicus. Our results show that a wide variety of strains belonging to Rhizobium,
Sinorhizobium (Ensifer), Mesorhizobium, and Bradyrhizobium isolated from legume
species non-related with the Lotea tribe were also able to induce L. japonicus and /or L.
burttii responses to inoculation. These root responses varied from only swellings or
pseudonodules to nitrogen fixing (Fix+/- or Fix+) nodules.
Session III
SIII-CP-18
Table 1. Lotus japonicus and L. burttii responses to inoculation with different Rhizobium, Sinorhizobium
(Ensifer), Mesorhizobium and Bradyrhizobium strains.
Genus
Specie
Strain
L. japonicus
L. burttii
leguminosarum
W290/TA1
Nod+, Fix+/Nod+, Fix+/bv trifolii
leguminosarum
ISL9
NodNod+
bv. viciae
giardinii bv
H152T
Nod+, FixNod+, Fix+/giardinii
etli
CE3
Nod+, Fix+/Nod+, Fix+/Rhizobium
gallicum
R602T
nd
Nod+, Fix+/indigoferae
CCBAU71714T
NodNod+/-, Fixhainanense
CCBAU57015T
NodNod+/-, Fixloessense
CCBAU71903T
NodNod+/-, Fixundicola
NodNod+/- Fix+/sullae
CC1337
Nod+/-, Fixnd
Rhizobium species that are Nod- with L. japonicus and L. burttii: R. galega
HH103
Nod+, FixNod+/Fix+
Sinorhizobium
fredii
SMH12
Nod+, Fix+
Nod+, Fix+
NGR234
Nod+/-, FixNod+/-, FixSinorhizobium species that are Nod- with L. japonicus and L. burttii: S. meliloti,
S. medicae, S. kostiense, S. morelense
australicum
LMG24608T
Nod+/-, FixNod+, FixMesorhizobium
loti
NZP2235
Nod+/Fix+
Nod+/Fix+
plurifarium
LMG11892T
NodNod+, Fix+ (1)
temperatum
CCBAU11018T
Nod+, FixNod+, Fix+ (1)
Mesorhizobium species that are Nod- with L. japonicus and L. burttii: M. amorphae, M. camelthorni,
M. chacoense, M. robiniae.
Bradyrhizobium
canariense
BTA-1T
nd
Nod+, Fix+
jicamae
nd
Nod+
liaoningense
nd
Nod+
pachyrhizi
PAC48T
nd
Nod+, Fix+/Bradyrhizobium species that are Nod- with L. japonicus and/or L. burttii: B. japonicus, B. elkanii, B.
spp. (peanut), B. betae.
Nod-: absence of macroscopic root responses. Nod+/-: swellings, pseudonodules or a few white nodules
are formed. Nod+: nodules of normal external morphology are formed. Fix-: absence of nitrogen fixation.
Fix+/-: weak or very weak nitrogen fixation. Fix+: presence of effective pink nodules. (1): acetylene
reduction was measurable. nd: not determined
ACKNOWLEDGMENT
This work was supported by grants of Andalusia Government, P07-CVI-02506 and P11-CVI-7500. We
thank Dr. Niels Sandal for providing Lotus seeds.
REFERENCES
Estrella, M.E., et al. (2009). Appl. Environ. Microbiol. 75: 1088-1098.
Lorite, M.J., et al. (2010). Proceedings XIII-SEFIN, pp.23-24, Zaragoza (Spain).
Madsen, L.H., et al. (2010). Nature. Doi: 10.1038/ncomms1009.
Sandal, N., et al. (2012). DNA Res. 19: 317-323.
Session III
SIII-CP-19
Los efectores secretados a través del T3SS suprimen la respuesta de
defensa en soja inducida por Ensifer (=Sinorhizobium) fredii HH103.
Cubo, M.T.*, Jiménez-Guerrero, I., Pérez-Montaño, F., Ollero, F.J., López-Baena, F.J.
Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla. Avda. de Reina Mercedes,
6.41012-Sevilla, España.
* [email protected]
RESÚMEN
La función de los efectores secretados a través del sistema de secreción de tipo 3 (T3SS)
simbiótico es aún desconocida. Por su similitud con efectores de patógenos, los
efectores rizobianos deberían también ser capaces de suprimir respuestas de defensa de
la leguminosa para favorecer la infección. Este trabajo pretende determinar qué
respuestas defensivas se disparan en la soja en presencia de un rizobio compatible,
Ensifer fredii HH103, y si la presencia del T3SS hace que esta estirpe sea capaz de
nodular esta planta más eficientemente.
INTRODUCCIÓN
Muchas bacterias patógenas Gram-negativas utilizan el T3SS para inyectar efectores al
interior de la célula hospedadora e inducir la enfermedad. Las plantas se defienden
induciendo una respuesta de defensa mediada por el reconocimiento de ciertas
moléculas del patógeno. Los efectores alterarían rutas de señalización en el hospedador
para suprimir estas defensas. Sin embargo, algunos efectores son reconocidos por
receptores específicos, disparándose una respuesta de defensa más fuerte que bloquea la
infección. Los mecanismos de defensa de las plantas incluyen la acumulación de calosa
y la producción de ROS, NO y proteínas PR y suelen estar asociados a la producción de
hormonas vegetales como el ácido salicílico (SA), ácido jasmónico (JA) y etileno (ET).
E. fredii HH103 (desde ahora HH103) es una estirpe de amplio rango de nodulación que
posee el T3SS y que es capaz de secretar una serie de proteínas, denominadas Nop, al
medio extracelular en respuesta a flavonoides. El papel de estos efectores en la
simbiosis es aún desconocido aunque algunos se han caracterizado bioquímicamente y
se ha demostrado que juegan un papel importante en la nodulación de ciertas
leguminosas.
Al inicio de la infección los rizobios son reconocidos como patógenos, pero la respuesta
de defensa es débil y transitoria. Sin embargo, en la interacción simbiótica HH103-soja,
se observa un aumento de la expresión del gen GmPR1 en la raíz en presencia de esta
bacteria. Esta expresión aumenta tras inocular con un mutante que no secreta Nops.
Es de destacar que se ha observado una restricción en la nodulación de la soja
dependiente del T3SS. Un ejemplo sería la especificidad de cultivar que impide la
nodulación de variedades americanas de soja por sólo algunas estirpes de E. fredii.
MATERIAL Y MÉTODOS
Los ensayos de nodulación en soja cv. Williams82 se realizaron según el método
descrito por de Lyra et al. (2006). La expresión de genes de soja por qPCR se cuantificó
según el método descrito por López-Baena et al. (2009). La determinación de las
hormonas vegetales, mediante HPLC, y la detección de acumulaciones de calosa se
realizaron siguiendo cualquier de los métodos descritos en la literatura.
López-Baena et al. (2009) determinaron que el gen de soja PR1 se inducía en raíces
inoculadas con HH103 en los estadíos iniciales de la nodulación. Esta inducción era
Session III
SIII-CP-19
mayor en ausencia de proteínas Nop. Para determinar si esta respuesta de defensa se
asociaba a una reducción en el número de nódulos se realizó una cinética de nodulación
que determinó que las plantas inoculadas con un mutante en el T3SS sufrían un retraso
claro en la formación de nódulos. Este menor número de nódulos se correlacionaba con
una menor expresión de los genes NIN, indicando que se estaban formando un menor
número de tubos de infección.
En este trabajo se estudió la expresión de genes relacionados con la patogénesis como el
gen PR1 y dos peroxidasas a 2, 4 y 8 d.p.i. La expresión de PR1 volvió a ser mayor en
raíces de plantas inoculadas con el mutante en T3SS pero en este caso, y a diferencia de
los resultados previamente publicados, se aumentó la densidad celular del inóculo. Este
aumento en el número de células provocó un adelanto en la respuesta de defensa. Las
dos peroxidasas estudiadas tuvieron un pico de expresión a 2 d.p.i. y posteriormente fue
decreciendo. Al igual que ocurría con la expresión de NIN, la transcripción de estos
genes fue menor en plantas inoculadas con el mutante en el T3SS, indicando que
probablemente se estuviera induciendo la formación de un menor número de nódulos.
La expresión de PR1 es dependiente de SA por lo que se estudió si se estaba
produciendo un aumento de la concentración de esta hormona en las raíces. Además de
SA, se cuantificaron otras hormonas vegetales como el ile-JA y el ácido abscísico
(ABA) a 2, 4 y 8 d.p.i. Los resultados indicaron un aumento en la concentración de SA
en las plantas inoculadas con el mutante en el T3SS, indicando que efectivamente un
aumento en la expresión de PR1 se asociaba a un aumento en la concentración de SA.
Por otro lado, los niveles de JA se mantuvieron similares en todos los tratamientos a 2 y
4 d.p.i. Sin embargo, a los 8 días se produjo un aumento drástico en la concentración de
la hormona en las plantas inoculadas con la estirpe parental HH103. En cuanto al ABA,
a pesar de no observarse diferencias entre tratamientos a los diferentes tiempos, sí hubo
un aumento claro de su concentración a los 2 días para posteriormente ir disminuyendo
con el tiempo.
Una vez determinado que se estaba produciendo una respuesta de defensa que estaba
siendo suprimida por los efectores de HH103, se intentó determinar qué molécula
bacteriana estaba induciendo esta respuesta de la planta. Los polisacáridos de superficie
suelen estar involucrados en la inducción de respuestas de defensa primarias en
patógenos por lo que estudiamos su papel en la inducción de la formación de depósitos
de calosa. Los resultados indicaron que si bien no había diferencias en la deposición de
calosa entre la estirpe silvestre y el mutante en el T3SS, en las raíces de plantas
inoculadas con los mutantes en superficie apenas se observaban depósitos. Esto podría
indicar que la superficie de la bacteria es responsable del reconocimiento inicial del
rizobio como agente patógeno.
AGRADECIMIENTOS
Este trabajo ha sido financiado con los proyectos AGL2009-13487-C04 del MEC y P11-CVI-7050 de la
Junta de Andalucía. El trabajo de Irene Jiménez-Guerrero ha sido financiado con un contrato predoctoral
del IV Plan Propio de la US.
REFERENCIAS
López-Baena, F.J., et al. (2009). Mol. Plant Microbe Interact. 22: 1445-1454.
Session III
SIII-CP-20
Pode Bradyrhizobium japonicum, em combinação com diferentes
densidades de plantio de soja alterar componentes do rendimento?
de Luca, M.J.1*, Nogueira, M.A.2, Hungria, M.2
1
Universidade Estadual de Londrina; Brasil, Londrina, PR. 2 Embrapa Soja, Brasil, Londrina, PR.
* [email protected]
RESUMO
A campo, baixas densidades de plantas de soja, de 80.000 pl./ha, estimulam a formação
de grãos por planta, mas não a produtividade; contudo, diferentes estirpes de
Bradyrhizobium não afetam esse parâmetro. Em casa de vegetação, densidades de
80.000 pl./ha incrementam o número de vagens/pl. em relação à densidade de 320.000
pl./ha, com diferenças entre estirpes.
INTRODUÇÃO
Baixas densidades de plantas resultam em incrementos na disponibilidade de luz na
parte inferior das plantas, onde a radiação é baixa e a abscisão de flores e vagens
pequenas é elevada, resultando em um aumento na retenção de vagens (Johnston et al.,
1969). Há relatos de que a relação infravermelho/vermelho afeta a ultra-estrutura dos
cloroplastos, a partição de carboidratos para as células, a eficiência fotossintética e a
concentração de vários metabólitos (Kasperbauer, 1987), bem como a nodulação e a
fixação biológica do nitrogênio (FBN) (Lie, 1964; 1969). O objetivo deste trabalho foi o
de verificar o efeito de diferentes densidades de plantio de soja, em combinação com
diferentes estirpes de B. japonicum, em componentes de rendimento.
MATERIAL E MÉTODOS
Foram realizados ensaios em casa de vegetação e a campo, em Londrina, PR, Brasil, na
estação experimental da Embrapa Soja. Utilizou-se a cultivar de soja BRS 284, de
hábito de crescimento indeterminado. Em ambos os experimentos foram avaliadas duas
densidades de semeadura, de 80.000 pl./ha e de 320.000 pl./ha. Sementes de soja foram
inoculadas com as estirpes de Bradyrhizobium japonicum SEMIA 5079 (=CPAC 15) e
CNPSo 2050, na dose de 1,2 milhões de células/semente. Foram incluídos controles não
inoculados sem N mineral e, a campo, também com N mineral.
No experimento em casa de vegetação foram utilizados vasos de 50 x 23 x 16 cm com
uma mistura de terra e areia (3:1) esterilizada. No tratamento simulando 80.000 pl./ha
cada vaso continha duas plantas separadas por 25 cm, enquanto no de 320.000 pl./ha,
oito plantas separadas por 6 cm. O delineamento experimental foi em blocos ao acaso,
com 30 repetições por tratamento. O ensaio foi coletado em R5. Avaliou-se o número
total de vagens por planta. Os ensaios de campo foram conduzidos por três anos (2010,
2011 e 2012), em parcelas de 4 m x 10 m, com desenho experimental em blocos ao
acaso, com seis repetições. Em V6 foram avaliados o número de grãos/pl. e o
rendimento; avaliaram-se, também, parâmetros de fixação biológica do nitrogênio,
como número (NN) e massa (M) de nódulos secos por planta e teor de N dos tecidos por
digestão sulfúrica. As análises estatísticas foram realizadas com o software Infostat (Di
Rienzo et al., 2009).
RESULTADOS E DISCUSÃO
No experimento em casa de vegetação, houve maior formação de vagens por planta na
menor densidade, bem como diferenças entre estirpes, sendo maior com a estirpe
CNPSo 2050 em ambas densidades (Tabela 1). Na maior densidade de plantas, os
tratamentos 4 e 6 foram iguais. Uma hipótese é de que baixas densidades e a estirpe
Session III
SIII-CP-20
CNPSo 2050 afetaram o balanço de CKs. Em soja, CKs podem incrementar a relação
fonte/dreno, promovendo a divisão celular nos ovários jovens e redirecionando o
movimento dos assimilados para os ovários em desenvolvimento, diminuindo a taxa de
aborto de vagens.
Tabela 1. Número de vagens por planta. obtido em ensaio em casa-de-vegetação. Embrapa Soja
(Londrina-PR)
Tratamento 1
T1
T2
T3
T4
T5
T6
113,3 b2
49,5 c
102,6 b
44,5 c
138,7 a
57,8 c
n° de vagens/pl.
1
Tratamentos: T1) testemunha (sem inoculação e sem N mineral), 2 pl./vaso; T2) testemunha 8 pl./vaso;
T3) SEMIA 5079, 2 pl./vaso; T4) SEMIA 5079, 8 pl./vaso; T5) CNPSo 2050, 2 pl./vaso; T6) CNPSo
2050, 8 pl./vaso.
2
Letras distintas indicam diferenças significativas (Teste de Fisher, p<0,10).
No experimento de campo, baixas densidades de plantas (80.000 pl./ha) estimularam a
formação de grãos por planta, mas não foram observadas diferenças no rendimento
(Tabela 2). Também não foram observadas diferenças entre estirpes. Em baixas
densidades houve um incremento nos parâmetros de NN/pl. e MNS/pl., mas não se
detectaram diferenças entre estirpes.
Tabela 2. N° grãos/pl.; teor de N na parte aérea [TEORN (%)], número de nódulos por planta (NN/pl.), e
massa de nódulos secos por planta (MNS mg/pl.) obtido em um ensaio conduzido no campo experimental
da Embrapa Soja (Londrina – PR).
Rendimento
Tratamento
(kg/ha)
N° grãos/pl TEORN (%)
NN/pl
MNS mg/pl
T1
2473 a1
343,6 a
3,2 a
81,8 a
264,8 a
T2
2798 a
127,5 b
3,1 a
54,8 b
222,0 bc
T3
2592 a
354,1 a
3,3 a
48,9 b
123,1 d
T4
2805 a
127,5 b
3,3 a
33,7 c
102,4 d
T5
2371 a
334,3 a
3,1 a
71,7 a
251,0 ab
T6
2655 a
135,8 b
3,2 a
46,4 b
196,7 c
T7
2427 a
329,4 a
3,0 a
70,1 a
242,4 ab
T8
2644 a
129,4 b
3,1 a
49,6 b
208,1 c
1
Tratamentos, T1) Testemunha sem inoculação, 80.000 pl/ha; T2) Testemunha sem inoculação, 320.000
pl/ha; T3) T + fertilizante nitrogenado (200 kg de N/ha, parcelados em duas aplicações), 80.000 pl/ha;
T4) T + fertilizante nitrogenado (200 kg de N/ha, parcelados em duas aplicações), 320.000 pl/ha; T5)
CNPSo 2050, 80.000 pl/ha; T6) CNPSo 2050, 320.000 pl/ha. T7) SEMIA 5079, 80.000 pl/ha; T8)
SEMIA 5079, 320.000 pl/ha.
2
Letras distintas indicam diferenças significativas (Teste de Fisher, p<0,10).
AGRADECIMENTOS
Trabalho executado com recursos de Embrapa Soja, INTA (Instituo Nacional de Tecnologia
Agropecuaria, Argentina) e CNPq-Repensa (562008/2010-1).
REFERENCIAS
Beaufils, E.R. (1973). Soil Science, Bulletin N° 1.
Di Rienzo, J.A., et al. (2009). InfoStat versión. FCA, Universidad Nacional de Córdoba.
Johnston, T.J,. et al. (1969). Crop Sci. 9:577-581.
Kasperbauer, M.J. (1987). Plant Physiol. 85: 350-354.
Lie, T.A. (1964). Wageningen University, Wageningen, 89 p.
Lie, T.A. (1969). Plant Soil 30:391-404.
Session III
SIII-CP-21
Effects of glyphosate-resistant gene and herbicides on biological
nitrogen fixation symbiotic efficiency and grain productivity of
soybean in Brazil.
Hungria, M.1, Mendes, I.C.2, Nakatami, A.S.1, 3, Reis-Junior, F.B.2, Fernandes, M.F.4*
1
Embrapa Soja, Londrina, PR, Brazil. 2 Embrapa Cerrados, Planaltina, DF, Brazil. 3 Pos-doc fellowship
from CNPq. 3 Embrapa Tabuleiros Costeiros, Aracaju, SE, Brazil.
* [email protected]
ABSTRACT
We reported the effects of transgenic glyphosate resistant soybean cultivars (GRC), glyphosate
and weed management strategies (glyphosate + GRC vs. conventional herbicides + non-GRC)
on biological nitrogen fixation (BNF), symbiotic efficiency (SyEf) and grain yield (SGY) in
soybean. Six variables related to BNF were pooled and analyzed as SyEf. Data were obtained in
field trials in six sites in Brazil and three growing seasons. Small effects of GRC and glyphosate
where observed on SyEf in some sites. SyEf did not differ between the two weed management
strategies. SGY was higher in the glyphosate + GRC management in three out of the six sites.
INTRODUCTION
Glyphosate-resistant soybean cultivars (GRC) were released in Brazil in 2003 and, in 2010,
have already accounted for 86% of the cultivated area in the country. In 2010, it was estimated
that the inoculation of soybean with N2-fixing bacteria led to savings of US$ 7 billion/year due
to the non-use of N fertilizers. The widespread use of glyphosate and GRC has raised concerns
whether the biological N fixation (BNF) would be affected. Results on the effects of glyphosate
or GRC on BNF are inconsistent (Bohm et al., 2009; Kremer and Means, 2009; Zablotowicz
and Reddy, 2004). When deleterious effects of glyphosate are observed on the BNF variables,
they have not been accompanied by decreases in soybean grain yields (Bohm et al., 2009). In
this work we reported the results of experiments carried out between 2003 and 2006 in six
major producing areas of Brazil. Our objective was to evaluate the effects of GRC, glyphosate
and weed management strategies on the BNF symbiotic efficiency and grain productivity in
soybean.
The experiments were set up under no-till in the 2003/2004; 2004/2005, and 2005/2006 growing
seasons at six sites in Brazil: Passo Fundo (RS); Ponta Grossa (PR); Londrina (PR); Uberaba
(MG); Planaltina (DF); and Luiz E. Magalhães (BA). Trials were conducted in a RBD, with 5
treatments x 3 cultivars, with 6 replicates. Treatments were 1) GRC + glyphosate; 2) GRC +
conventional herbicides; 3) non-GRC + conventional herbicides; 4) GRC + hand weed control;
5) non-GRC + hand weed control. Three pairs of cultivars, each including the non-GRC and its
nearly-isogenic GRC, were cropped in each site. Soybean was inoculated with Bradyrhizobium
elkanii SEMIA 587 and B. diazoefficiens SEMIA 5080. At the V4 stage, plants were evaluated
for nodule dry weight (NDW), and at R2, for NDW, shoot total N and N concentration, total Nureide and percentage of N-ureide. SyEf was described by these six variables pooled and
analyzed by multivariate techniques. SGY was determined at harvest. Means contrasts were
used to evaluate the effects of transgenic trait, type of herbicide in GRC, and weed management
strategies on SGY and SyEf. Multiresponse permutation procedures (MRPP, Mielke and Berry,
2000) were used to test for differences in SyEf between treatments.
RESULTS E DISCUSSION
Biological Nitrogen Fixation Symbiotic Efficiency (SyEf).
SyEf was affected by the type of cultivar (Contrast 1) in four out of six areas, and by the
type of herbicide (Contrast 2) only in Passo Fundo (Table 1). In all these cases, effects
were very small, as observed by the chance-corrected within-group agreements (A
Session III
SIII-CP-21
values) (see footnotes in Table 1). SyEf did not differ between the two weed
management strategies (Contrast 3, Table 1).
Table 1. Statistical significance (p) and effect size (A value)1 of means contrasts2 comparing transgenic
GR trait, type of herbicide and weed management strategy on BNF symbiotic efficiency in six areas in
Brazil.
p values (A values)
Site
Contrast 1
Contrast 2
Contrast 3
Passo Fundo
0.040 (<0.01)
0.005 (0.023)
0.069 (<0.01)
Ponta Grossa
0.100 (<0.01)
0.251 (<0.01)
0.418 (<0.01)
Londrina
0.003 (0.011)
0.073 (<0.01)
0.611(<0.01)
Uberaba
0.716 (<0.01)
0.470 (<0.01)
0.473 (<0.01)
Planaltina
0.009 (0.013)
0.484 (<0.01)
0.408 (<0.01)
L. E. Magalhães
0.047 (<0.01)
0.179 (<0.01)
0.554 (<0.01)
1
A value represents the chance-corrected within-group agreement. When A equals zero, in a scale from 0
to 1, the within-group heterogeneity of the samples equals that observed by chance.
2
Contrast 1 compares GR and non-GR cultivars; Contrast 2 compares glyphosate and conventional
herbicide; Contrast 3 compares GR-cultivars + glyphosate and non-GR cultivars + conventional
herbicide.
Grain Yield.
GRC reduced SGY only at Passo Fundo (-21 %). In GRC, the use of glyphosate resulted
in SGY increases (+ 8 to 33%) in four out of the six areas evaluated. SGY was higher in
the glyphosate + GRC-based weed management in three of the six regions (+10 to 12%)
(Table 2).
Table 2. Soybean grain yield as affected by gliphosate resistant cultivar (GRC) (Contrast 1, C1), herbicide
type (Contrast 2, C2) and weed management strategy (Contrast 3, C3) in six major producing areas in
Brazil
Passo
Ponta
Luis E.
Fundo Grossa Londrina Uberaba Planaltina Magalhães
C1 GRC
859
2545
2230
2957
3378
2550
Non-GRC
1094
2512
2249
3023
3442
2572
p
<0.01
ns
ns
ns
ns
ns
C2 GRC with glyphosate
1089
2747
2389
2939
3572
2766
GRC with conventional
herbicide
815
2474
2200
2931
3405
2448
p
<0.01
<0.05
<0.05
ns
ns
<0.05
C3 GRC + glyphosate
1089
2747
2389
2939
3572
2766
Non-GRC + conventional
herbicide
1067
2466
2178
2984
3387
2462
p
ns
<0.05
<0.01
ns
ns
<0.05
ACKNOWLEGMENTS
REFERENCES
Kremer, R., and Means, N. (2009). Eur. J. Agron. 31: 153-161.
Zablotowicz, R., and Reddy, K. (2004). J. Environ. Qual. 33: 825-831.
Bohm, G.M.V., et al. (2009). Soil Biol. Biochem. 41: 420-422.
Mielke, P.W., and Berry, K.J. (2000). Permutation methods: a distance function approach. Springer, NY,
USA, 352 pp.
Session III
SIII-CP-22
Diversidade genética de bactérias que colonizam nódulos radiculares
de Phaseolus vulgaris L. cultivado em campo e em casa de vegetação.
Oliveira-Francesquini, J.P.1*, Hungria, M.2, Glienke, C.1, Kava-Cordeiro, V.1, GalliTerasawa, L.1
1
Departamento de Genética, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba,
Paraná, Brasil. 2 Embrapa Soja, Londrina, Paraná, Brasil.
* [email protected]
RESUMO
Foi realizado o sequenciamento parcial dos genes 16S rRNA e glnII de seis isolados de
nódulos radiculares de feijoeiro comum (Phaseolus vulgaris L.), sendo três de plantas
cultivadas a campo (LGMB10, LGMB57 e LGMB58) e três de plantas cultivadas em
casa de vegetação (LGMB73, LGMB88 e LGMB99). Foi observada uma preferência de
colonização de acordo com o experimento avaliado.
INTRODUÇÃO
A análise da diversidade genética de bactérias capazes de nodular o feijoeiro comum
auxilia no processo de desenvolvimento de tecnologias de baixo custo para a produção
desta cultura, como o desenvolvimento de inoculantes contendo espécies fixadoras de
nitrogênio. A associação entre a planta e o microssimbionte obedece uma estreita
relação, regulada geneticamente. No entanto, bactérias nativas isoladas a partir de
nódulos de Phaseolus vulgaris mostram uma diversidade genética considerável,
sugerindo que várias espécies de Rhizobium podem se associar com o feijoeiro (Eardly
et al., 1995; Piñero et al, 1988). Ainda, variações na diversidade em condições de
campo e em solos diluídos foram observadas por Alberton et al. (2006) e Oliveira et al.
(2011), baseadas no estudo de marcadores moleculares. No presente trabalho, essas
diferenças foram demonstradas através da análise de sequências de DNA.
MATERIAL E MÉTODOS
Sequenciamento do gene 16S rRNA
A amplificação inicial e o sequenciamento do gene foram realizados de acordo com
Menna et al. (2006).
Sequenciamento do gene glnII
A amplificação inicial e o sequenciamento do gene foram realizados de acordo com
Stepkowski et al. (2005).
Análise filogenética
Para cada gene, as sequencias obtidas foram alinhadas com sequencias de linhagens já
identificadas, dentre elas as linhagens tipo. O alinhamento foi realizado no programa
MAFFT (http://mafft.cbrc.jp/alignment/server/) e a construção da árvore filogenética no
programa MrBayes (Ronquist and Huelsenbeck, 2003), utilizando o GTR (General
time-reversible) como modelo evolutivo.
Por meio da análise da árvore filogenética gerada com os dados do 16S rRNA (dados
não mostrados) não foi possível inferir uma classificação precisa para os isolados a
nível de espécie, uma vez que alguns deles formaram grupos com mais de uma
linhagem tipo. Por outro lado, a árvore gerada com os dados do glnII (dados não
mostrados) apresentou uma melhor resolução para as espécies avaliadas, dando um bom
suporte na análise das sequencias concatenadas (Figura 1).
Session III
SIII-CP-22
Apesar das linhagens de Rhizobium leguminosarum, R. etli e R. phaseoli não estarem
bem resolvidas nesta análise, pode-se observar que os isolados de plantas cultivadas em
casa de vegetação formaram um grupo com 100% de suporte com a espécie
R. miluonense e os isolados provenientes do experimento a campo agruparam (100% de
suporte na Inferência Bayesiana) com R. leguminosarum, o que oferece um forte indício
para sua classificação. Desta forma, a especificidade de colonização de acordo com as
condições ambientais fornece subsídios para o desenvolvimento de inoculantes
comerciais com linhagens que sejam capazes de se adaptar ao ambiente em que serão
utilizadas.
A caracterização genética através do gene 16S rRNA tem sido utilizada como ferramenta
de classificação para bactérias, no entanto sua análise isolada não é suficiente para
diferenciar espécies estreitamamente relacionadas. Sendo assim, outros genes têm sido
propostos para auxiliar na realização de análises filogenéticas, dentre eles o glnII
(Gevers et al., 2005). No presente estudo, foi possível realizar uma melhor
discriminação entre as espécies, baseada na análise conjunta das duas sequências
gênicas. No entanto, a avaliação de um maior número de genes, além de um estudo
polifásico, é recomendada para uma classificação mais precisa.
Figura 1. Árvore filogenética baseada nas sequencias de glnII e 16S rDNA de rizóbios isolados de
nódulos radiculares de feijoeiro comum e linhagens de referência. A árvore foi gerada no programa
MrBayes, utilizando GTR como modelo evolutivo.
AGRADECIMENTOS
Este trabalho teve apoio do CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico,
Brasil), CNPq-Repensa (56008/2010-1).
REFERÊNCIAS
Alberton, O., et al. (2006). Soil Biol. Biochem. 38: 1298-1307.
Eardly, B.D., et al. (1995). Appl. Environ. Microbiol. 61: 507-512.
Menna, P., et al. (2006). Syst. Appl. Microbiol. 29: 315-332.
Gevers, D., et al. (2005). Nat. Rev. Microbiol. 3: 733-739.
Oliveira, J.P., et al. (2011). World J. Microbiol. Biotechnol. 27: 643-650
Piñero, D., et al. (1988). Appl. Environ. Microbial. 54: 2825-2832.
Ronquist, F., and Huelsenbeck, J.P. (2003). Bioinformatics. 19: 1572-1574.
Stepkowski, T. et al. (2005). Appl. Environ. Microbial. 71: 7041-7052.
Session III
SIII-CP-23
Symbionts of Mimosa spp. in ultramafic soils in central Brazil.
Reis Junior, F.B.1*, James, E.K.2, Hertel Júnior, C.R.1, Lopes, A.A.C.1, Alves, M.R.P.1,
Souza, L.M.1, Baura, V.A.3, Mendes, I.C.1, Andrade, L.R.M.1
1
Embrapa Cerrados. Planaltina, Distrito Federal, Brazil. 2 The James Hutton Institute. Dundee, Scotland. 3
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná (UFPR). Curitiba,
Paraná, Brazil.
* [email protected]
ABSTRACT
In this study we investigated the diazotrophic symbionts associated with Mimosa in the
metal-rich soils from Barro Alto – GO, Brazil. Our results indicated Burkholderia spp.
as their symbionts in this peculiar environment, and that these symbioses could
probably play an important role in the restoration of ultramafic areas degraded by
mining activity.
INTRODUCTION
Recent studies have shown that species of Mimosa, native to the Brazilian Cerrados,
form symbiosis with diazotrophic bacteria known as β-rhizobia, particularly
Burkholderia spp., and seems to play a prominent role in N cycling in this ecosystem
(Reis Junior et al., 2010). These same studies suggest that environmental characteristics
rather than the host species are responsible for determining the distribution of
Burkholderia species. Previous reports have shown that acidic soils, which is not the
case of the ultramafic areas, could be more conducive for the survival of Burkholderia
symbionts. The neutral to basic pH is more favorable for Cupriavidus, another symbiont
of Mimosa spp., but not yet found in Brazil nodulating these plants in natural systems.
Cupriavidus is also known as a metal-loving genus (Klonowska et al., 2012), so it might
be expected to encounter endemic Mimosa spp. that have co-evolved with similar
bacteria in the ultramafic soils, as these contain an excess of metals such as cobalt,
chromium, copper, and especially nickel.
The aim of this study was to investigate the diazotrophic symbionts associated with M.
somnians and M. claussenii in the peculiar metal-rich soils of Barro Alto – GO, Brazil.
This is the first step in a bigger project that aims to give greater knowledge in the
symbiosis between diazotrophs and the legumes that grow in these soils with high metal
concentrations. In addition, the results obtained can provide information to assist in
restoration programs of areas affected by Ni mining.
Two sites were chosen for this study corresponding to different Ni bioavailabilities, and
where M. somnians and M. claussenii were observed to grow. Nodules were sampled
from Mimosa collected in the above sites, and these were taken for “rhizobial” isolation
and microscopy according to standard protocols (Reis Junior et al. 2010). Nodules that
were sub-sampled for microscopy were analyzed by immunogold labeling with
antibodies specific to the genera Burkholderia and Cupriavidus (Reis Junior et al.
2010). The 16S rRNA from two isolates of M. claussenii that morphologically
represented a large part of the isolates were amplified and sequenced for bacterial
identification. Symbiotic efficiency was assessed using plants of M. pudica grown in
pots containing a mixture of sand and vermiculite (50/50) and supplemented with
nitrogen-free nutrient solution. The dry weight of shoots and roots were measured at 45
days post inoculation in a greenhouse.
Session III
SIII-CP-23
(A)
(B)
(C)
Figure 1. Light micrographs of M. claussenii (A) and M. somnians (B) nodules from ultramafic soil that
have been immunogold-labeled with an antibody against B. phymatum, and of M. claussenii with an
antibody against C. taiwanensis (C).
The majority of the nodules examined from both sites were found to be effective in
appearance when examined under the light microscope. It was shown that almost all
Mimosa nodules reacted positively with the B. phymatum antibody (Figs. 1A and 1B).
No nodules reacted with the antibody against C. taiwanensis (Fig 1C).
The 16S rRNA sequencing of the two isolates of M. claussenii confirmed that the
isolates belong to the Burkholderia genus.
In general, inoculation promoted significant increases in shoot (up to 2100%) and root
(up to 400%) dry matter production of M. pudica, which indicates that the symbiosis
was efficient and shows its importance for plant growth (Figs. 2A and 2B).
(A)
(B)
Figure 2. M. pudica inoculated with isolates from M. claussenii (47) and M. somnians (43) from sites
with lower (A) and higher (B) Ni availability, respectively, compared with uninoculated control (1).
Recent studies have shown that as with nodulation, the degree of endemism apparently
has no effect on the general choice of Burkholderia as a symbiont by the Mimosa spp.
(Reis Junior et al., 2010). Our preliminary results indicate that even with soil
characteristics apparently not suitable for these bacteria, such as the ultramafic soils
from this study, Burkholderia remains as the main and preferred symbiont of Mimosa in
Brazil. Additionally, we can suggest that this symbiosis could be an important factor for
the restoration of ultramafic areas degraded by mining activity.
ACKNOWLEGMENTS
This work was supported by Anglo American Brazil, National Counsel of Technological and Scientific
Development (CNPq) and INCT-FBN.
REFERENCES
Klonowska, A. et al. (2012). FEMS Microbiol. Ecol. 81: 618-635.
Reis Junior, F.B., et al. (2010). New Phytol. 186: 934-946.
Session III
SIII-CP-24
Cloning, Expression and Characterization of a Chitinase class III from
Casuarina glauca nodules.
Graça, I.1, 2*, Liang, J.1, 3, Guilherme, M.2, Ferreira-Pinto, M.3, Ribeiro, A.1, 3, Pereira,
A.S.2
1
Centro BIOTROP/IICT, Oeiras, Portugal. 2 Requinte/CQFB, Departamento de Química, FCT/UNL,
Monte da Caparica, Portugal; 3ITQB/UNL, Oeiras, Portugal.
* [email protected]
ABSTRACT
Actinorhizal plants have become increasingly important as climate changes threaten to
remake the global landscape over the next decades. These plants are able to grow in
poor and disturbed soils and are important elements in plant communities worldwide.
Besides that, most actinorhizal plants are capable of high rates of nitrogen fixation due
to their capacity to establish root nodule symbiosis with N 2-fixing Frankia strains.
Nodulation is an ontogenic process that requires a sequence of highly coordinated
events. One of these mechanisms is the induction of defence-related events, whose
precise role is not clear. Here we report on the biochemical and biological
characterization of a class III chitinase from Casuarina glauca (CgCHI3) activated
specifically in nodules as compared with roots and leaves. According to our results, this
protein seems to be involved in nodule development, most likely in the formation of the
infection thread or in any other cell modification to accommodate the symbiotic
bacteria.
INTRODUCTION
Actinorhizal plants are a group of perennial dicots distributed over 8 different families
and 25 genera. These plants are essentially woody shrubs or trees (Benson and Silvester
1993). Ecologically, the majority of actinorhizal plants are pioneers on nitrogen-poor
open sites, being of great economical and ecological relevance in the recovery and
stabilisation of degraded ecosystems (Diem and Dommergues 1990).
Like in the case of legumes, actinorhizal symbiosis results in the formation of a new
organ, the root nodule, where bacteria are hosted and N 2 fixation takes place
(Pawlowski and Bisseling 1996). Nodulation is a highly complex and coordinated
process that involves the induction and suppression of several genes and their respective
products. Amongst others, defence genes are activated during nodulation, which may be
involved in nodule protection or development (Fortunato et al. 2007; Santos et al.
2010).
Chitinases are an important group of enzymes up-regulated during N2-fixing rootnodule symbioses where they might play multiple roles (reviewed by Santos et al.
2008). Here we report on the biochemical and biological characterization of CgCHI3, a
class III chitinase activated specifically in Casuarina glauca nodules induced by
Frankia, and discuss its possible role during the symbiotic process.
Recombinant protein expression.
The Open Reading Frame of cgchi3 (accession number: EF134410) was cloned into the
pET-28a expression vector (Novagen) and expressed in Escherichia coli strain BL21
(DE3) for protein expression. BL21 (DE3) cells containing the pRec-cDNA-ORF were
grown in 1L LB medium at 37ºC, during 2h. The expression of the recombinant protein
(CgCHI3) was then induced by IPTG during 3h. CgCHI3 was purified from inclusion
bodies at 15ºC as described by Kirubakaran & Sakthivel (2007) with slight
Session III
SIII-CP-24
modifications: β-mercaptoethanol was replaced by the redox buffer GSSG/GSH.
Biochemical assays included chitinase, chitosanase, glucanase, lisozymatic and
cellulase activities. Biological assays against bacteria (Frankia alni, Rhizobia, E.coli,
Paracoccus denitrificans and Bacillus subtilis) and phytopathogenic fungal species,
Botritys cinerea (Jatropha curcas), Colletotrichum gloeosporioides (Manihot
esculenta), Furasium oxysporum (Solanum lycopersicum), Trichoderma viridae
(Solanum lycopersicum), were performed with the disc diffusion method (Bauer et al
1996).
The recombinant CgCHI3 (32 kDa) was mainly produced as insoluble inclusion bodies.
Therefore, it was further solubilized, purified and refolded in an urea gradient. The
purified protein was unable to use chitin and chitosan as substrates, but has successfully
metabolized β-1,4-endoglucanase, β-1,3-glucan and lysozyme. Biological assays with
symbiotic Frankia and rhizobia did not affect the symbiotic process, suggesting that
CgCHI3 is not directly involved in the infection process. Additionally, CgCHI3 was
unable to impair or promote growth of pathogenic bacteria as well as pathogenic fungi.
According to Fortunato et al (2007), cgchi3 is an early nodulin gene, being expressed in
the meristem and in the uninfected cortical cells of young nodules, and is probably
associated with the process of nodule development. Our results support this hypothesis,
suggesting that most likely CgCHI3 is involved in the formation of the infection thread
or in any other cell modification to accommodate the symbiotic bacteria.
ACKNOWLEDGEMENTS
This work was supported by grants PPCDT/AGR/55651/2004 and PTDC/AGRFOR/4218/2012 from Fundação para a Ciência e Tecnologia (FCT), co-financed by the
European Fund FEDER, and by FCT grant SFRH/BD/41589/2007, co-financed by the
Portuguese PIDDAC program and European Social Fund, under the 3 rd framework
program.
REFERENCES
Bauer, A.W. et al. M (1996). Am. J. Clin. Pathol 45: 493-496.
Benson, D.R., and Silvester, W.B. (1993). Microbiological Reviews, Washington. 57: 297-319.
Diem, H.G., and Dommergues, Y. (1990). In:, The Biology of Frankia and Actinorhizal Plants, Academic
Press Inc., New York. Schwintzer C.R., Tjepkema J.D., eds, pp. 317-342.
Fortunato, A, et al. (2007). Physiol. Plantarum 130: 418-426
Pawlowski, K., and Bisseling, T. (1996) The Plant Cell 8: 1899-1913.
Santos, P., et al. (2010).. Symbiosis 50: 27-35. doi:10.1007/s13199-009-0031-0
Santos, P., et al. (2008). Plant Biotech. 25, 299-307. doi:10.5511/plantbio technology.25.299.
Singh, A., et al. (2007). Protein Expr. Purif. 56:100-109.
Session III
SIII-CP-25
Negative short-term salt effects on the soybean-B. japonicum
interaction and partial reversion by calcium addition.
Muñoz, N.1, 2, Rodriguez, M.1, Robert, G.1, 2, Lascano, R.1, 2*
1
Instituto de Fisiología y Recursos Genéticos Vegetales, Centro de Investigaciones Agropecuarias-INTA,
Camino a 60 Cuadras Km 5 y ½, Córdoba Argentina. 2 Cátedra de Fisiología Vegetal, Facultad de
Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 299,
Córdoba Argentina.
*
[email protected]
ABSTRACT
The short-term (2 h) effects of salt stress (50 and 150 mM NaCl), on early events of
soybean-B. japonicum interaction were analyzed determining the following parameters
in root hair with o without calcium addition: deformation, apoplastic superoxide radical
production (O2.-), root hair death and sodium/potassium ion content. We also analyzed
whether this short-term salt stress influenced later formation of crown and noncrown
nodules determining number and weight of nodules. The negative effect of salt stress on
root hair deformation, O2.- production, ionic homeostasis, root hair death and crown
nodulation were attenuated by the addition of 5 mM CaCl2.
INTRODUCTION
Soybean-rhizobia symbiotic interaction is severely affected by salt stress. Our group has
characterized early negative effects of salt stress on root hair deformation and on the
production/degradation of reactive oxygen species (ROS), which are fundamental
responses during early events of legume-rhizobia interaction. Decreased root hair
deformations and root hair death were observed 2 h post inoculation in saline
treatments. Some of the responses observed in combinations of salt stress and the
rhizobia are similar to pathogenic interactions. The addition of calcium, a common
agricultural practice to reverse salt effects, also has a positive effect on the soybeanrhizobia symbiotic interaction. The positive effect of calcium addition on soybean under
saline conditions is first reported.
Early effects of salt stress treatments (50 and 150 mM NaCl) were evaluated
determining deformation, apoplastic superoxide radical production (O 2 -), root hair death
and sodium/potassium ion content (Muñoz et al., 2012) with or whitout the calcium
addition (5 mM CaCl2). The influences of this short-term salt stress on later stages were
evaluated determining formation of crown and noncrown nodules (number and weight
of nodules).
Short-term salt stress on ionic homeostasis, root hair deformation and death: effect of
calcium addition.
Na+/K+ ratios in root hair was increased in a dose-dependent manner during saline
treatments and independently of the symbiont presence, and did not change in osmotic
treatments. This change in the Na + /K+ ratios is due to sodium influx from the
extracellular medium to the intracellular environment and not to an outflow of
potassium and these responses were partially reverted with the addition of calcium.
Root hair deformation was not affected under 50 mM NaCl treatment, although it was
significantly delayed. Under 150 mM NaCl, root hair deformation was significantly
reduced. Interestingly, these responses were partially reverted with the addition of
Session III
SIII-CP-25
calcium. Root hairs death was evaluated by observation of nuclear morphology. Loss of
nuclear integrity (chromatin condensation and DNA fragmentation) was observed in 50
mM saline treatments inoculated and 150 mM regardless of the symbiont presence. The
differences observed in the nucleus morphology, particularly associated with the
increase of nucleus with chromatin condensation and the decrease of nucleus with DNA
fragmentation in saline treatments supplemented with calcium, with respect to the
unsupplemented, indicates that the addition of calcium to saline treatments inhibited or
at least delayed death process (Figure 1). This inhibition or delay in the death progress,
given by calcium addition, could help to sustain the rhizobia infection and subsequent
nodule formation.
Short-term salt stress on nodulation: effect of calcium addition.
The negative effect of short-term saline treatments on crown nodule formation was
dose-dependent and was produced by the ionic component of salt stress, since the
number of nodules remained unaltered in the osmotic controls of these treatments
(Figure 2). This result suggests that ionic homeostasis of root hair during the early event
of symbiotic interaction has a key role on later events, such as nodule formation. The
addition of calcium helped to partially reverse the negative effects of salt on nodule
formation.
Figure 1. Percentages of nuclei with
chromatin
condensation
or
DNA
fragmentation after two hours under
different treatments: NaCl (50 or 150
mM), and NaCl (50 or 150 mM)
supplemented with 5 mM CaCl2, all of
them inoculated and noninoculated with
B. japonicum.
Figure 2. Nodule number in soybean
plants treated at germinated seed stage
for two hours with: NaCl (50 or 150
mM), Sorbitol (100 or 300 mM) and
NaCl (50 or 150 mM) supplemented
with 5 mM CaCl2 all of them
inoculated with B. japonicum.
ACKNOWLEDGEMENTS
This work was supported by grants from the Agencia de Promoción Científica y Tecnológica, Argentina,
and the Instituto Nacional de Tecnología Agropecuaria (INTA).
REFERENCES
Muñoz, N., et al. (2012). Environ. Exp. Bot. 78: 76-83.
Session III
SIII-CP-26
Expresión de supresores de muerte celular en raíces en cabellera de
soja: efectos bajo condiciones de estrés y en la interacción con
Bradyrhizobium japonicum.
Robert, G.1, 2*, Muñoz, N.1, 2, Lascano, R.1, 2
1
Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV-INTA), Camino a 60 Cuadras Km 5 ½,
X5020 ICA, Córdoba, Argentina. 2 FCEFyN, Universidad Nacional de Córdoba, Cátedra de Fisiología
Vegetal. Avda. Vélez Sarsfield 290. CP 5000, Córdoba, Argentina.
* [email protected]
RESUMEN
La expresión del supresor de muerte celular programada, Ced-9, del nemátodo
Caenorhabditis elegans en raíces en cabellera de plantas de soja (Glycine max), retrasó
los procesos de muerte celular inducidos por i) inoculación con Bradyrhizobium
japonicum bajo condiciones de estrés salino moderado (NaCl 50 mM) y ii) estrés salino
severo (NaCl 150 mM), los cuales mostraron características de procesos de muerte
ordenada y necrótica, respectivamente. Asimismo, paradójicamente la expresión de
Ced-9 inhibió la nodulación. En este trabajo se presentan evidencias de que estos
efectos podrían estar relacionados con alteraciones en el proceso de autofagia.
INTRODUCCIÓN
Los supresores de muerte celular de origen animal no presentan homólogos en el reino
vegetal, no obstante, la expresión de estos genes ectópicos afectan los eventos de
muerte/vida en las plantas. Un aspecto importante a destacar es que si bien se ha
evaluado la conservación de función de éstas proteínas entre los reinos, no se han
esclarecido los mecanismos a través de los cuales ejercen sus efectos en las plantas.
En este trabajo, evaluamos posibles mecanismos de acción a través de los cuales CED-9
podría estar ejerciendo sus efectos sobre los eventos de muerte y la nodulación en raíces
en cabellera de soja.
MATERIAL Y MÉTODOS
Las raíces en cabellera fueron obtenidas mediante la infección con Agrobacterium
rhizogenes K599 y fueron sometidas a los distintos tratamientos durante 3 h.
La determinación del contenido de malondialdehido (MDA) se realizó según Heath y
Packer (1968). Los niveles de ATP se determinaron según Rodriguez et al. (2010) y el
contenido de iones se cuantifico por HPLC.
El análisis de expresión de genes fue realizado mediante PCR tiempo real.
Efectos de la expresión del supresor de muerte celular Ced-9 en raíces en cabellera
sometidas a condiciones de estrés.
Raíces en cabellera fueron sometidas a condiciones de estrés inductoras de muerte,
previamente identificadas por nuestro grupo (Muñoz et al., 2012): inoculación con B.
japonicum en presencia de estrés salino moderado (50 mM NaCl) y estrés salino severo
(150 mM NaCl). Asimismo, estos eventos de muerte ocurrieron en presencia y ausencia
de generación de especies activas del oxígeno (EAO), respectivamente.
Se evaluaron los niveles de malondialdehido (MDA), los contenidos de ATP y de los
iones Na+, K+ y Ca2+, y se analizó actividad autofágica a través del estudio de las formas
lipidizadas de la proteína ATG8 (ATG8-PE) mediante western blot. Los resultados
obtenidos sugieren que bajo condiciones de inoculación en presencia de estrés salino,
los eventos de muerte celular en raíces en cabellera silvestres ocurren de manera
Session III
SIII-CP-26
ordenada (incrementos de ATP y de actividad autofágica, Figura 1) y presentan daño
oxidativo (altos niveles de MDA y generación de EAO). Contrariamente, bajo
condiciones de estrés salino severo, los niveles de ATP y MDA no mostraron
diferencias respecto al tratamiento control, pero presentaron alteraciones en la
homeostasis iónica, principalmente pérdida de K+, sugiriendo eventos de muerte celular
no ordenada o necrótica. Bajo estos tratamientos, la expresión de Ced-9 inhibió o al
menos retardó la muerte celular en las raíces en cabellera. Bajo las condiciones de estrés
por B. japonicum + 50 mM NaCl, las raíces Ced-9 no presentaron incrementos en el
contenido de MDA y la actividad autofágica se vio afectada (Figura 1); mientras que en
los tratamientos de 150 mM NaCl, la expresión de Ced-9 previno la pérdida de K+ y
Ca2+ e incrementó los contenidos de ATP.
Estos resultados demuestran el rol del supresor de muerte celular de origen animal en el
control de la homeostasis iónica y del metabolismo celular en las plantas, como así
también su participación en el control del proceso de autofagia.
Número de nódulos
Efectos de la expresión del supresor de muerte celular Ced-9 en raíces en cabellera
sobre la nodulación.
Por otro lado, se estudiaron los efectos de la expresión de Ced-9 sobre la nodulación de
las raíces en cabellera. Curiosamente, las raíces Ced-9 tuvieron inhibida su capacidad de
nodulación y mostraron una reducción significativa en los niveles de expresión de genes
claves del proceso de autofagia. Con el fin de evaluar la participación de autofagia en la
interacción simbiótica, se evaluó la nodulación en raíces en cabellera con silenciamiento
post-transcripcional de ATG6, proteína clave para la inducción del proceso. Estas raíces
presentaron una disminución dramática en el número de nódulos (Figura 2), postulando
así la importancia del proceso de autofagia en la interacción soja-B. japonicum.
60
50
40
30
20
10
0
k599 vacía
Figura 1. Evaluación de los niveles de proteína
ATG8 en raíces en cabellera K599-vacía y pBECed-9 mediante anticuerpos anti-AtATG8a.
ATG6i
Figura 2. Nodulación en raíces en cabellera
silvestres (K599 vacía) y con silenciamiento
post-transcripcional de Atg6 (ATG6i)
BIBLIOGRAFÍA
Rodriguez, M., et al. (2010). J. Plant Physiol. 167: 1137-1144.
Muñoz, N., et al. (2012). Environ. Exp. Bot. 78: 76-83.
Heath, R., and Packer, L. (1968). Arch. Biochem. Biophys. 125: 180-198.
Session III
SIII-CP-27
Efecto del contenido de taninos condensados (TC) sobre la formación
de nódulos en especies del género Lotus.
Escaray, F.J.1*, Estrella, J.1, 2, Paolocci, F.3, Damiani, F.3, Ruiz, O.A.1
1
IIB-INTECH/CONICET-UNSAM, Chascomús, Bs. As., Argentina. 2 Investigadora adjunta, Comisión
de Investigación Científica (CIC). 3 National Research Council, Plant Genetics Institute-Perugia (CNRIGV), Perugia, Italy.
*
[email protected]
RESUMEN
Cuatro genotipos de L. corniculatus cv. Leo, con diferente contenido de TC en raíces, se
inocularon con dos cepas de rizobios de estrecho rango de nodulación (Mesorhizobium
loti NZP2213 y Bradyrhizobium loti NZP2309). No se observó interacción entre los
genotipos y los tratamientos de inoculación para todos los parámetros medidos,
demostrando que el contenido de TC no es un factor determinante para la nodulación en
L. corniculatus.
INTRODUCCIÓN
Algunos autores han sugerido que la composición y los niveles de taninos condensados
(TC) presentes en raíces de plantas del género Lotus forman parte de los mecanismos de
determinación del rango de hospedante para la nodulación en la simbiosis mutualista
con rizobios (Pankhurst et al., 1982; Cooper and Rao, 1992). Sin embargo, estos autores
basaron sus resultados en estudios realizados con diferentes especies de Lotus, con lo
cual, no se puede descartar que hayan participado otros factores especie-específicos
además de los TC. Para poner a prueba la hipótesis de que los TC en el género Lotus,
son factores determinantes para la formación de nódulos, nos propusimos realizar
ensayos de nodulación con cepas de rizobios que tienen distinto rango de hospedante y
plantas isogénicas de L. corniculatus que presentan diferentes niveles de TC en sus
raíces.
MATERIAL Y MÉTODOS
Se utilizaron clones de cuatro genotipos de L. corniculatus cv. Leo transformados con la
construcción CaMV35S-Sn, que expresa el gen Sn, el cual codifica para un factor de
transcripción relacionado con la síntesis de TC (Robbins et al., 2003; Paolocci et al.,
2007). Dos de esos genotipos tienen bajo contenido de TC en raíces (Sn6 y Sn9), uno
tiene alto contenido de TC (Sn10) y uno transformado con el vector vacío que tiene
contenido intermedio (121.1). Los clones fueron cultivados durante 30 días bajo
condiciones controladas, en jarras Leonard que contenían arena:perlita (1:1) e irrigadas
con solución Evans 1x sin nitrógeno. Se aplicaron tres tratamientos: a) inoculación con
la cepa M. loti NZP2213, b) inoculación con la cepa B. loti NZP2309 y c) sin inocular
(controles); con 6 repeticiones biológicas. La inoculación se realizó 10 días después de
la siembra con 1 ml del cultivo de la cepa en YEM líquido (DO = 0,8). La tinción y
cuantificación de TC se realizó de acuerdo a la metodología descripta por Escaray et al.,
(2012).
Al finalizar el ensayo se pudo observar que todos los clones de los genotipos cultivados
bajo condiciones control o inoculados con la cepa B. loti presentaban un aspecto
clorótico en comparación a los inoculados con la cepa M. loti. Por otra parte, se observó
un mayor peso seco y longitud de tallo principal en aquellos genotipos inoculados con
la cepa M. loti; sin embargo, no observó interacción entre los factores genotipo x
tratamiento aplicado (p>0,5).
Session III
SIII-CP-27
Bajo la condición control ningún clon presentó nódulos. Por su parte, se observó que
todos los clones inoculados con la cepa M. loti, independientemente del nivel de TC que
expresaban en raíz, formaron nódulos de tamaño normal y color rosado. Por su parte,
los clones inoculados con la cepa B. loti formaron nódulos de menor tamaño, color
blanquecino y en número marcadamente mayor (Figura 1A y 1B). Considerando que el
aspecto rojizo de los nódulos se encuentra relacionado generalmente con su capacidad
fijadora de nitrógeno (Lodwig et al., 2003) y que la inoculación con cepas deficientes
para la fijación de nitrógeno (denominadas “Fix -“) genera un fenotipo generalmente
hipernodulante (Lohar and VandenBosch, 2005), el fenotipo de nodulación observado,
junto con los parámetros evaluados en la parte aérea, indican que la inoculación con la
cepa B. loti forma nódulos Fix- en los cuatro genotipos evaluados.
Independientemente del contenido basal de TC en raíces, se observó un mayor
contenido de estos compuestos cuando la inoculación se realizó con la cepa de B. loti y
un menor contenido en aquellas inoculadas con M. loti (Tabla en la Figura 1). Se pudo
observar a través de la tinción de TC en raíces noduladas, que existe una mayor
acumulación de estos compuestos en el nódulo formado por la cepa B. loti (Figura 1C y
D), resultados concordantes con el trabajo de Pankhurst et al. (1979).
Figura 1. Clones nodulados y tinción y contenido de TC en raíces con nódulos. Clones del genotipo Sn10
inoculado con M. loti NZP2213 (A y C) y B. loti NZP2309 (B y D). C y D: el color azul indica la
presencia de TC (tinción con DMACA:HCl). A la derecha se muestra la tabla con el contenido de TC en
raíces.
En función de los resultados obtenidos se puede concluir que el contenido de TC de L.
corniculatus no es un factor determinante del rango de hospedante para la formación de
nódulos, en la simbiosis mutualista con rizobios.
BIBLIOGRAFÍA
Cooper, J.E., and Rao, J.E. (1992). Plant Physiol. 100: 444-450.
Escaray, F.J., et al. (2012). Phytochem. Lett. 5: 37-40.
Lodwig, E.M., et al. (2003). Nature 422: 722-726.
Lohar, D.P., and VandenBosch, K.A. (2005). J. Exp.Botany 56: 1643-1650.
Pankhurst, C.E., et al. (1979). J. Exp.Botany 30: 1085-1093.
Pankhurst, C.E., et al. (1982). J. Gen. Microbiol. 128: 1567-1576.
Paolocci, F., et al. (2007). Plant Physiol. 143: 504-516.
Robbins, M.P., et al. (2003). J. Exp.Botany 54: 239-248.
Session III
SIII-CP-28
Alteraciones en el proceso de nodulación en ausencia y presencia de
fuente de nitrógeno externa de un mutante deficiente en el transporte
de nitrato de la leguminosa Lotus japonicus.
García-Calderón, M.1*, Pal’ove-Balang, P.2, Pérez, C.M.1, Betti, M.1, Marquez, A.J.1
1
Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Universidad de
Sevilla, C/ Profesor García González, 1, 41012, Sevilla. 2 Institute of Biology and Ecology, P. J. Šafárik
University, Mánesova 23, SK-04001 Košice, Slovak Republic.
*
[email protected]
RESUMEN
Diferentes estudios de nodulación se realizaron utilizando plantas silvestres y mutantes
resistentes a clorato (Ljclo1) a dos estadíos diferentes tras la inoculación con
Mesorhizobium loti. El proceso de nodulación de las plantas mutantes fue similar al de
las plantas silvestres en ausencia de fuente de nitrógeno externa, sin embargo, la
biomasa específica de nódulos respecto a la de raíz disminuyó considerablemente en
plantas mutantes cuando se cultivaron en presencia de nitrato o amonio en el medio de
riego. Estos resultados sugieren la existencia de alteraciones en el proceso de
señalización por nitrógeno relacionado con la nodulación de las plantas.
INTRODUCCIÓN
Estudios recientes han mostrado que una amplia variedad de mecanismos regulatorios
están implicados en la modulación por nitrógeno de la organogénesis del nódulo en
plantas leguminosas (Omrane and Chiurazzi, 2009). Si los efectos represivos del NO 3 se deben al ión por sí mismo, a los productos de su asimilación o a ambos, no está
claramente demostrado (Morere-LePaven et al., 2011). Un mutante resistente a clorato
(Ljclo1) fue identificado de la leguminosa modelo Lotus japonicus, capaz de crecer
considerablemente bien durante una semana en medios con una concentración entre 0,510 mM KClO3, mientras que las plantas silvestres no eran capaces de sobrevivir en estas
condiciones. Estudios previos habían indicado que dicho mutante posee niveles
normales de nitrato reductasa pero está afectado en el transporte de nitrato y/o clorato de
baja afinidad (Márquez et al., 2005). Estas plantas mutantes fueron utilizadas para
examinar el proceso de nodulación en ausencia y presencia de distintas fuentes de
nitrógeno.
MATERIAL Y MÉTODOS
El cultivo de las plantas así como la inoculación con rizobios se realizó como se
describe en García-Calderón et al. (2012). Las plantas control se regaron con medio
Hornum (Handberg and Stougaard, 1992). Se utilizaron medios conteniendo KNO 3 o
NH4Cl como única fuente de nitrógeno a las concentraciones indicadas en cada
experimento. 3 mM de KCl se añadió al medio de amonio para compensar la falta de K.
Se realizaron diferentes estudios de nodulación de plantas silvestres y mutantes a dos
estadíos de tiempo tras la inoculación (45 y 65 días). Se determinó el número total y el
peso fresco de nódulos, la relación de peso fresco de nódulos por número de nódulos así
como el peso fresco de nódulos por peso fresco de raíz. No se obtuvieron diferencias
significativas de estos parámetros ni a 45 ni a 65 días cuando el proceso de nodulación
se realizó en ausencia de fuente de nitrógeno externa. Sin embargo, sí se obtuvieron
diferencias cuando los experimentos se realizaron en presencia de nitrato o amonio. Se
observó una fuerte inhibición de la ratio peso fresco de nódulos por peso fresco de raíz
Session III
SIII-CP-28
tanto en plantas silvestres como mutantes Ljclo1, a todas las concentraciones
examinadas. Los resultados obtenidos en plantas mutantes mostraron una mayor
inhibición del proceso de nodulación a concentraciones de 1-2 mM de nitrato
comparado con plantas silvestres. De hecho, no se obtuvieron nódulos a 45 días en
plantas mutantes en presencia de 1 ó 2 mM de nitrato, mientras que sí se obtuvieron en
las plantas silvestres (resultados no mostrados). Además, los datos obtenidos después de
65 días tras la inoculación mostraban una inhibición del 70% en la ratio de peso fresco
de nódulos por peso fresco de raíz en plantas mutantes a 1 mM de nitrato, mientras que
en el caso de plantas silvestres esta inhibición fue sólo del 30% (Figura 1). El reducido
número de nódulos presentes a concentraciones de 2 mM de nitrato fue casi el doble en
plantas silvestres que en mutantes. También se obtuvieron diferencias en las plantas
mutantes Ljclo1 respecto a las plantas silvestres cuando se estudió el proceso de
nodulación en presencia de 1-2 mM de NH4+, aunque en menor grado que en presencia
de nitrato. Por tanto, las diferencias observadas en el proceso de nodulación no parecen
ser una respuesta específica del mutante en presencia de nitrato sino más bien es
indicativo de un defecto general de la señalización por N característica del mutante
Ljclo1. Experimentos de qRT-PCR han demostrado alteraciones en los niveles de
expresión de algunos genes implicados en la asimilación de nitrógeno en respuesta a la
presencia/carencia de nitrógeno, sugiriendo también un posible defecto en la
señalización por nitrógeno en las plantas mutantes Ljclo1.
0.8
WT
Ljclo1
Pfresco Nódulos/Pfresco raíz
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-
sin N
0,5mM NO
3
-
1mM NO
3
-
2mM NO
3
+
0,5mM NH
4
+
1mM NH
4
+
2mM NH
4
Figura 1. Ratio del peso fresco de nódulos por peso fresco de raíz de plantas silvestres y mutantes Ljclo1
de Lotus japonicus a 65 días tras la inoculación.
Los valores representan la media de siete réplicas biológicas ± error standard. indica diferencias
significativas entre plantas WT y mutantes, determinadas mediante el test de Student (P<0,05).
AGRADECIMIENTOS
Agradecemos la financiación de la Junta de Andalucía al grupo BIO-163 y proyecto CVI-P10-6368 así
como de la Unión Europea (proyecto EXPERT).
BIBLIOGRAFÍA
García-Calderón, M., et al. (2012). Mol. Plant Microbe Interact. 25: 211-219.
Handberg, K., and Stougaard, J. (1992). Plant J. 2: 487-496.
Márquez, A.J. et al. (2005). J. Exp. Bot. 56: 1741-1749.
Morere-Le Paven, M.C., et al. (2011). J. Exp. Bot. 62: 5595-5605.
Omrane, S., and Chiurazzi, M. (2009). Plant Signal. Behav.4: 1066-1068
Session III
SIII-CP-29
Phylogenetic diversity and structure of sebacinoid fungi associated
with plant communities along an altitudinal gradient.
Garnica, S.*, Riess, K., Bauer, R., Oberwinkler, F.
Eberhard Karls Universität Tübingen, Institute of Evolution and Ecology, Plant Evolutionary Ecology,
Auf der Morgenstelle 1, 72076-Tübingen, Germany.
*
[email protected]
ABSTRACT
The phylogenetic diversity and patterns in the structure of Sebacinales communities
from eight vegetation types with different degrees of disturbance along an altitudinal
gradient were studied. Ecosystem disturbances and environmental factors proved to be
the major forces influencing the diversity and structure of Sebacinales communities.
INTRODUCTION
Sebacinales form a wide spectrum of symbiotic associations with plants, including
orchioid, ericoid and arbutoid mycorrizas; ectomycorrizas; endophytic associations with
herbs and interactions with mosses (Oberwinkler et al. 2013). Only few of these fungi
have been isolated and cultivated on standard media showing a great potential for
enhancing plant growth under controlled conditions. In order to better understand the
diversity and how the Sebacinales communities are structured in relatively undisturbed
plant communities as well as communities with various degrees of disturbance (Garnica
et al. 2013), we addressed the following specific questions: What are the phylogenetic
diversity and structural patterns that characterise the Sebacinales communities in the
studied plant communities? Do soil factors, altitude and/or plant communities affect the
phylogenetic structure of the Sebacinales communities?
In our study, we analysed the phylogenetic diversity and structure of the Sebacinales
communities from 456 thalli or roots from eight different vegetation types under various
degrees of disturbance along an altitudinal gradient in the Bavarian Alps (Germany). To
amplify the 3' region of the 18S region, intergenic transcribed spacers (ITS1 and ITS2),
the 5.8S ribosomal subunit and the D1/D2 regions of the nucLSU of Sebacinales, we
used various sets of specific and universal fungal primers. Phylogenetic, diversity and
community structure analyses were performed using several approaches.
A total of 264 sebacinoid sequences spanning the ITS, 5.8S and D1/D2 domains
(approx. 1300 bp) of the 28S rRNA gene were amplified by mainly using a nested PCR
approach. a 97% sequence similarity, 73 molecular taxonomic units (MOTUs) of
Sebacinales were detected from 70 host species belonging to 44 plant families. A total
of 26 MOTUs proved to be singletons.
Although Sebacinales were shown to occur in low abundance in the roots of the plants,
these microorganisms are phylogenetically very diverse and widely distributed in the
ecosystems analysed. Ordination analyses showed that the land use, pH and humus
content strongly influenced the diversity and composition of Sebacinales. In most cases,
the Sebacinales communities in ecosystems with extreme soil conditions or with
extremely large anthropogenic use exhibited significant phylogenetic clustering,
whereas in undisturbed plant communities, no trends were observed. These findings
Session III
SIII-CP-29
suggest that disturbance of the ecosystems and environmental forces have influenced
the diversity and structure of Sebacinales communities at local and spatial scales.
Finally, we discuss the promising potential of locally adapted Sebacinales strains and
their use as biofertilizers, e.g. in the stimulation of the growth of crop plants and/or in
the restoration of degraded ecosystems.
ACKNOWLEGMENTS
This research was supported was supported by the German Research Foundation (grant OB 24/30).
REFERENCES
Garnica, S., et al. (2013). FEMS Microbiol. Ecol. 83: 265-78.
Oberwinkler, F., et al. (2013). Mycol. Progress 12: 1-27.
Session III
SIII-CP-30
Grain yield of cowpea in Ghana responds to Rhizobium inoculation.
Atakora, W.K.1, Guimarães, A.P.2*, Fosu, M.1 Boddey, R.M.2, Xavier, G.R.2
1
Embrapa Agrobiologia, BR 465, km 07, Seropédica, 23891-000, Rio de Janeiro, Brazil. 2 Savanna
Agricultural Research Institute (SARI), P.O. Box 52, Tamale, Northern Region, Ghana.
* [email protected]
ABSTRACT
Over a two-year period two replicated plot experiments were performed at the SARI
filed station in Tamale northern Ghana and 31 unreplicated on-farm trials in the nearby
districts. The field station experiments tested three different Rhizobium strains on a
polymer based carrier. Without inoculation, grain yields were between 1060 kg ha -1
(Exp.1) and 1370 kg ha-1 (Exp 2) and inoculation increased these to over 2000 kg ha -1
in both experiments. These results were confirmed on the trials on smallholder´s
properties where inoculation with two different Rhizobium strains increased average
grain yields by 430 to 560 kg grain ha -1 at the June planting and between 495 and 570
kg ha-1 at the August planting.
INTRODUCTION
Cowpea (Vigna unguiculata, Walp.) is the foremost grain legume crop in Africa where
the crop is estimated to occupy over 11 million ha (4 million ha in Nigeria alone) with a
mean yields between 450 and 500 kg grain ha -1. In Ghana the crop occupies ~200,000
ha with mean grain yield of~450 kg/ha. Cowpea nodulates well in almost all soils of the
tropics and other regions and until recently both Brazil and in Africa it was assumed
that as Rhizobium capable of nodulating this crop were found in the soils of these
regions it was not worth inoculating the crop with selected strains. However, results
from trials in Brazil over the last decade have shown frequent, significant and
considerable increases in grain yield to inoculation of selected Rhizobium strains. The
objective of the present study was to test on a field station in Northern Ghana and in
smallholder properties in the same region the technology of the inoculation of cowpea
with strains of Rhizobium selected in Brazil with inoculants made with the CMC
polymer used as carrier.
In August 2011 a set of un-replicated trials were established on 15 smallholder
properties with four treatments: non-inoculated with zero and 40 kg N of ammonium
sulphate and two inoculated with Rhizobium strains BR 3267 and BR 3299, Rhizobium
strains from Brazil with no phosphate addition. Similar on-farm trials, 16 each, were
planted in June and August 2012 with the same four treatments and all plots were
amended with 60 kg P ha-1 as triple super phosphate
Two field experiments were established with seven treatments in a randomized block
design (5 replicates) at two different sites at the SARI field station planted in June and
August of 2012, respectively, again with phosphate addition (60 kg P ha-1). The treatments
consisted of inoculation with three selected, BR 3262, BR 3267 and BR 3299 and a fourth
treatment with a mixture of the strain BR 3267 and BR 3299, and three non-inoculated
treatments fertilized with 0, 40 and 80 kg N, respectively, as ammonium sulphate.
Harvests for assessing nodule weight were made at 35 days after planting (DAP) and for
final dry matter accumulation and grain yield at approximately 70 DAP.
Session III
SIII-CP-30
For the on-farm trials nodule weight and grain yield responded positively to the
application of all Rhizobium inoculants. In the first set of trials of 2011 when P was not
added grain yields increased from an average for all trials of 675 kg to 859 and 1062 kg
grain ha-1 for the strains BR 3299 and BR 3267, respectively.
In the on-farm trials in 2012 where P fertilizer was applied nodule dry weight increased
from 322 to 482 and 522 mg plant-1 for strains BR 3299 and BR 3267 respectively, but
N fertilizer reduced nodule weight to 138 mg plant -1 (Table 1). Mean grain yield was
increased from 1162 to 1722 and 1594 kg grain ha -1
In the second set of on-farm trials planted in August similar results were obtained the
Rhizobium inoculants increased yields from 891 to approximately 1400 kg ha -1.
The field station experiments also showed large responses of nodule mass and grain
yield to Rhizobium inoculation, such that yields increased from approximately 1300 kg
grain ha-1 to over 1650 kg ha-1 at the June planting and from 1000 to over 2000 kg grain
ha-1.
Table 1. On farm trials first planting 2012. Nodule weight at 35 days after planting and yield
parameters a final harvest (70 DAP)
Treatment
Non Inoculated
40 Kg N ha-1
Strain 3267
Strain 3299
CV (%)
35 DAP
Nodule weight
mg DM plant
322 B
138 C
522 A
482 A
23
-1
DM shoot
Final Harvest 70 DAP
Dry weight pods
Grain yield
-1
-------------------------- kg ha ------------------------2510 B
1882 AB
1162 B
5844 A
1185 B
1086 B
2563 B
3724 A
1722 A
2401 B
2105 AB
1594 A
11
18
35
Values are means of eleven replicates. Means within the same column followed by the same letter are not different at p< 0.0 5
(Student ‘t’ test)
In all cases N fertilizer had a highly significant positive effect on total shoot dry matter
but this was not reflected in grain yield.
The large increases in grain yield obtained in 2 field experiments and 22 on-farm trials
to the application of the polymer-based inoculant of selected Rhizobium strains show
that an extremely small investment (US$ 2 to 3 per ha) between 300 and almost 1200 kg
of extra grain can be harvested which illustrates the huge potential of this simple
technology (Fernandes Júnior et al., 2012).
ACKNOWLEDGEMENTS
This work was funded by the Africa/Brazil Agricultural Innovation Marketplace supported by Embrapa,
The Bill and Melinda Fates foundation, DfID (UK).
REFERENCES
Fernandes Júnior, P.I., et al. (2012). African J. Biotech. 11: 2945-2951.
Session III
SIII-CP-31
Pseudomonas fluorescens enhances photosynthesis and improves
bioactive profiles and antioxidant potential in blackberry (Rubus sp.
Var. Lochness) in field conditions.
García-Seco, D., Bonilla, A., Algar, E., García-Villaraco, A., Gutiérrez Mañero, F.J. *,
Ramos Solano, B.
Faculty of Pharmacy, Universidad CEU San Pablo, Ctra. Boadilla del Monte Km 5.3, 28668 Boadilla del
Monte, Madrid, Spain.
* [email protected]
ABSTRACT
Inoculation of P.fluorescens N21.4 to blackberry plants (Rubus spp. var Lochness)
throughout its phenological cycle in field conditions resulted in improved
photosynthetic yield during vegetative growth. This improvement was also detected in
fruits that showed improved antioxidant potential and higher flavonoid and anthocyanin
contents.
INTRODUCTION
Blackberry (Rubus spp. var Lochness) is an aggregate fruit, composed of small
drupelets, belonging to the Rosaceae family. Blackberry represents an important food
for human health in prevention of chronic diseases due to the presence of numerous
bioactive compounds. These bioactive compounds (anthocyanins, phenolic acids and
flavonoids all with antioxidant activity) are products of secondary metabolism designed
to provide maximal adaptation for the plant against environmental changes; therefore,
secondary metabolism is highly inducible and is subjected to strong fluctuations due to
environmental conditions (Boué et al., 2008), and therefore, the potential benefits for
health through the diet are not consistent. Among the factors that are able to trigger
secondary metabolism are beneficial bacteria able to induce systemic resistance (Ramos
Solano et al., 2010b). This lack of reproducibility may be overcome by means of
elicitation during plant growth, which will be reflected in secondary metabolites
contained in the fruit (Capanoglu, 2010). In a previous study, this strain demonstrated
its ability to trigger secondary metabolism with a low number of plants (García-Seco et
al., 2013). The aim of the present study was to confirm this ability in a larger sample
area, evaluating photosynthesis, as a marker of systemic resistance, and fruit quality to
confirm the potential of this strain to palliate changes in concomitant to environmental
changes.
Pseudomonas fluorescens N21.4 (CECT 7620) was isolated from the rhizosphere of
Nicotiana glauca (Ramos Solano et al., 2010a). Three greenhouses (360 plants) of R.
fruticosus var. Lochness were defined within the production area. Plants were grown
from November through May under natural light conditions. Four blocks were defined
in each greenahouse, N21.4 inoculated plants, N21.4 plus the usual chemical treatments,
and the non-inoculated controls (Control and Chemical-Control). Inoculations were
delivered by soil drench every two weeks.
Total flowering buds and branches were recorded at two time points and photosynthesis
was analysed by a Fluorescence Monitoring System (FMS; Hansatech).
Fruits were harvested and bioactive contents were determined at the maximum point of
production. The total phenolic content was determined with Folin-Ciocalteau reagent
(Sigma-Aldrich, St Louis, MO) by colorimetry with modifications (Xu and Chang,
2007). Total flavonoid content was measured by the aluminum chloride colorimetric
Session III
SIII-CP-31
assay (Zhishen et al., 1999). Total anthocyanin content was determined quantitatively
by the pH differential method of Giusti and Wrolstad (2001). The radical scavenging
activity of R. fruticosus extract against DPPH free radical (antioxidant potential) was
measured using the method of Brand-Williams et al. (1995). Epicatechin was
determined by Capillar Electrophoresis according to Piovenzal et al. (2013).
Photosynthesis was improved in the first sampling time (January) on inoculated plants
as compared to controls, as indicated by the significantly lower values of Fo, and
greater φPSII, and Fv/Fm. These physiological values were consistent with higher
number of flowering branches and flowering buds per branch. However, during the
second sampling time (May, maximum production), this significant difference
disappeared, indicating that inoculated plants had accelerated growth probably due to
increased photosynthesis, a mechanism that has been proposed for growth promotion of
some PGPR strains (Zang et al., 2008).
Fruits from plants inoculated with N21.4 showed increased antioxidant potential, and
higher flavonoids and anthocyanins. Total flavonoids levels in controls were under 65
mg catechin equivalents per 100 g FW, while inoculated fruits were over 80 mg
catechin equivalents (100 g FW), being these differences significant. However, the
flavonoid (+)-epicatechin concentration was higher on inoculated plants but not
significant. Total antocyanins were arround 90 mg of cyanidin-3-glucoside equivalents
in controls and increased up to 120 mg of cyanidin-3-glucoside equivalents, on
inoculated fruits.
ACKNOWLEDGEMENTS.
The authors would like to thank Ministerio de Ciencia e Innovación for granting DGS (BES-2010038057) and projects AGL 2009-08324, CM S2009/AMB-1511, and Agricola El Bosque, S.L.
REFERENCES.
Boué, S.M., et al. (2008). J. Agric. Food Chem. 57: 2614-2622.
Brand-Williams, W., et al. (1995). Lebensm. Wiss Technol. 28: 25-30.
Capanoglu, E. (2010). Trends Food Sci. Technol. 21: 399-407.
Garcia-Seco, D., et al. (2013). Agron. Sustain. Dev. 33: 385-392.
Giusti, M., and Wrolstad, R. (2001). Current protocols in food analytical chemistry. pp 121-129.
Piovenzal et al. (2013). Electrophoresis, in press.
Ramos Solano, B., et al. (2010a). Plant soil 334: 189-197. doi: 10.1007/s11104-010-0371-9
Ramos Solano, B., et al. (2010b). J Agric Food Chem 58:1484–1492. doi: 10.1021/jf903299a
Xu, B.J., and Chang, S.K.C. (2007). J. Food Sci. 72: 159-166.
Zang, H., et al. (2008). Plant J 56: 264–273. doi: 10.1111/j.1365-313X.2008.03593.x
Session III
SIII-CP-32
Improving opium poppy yield through MAMPs from selected
beneficial bacterial strains in field trials.
Bonilla, A.1, Garcia-Seco, D.1, Muñoz Ledesma, F.J.2, Lucas, J.A.1*, Ramos Solano,
B.1, Gutiérrez Mañero, F.J.1
1
Faculty of Pharmacy, Universidad CEU San Pablo, Ctra. Boadilla del Monte Km 5.3, 28668 Boadilla
del Monte, Madrid, Spain. 2 ALCALIBER I+D+i, S.L.U. Ctra. Carmona-El Viso del Alcor, Km.1,8.
41410 Carmona, Sevilla, Spain
* [email protected]
ABSTRACT
Three beneficial PGPR strains were inoculated at two different physiological status of
Papaver somniferum and in both status, in experimental plots in field conditions in
spring 2010 and 2011. Foliar applications of two strains resulted in increased yield due
to an increase in opium poppy capsule weight. The increases detected under
Chryseobacterium balustinum Aur 9 were due to increased seed production while
Stenotrophomonas maltophila N5.18 caused an increase in straw, not in seeds. The
effects of N5.18 at the end of the flowering period was consistent in the two
experiments. This was coupled to increases in alkaloids since they accumulate mainly in
the capsule straw and also to an increase in photosynthetic yield.
INTRODUCTION
Field production of opium poppy is the only economically feasible way to obtain
morphinane alkaloids, being morphine the best analgesic to date. Legal production of
Papaver sominferum worldwide is carried out in Australia, France, Spain and Turkey,
with Spain accounting for 73 MT of 440.3 (INCB, 2012), being under strict control by
the Ministerio de Sanidad.
Benzylisoquinoline alkaloids (BIAs) are a large and structurally diverse group of
nitrogenous compounds related with plant defence. Among the Papaveraceae family
only Papaver somniferum is able to produce significant amounts of alkaloids
accumulated only in the capsules. However, due to the inducible nature of secondary
metabolism, alkaloid levels change according to environmental conditions, and
therefore, yield is highly compromised due to different factors. This lack of
reproducibility may be overcome by the means of elicitation with PGPR (Ramos Solano
et al., 2010). Plants possess an active secondary metabolism that can be actively
expressed in response to stress. Hence, triggering plant’s metabolism by the means of a
Microbial Associated Molecular Pattern (MAMP) (Erbs and Newman, 2012) or a plantgrowth-promoting rhizobacteria (PGPR) could increase levels of secondary metabolites
The aim of this study was to evaluate the effects of inoculating 3 PGPR strains in
Papaver somniferum plants to enhance secondary metabolism of BIAs. To evaluate the
systemic stimulation of poppy metabolism yield parameters were determined, as well as
photosynthesis and alkaloid contents, as metabolic markers of the induction.
Field trials were conducted on 15 m 2 subplots within two Papaver somniferum
production areas, as indicated by Alcaliber I+D+I. Three bacterial strains were used
Aur9 (Chryseobacterium balustinum), N21.4 (Pseudomonas fluorescens) and N5.18
(Stenotrophomonas maltophilia); each of them was delivered by foliar spray right
before flowering stem grows (cabbage state) and two weeks after when flowers are
formed (flowering state), and in both status. Photosynthesis was measured 2 weeks after
inoculation. When plants were dryed, one subsample (1 m2) was defined within each
Session III
SIII-CP-32
plot and plant number, capsule number were recorded. Capsules were brought to the lab,
seeds were separated from straw and weight was determined. Alkaloids were extracted
and determined by HPLC. ANOVAs were used to determine statistical significance of
data and LSD
Among the three strains assayed, only N5.18 delivered at the flowering state was able to
trigger Papaver somniferum metabolism, causing significant increases as compared to
non-inoculated control plants. The most affected parameter was total capsule weight,
due to significant increases in straw and seed dry weight. This increase in biomass is
supported by the improvement in photosynthetic parameters, a mechanism reported for
some PGPR to improve plant yield (Zhang et al., 2008; Zulak et al., 2008)
Total alkaloid contents (mg/g straw) were not significantly increased by this strain, and
there was only an increasing trend in morphine. Despite the reported role of BIAs as
phytoalexins (Facchini et al., 1996), our results are based only in 4 alkaloids (morphine,
codein, thebain and oripavine) so the possibility of other BIAs being increased may not
be ruled out. Consistently, an increase in sanguinarine after Botrytis PAMPs (PathogenAssociated Molecular Patterns) has been reported (Facchini et al., 1996). Furthermore,
after elicitation with certain MAMPs or PAMPs, metabolic pathways leading to
phytoalexins may be detoured to one specific branch by triggering specific branching
points (Rühmann et al., 2013). This becomes especially relevant in alkaloid biosynthetic
pathways which are highly compartimented in different tissues and subcellular fractions
(Facchini et al., 1996).
Considering effects on biomass increase by MAMPs of N5.18, together with alkaloids
results in an encouraging result for production since the increase in total straw will
result in higher alkaloid yields when considering surface under production.
ACKNOWLEDGEMENTS
The authors thank Alcaliber I+D+I, for funding and technical help. This research was supported by
Ministerio de Ciencia y Tecnología AGL2009-08324 and CAM S-0505/AMB/000321.
REFERENCES
Erbs and Newman.(2012). Mol. Pl. Pathol. 13: 95-104.
Facchini P.J., et al. (1996). Plant Physiol. 111: 687-697.
INCB (2012). ISBN: 978-92-1-048152-6.
Ramos-Solano B., et al. (2010). J. Agri. Food Chem., 58:1484-1492.
Rühmann S., et al. (2013) Pl. Biol. Biochem. http://dx.doi.org/10.1016/j.plaphy.2013.03.011.
Zulak K.G., et al. (2008). BMC Plant Biol. 8: 5-23.
Zang et al. (2008). Plant J. 56: 264–273. doi: 10.1111/j.1365-313X.2008.03593.x.
Session III
SIII-CP-33
Rhizobial cellulase CelC2 and its Ensifer homolog play an important
role in plant and nodule development.
Menéndez, E.*, Robledo, M., Velázquez, E., Martínez-Molina, E., Rivas, R., Mateos,
P.F.
Department of Microbiology and Genetics. CIALE. University of Salamanca. Spain.
* [email protected]
ABSTRACT
Cellulolytic enzymes are widespread in bacteria establishing root-nodule symbioses.
Genes encoding these cellulases are present in all rhizobia genera. Among them, celC
gene from Rhizobium leguminosarum bv trifolii ANU843 encodes a ß1-4D
endoglucanase (GH8), called CelC2, that is shown to be essential for primary symbiotic
infection in legume host roots and shows host specificity for Trifolium repens root hair
tips (Robledo et al., 2008). Furthermore, there are celC orthologs in the genera
Rhizobium and Ensifer (Robledo et al., 2011, 2012). Here we report the effects of
rhizobial CelC2 and its E. medicae WSM419 homolog in plant and nodule
development.
INTRODUCTION
Among others, Rhizobia-legume symbiotic association is one of the most important
plant-microbe interactions and provides the most efficient assimilable Nitrogen source
for agricultural crops. The nitrogen-fixing endosimbiotic bacteria can invade their plant
hosts though colonization of intercellular epidermal spaces, crack entry at emerging
lateral roots or intracellular infection of root hairs. The infection process is tightly
regulated in legumes to ensure appropriate bacterial symbiont penetration to establish a
nitrogen-fixing, intracellular state within the host. Preliminary studies have shown
rhizobial cellulase CelC2 is essential in primary symbiotic infection and its tightly
regulated production is required for root hair and nodule functional development in its
host, Trifolium repens (Robledo et al., 2008, 2011). Besides that, few are known about
cellulases implications in other symbiotic systems, as the model interaction MedicagoEnsifer. In this study we show the role of CelC2 and its Ensifer homolog when are
homo/heterologous expressed in this model system.
Bacterial strains and plant material.
M. truncatula Jemalong line A17 seeds were used as plant material. E. meliloti 1021, E.
medicae WSM419, A. rhizogenes strain ARqua1 and its derivatives were used as
bacterial inoculants.
Engineering celC-expressing and gfp-tagged derivative strains.
Construction of E. meliloti 1021 and E. medicae WSM419 derivative strains was
performed introducing vector pJZC2 (Robledo et al. 2008) and pEMC2 in wild-type
strains by triparental matting. To generate GFP-expressing bacteria, plasmid pHC60
(Cheng and Walker, 1998) was introduced into wild type and derivative strains as
described.
A.rhizogenes-mediated root transformation.
Seedlings were transformed with A. rhizogenes strain ARqua1 carrying the appropriate
binary vector using standard protocols (Boisson-Dernier et al., 2001). Composite plants
were inoculated with 1 mL of E. meliloti 1021 suspension (OD600 0.5). Nodules were
collected 4 weeks after inoculation and sectioned for inmunoblotting assays.
Session III
SIII-CP-33
CelC2 heterologous expression in Medicago and Ensifer has similar phenotypes.
To analyze heterologous expression effects, we have introduced the rhizobial celC gene
into E. meliloti 1021 and M. truncatula A17, which contain no homologs in their
genomes. E. meliloti 1021C2+ produces a delay in plant development as shown by plant
shoot length and dry weight, in Nitrogen-free conditions. Nodule sections showed
abnormal morphology and premature senescence. Moreover, ANU843 celC
overexpression in E. medicae WSM419 has similar results (Table 1).
At the same time, CelC2 root-transformed M. truncatula plants and inoculated with E.
meliloti 1021 showed similar phenotype to wild type plants inoculated with the CelC2
derivative strain (Table 1). Although nodule inmunohistological sections show CelC2
expression in the whole host root, only infection zone is affected, mainteining rest of
root structures intact.
celC of E. medicae WSM419 overexpression enhances plant development in Medicago.
Moreover, we have introduced R. leguminosarum bv trifolii ANU843 and E. medicae
WSM419 celC in wild type WSM419 strain. Greenhouse assays show a significative
increase in plant development when inoculated with its own cellulase overproducing
derivative, as revealed by plant shoot lenght data (Table 1)
Table 1. Differences in M. truncatula plant shoot length (cm) 3 months after inoculation, when grown
under N2-free conditions (±S.E.*)
E. meliloti 1021
Uninoculated
0,02±0,001 a
Wild-type
16,1±0,98 b
1021C2+
7,8±0,51 c
-
E. medicae WSM419
Uninoculated
3,95±0,17 a
Wild-type
28,74±0,65 b
EV
27,69±0,52 b
celC+ (843)
12,04±1,05c
celC+ (WSM)
34,57±0,57 d
M. truncatula
EV
4,3±0,44 a
EV 1021
4,97±0,87 a
celC
3,11±0,31 b
celC1021
3,13±0,3 b
-
Values followed by the same letter in each treatment are not significantly different from each other at P =
0.05 according to Fisher’s Protected LSD (Least Significant Differences). S.E. = Standard Error.
*From each treatment 3 replicates with at least 35 plants each were analyzed.
ACKNOWLEDGEMENTS
M.E. acknowledges FPI-MICINN PhD fellowship. This work was supported by MICINN AGL200803360 and AGL2011-28227 grants and Junta de Castilla y León project SA306A11-2 .
REFERENCES
Boisson-Dernier, A., et al (2001). Mol. Plant Microbe Interact. 14: 695-700.
Cheng, HP and Walker, G.C. (1998). J. Bacteriol. 180: 5183-91.
Robledo, M., et al (2008). Proc. Natl. Acad. Sci. USA 105: 7064-7069.
Robledo, M., et al (2011). Mol. Plant Microbe Interact. 24: 798-807.
Robledo, M., et al (2012). Microbial Cell Factories 11: 125.
Session III
SIII-CP-34
Ocorrência de fungos micorrízicos arbusculares na cultura da palma
no semiárido de Pernambuco.
Mergulhão, A.C.E.S.*, Silva, M.L.R.B., Figueroa, C.S., Lyra, M.C.C.P.
Instituto Agronômico de Pernambuco - IPA. Laboratório de Genoma-Avenida General San Martin, 1371,
Bongi, CEP. 52761000 Recife-Pernambuco. Brasil.
*
[email protected]
RESUMO
A palma forrageira tem grande importância econômica no Nordeste do Brasil por ser o alimento
principal dos animais e por possuir alta capacidade de adaptar-se a região semiárida. Os fungos
micorrízicos arbusculares (FMA) são importantes por beneficiarem o crescimento de culturas de
interesse econômico, aumentando absorção de nutrientes de baixa mobilidade no solo, o que
resulta em vegetais mais tolerantes a entraves bióticos e abióticos. A falta de conhecimento
sobre a ocorrência dos FMA na cultura da palma é um enigma que precisa ser investigado para
entender melhor a íntima relação que desenvolvem estes fungos com a planta hospedeira. Este
trabalho teve como objetivo ampliar os conhecimentos sobre a ocorrência e diversidade dos
FMA coletados da rizosfera de palma forrageira como também de palma frutífera em duas áreas
dos municípios de Caruaru (CA) e Arcoverde (AR) no Agreste e Sertão de Pernambuco
respectivamente. Maior esporulação e ocorrência de FMA ocorreram na rizosfera de palma em
Arcoverde quando comparado a Caruaru. Foram identificadas 22 espécies de FMA nas áreas
estudadas. O gênero Glomus foi o mais comumente encontrado nas áreas estudadas podendo
assim ser testado em benefício do solo e das palmas forrageiras e frutíferas no Agreste e Sertão
de Pernambuco.
INTRODUÇÃO
Os fungos micorrízicos arbusculares (FMA) destacam-se pela grande importância nos
habitats ecológicos ocupados pelas plantas. Estes fungos promovem melhoria no
crescimento dos hospedeiros pelo aumento da absorção de nutrientes e contribuem
consequentemente para a sua sobrevivência. Devido a essas características e à ampla
distribuição, os FMA desempenham importante papel no equilíbrio das comunidades.
Esses simbiontes proporcionam ao vegetal maior rapidez no crescimento, melhoria geral
no estado nutricional e na produção de biomassa (Cavalcante et al., 2009). A cultura da
palma forrageira no Nordeste do Brasil constitui um dos suportes básicos de
subsistência da pecuária leiteira, sobretudo em zonas áridas e semiáridas (Mergulhão et
al., 2012). Estudos com esta cultura e FMA são necessários, visto que, o conhecimento
sobre quais fungos micorrízicos está presente na cultura da palma não é conhecido.
Essas informações podem ser utilizadas para a seleção de isolados eficientes para
aplicação em processos biotecnológicos de interesse agrícola ou ambiental na cultura da
palma. Desta forma esse trabalho teve como objetivo ampliar os conhecimentos sobre a
ocorrência e diversidade dos FMA coletados da rizosfera de palma forrageira como
também de palma frutífera em duas áreas dos municípios de Caruaru (CA) e Arcoverde
(AR) no Agreste e Sertão de Pernambuco respectivamente.
MATERIAL E MÉTODOS
Foram realizadas coletas de solos em 15 plantas de palma forrageira como também de
palma frutífera (existentes no Banco Ativo de Germoplasma (BAG) do Instituto
Agronômico de Pernambuco - IPA) em duas áreas dos municípios de Caruaru (CA) e
Arcoverde (AR) no Agreste e Sertão de Pernambuco respectivamente (Tabela 1). Em
cada área amo st ras de so lo (5 -20 cm de profundidade) foram co let adas
preferencialmente na rizosfera das plantas, sendo os pontos definidos aleatoriamente. Os
esporos de FMA foram extraídos do solo por peneiramento úmido (Gerdemann e
Nicolson, 1963) e centrifugação em água e sacarose a 50% (Jenkins, 1964), contados
Session III
SIII-CP-34
em microscópio estereoscópico e também montados em lâminas com PVLG ou reagente
de Melzer + PVLG (1:1) e observados ao microscópio. Para identificação taxonômica
foram consultados Schenck e Pérez (1990) e a home page http://invam.caf.wvu.edu. O
número total de esporos foi submetido à análise de variância (ANOVA) considerando
os quinze pontos amostrais, sendo sete pontos em Arcoverde e oito pontos em Caruaru
como repetições. As médias das diferentes áreas foram comparadas pelo teste de Tukey
(P≤0,05) utilizando o programa Sanest (Zontal et al., 1984), para minimizar a variância
o número de esporos foi transformado em Log (X+1,5).
A quantidade de esporos de FMA em Arcoverde foi maior (média de 4,9 esporos g -1 de
solo) do que na área de Caruaru com média de 2,6 esporos g -1 de solo (Tabela 1). Valor
inferior a estes foram encontrados em área de caatinga nativa na Bahia por Silva et al.
(2001) onde o número de esporos foi de 1,6 g -1 de solo. Segundo Dodd et al. (1983)
apesar da quantidade de esporos no solo não fornecer o valor real de sua infectividade,
pode dar indicação do nível populacional dos FMA. Foram identificadas vinte e duas
espécies de FMA, sendo quatorze espécies de FMA encontradas em AR e oito espécies
em CA. O maior número de espécies (8) de FMA identificadas pertence ao gênero
Glomus e representam 36% do total de espécies encontradas. A diversidade de espécies
de FMA neste trabalho foi considerada média, porém são necessários mais estudos para
permitir o registro inclusive daquelas espécies ainda não conhecidas para a ciência.
Tabela 1. Espécies vegetais (Palmas forrageiras e frutíferas), fungos micorrízicos e número de esporos
registrados em duas áreas Arcoverde (AR) e Caruaru (CA) no Agreste e Sertão de Pernambuco.
*
Médias originais, transformadas em Log (X+1,5) para análise de variância, CV(%)=30
CONCLUSÕES
Maior ocorrência de FMA foi verificada na rizosfera de palmas em Arcoverde. O
gênero Glomus foi o mais comumente encontrado nas áreas estudadas podendo assim
ser testado em benefício do solo e das palmas forrageiras e frutíferas no Agreste e
Sertão de Pernambuco.
REFERENCIAS
Cavalcante, U.M.T., et al. (2009). Aspectos da simbiose micorrízica arbuscular. 28 p.
Dodd, J., et al. (1983). Israel J. Botany 32: 10-21.
Gerdemann, J.W., and Nicolson, T.H. (1963). Transactions of the British Mycological Society, 46: 235244.
Jenkins, W.R. (1964). Plant Dis. Rep. 48: 692 p.
Mergulhão, A.C.E.S., et al. (2012). Pes.Agropecu. Pernambucana 17: 78-82.
Schenck N.C., and Pérez, Y. (1990). Manual for the identification of VA mycorrhizal fungi, 3 ed.
Silva, G.A., et al. (2001). Rev. Bra. Bot. 24: 135-143.
Zonta, E.P., et al. (1984). Sistema de análise estatística para microcomputadores.
Session III
SIII-CP-35
Bactérias diazotróficas associadas a cladódios de palma: solubilização
de fosfato inorgânico e tolerância à salinidade.
Silva, M.L.R.B.*, Figueroa, C.S., Mergulhão, A.C.E.S., Lyra, M.C.C.P.
Instituto Agronômico de Pernambuco-IPA. Laboratório de Genoma. Avenida General San Martin, 1371
Bongi Recife. Pernambuco. Brasil.
*
[email protected]
RESUMO
A cultura da palma forrageira no Nordeste do Brasil tem uma grande importância
econômica por ser o alimento principal dos animais na região semiárida. Dessa forma,
para propiciar boas condições de desenvolvimento e produtividade, deve-se dar grande
importância ao suprimento de fósforo. Esse trabalho teve como objetivo selecionar
bactérias pela sua capacidade de solubilização de fosfato inorgânico e crescimento em
meio de cultura livre de fonte nitrogenada sob diferente concentrações de NaCl. A
capacidade de solubilização de fosfato foi avaliada pelo índice de solubilização (IS).
Para avaliação da influencia de sal sobre o crescimento bacteriano em meio de cultura
livre de fonte nitrogenada cultivadas em meio NFb semissólido, acrescido de 2,5 e 5,0
de NaCl. As bactérias diazotróficas associadas à palma, cultivadas no agreste e sertão
de Pernambuco, apresentaram a capacidade de solubilizar fosfato inorgânico e crescer
em meio de cultura livre de fonte nitrogenada sob concentrações de 2,5% e 5% de NaCl,
tornando-se candidatas a promoção de desenvolvimento vegetal em solos salinos.
INTRODUÇÃO
A cultura da palma forrageira no Nordeste do Brasil tem uma grande importância
econômica por ser o alimento principal dos animais na região semiárida. Dessa forma,
para propiciar boas condições de desenvolvimento e produtividade, deve-se dar grande
importância ao suprimento de fósforo. De acordo com Kuklinsky-Sobral et al. (2004) a
habilidade das bactérias endofíticas em relação à solubilização do fosfato inorgânico
vem despertando grandes interesses. Uma vez que as bactérias endofíticas durante o
processo de colonização, pode inicialmente colonizar superficialmente o hospedeiro e,
consequentemente, fornecer o nutriente fósforo para o desenvolvimento das plantas. As
bactérias solubilizadoras de fósforo, quando utilizadas como biofertilizante, propiciam
muitas vantagens para a planta e sua produtividade. Desta forma esse trabalho teve
como objetivo selecionar bactérias pela sua capacidade de solubilização de fosfato
inorgânico e o crescimento em meio de cultura livre de fonte nitrogenada sob diferente
concentrações de NaCl.
MATERIAL E MÉTODOS
Solubilização de fosfato inorgânico.
Para a solubilização, os isolados foram crescidos em 5 mL do meio DYGS liquido
(Rodrigues Neto et al., 1986) durante 48 h, a 28º C, sob agitação de 150 rpm. À
capacidade de solubilização de fosfatos, foi realizada segundo Verma et al. (2001),
modificado (Santos et al., 2012) fosfato insolúvel (10g/l de glicose; 5 g/l de NH4Cl; 1
g/l de NaCl; 1g/l de MgSO4.7H2O; 4g/l de CaHPO4; 15 g/l de agar; pH 7,2). Em cada
placa, foi colocado 5 μL do crescimento bacteriano com três repetições em pontos
equidistantes. As placas foram incubadas a 28 °C por 72 h e, em seguida, foi verificada
a presença de área solubilizada (capacidade de solubilizar) e feita à medição do
diâmetro dessa área e da colônia. A partir destes dados, foi obtida a relação entre os
diâmetros do halo e da colônia, utilizado na avaliação do potencial de solubilização.
Session III
SIII-CP-35
Salinidade sobre o crescimento bacteriano.
A influência da salinidade sobre a capacidade de bactérias diazotróficas em se
desenvolver em meio de cultura livre de fonte nitrogenada foi avaliada por meio da
inoculação de colônias isoladas em meio NFb semissólido (Döbereiner et al., 1995),
acrescido dos seguintes tratamentos: 0% de NaCl; e as concentrações 2,5 e 5% de NaCl
designadas para caracterizar bactérias halo tolerantes (Santos et al., 2012). Os
experimentos foram realizados em duplicata, incubados a 28°C por oito dias, sendo o
experimento repetido mais uma vez. O crescimento bacteriano foi avaliado observandose a presença ou ausência de uma película típica (halo) do crescimento bacteriano no
interior do meio de cultura.
Quando avaliado o índice de solubilização (IS), foi observado que os 11 isolados
bacterianos capazes de solubilizar fosfato inorgânico, in vitro, expressaram valores de
IS variáveis, indicando diferenças quanto ao potencial de solubilização em meio sólido.
Cinco isolados diazotróficos endofíticos apresentaram valores de IS baixos entre 1,00 a
2,00, enquanto que 06 isolados apresentaram IS médios entre 2,00 a 3,43. Os isolados
LG-P2 e NFB P64 foram consideradas não solubilizadoras por não apresenta a
formação de halo no meio com fosfato após 18 dias de crescimento (Figura 1). O
entendimento da capacidade e da eficiência de microrganismos, em solubilizar fosfatos
pode levar à seleção de isolados com alto potencial de uso para a inoculação em plantas
(Souchie et al., 2005).
Dentre as bactérias diazotróficas avaliadas, 100% formaram película de crescimento
bacteriano no meio de cultura no tratamento de 0% de NaCl, 90% em 2,5% e 60% em
5% de NaCl. Estes micro-organismos halo tolerantes, utilizam, em geral, estratégias de
osmoadaptação flexíveis que lhes permitem responder rapidamente a flutuações de
salinidade do meio exterior, como a acumulação de íons inorgânicos em concentrações
elevadas, mantendo dessa forma a integridade celular (Santos et al., 2012),
possibilitando a sobrevivência destes em solos salinos e sódicos (Stamford et al., 2005).
O mecanismo de promoção de crescimento vegetal por microrganismos endofíticos
ainda necessita de mais estudos para um melhor entendimento dos fatores envolvidos.
A
B
Figura 1 . (A) NFB -P33 bactéria solubilizadora
de fosfato inorgânico, (B) LG P2 bactéria não
solubilizadoras por não apresenta a formação de
halo no meio de cultura.
CONCLUSÕES
Bactérias diazotróficas associadas à palma, cultivadas no agreste e sertão de
Pernambuco, apresentaram a capacidade de solubilizar fosfato inorgânico e crescer em
meio de cultura livre de fonte nitrogenada sob concentrações de 2,5% e 5% de NaCl,
tornando-se candidatas a promoção de desenvolvimento vegetal em solos salinos.
REFERENCIAS
Döbereiner, J., et al. (1995). Como isolar e identificar bactérias diazotróficas de plantas não-leguminosas.
60p.
Kuklinsky-Sobral, J., et al. (2004). Environ. Microbiol. 6: 1244-1251.
Rodríguez, H., and Fraga, R. (1999). Biotech. Adv. 17: 319-339.
Santos, I. B., et al. (2012). Biosci. J. 28: 142-149.
Souchie, E. L., et al. (2005). Pesqui. Agropecu. Bras. 40: 1149-1152.
Stamford, N.P., et al. (2005). Microbiota dos Solos Tropicais. 399 p.
Session III
SIII-CP-36
Genotypic characterization of natural rhizobial populations isolated
from pea and lentil in two eco-climatic subhumid and semi-arid zones
in Algeria.
Riah, N.1, 2*, Djekoun, A.1, Heulin, K.2, Laguerre, G.2
1
Laboratoire de Génétique, Biochimie et Biotechnologies Végétales.Université Mentouri Constantine,
Algérie. 2 CIRAD, UMR 113 Symbioses Tropicales et Méditerranéennes, F-34398 Montpellier, France.
* [email protected]
ABSTRACT
The genetic diversity of 237 isolates from root nodules of pea and lentil plants sampled
in six study sites representing two contrasting eco-climatic conditions subhumid and
semi-arid in East Algeria. Rhizobial isolates were characterized by PCR/RFLP of 16S23S ribosomal intergenic region (16S-23S IGS) and of nodD-F symbiotic region, and
sequence analysis 16S ribosomal DNA gene. 26 IGS/nod genotypes assigned to the
Rhizobium leguminosarum species were identified. The diversity of genotypes is mainly
accounted for by the diversity of the IGS types. The structure of the population is
affected by the host plant of origin but even more by the geographic effect, which is
especially marked in the semi-arid zone.
INTRODUCTION
In Algeria food and forage legumes are, with cereals, major challenges of agriculture.
Legume plants have the capacity to enter a nitrogen fixing symbiosis with rhizobial soil
bacteria. Water déficit, rain irregularity and salt constraint represent major limitations
for plant growth and agronomic production in Mediterranean zone, particularly in
Algeria (L’Taief et al., 2009). Rhizobial adaptation to drough stress is variable
(BenRomdhane et al., 2009). In this work we examined structure and genetic diversity
of natural rhizobial populations isolated from pea and lentil in study sites representing
two contrasting eco-climatic conditions subhumid and semi-arid in East Algeria.
The isolates were characterized by restriction fragment length polymorphism analysis
of polymerase chain reaction (PCR-RFLP), amplified DNA fragments with
restriction enzyme HaeIII (Laguerre et al., 2003). Two DNA regions were targeted
using a chromosomal marker, the intergenic spacer region between 16S and 23S
rDNA (IGS) and a functional marker, the nodulation gene region nodD-F. The rDNA
16S gene was amplified using universal primers fD1 and rD1 as described by
Laguerre et al. (1994). and primer 16S-1080r (Zakhia et al., 2006).
Rhizobial population genetic diversity.
A total of 237 bacteria were isolated from root nodules of proteaginous pea, forage pea
and lentil, approximately 80 isolates from each host plant. PCR-RFLP analysis
distributed these isolates in 14 IGS types and 10 nod types. The combination of these
haplotypes allowed to classify the isolates into 26 distinct IGS/nod genotypes. The
diversity of IGS/nod genotypes is mainly accounted for by the diversity of the IGS
types with 5 predominant types representing a total of 87% isolates.
Session III
SIII-CP-36
Genetic Structure of the rhizobial populations.
Rhizobial genotype distributions in eco-climatic zone, in different sites and from host
plant of origin were analysed by AMOVA for comparison. Differentiation is high
between the populations from both zones, due to their contrasted dominant genotype
frequencies. Significant differences are also observed between sites in a given ecoclimatic zone. The structure of the population is affected by the host plant of origin but
even more by the geographic effect, which is especially marked in the semi-arid zone.
Some dominant genotypes were observed only within the populations of the cultivated
lentil in semi-arid zone. The host plant of origin does not influence the distribution of
the IGS types.
16S rRNA Sequence-based phylogeny.
The partial sequences of the 16S rRNA representing IGS/nod genotype, show little
variation. These sequences have high (99% to 100%) similarity with sequences of
Rhizobium leguminosarum strains available in the databases. From the phylogenetic tree
Algerian isolates may be regarded as Rhizobium leguminosarum (Figure. 1).
0.01
B. japonicum ATCC 10324T
E. morelense Lc04T
E. terangae LMG 6463
100
E.meliloti LMG 6133T
89
E. saheli LMG 7837T
R.sullae IS123T
95
R.indigofera CCBAU 71042T
R .mongolense USDA 1844T
72
R. gallicum R602spT
97
76 R. yanglingense SH22623T
Rhizobium sp. BLR195 (Lens culinaris, Bangladesh)
71
Rhizobium sp. BLR29 (Lens culinaris, Bangladesh)
94
R.etli CFN42T
89
R.pisi DSM 30132T (Pisum sativum)
R.phaseoli ATCC 14482
R.etli WzP15 (Pisum sativum, China)
76 R.etli WzP3 (Pisum sativum, China)
R.fabae CCBAU 33202T (Vicia faba, China)
72
Ag. rhizogenesT
100
R. lusitanum P1-7T
R. hainanense I66T
100
99
R. tropici CIAT8899T
Isolate CL4
85
Isolate BP25 and 21 other isolates
T
79 R. leguminosarum bv. viciae USDA 2370 (Pisum sativum, USA)
82 R. leguminosarum bv. trifolii ATCC 14480
R. leguminosarum bv. viciae XtP1 (Pisum sativum, China)
Isolate SL15
Figure 1. Phylogenetic tree (neighbour-joining) based on 907 bp alignment of nucleotide sequences of
the 16S rDNA. Only bootstrap probability values of ≥70% (over 100 replicates) are indicated at the
branching points. The Algerian isolates are shown in bold. The letter “T” indicates the type strain of the
species. The sequence of isolate BP25 is identified with sequences of 21 other Algerian isolates. The host
plant of origin and geographic origin of the strains are indicated between parentheses. B.:
Bradyrhizobium; E.: Ensifer; R.: Rhizobium; Ag.: Agrobacterium. The scale bar indicates the number of
substitutions per site.
REFERENCES
L’Taief, B., et al. (2009). Biotechnol. Agron. Soc. Environ. 13: 537-544.
Zakhia, F., et al, (2006). Microb. Ecol. 5: 375-393.
Ben Romdhane, S., et al. (2009). Soil. Biol. Biochem. 41: 2568-2572.
Session III
SIII-CP-37
Native bradyrhizobia isolated from Lupinus mariae-josephae possess
an essential T3SS for symbiosis.
Durán, D.1, Pastor, V.1, Zehner, S.2, Göttfert, M.2, Imperial, J.
Argüeso, T.1*
1, 3
, Rey, L.1, Ruiz-
1
Departamento de Biotecnología (ETS de Ingenieros Agróomos) and Centro de Biotecnología y Genética
de Plantas (CBGP). Universidad Politécnica de Madrid. 28040 Madrid, Spain. 2 TU Dresden, Institute of
Genetics, Dresden, Germany. 3 CSIC-Madrid, Spain.
* [email protected]
ABSTRACT
Analysis of the genome sequence of bradyrhizobia strains isolated from root nodules of
Lupinus mariae-josephae revealed the presence of a type III secretion system (T3SS).
Mutagenesis of ttsI gene that codes for the transcriptional activator (TtsI) resulted in the
formation of white, non-fixing nodules in L. mariae-josephae. The T3SS cluster
includes a gene coding for a NopE-like protein with an autocleavage motif. The NopE
protein is an effector in the Bradyrhizobium-soybean symbiosis (Wenzel et al., 2010).
The autocatalytic properties of the purified NopE-like protein have been studied.
INTRODUCTION
Lupinus mariae-josephae is an endemic lupine found in basic soils from a small area in
Eastern Spain. Endosymbiotic bacteria that form nodules with L. mariae-josephae have
been isolated from this area and characterized by multilocus phylogenetic analysis as
belonging to Bradyrhizobium genus. These bradyrhizobia gather in six different
operational taxonomic units (OTUs) unrelated to any other isolated from lupines (Duran
et al. 2013). Draft genomic sequences from representative strains corresponding to the
different OTUs have been obtained and analyzed in this work.
Genome draft.
Genome sequencing was performed with a HiSeq2000 instrument (500bp library,
PE100, 100 x coverage) at BGI (Beijing Genomics Institute, Shenzhen, China), and the
reads were assembled into several contigs and scaffolds using the Short
Oligonucleotides Alignment Program (SOAPdenovo).
Construction of a ttsI mutant.
For mutant construction, a DNA fragment containing an internal deletion of ttsI gene
was cloned into plasmid pK10mobSac and then conjugated to the wild-type strain.
NopE expression and purification.
Plasmid pT7.7 was used for the expression of NopE-Strep in E. coli. Cloning of LmjC
nopE gene, modified to code for a C-terminal Strep-tag, was performed by PCR using
specific primers. NopE was purified by StrepTactin affinity chromatography.
Bradyrhizobia isolated from Lupinus mariae-josephae contains a T3SS.
Analysis of the genome drafts of several isolates from L. mariae-josephae (Lmj strains)
revealed the presence of a type III secretion system (T3SS) in a cluster of about 30
genes. Genes belonging to this cluster encode the transcriptional activator TtsI,
structural components of the secretion apparatus, secreted proteins including a NopElike found among a few uncharacterized proteins of plant -associated bacteria
(Schirrmeister et al. 2011), as well as hypothetical and unknown proteins. The highest
Session III
SIII-CP-37
gene conservation was observed with the genes encoding T3SS structural components
of B. diazoefficiens USDA110, B. japonicum USDA6 and B. elkanii USDA61.
However, the genes of non-structural proteins from Lmj strains are not conserved in
other bacterial species.
T3SS of Bradyrhizobium sp. LmjC is essential for symbiosis with L. mariae-josephae.
TtsI has been described as the positive regulator of the T3SS. In order to ascertain the
relevance of T3SS in Lmj strains, a ttsI mutant was generated in strain LmjC. Symbiosis
of L. mariae-josephae with the mutant appeared to be severely impaired, since plants
were significantly smaller than those inoculated with the wild type strain and since
nodules produced by the mutant were white and unable to reduce acetylene. This
suggests that the T3SS from strain LmjC is required for effective symbiosis with L.
mariae-josephae.
Autocleavage of purified LmjC NopE protein in the presence of different cations.
NopE-like protein from LmjC strain presents just one autocatalytic motif (DUF1521)
unlike NopE1 and NopE2 proteins secreted by the T3SS of B. diazoefficiens that
contain two cleavage sites that play a role in the symbiotic host-range definition
(Schirrmeister et al. 2011). Consistent with this trait, LmjC NopE protein, purified from
E. coli, was autocleaved in two fragments of the predicted size in the presence of Ca 2+,
Cu2+, Cd2+, Zn2+ and Mn2+. In contrast, autocleavage did not take place in the presence
of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif of LmjC NopE
in E. coli abolished the in vitro self-cleavage. The role of NopE in LmjC strain is
unknown, and the behavior of the NopE-DUF1521 mutant in LmjC strain is required.
ACKNOWLEDGMENTS
This work was supported by FBBVA (Contract BIOCON08-078 to TRA), MICINN (CGL2011-26932 to
JI), and UPM (AL12-PI+D05 to LR).
REFERENCES
Duran, D. et al. (2013). Syst. Appl. Microbiol. 36: 128-136.
Schirrmeister, J., et al. (2011). J. Bacteriol. 193: 124-129.
Wenzel, M., et al. (2010). MPMI 23: 124-129.
Session III
SIII-CP-38
Which phosphatases may contribute to P use efficiency for N 2 fixation
in the rhizobial symbiosis with legumes ?
Drevon, J.J.1*, Amenc, L.1 , Pernot, C.1 , Abadie, J.1 , Blair, M.2, Lazali, M.1, Bargaz, A.1,
3
, Ghoulam, C.4, Ounane, S-M.5, Zaman-Allah, M.1, 6
1
INRA, UMR Eco&Sols, 1 Place Pierre Viala, 34060 Montpellier, France. 2 Cornell University, 242
Emerson Hall, Ithaca, NY 14853, USA. 3 Swedish University of Agricultural Sciences (SLU), Box 103,
SE-23053 Alnarp, Sweden. 4 Faculté des Sciences et Techniques Guéliz, BP 549, 40000 Marrakech,
Maroc. 5 ENSA, Département de phytotechnie. Avenue Hassan Badi, El Harrach 16200 Alger, Algérie.
6
ICRISAT, International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India.
* [email protected]
ABSTRACT
In order to assess the contribution of phosphatases activity to P use effeciency for N 2
fixation in legume nodules, an in situ RT-PCR methodology was used to localize and
quantify the transcripts of candidate phosphatases genes in nodules of common bean in
hydro-aeroponic culture under deficient vs sufficient P supply. The transcript
localization of phytase, phosphoenolpyruvate-, fructose 1,6 bisphosphate- and trehalose
6-phosphate- phosphatases was found to be tissue specific and to differ among Pase
genes, P treatment and legume genotype. It is concluded that these phosphatases
contribute differently to the use of organic P for N2 fixation and the increase in nodule
permeability to O2 under P deficiency.
INTRODUCTION
Symbiotic nitrogen fixation (SNF) by legumes may provide an ecologically acceptable
complement or substitute for mineral nitrogen fertilizers that farmers cannot afford for
economic limitation or try to minimize for environment sake. However, P deficiency is
a major limiting factor for legume-rhizobia symbioses, particularly in acidified or
calcareous soils. Nevertheless the expression of legume SNF potential under P
deficiency may be improved (Pereira and Bliss, 1987; Vadez et al., 1999). In this study
we have compared the expression of various acid phosphatases (APases) in nodules of
common bean recombinant inbred lines (RILs) that were previously selected for their
contrast in P use efficiency (PUE) for SNF (Drevon et al., 2011).
Common bean RILs were selected and grown in hydro-aeroponic culture as described
by Hernandez and Drevon (1991). The in situ RT-PCR was performed as described in
Bargaz et al. (2012) on sections of standard nodules with 3 mm diameter that were
harvested at flowering stage, and immediately fixed. Specific primers for APase genes
were designed online at the National Center of Biotechnology Information (NCBI,
http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the known mRNA sequence of the
homologous genes of Medicago and Glycine spp.
All candidate genes of APases were intensely marked by fluorescence in the layers of
inner-cortex cells, i.e. the tissue localized between vascular traces and the infected zone
of nodule (Figure 1). This reveals an intense metabolism of organic P in this tissue. It
may supply Pi for the energy-demanding cell turgescence for permeability of nodule
cortex to gases (Serraj et al., 1995; Schultze and Drevon, 2005) and/or for the
catabolism of sucrose from vascular traces into organic acids for bacteroids in the
infected zone.
Session III
SIII-CP-38
The higher increase in phytase signal (Lazali et al., 2013) and activity (Araújo et al.,
2008) under P deficiency for the RIL115 compared to the less efficient RIL147
suggests that the use of phytate in nodule cortex may contribute to the adaptation of the
rhizobial symbiosis to low-P soils.
In the outer-cortex, the expression of TPP might contribute to the control of various
abiotic constraints by trehalose release (Bargaz et al., 2013), and that of PEPase
suggests Pi mobilization from senescent cells (Bargaz et al., 2012). Both TPP and PEP
genes expression increased under P deficiency. In the infected zone these genes may
supply Pi for bacteroidal metabolism. In the middle cortex, surrounding the internal
cortex, the PEPase activation under P deficiency would not only supply additional Pi
but also reduce active oxygen species through a non-enzymatic oxidative
decarboxylation of pyruvate by hydrogen peroxide (Bargaz et al., 2012).
Figure 1. Transcript localization of phytase (left), phosphoenolpyruvate phosphatase (center), trehalose
6-phosphate phosphatase (right) in nodule transversal section of the efficient common-bean RIL115.
In conclusion, the increase of APases transcripts under P-deficiency predicts not only an optimal
intracellular Pi scavenging, but also opens up a challenge to understand whether their specific tissular
localization in nodules is also related with responses to environmental constaints, such as oxidative stress,
and the osmo-regulation of nodule gas diffusion.
ACKNOWLEGMENTS
under the reference ID 1001-009 and the EU AVERROES scholarship programme and INCOMED
Aquarhiz project for the internships of Adnane Bargaz and Mainassara Zaman-Allah in Montpellier, and
AUF-PCSI 63113PS012 of Algeria-French cooperation for the internship of Mohamed Lazali.
REFERENCES
Araújo, A.P., et al. (2008). Plant Soil 312: 129-138.
Bargaz, A, et al. (2012). J. Exp. Bot. 63: 4723-4730.
Bargaz, A, et al. (2013). Planta: DOI 10.1007/s00425-013-1877-1.
Drevon, J.J., et al. (2011). Proc. Environ. Sci. 9: 40-46.
Hernandez, G. and Drevon, J.J. (1991). J. Plant Physiol. 138: 587-590.
Lazali, M., et al. (2013). Planta: DOI: 10.1007/s00425-013-1893-1.
Pereira, P.A.A. and Bliss, F.A. (1987). Plant Soil 104: 79-84.
Serraj, R., et al. (1995). Plant Cell Environ. 18: 455-462.
Vadez, V., et al. (1999). Euphytica 106: 231-242.
Session III
SIII-CP-39
nod gene inducers released by Mimosa flocculosa seeds and its effects
on common bean growth and yield under Brazilian cerrado soil
conditions.
Mercante, F.M.1*, Otsubo, A.A.1, Cunha, C.O.2
1
Embrapa Western Agriculture, Dourados, MS, Brazil.
Technological Development-CNPq, Brasília, DF, Brazil.
* [email protected]
2
National Council for Scientific and
ABSTRACT
The nod gene-inducing effect of M. flocculosa seed rinse (SR) was assayed by betaglucuronidase activity of Rhizobium tropici CIAT899 harbouring plasmid pGUS32
(containing Sinorhizobium sp. BR816 nodABC promoter fused to the gusA reporter
gene); SR of Phaseolus vulgaris cv. Pérola was used as a positive control. M. flocculosa
SR was also tested as a rhizobial inoculant additive in a common bean field trial; the
commercially recommended strains R. tropici CIAT899 and Rhizobium sp. PRF 81
were used in inoculants. For both rhizobial strains assayed, significant gains in common
bean grain yield were obtained when M. flocculosa SR was used as an inoculant
additive.
INTRODUCTION
Common bean (Phaseolus vulgaris), as well as other leguminous crops, is able to
establish a symbiotic relationship with rhizobia. The early steps of symbiotic nodule
formation by rhizobia on plants require the coordinate expression of several nodulation
(nod) gene operons, which are induced by molecules released from seeds and plant
roots, mainly flavonoids (Peters et al., 1986; Ghasem et al., 2012). Our previous studies
(Mercante and Franco, 2000) revealed that a mixture of seed rinses from common bean
and M. flocculosa had synergistic effect on nod gene inducing activity; this synergistic
effect was observed with R. tropici strains CIAT899 and F98.5, as well as with R. etli
strain CFN42. The aim of this study was to evaluate the effects of M. flocculosa SR,
alone or in combination with common bean SR, on nod gene activity of R. tropici
CIAT899/pGUS32; comparing in vitro nod gene expression with the effects of SR (as
an inoculant additive) on common bean growth and grain yield under Brazilian Cerrado
soil conditions.
Seed rinse preparation.
M. flocculosa SR was obtained from previously scarified (H2SO4 for 5 minutes) seeds
(10 g), by adding 100 mL of sterile distilled water and incubating, in the dark, at 30°C
for 24 h, under agitation (80 rpm). Common bean SR was obtained by adding 100 mL
of sterile distilled water to 100 g of seeds and incubating, in the dark, at 30°C for 24 h,
under agitation (80 rpm). SR was filter-sterilized (0.22 μm Millipore filters) before use.
Expression of nod genes in Rhizobium tropici CIAT899.
The expression of nod genes was studied in Rhizobium tropici CIAT899/pGUS32 and
was evaluated by measuring the activity of the enzyme β-glucuronidase, using as
substrate p-nitrophenyl-β-D-glucuronide at a concentration of 1000 μg mL -1. Inductions
were carried out by incubating 20 μL of bacteria culture with 180 μL of diluted filtersterilized SR. After a induction period of 48 h at 30°C, reactions were carried out at
37°C, after addition of 100 μL of "GUS buffer", as described by Mercante and Franco
(2000).
Session III
SIII-CP-39
Grain yield under field conditions.
Experiment was conducted in a Brazilian Cerrado Oxisol, in the City of Dourados, Mato
Grosso do Sul State, Brazil. Treatments consisted of: (1) control (without rhizobia
inoculation and N fertilizer), (2) N-control (80 kg ha-1 of N-urea : 20 kg ha-1 at sowing;
30 kg ha-1, 20 days after emergency-DAE; 30 kg ha-1, 40 DAE), (3) inoculation with R.
tropici CIAT899; (4) inoculation with Rhizobium sp. PRF 81. Seeds of Phaseolus
vulgaris cv. Pérola were inoculated with a peat-based rhizobial inoculant (109 cells of
rhizobia per gram of peat). Rhizobial inoculants were added to common bean seeds in
the proportion of 500 g to 50 kg of bean seeds. Subsequently, seeds were treated with
different combinations of SR, in the proportion of 300 mL kg -1 of common bean seeds.
The highest values of β-glucuronidase activity were observed when common bean SR
was used alone, followed by the 1:1 (v:v) combination of common bean SR and M.
flocculosa SR (Figure 1A). Even though the combination of M. flocculosa SR and
common bean SR did not result in a synergistic induction of nod genes in R. tropici
CIAT899 in this assay; the use of M. flocculosa SR, at a concentration of 10% (10 g of
M. flocculosa seeds in 100 mL of sterile distilled water), significantly increased
common bean grain yield when seeds were inoculated with either CIAT899 or PRF 81
strains, up to around 3000 kg ha-1 (Figure 1B). In addition, the use of M. flocculosa SR
significantly increased common bean grain yield supplied with mineral N, but did not
affect control plants. Interestingly, in earlier experiment, common bean nodulation was
not inhibited by suppling mineral N if M. flocculosa SR was used as an inoculant
additive (Mercante and Franco, 2000).
(A)
(B)
Figure 1. Activity of β-glucuronidase enzyme, resulting from induction of nod genes in strain CIAT899
(A) and grain yield of common bean inoculated with CIAT899 or PRF 81 strains (B). Values are means
of 6 replicates. Different letters on the bars indicate contrast by Duncan test, 5%.
ACKNOWLEGEMENTS
The study was partially supported by National Council for Scientific and Technological Development
(CNPq) and Fabio M. Mercante is a research fellow from CNPq.
REFERENCES
Ghasem, F., et al. (2012). J. Agr. Sci. Tech. 14: 1255-1264.
Mercante, F.M., and Franco, A. A. (2000). R. Bras. Ci. Solo 24: 301-310.
Peters, N.K., et al. (1986). Science 233: 977-980.
Session III
SIII-CP-40
Diversidade de fungos micorrízicos arbusculares e colonização do solo
em sistema de cultivo consorciado cafeeiro (Coffea arábica L.) e
seringueira (Hevea brasiliensis).
Colozzi-Filho, A.1*, Bugatti, E.P.1, Garbossi, A.1, Scaramal, A.2, Machineski, O.1,
Carrenho, R.3
1
Laboratório de Microbiologia de Solos, Instituto Agronômico do Paraná, Londrina-Pr., Brasil. 2 Bolsista
do Laboratório de Microbiologia do Solo-IAPAR. 3 Departamento de Agronomia, Universidade Estadual
de londrina, Londrina, Pr. Brasil.
* [email protected]
RESUMO
Avaliou-se a diversidade de Fungos Micorrizicos Arbusculares (FMA) e a produção de
micélio externo total (MET), em solo sob cultivo de cafeeiro e seringueira, em
monocultivo ou consórcio. Foram identificadas trinta espécies de FMA no sistema,
distribuídas em seis gêneros. A maior diversidade foi observada na seringueira solteira.
A menor diversidade observada no cafeeiro solteiro. Quanto menor a densidade de
seringueiras no cafeeiro, maior a diversidade de FMA no solo. A maior colonização do
solo por FMA foi observada no tratamento com maior densidade de raízes. O cultivo
consorciado cafeeiro-seringueira proporcionou maior diversidade de FMA e influenciou
positivamente a atividade dos FMA para produção de MTE no solo.
INTRODUÇÃO
O cultivo do cafeeiro (Coffea arabica L.) sombreado com seringueira (Hevea
brasiliensis) pode reduzir o stress de cultivo a pleno sol, aumentando a produção do
cafeeiro além de proporcionar renda extra com a extração do látex da seringueira. Os
fungos micorrízicos arbusculares (FMA) são de ocorrência natural no solo e
estabelecem relações de simbiose mutualística com o cafeeiro. O cultivo da seringueira
nas entrelinhas do cafeeiro pode alterar a diversidade e a ocorrência dos FMA no solo,
com efeito sobre a simbiose micorrízica e até sobre a produtividade do cafeeiro.
MATERIAL E METODO
As avaliações foram realizadas em um experimento conduzido em campo desde 2001,
na Estação Experimental do Instituto Agronômico do Paraná (IAPAR) em Londrina-PR,
em um latossolo vermelho distroférrico textura argilosa. Os tratamentos são 3
espaçamentos de seringueira entre as linhas de cafeeiro (13.0m;16.9m e 22.1m por 4m
de distancia entre as seringueiras na linha do cafeeiro), mais os controles cafeeiro e
seringueira solteiros, nos espaçamentos de 2.5mx1.3m e 8.0mx2.5m, respectivamente.
O delineamento experimental é de blocos casualizados com 4 repetições. Amostrou-se o
solo na profundidade de 0-20 cm. Os esporos foram extraídos do solo por peneiramento
úmido e centrifugação em água e sacarose e identificados morfológicamente sob
microscópio a nível de gênero e espécies. A extração do micélio externo total (MET) foi
realizada segundo a metodologia descrita por Melloni et al. (1999). Determinaram-se os
índices de diversidade de Shanon.
Foram identificadas trinta espécies de FMA no sistema, distribuídas em seis gêneros. A
maior diversidade (20 espécies) foi observada na seringueira solteira. A menor
diversidade (13 espécies) foi observada no cafeeiro solteiro. Quanto maior o
espaçamento de seringueiras no cafeeiro, maior a diversidade de FMA no solo. As
espécies Acaulospora aff. Sieverdingii, A. koskei, A. mellea, A. morrowiae, A.
Session III
SIII-CP-40
scrobiculata, Diversispora spurca, Glomus claroideum, G. etunicatum e G.
macrocarpum tiveram ocorrência generalizada, independente dos tratamentos. Por outro
lado, observou-se também que 11 espécies apresentaram ocorrência restrita em apenas 1
tratamento. O cafeeiro possui alta dependência micorrízica, na fase de mudas (Siqueira
e Colozzi-Filho, 1986) e também em plantas adultas a campo (Cardoso et al., 2003).
Lopes et al. (1983), Nogueira, 2007) identificaram vinte e duas espécies de FMA em
raízes de cafeeiro, os gêneros mais encontrados foram: Acaulospora e Glomus. O MET
foi superior no tratamento com o menor espaçamento de seringueira em consórcio,
diferindo significativamente dos demais tratamentos. O tratamento com a seringueira
solteira foi o que apresentou o menor comprimento do MET (Figura 1). O cultivo
consorciado cafeeiro-seringueira proporcionou maior diversidade de FMA e influenciou
positivamente a atividade dos FMA para produção de MTE no solo.
Figura 1. Hifas de fungo em monocultivo de cafeeiro, seringueira ou em
cultivo consorciado de ambos com diferentes densidades de seringueira.
AGRADECIMENTO
Ao Programa CAFÉ do IAPAR.
REFERENCIAS
Cardoso, E.J.B.N,. et al. (2003). Rev. Bra. Ciên. Solo 27: 415-423.
Colozzi Filho, A. e Nogueira, M.A. (2007). Microbiota do solo e qualidade ambiental, p. 39-56.
Melloni, R., et al. (1999). Rev. Bra. Ciên. Solo 24: 767-775.
Rillig, M.C., et al. (2006). New Phytol. 171: 41-53.
Siqueira, J.O. e Colozzi Filho, A. (1986). Rev. Bra. Ciên. Solo 10: 207-211.
Session III
SIII-CP-41
Effect of mycorrhizal inoculation on wheat and barley genotypes
differing in Al-tolerance growing at phytotoxic Al level.
Meier, S., Curaqueo, G., Seguel, A., Aguilera, P., Cornejo, P., Borie, F. *
Scientific and Technological Bioresources Nucleus BIOREN-UFRO, Universidad de la Frontera, P.O
Box 54-D, Temuco, Chile.
* [email protected]
ABSTRACT
The aim of this study was to test the effect of arbuscular mycorrhizal (AM) inoculation
on wheat and barley genotypes differing in Al tolerance when growing at phytotoxic Al
level. The experiment was carried out in a growth chamber using soiless substrate, Altolerant and Al-sensitive plants genotypes, a native mycorrhizal inoculum and a mineral
solution additioned or not with 200 uM of Al. After five weeks of growing, plants were
harvested and biomass, root colonization, mycorrhizal spores, glomalin and Al in shoots
and roots were measured. Results showed that spore number increased in all cultivars
when subjected to Al following the same trend that glomalin production. Al-sensitive
wheat cultivar “Tukan” as well the Al-sensitive barley cultivar “Scarlett” significantly
increased the glomalin content suggesting an important role in Al detoxification in the
rhizosphere.
INTRODUCTION
Chilean cereal production is chiefly supported in acidic soils from volcanic origin. Soil
acidity is one of the main constraints for the productivity of agricultural plants at
worldwide level. The excess of protons ( H+) in soils involves an increase of phytotoxic
Al levels together a decrease in the availability of P, Ca, Mg and Mo. Therefore, plant
roots growing in acid soils subjected to such stressful environments reduce seriously
their capacity of nutrient acquisition (Tang and Rengel, 2003) thus affecting crop
productivity.
For overcoming the negative effects on productivity of acid soils farmers apply some
agricultural management including lime application, phosphate fertilizers and/or the use
of Al tolerant plant cultivars. Some of such practices are sometimes economical
unfeasible (lime) or have potential environmental risks (P-fertilizers). Accordingly, it
appears important to deep in how to increase Al tolerance in cultivars habitually used in
local extensive agriculture. However, a key factor such as the presence of associated
microorganisms in its rhizosphere has not been considered.
The AM symbiosis plays an important role protecting the roots from deleterious Al
effects conferring to the plant hosts a higher Al tolerance through an enhanced P, Ca or
Mg acquisition (Marschner, 1995) as well as an increased exudation of organic anions
with Al chelation capacity (Klug and Cumming 2007; 2009). In both mechanisms a
decrease on the deleterious effects of Al in the rhizosphere is produced. In addition,
Aguilera et al. (2011) have evidenced the Al capacity to form stable chelates with
glomalin (expressed as GRSP), a glycoprotein released by AM fungal structures,
suggesting another way of decreasing the toxicity of free Al. More recently, Seguel et
al. (2013) have reviewed the role of AM on decreasing Al phytotoxicity in acidic soils
depicting an integrated model for induced Al resistance to plants.
In consequence, the aim of this study was to determine the effect of mycorrhizal
inoculation on grow wheat and barley cultivars differing in Al tolerance when subjected
to high Al phytotoxic level.
Session III
SIII-CP-41
The experiment was carried out in 300 mL polypropilene pots containing vermiculiteperlite (2:1) as substrate inoculated or not with AM fungi spores from a pot culture with
sudangrass originated from a soil with 70% Al-saturation (300-500 spores). Wheat
seeds of Al-tolerant genotype “Porfiado” and Al-sensitive “Tukan” together barley
seeds of Al-tolerant genotype “Aurora” and Al-sensitive “Scarlett” were sown in each
pot. Additionally, the Al-tolerant wheat genotype “Atlas 66” was included as a
comparison treatment. Seedlings were irrigated with modified Hoagland mineral
solution during two weeks. Lately, Al was added to mineral solution (0-200 µM as
Al2(SO4)3) and plants grown for other 3 weeks. At harvesting, biomass production,
spore number, root colonization (%), glomalin (Wright and Upadhyaya, 1996) and Al in
shoots and roots were measured. The experiment was carried out with four replicates.
It is recognized that Al damage is mainly produced at root level, particularly rootlets by
shortening and thickening of them interfering in nutrient absorption. Hence, for testing
Al tolerance or sensitivity is better to use root length instead root weight. Consequently,
we did not found significant differences between cultivars. In general, as species, barley
is more Al-sensitive than wheat; however, barley “Aurora” appears to have a high Al
tolerance sometimes similar or higher than wheat cultivars.
Arbuscular mycorrhizal spore number increased significantly in all the cultivars tested
when plants were subjected to Al. The lowest spore number were obtained in “Atlas”
cultivar probably due to this is a foreign cultivar not habitually cropped in volcanic acid
soils and non adapted to such conditions. In addition, results showed that GRSP content
increased in all cultivars when subjected to Al following the same trend that spore
number, which reinforces the role played by this glycoprotein in conferring higher Al
tolerance to host plants when they live in symbiosis with AM fungi. Al-sensitive wheat
cultivar “Tukan” as well the Al-sensitive barley cultivar “Scarlett” increased in a higher
degree the GRSP levels suggesting that its role in Al detoxification in the rhizosphere is
more important in Al-sensitive than Al-tolerant plants. This statement deserves more
and deeper studies in the near future.
ACKNOWLEDGEMENTS.
This study was supported by CONICYT Grant 1100642.
REFERENCES
Aguilera, P., et al. (2011). Soil Biol. Biochem. 43:2427-2431.
Klugh, K., and Cumming, J. (2007). Tree Physiol. 27: 1103-1112.
Klugh, K., and Cumming, J. (2009). Soil Biol. Biochem. 41: 367-373.
Marschner, H. (1995). Academic Press Inc. San Diego, CA, 889p.
Seguel, A., et al. (2013). Mycorrhiza 23: 167-183.
Tang, C., and Rengel, Z. (2003). Handbook of Soil Acidity. Marcel Dekker, N.Y, pp-57-81.
Wright, S., and Upadhyaya, A. (1996). Soil Sci., 161: 575-586.
Session III
SIII-CP-42
Origin of arbuscular mycorrhizal fungi determines plant development
and resource distribution between Phaseolus vulgaris L. and Oriza
sativa in intercropping.
Ramanankierana, H.1*, Rasamiarivelo, A.V.2, Razafimbelo, T.3, Becker, T.4,
Razakatiana, A.T.E.2, Manitriniaina, H.3, Rabeharisoa, L.3, Randriambanona, H.1,
Drevon, J.J.4, Duponnois, R.5
1.
Laboratoire de Microbiologie de l’Environnement, Centre National de Recherches sur l’Environnement,
BP 1739 Fiadanana Antananarivo. Madagascar. 2 Laboratoire de Biotechnologie-Microbiologie, Faculté
des Sciences BP 906, Université d’Antananarivo. Madagascar. 3 Laboratoire des Radioisotopes,
Université d’Antananarivo BP 3383 Antananarivo. Madagascar. 4 IRD, UMR 210 Eco&Sols, CIRADINRA-SupAgro, 2 place Viala, Batiment 12, 34060, Montpellier Cedex France. 5 Laboratoire des
Symbioses Tropicales et Méditerranéennes (LSTM) UMR 113 IRD/CIRAD/SupAgro/UM2/USC INRA,
TA A-82/J 34398 Montpellier cedex 5 France.
*
[email protected]
ABSTRACT
To illustrate the effect of arbuscular mycorrhizal fungi (AMF) origin on plant
development and resource distribution between rice and bean in intercropping, three
groups of AMF were used from soil rhizospheric of these two plants. The combination
of AMF from rice and common bean stimulated the development of both cultivated
plants and increase the concentration of P and N mineral on aerial part. Our work
suggests that the origin of AMF contributes to increase yield and to reduce the use of
organic, mineral or chemical amendment in rice-bean intercropping.
INTRODUCTION
Plant species coexistence constitutes one of biological tools which can be valorized to
improve soil fertility management. However, environmental factors determining the
distribution of resource between these plant species are few documented. This study
shows the effect of arbuscular vesicular mycorrhizal fungi origin on plant development
and resource distribution between P. vulgaris and O. sativa planted separately or
together in low fertility ferralitic soil of Madagascar.
Plant and fungal material.
The plants and AMF used in this study occur in a field culture situated on Malagasy
highland plateau where common bean P. vulgaris L. and O. sativa are usually cultivated
on red ferralitic soil. The three used AMF groups were collected from root system of P.
vulgaris and/or O. sativa cultivated separately or associated in a field [(F1) from P.
vulgaris, (F2) from O. sativa and (F3) a combination of the two AMF groups]. Each of
the three AMF groups was used to inoculate P. vulgaris and/or O. sativa cultivated
together or separately in 2 litter plastic pot culture containing 1950 g of an autoclaved
soil mixture of sand and ferralitic soil (50:50 v:v). Ferralitic soils were amended or not
with organic amendment and/or phosphate fertilizer (20 Kg P ha-1 of manure or M20, 20
Kg P ha-1 of triple superphosphate ferlitizer or TSP 20, and both manure and triple
superphosphate or TSP 20_M20). Control was established with ferralitic soil without
amendment (manure or triple superphosphate). The plants were maintained in the
glasshouse conditions (daylength of 12 h per day) and no other nutrients were given
during the experiment.
Session III
SIII-CP-42
Plant development, resource distribution, and mycorrhizal/rhizobial infection
assessment.
The plants were harvested after 60 days growing time. For each plant, the mycorrhizal
rate was assessed by the method of Phillips and Hayman (1970) and the number of
nodule was counted for each common bean individual plant. Dried plant material was
ground in a ball mill, mixed thoroughly, and P and N concentration of the shoots of
each plant species were determined. Phosphate concentrations were determined by the
molybdate blue ascorbic acid method (Watanabe and Olsen, 1965) and N concentration
with HCN analyser. The total dry eight of each plant was determined (85°C, 24 hours).
Inoculation with one of the three AMF groups and fertilization with TSP 20 and/or M
20 change the biomass produced in each pot culture with an increasing of plant
development in co-culture system. Co-culturing common bean and rice in the pots
amended or not with manure or triple phosphate stimulated significantly the
development of biomass (Figure 1). The effect of the AMF group on the development of
plant was more significant for the combination of rice and common bean AMF than for
the use of the two AMF groups separately.
Figure 1. Development (total biomass) of rice and common bean inoculated with the three AMF groups :
Common bean in monoculture;
in co-culture.
Common bean in co-culture;
Rice in monoculture ;
Rice
Phosphorus and N mineral content of co-culturing plants inoculated with the
combination of rice and common bean AMF was significantly higher than those
measured on plant inoculated with the two groups of AMF separately. The occurrence
of the two groups of AMF stimulated the establishment of symbioses structure
(mycorrhizas and nodules of nitrogen fixing bacteria) on root systems of the two
cultivated plants.
ACKNOWLEGMENTS
This work was supported by the project Fabatropimed (Agropolis Foundation under the reference ID
1001-009)
REFERENCES
Phillips, J.M., and Hayman, D,S. (1970). Trans Br Mycol Soc 55: 158-160.
Watanabe, F.S., and Olsen, S.R. (1965). Soil Sci. Soc.Am; Proc. 29: 67- 678.
Session III
SIII-CP-43
Asociación Española de Leguminosas
De Ron, A.M.1, De la Cuadra, C.2, Millán, T.3*
1
Dpto de Recursos Fitogenéticos. Misión Biológica de Galicia. CSIC.Pontevedra. 2 Centro de Recursos
Fitogenéticos. INIA. Apdo. 1045 Alcalá deHenares. Madrid. 3Dpto de Genética, Universidad de Córdoba.
Campus de Rabanales. Córdoba
* [email protected]
RESUMEN
LaAsociación Española de Leguminosas (AEL), es una entidad sin ánimo de lucro,
constituida para la promoción de las Leguminosas en España. Trata de contribuir a la
coordinación de la investigación con el sector agrícola español, promover foros de
discusión con los interlocutores sociales, defender la variabilidad y fomentar nuevos
usos de las leguminosas.
INTRODUCCIÓN
LaAEL fue creada como resultado de la iniciativa de un grupo nacional de
investigadores de leguminosas. La idea partió de la Primera Conferencia Técnica sobre
Leguminosas Grano celebrada en 1992 en Palencia reuniendo a 90 personas interesadas
en la producción y el consumo de las leguminosas ibéricas procedentes del campo de la
investigación, del mundo empresarial y de la administración pública. Ocho años
después, dentro de las actividades de la 3ª Conferencia sobre Leguminosas Grano
celebrada en Valladolid, surgió la decisión de constituir una Asociación, entendida
como un puente desde la investigación hacia productores, empresas y administración.
La AEL fue constituida en febrero del 2000 y registrada en mayo del mismo año.
OBJETIVOS
Los objetivos de esta asociación son: a) Promover el desarrollo de las leguminosas en
España. b) Coordinar la investigación con el sector agrícola español. c) Servir de foro de
discusión de investigación y sector con los interlocutores sociales. d) Defensa de la
variabilidad de las leguminosas. e) Fomento de nuevos usos de las leguminosas.
ACTIVIDADES
Esta asociación ha organizado cinco jornadas, una cada tres años, a partir del año 2000,
y las Jornadas de Difusión de Nutrición y Salud en Madrid (2002).las contribuciones
presentadas en las diferentes jornadas se han publicado en Actas de la asociación y otras
publicaciones (http://www.leguminosas.es/actas-de-la-ael.html). La amplia distribución
geográfica de los miembros de la AEL ha permitido poner en contacto los grupos de
investigación de diferentes áreas con el objetivo común de las leguminosas. Esta
asociación tiene también una proyección internacional, ha participado en el apartado de
difusión de resultados en los proyectos Europeos PHASELIEU y GRAINLEGUMES y
ha colaborado en la organización de congresos internacionales como EUFABA y
ASCOCHYTA2012.
La AEL está conectada con instituciones nacionales, como los Ministerios de Educación
y Agricultura, Alimentación y Medio Ambiente, asociaciones de productores de
semillas y de desarrollo rural. Pertenece a la COSCE (Confederación de Sociedades de
Científicos de España , http://www.cosce.org/ ) y a la Plataforma Tecnológica de
Session III
SIII-CP-43
Agricultura Sostenible (http://www.agriculturasostenible.org), foro de encuentro entre los
agentes del sistema ciencia-tecnología-empresa en el ámbito del sector agrario.
La amplia distribución geográfica de sus miembros ha permitido poner en
contacto los grupos de investigación de diferentes áreas pero con el objetivo común de
las leguminosas.
DIFUSIÓN
Los objetivos y actividades de la asociación se difunden a través de su página
web (http://www.leguminosas.es/) donde se pueden descargar publicaciones
relacionadas (revista Grain Legumes, descriptores, catálogos, recetarios) y las Actas de
las cinco reuniones organizadas por la asociación.
A través de nuestro blog (http://leguminosas.wordpress.com/) se pueden
comentar las noticias de actualidad relacionadas con leguminosas.
FUTURO
Entre nuestros objetivos para el futuro se contempla la organización de un
Foro sobre Leguminosas y Jornadas Técnicas (Sur y Norte de España). Pretendemos
estrechar la relación con otras sociedades afines, como la SEFIN, y asociaciones
relacionadas con la nutrición y alimentación. También pretendemos ampliar nuestra
relación con países vecinos como Portugal.
Session IV
Associative interaction plant/beneficial
microbe
Session IV
SIV-P-1
Biotechnological applications of the plant-microorganism interaction:
health, environment and agriculture.
Gutierrez Mañero, F.J.*
Universidad CEU-San Pablo. Facultad de Farmacia. Madrid. España.
*
[email protected].
Nowadays, the rhizosphere is an ecosystem, although it was not considered as such until
the late 20th century. It’s a unique ecosystem, probably the largest in the Earth, of
outmost complexity since three partners (plant, soil and microorganisms) are involved.
Understanding of this system calls for the previous knowledge of each of the three parts,
to then learn about their interaction. Today, the complex system of microbial
communities associated to plant roots is considered the second genome of the plant,
with a key role on its physiology and health (Berendsen, 2012).
As a matter of fact, research in the plant microorganism interaction is currently one of
the hot-spots in biology research. The study of the rhizosphere is resulting in an
enormous array of biotechnology-based applications with successful results for the food
market, agriculture, pharmacy and environment.
A multidisciplinary approach is absolutely necessary to study the rhizosphere, calling
for multiphase methodologies involving all areas of knowledge in biology. All
approaches, from the most basic knowledge starting at biophysics and biogeochemistry,
to molecular levels, namely communication strategies, metabolic adaptations and
multitrophic interactions; and finally, studying the physiology of complex systems such
as symbiotic relations (mycorrhiza and N fixation), growth promotion and plant fitness.
Each of the before mentioned approaches to the rhizosphere, with no exception, have
contributed to considerable improvements of knowledge from a basic or applied point
of view.
The rhizosphere is probably the area where the most specialized signal exchange
between plant and microorganism takes place. The first communication process studied
within this area was the symbiotic interaction, and from there on, it was extended to
plant-pathogenic microorganism and plant-beneficial microorganism interactions,
specifically with Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper et al.,
1989). This study extends to communication among microorganisms in order to
organize their relations with other living organisms within the rhizosphere, including
other microorganisms and extending to the plant and how its physiology is affected.
Identification and knowledge about bacterial communication has contributed to
understand some mechanisms used by certain PGPR to improve plant fitness. The
interaction between plants and beneficial bacterial strains is one of the most complex
processes known due to the exchange of information between two organisms that do not
share the same cellular compartment. The relevance of this exchange is such that the
more accessible partner, the microorganism, has become the target for manipulation, in
order to manipulate the most complex partner, the plant, in our benefit.
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Figure 1. Model of the interactions among beneficial, commensal and pathogenic microorganisms, and
within these and the plant. Effects of microorganisms on the plant.
Based on the above, and considering the actual knowledge of the interaction mechanism
that deals with:
 Defensive systems in the plant
 Plant secondary metabolism involved in defense
 Physiology of plant growth and development
 Communication and elicitation systems
 Signal transduction mechanisms
 Microbial metabolism involved in cell to cell communication outside the plant
(basically quorum sensing)
 Microbial metabolism involved in biogeochemical cycles
A number of biotechnology based procedures are currently being used, all of them
based on this knowledge of the plant-microorganism interaction. Some are currently
being used with great profit, and are named below.
 Agrifood technology, including plant production both, from the classical
agronomic point of view, that is evaluating yield improvment, and from a
modern point of view, that is considering production with low chemical inputs,
environmentally friendly and sustainable agriculture and evaluating food quality.
 Pharmaceuticals and nutraceuticals, improving bioactive concentration in foods
and phytopharmaceuticals in medicinal plants.
 Phytorrizorremediation, use of biological systems to clean up toxic waste and
recovery of degraded or polluted ecosystems.
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A.
B.
Figure 2. A. Workplan to isolate beneficial microorganisms from rhizosphere systems. B. Factors that
affect plant’s secondary metabolism, the potential biotechnological targets to improve the plant’s
adaptative capacity to stress and applications.
The rhizosphere of wild plants has been shown as an excellent source of specialized
PGPR due to the co-evolution processes that have taken place throughout the years
(Gutierrez Mañero et al., 2003). Some rhizosphere strains are able to trigger plant’s
metabolism, a process known as elicitation. Biological elicitation is a useful strategy to
improve plant production (biomass) and/or to trigger secondary metabolism, which is
especially relevant for the pharmaceutical and food industry (Zhao et al., 2005; Boué et
al., 2009; Nosov, 2012). The use of PGPR to trigger secondary metabolism is an
extremely useful tool to increase concentration of bioactive compounds in the plant,
which results in a raw material of high quality, that can be used either as a food product,
naturally rich in bioactive compounds, or for the pharmaceutical industry, to develop
new drugs or food supplements.
Systemic induction of plant secondary metabolism involved in defense has been shown
in a number of plant species (van Loon, 2007). However, the nature of the molecules
involved in triggering the response is not clear yet. Several bacterial determinants have
been proposed and their role demonstrated in induction of systemic resistance in several
plant species, such as bacterial cell wall lipopolysaccharides (LPS) or flagellin, the
structural protein from bacterial flagelum (Ramos Solano et al., 2008b; van Wees et al.,
2008). This fact suggests that there is certain specificity on the recognition process that
is mediated by plant receptors able to trigger a signaling cascade to activate defense
mechanisms. A common feature of elicitors is that they are conserved components of
cell surface, or metabolites released by the microorganism (siderophores, antibiotics,
biosurfactants, etc.), and a single microorganism may show different elicitors.
Currently, elicitors from pathogenic microorganisms are termed PAMPs, from
pathogen-associated molecular patterns, or MAMPs from microbe-associated molecular
patterns (Erbs and Newman, 2011).
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B.
Figure 3. A. Biotechnological applications of the plant-microorganism interaction targetted to plant
secondary metabolism. B. Metagenomic approach to the rhizosphere: isolation of genes from nonculturable rhizosphere microorganism should result in obtention of effective molecules beneficial to
plant fitness, avoiding the use of the microorganism.
REFERENCES
Berendsen, R.L., et al. (2012). Trends in Plant Science, 17: 478-486.
Boué, S.M., et al. (2009). J Agr Food Chem, 57: 2614-2622.
Erbs, G., and Newman, M.-A. (2011). Molecular Plant Pathology, 13: 95-104.
Gutiérrez Mañero et al. (2003). J.Plant Physiology, 160: 105-113.
Kloepper, J.W., et al. (1989). Trends Biotechnol, 7: 39-43.
Nosov, A.M. (2012). Applied Biochemistry and Microbiology. 48: 609-624
Ramos Solano, B., et al. (2008b). Phytopathology, 98: 451-457.
van Loon, L.C. 2007. Eur J Plant Pathol, 119: 243-54.
Van Wees, S. C., et al. (2008). Current Opinion in Plant Biology, 11: 443-448.
Zhao, J., et al. (2005). Biotechnol Adv, 23: 283-333.
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Aspectos bioquímicos y moleculares de la interacción Delftia-RizobioAlfalfa: un lenguaje de señales químicas.
Castro-Sowinski, S.*
Sección Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de la República, Igua 4225,
Montevideo, Uruguay
*
[email protected]; [email protected]
Las rizobacterias pueden ejercer efectos neutros, beneficiosos o desfavorables sobre el
crecimiento y desarrollo de las plantas. Estos efectos son el resultado de un intercambio
de señales donde existe un reconocimiento mutuo de moléculas difusibles producidas
por la planta y el microorganismo. Comprender esta compleja red de señales constituye
una gran oportunidad para utilizar esta información para el diseño de estrategias que
mejoren la productividad y sustentabilidad de los cultivos. Las plantas y los
microorganismos producen flavonoides, fitohormonas, receptores de membrana e
intracelulares, nodulinas, lectinas, enzimas, lipoquito-oligosacáridos, exopolisacáridos,
aminoácidos, ácidos grasos, vitaminas y moléculas volátiles, entre otras, como parte del
diálogo molecular.
La interacción más estudiada es la que establecen las plantas y las rizobacterias
promotoras del crecimiento vegetal (PGPR, del inglés Plant Growth Promoting
Rhizobacteria) diazótrofas, siendo los ejemplos más remarcables las interacciones
establecidas con miembros de los géneros Azospirillum y Rhizobium.
La estrategia de señalización entre los rizobios y las leguminosas se basa principalmente
en la producción de carbohidratos, tales como los Factores de Nodulación y
exopolisacáridos (EPS), entre otros. Pero las raíces y los microorganismos también
producen cócteles específicos de moléculas, diferentes a carbohidratos, que son
fundamentales durante una interacción exitosa. Las raíces secretan fitohormonas y
flavonoides (que actúan como moléculas atrayentes) que inician el diálogo químico del
par simbiótico. Los rizobios reconocen los flavonoides por su unión a receptores de
membrana conocidos como NodD, quienes funcionan como un sensor ambiental y
activador transcripcional de los genes que se encuentran aguas debajo de la caja nod.
En respuesta, los rizobios producen y secretan los Factores de Nodulación
(lipoquitooligosacárido), quienes actúan como ligandos de receptores ubicados en los
pelos radicales emergentes. Estos receptores se conocen como NFR-LK (por su siglas
en inglés, Nod Factor Receptor-like kinase). Luego del reconocimiento NFR-LKligando, se produce la depolarización de la membrana plasmática como consecuencia de
un flujo alterado de iones, seguido de oscilaciones periódicas en los niveles
intracelulares de calcio (calcium spiking o firma de calcio). El calcio actúa entonces
como un segundo mensajero que afecta una gran variedad de eventos celulares. Luego
del reconocimiento del Factor de Nodulación, se activan varios receptores quinasa que
funcionan activando una cascada de transducción de señales que controlan la progresión
del hilo de infección, la organogénesis del nódulo y la fijación de nitrógeno. Se produce
entonces la deformación de los pelos radicales, se inicia la formación del hilo de
infección, se rearregla el citoesqueleto, se produce la expresión de las nodulinas
tempranas, y finalmente se desarrolla el nódulo (Figura 1).
Algunos rizobios producen proteínas involucradas en su especificidad por el hospedero
y su eficiencia simbiótica. Tal es el caso de los sistemas de secreción del tipo III, T3SS,
capaces de inyectar proteínas (conocidas como Nops, nodulation outer proteins)
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directamente dentro de la célula hospedera. Por ejemplo, NopL y NopP interfieren con
las vías de señalización de la planta, actuando como un efector positivo que ayuda a la
formación del nódulo. Otras proteínas Nop contribuyen a la supresión de la respuesta
inmune de la planta, facilitan la liberación de los rizobios desde el hilo de infección,
inician la simbiosis, y participan en la persistencia del bacteroide.
Figura 1. Vista simplificada de la interacción rizobio-leguminosa. a) la planta secreta flavonoides que
inducen la expresión de los genes nod de rizobios, con producción del Factor Nod (FN). b) Percepción del
FN por parte de los receptores NFR-LK de raíz, oscilación de los niveles de Ca2+ que produce la síntesis
localizada de citoquinas (CK). CK induce la producción de ENOD40 y la cascada de señalización que
activa la respuesta simbiótica y la organogénesis del nódulo. c) deformación de los pelos radicales y
formación del hilo de infección (HI); los rizobios se movilizar por el HI. d) los rizobios penetran a las
células corticales, y se liberan en el citoplasma del hospedero, transformándose en simbiosomas, ubicados
dentro del bacteroide. e) se sintetiza el péptido CLE en el nódulo, y reconocimiento del mismo por los
recpetores específicos LRR-RLK; producción del inhibidor SDI que regula el número de nódulos (AON).
f) los nódulos indeterminados producen el péptido NCR, que induce la diferenciación del bacteroide.
Adaptado de Morel y Castro-Sowinski (2013).
El rizobio se traslada por el hilo de infección hasta que penetra a la célula vegetal por
endocitosis, y se rodea de la membrana peribacteroidea, de origen vegetal. Las
fitohormonas y péptidos señal de origen vegetal (ENOD40, CLE, NCR) inducen
entonces la organogénesis del nódulo, la proliferación celular y dediferenciación, y
diferenciación del bacteroide. El péptido CLE actúa como una señal que asciende desde
las raíces hacia sus receptores LRR (leucine-rich repeat), quienes controlan múltiple
aspectos del desarrollo del nódulo y la producción a nivel de las hojas de un inhibidor
(SDI, shoot-derived inhibitor) que regula el número de nódulos de la raíz, el cual forma
parte de la vía de señalización de autorregulación de la nodulación (AON,
autoregulation of nodulation), que controla la hipernodulación (Figura 1).
El Factor de Nodulación, así como la citoquina (mensajero secundario que se sintetiza
en las células epidérmicas, y se traslada a la células corticales iniciando la formación del
nódulo primordial) activan la expresión de los genes correspondiente a las nodulinas
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tempranas, como ENOD12 y ENOD40. Esta última participa en la movilización de
nutrientes a las células corticales y la maduración del tejido del nódulo, entre otras.
Las leguminosas como Medicago, Pisum, Vicia y Trifolium desarrollan meristemas
apicales activos que producen nódulos indeterminados, consecuencia de la
diferenciación irreversible mediada por el péptido NCR (nodule-specific cystein-rich).
Las células del hospedero producen los NRCs, y estos interfieren con el ciclo celular del
rizobio, afectando la diferenciación terminal de la bacteria.
La simbiosis rizobio-leguminosa puede favorecerse por la presencia de otras PGPR,
diferentes a los rizobios, que producen fitohormonas que estimulan el crecimiento de la
raíz, producen un cambio cualitativo y/o cuantitativo del perfil de flavonoides
secretados por las plantas, y/o producen sustancias que solubilizan nutrientes de baja
biodisponibilidad en el suelo (principalmente fósforo), entre otras. Probablemente, la
producción de fitohormonas sea el mecanismo que mejor explica este efecto, pero se
postula que el efecto se debería de la suma de diferentes mecanismos. En el caso de la
coinoculación de leguminosas con rizobios y Azospirillum spp., o Pseudomonas spp., se
induce la síntesis de flavonoides por las raíces de las leguminosas. Sin embargo, no
parece ser estrictamente necesaria la presencia del microorganismo. La aplicación de
exudados libres de bacterias a las leguminosas produce un efecto similar a la
coinoculación. La lista de metabolitos capaces de mejorar la asociación plantamicroorganismo incluye también: 1) las vitaminas que actúan como suplementos
nutricionales para los rizobios; b) enzimas hidrolíticas que ayudan al rizobio durante su
penetración al pelo radical o atacan hongos fitopatógenos, c) ácidos que solubilizan
fosfatos e incrementan su biodisponibilidad.
En los últimos años han sido publicados varios trabajos que posicionan a las bacterias
del género Delftia como PGPR (diazótrofo con actividad biocontroladora). Estas
bacterias fijan nitrógeno (actividad reductora de acetileno) en vida libre cuando se las
ensaya en medio semisólido libre de nitrógeno suplementado con vanadio; producen
sideróforos y la auxina ácido indol-3-acético (AIA); promueven el crecimiento de
alfalfa, trébol y soja en condiciones con bajo riego de nitrógeno; y actúan como
bacterias que asisten durante la interacción entre los rizobios y la leguminosa,
aumentando el número de nódulos por planta, la velocidad de nodulación y el peso seco
de la planta, en condiciones gnotobióticas. Este efecto también se evidenció cuando los
ensayos de plantas se realizaron en condiciones de invernáculo, utilizando
tierra:arena:vermiculita como soporte. Curiosamente, estas bacterias resisten Cr(VI) y
Pb(II), dejando pendiente el estudio de su potencial uso en la promoción del crecimiento
de plantas bioacumuladoras de metales pesados.
Con el objetivo de contribuir al entendimiento del diálogo químico involucrado en la
comunicación entre las leguminosas y sus PGPR, se estudió la composición de las
moléculas secretadas por plantas de alfalfa y las bacterias en condiciones de hidroponia.
Se cultivaron plantas de alfalfa y se inocularon y coinocularon con Sinorhizobium
meliloti U143 (inoculante comercial) y Delftia sp. JD2, y se colectaron las soluciones de
hidroponia (desde ahora, rizodeposiciones) a varios tiempos. La composición de las
rizodeposiciones se analizó por cromatografía gaseosa y líquida, acopladas a
espectrometría de masas y FID. Se destaca la gran producción de ácidos grasos,
principalmente ácido lignocérico (C24:0; derivado de la degradación de lignina), así
como una secreción aumentada de luteolina en las rizodeposiciones de plantas
coinoculadas, comparada con las rizodeposiciones obtenidas durante la inoculación
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simple con U143. Las grandes diferencias se detectaron a los 4 días de inoculación, con
disminución de la concentración de señales ya a los 7 días de inoculación. El aumento
en la secreción de luteolina puede explicarse porque los flavonoides tienen un efecto
importante en las primeras etapas de la interacción, induciendo la expresión de los genes
de nodulación y la atracción de los microorganismos hacia las raíces; además, protegen
a las células en división del daño oxidativo (poder antioxidante). También se detectó la
secreción aumentada de flavonoides no involucrados directamente en la inducción de la
expresión de los genes de nodulación. Este tipo de flavonoides suelen actuar como parte
del mecanismo de contención de un fenotipo hipernodulante. Se detectaron terpenos
(como el antioxidante limoneno), posiblemente involucrados en la interacción
microorganismo-planta.
Se evidenció la presencia de triptófano (precursor de la síntesis de AIA) en los exudados
colectados a los 4 días de inoculación (sin detección de AIA), mientras que a los 7 días
de inoculación se detectó AIA, concomitantemente con la desaparición del triptófano,
posiblemente debido a su utilización durante la biosíntesis de la auxina. Finalmente, las
rizodeposiciones (libres de microorganismos por filtración) producidas durante la
coinoculación, mostraron efectos promotores del crecimiento de alfalfa, sugiriendo que
las mismas contienen sustancias promotoras.
La detección de otros metabolitos será discutida durante la comunicación oral de María
Morel (Morel, M.A., Cagide, C., Dardanelli, M.S., Castro-Sowinski, S.: Caracterización
de la interacción Delftia-Sinorhizobium en alfalfa.)
CONTRIBUCIONES Y AGRADECIMIENTOS
Los resultados acá presentados son el resultado de trabajos de grado y posgrado (Mag.
María Morel, Lic. Victoria Braña, Est. Célica Cagide) financiados por Pedeciba y ANII.
Se agradece a la Dra. MS Dardanelli (Universidad Nacional de Río Cuarto, Río Cuarto,
Argentina), quien supervisó varios de los estudios realizados sobre las rizodeposiciones.
BIBLOGRAFÍA
Morel, M.A., and Castro-Sowinski, S. (2013). En: Plant microbe symbiosis-Fundamentals and Advances,
NK Arora (Ed.), Springer India. DOI 10.1007/978-81-322-1287-4_6 (en prensa).
Morel, M.A., et al. (2012). En: Crop Plant, Dr A. Goyal (Ed.) ISBN: 978-953-51-0527-5, InTech, DOI:
10.5772/37413. http://www.intechopen.com/books/crop-plant/legume-crops-importance-and-use-ofbacterial-inoculation-to-increase-production.
Morel, M.A., et al. (2011). Arch. Microbiol. 193: 63-68.
Mortier, V., et al. (2012). En: Plant signaling peptides. H.R. Irwing, and C. Gehring (Eds.) DOI
10.1007/978-3-642-27603-3_8.
Ubalde, M.C., et al. (2012). Curr. Microbiol. 64: 597-603.
268
Session IV
SIV-CO-1
Martínez-Hidalgo, P. 1, 2*, Martínez-Molina, E. 1, 2, Pozo, M.J.3
1
Departamento de Microbiología y Genética, Universidad de Salamanca, España. 2 GIR "Interacciones
mutualistas planta-microorganismo", Unidad Asociada al IRNASA. CSIC, Salamanca. España. 3
Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín. CSIC,
Granada, España.
*
[email protected]
ABSTRACT
In this study the potential for biological control of Micromonospora has been analysed,
in the context of the new agricultural sustainability that has been outlined by the
European Union and accepted by our country.
Results show that Micromonospora effectively protects tomato plants from grey mould,
caused by Botrytis cinerea. The quantitative analysis for gene expression in the
defensive routes of the plant shows that Micromonospora increases immune response of
the plant in what is now known as priming. The defensive response of the plant
inoculated with Micromonospora is different and stronger than the plants not inoculated
and it only appears when the pathogen is present.
INTRODUCTION
Agriculture derived from the green revolution that is defined by the use of pesticides
fertilizers and herbicides of chemical origin, together with genetic improvement of plant
germoplasm, produced an increase in agricultural productivity. Decades ago, the cost
and risks derived of this kind of agriculture were revealed and as a consequence, a new
agricultural revolution is starting to develop in which probiotic microorganisms are an
alternative to chemicals. However, it is necessary to make an adequate analysis,
evaluation and selection of the strains used in order to obtain the desired effect.
In our laboratory, strains of Micromonospora have been isolated from healthy plant
nodules in a variety of genera of leguminous plants. The main goal of this study was to
determine their function as probiotic bacteria as there is scarce information about them
even though their biotechnological potential and also their impact in this new
agriculture are relevant.
Plant culture, mRNA extraction, gene expression analysis by means of RT-PCR (qPCR)
and the statistical analysis were performed as shown in López-Raez et al. (2010).
Studies performed with Micromonospora to determine its effectiveness in tomato plant
defence against Botrytis gave excellent results. This effectiveness was observed in
whole plant and experiments in which leaves were detached before applying the
pathogen (detached leaf assay). It became clear that this microorganism, when
inoculated in plant roots, is capable of reducing the symptoms the fungi causes in leaves
(Figure 1).
Results for the quantitative analysis of gene expression in genes used as markers for
defence pathways in tomato, showed that the bacteria does not have a direct effect in the
induction of said defence mechanisms, nor in jasmonic or salicylic regulated response.
The pathogen (Botrytis) triggers the expression of the salicylic acid pathway (Figure 2).
269
Session IV
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Figure 1. Effect of Micromonospora in plant defence against Botrytis. A) Diameter of the necrotic halo in
treatments using whole plants. B) Necrosis caused by Botrytis in detached leaves. Treatments: C: Control,
inoculated only with Botrytis. PR18: Inoculated with Micromonospora strain PR18 a month before
infection with Botrytis. B5: Inoculated with Micromonospora strain B5 a month before infection with
Botrytis. PR181D: Inoculated with PR18c a day before infection with Botrytis. B51D: Inoculated with B5
a day before infection with Botrytis.
Figure 2. Quantitative PCR results for PR1, LOXA and PinII normalized against EF (Elongation Factor).
Treatments: C: Uninoculated plants. PR18: Inoculated with Micromonospora strain PR18. C+Bot:
Inoculated only with Botrytis. PR+Bot: Inoculated with Micromonospora strain PR18 and Botrytis.
Micromonospora inoculation was performed a month before detaching the leaves and infecting them with
Botrytis.
Plants treated with Micromonospora and later infected with Botrytis cause an activation
of LOXA and PinII genes (from the jasmonic acid pathway) and confers resistance
against this pathogen (El Oirdi et al., 2011) while downregulating the salicylic acid
pathway, which is the same one that becomes upregulated when Micromonospora is not
present (Figure 2). In conclusion, Micromonospora has a priming effect in tomato when
inoculated in the root. Defensive response against Botrytis is higher when a previous
treatment with Micromonospora was applied but it is only triggered when the pathogen
is infecting the plant. According with the results, Micromonospora is a good candidate
for biocontrol in order to protect crops against phytopathogenic fungi. Using this
bacteria also has the advantage of being able to be inoculated while sowing, since the
results did not vary when the plants were inoculated with Micromonospora one day or
one month before Botrytis, which means that the effectiveness of Micromonospora
inoculation lasts at least one month since its application.
ACKNOWLEDGEMENTS
This work has been done thanks to the funds from the Ministerio de Ciencia y Tecnología and the Junta
de Castilla y León. P.M-H. received a predoctoral fellowship from the CSIC (Programa JAE).
REFERENCES
López- Raez, J.A., et al. (2010). J. Exp. Bot. 61: 2589-2601.
El Oirdi, M., et al. (2011). Plant Cell 23: 2405-2421.
270
Session IV
SIV-CO-2
Movimiento y quimiotáxis en Pseudomonas fluorescens F113,
influencia en una colonización competente de la rizosfera de las
plantas.
R., Martín, M. *
Departamento de Biología. Facultad de Ciencias. Universidad Autónoma de Madrid. Madrid. España.
*
[email protected]
RESUMEN
Se presenta el análisis de la regulación de la funcionalidad del flagelo a través del
diGMPc y el posible papel de tres sistemas de quimiotaxis encontrados en la secuencia
de P. fluorescens F113.
INTRODUCCIÓN
Pseudomonas fluorescens F113 fue aislada de la rizosfera de remolacha y su capacidad
para producir compuestos con actividad antibiótica y antifúngica la convierte en una
estirpe de interés en biocontrol. Los usos de F113 en sistemas integrados
planta/microorganismo dependen de la eficacia de la colonización de la rizosfera. Uno
de los caracteres de interés en la colonización competitiva de la rizosfera es la capacidad
de movimiento (Capdevila et al., 2004; Martínez-Granero et al., 2006). El conocimiento
adquirido sobre la regulación del movimiento en F113 (Redondo-Nieto et al., 2008;
Navazo et al., 2009; Barahona et al., 2010; Martínez-Granero et al., 2012) nos ha
permitido diseñar estirpes que se mueven mucho más y que presentan muy disminuida
la capacidad para formar biopelículas (Barahona et al., 2010). Estas estirpes
hipermóviles son más competitivas y presentan sustancialmente mejorada la capacidad
de biocontrol en dos patosistemas distintos (Barahona et al., 2011). En P. fluorescens
F113, hemos observado la existencia de dos aparatos flagelares uno típico de todas las
Pseudomonas y otro similar al de A. vinelandi y existen al menos seis señales (5
ambientales y 1 citoplasmática) que independientemente regulan el movimiento a nivel
de síntesis de cada uno de los aparatos flagelares y de su funcionalidad (Navazo et al.,
2009; Martínez-Granero et al., 2012). Por otro lado, sabemos que el movimiento
quimiotáctico es esencial para asentarse y colonizar competentemente la rizosfera de las
plantas por parte de las rizobacterias (de Weert et al., 2002). Las pseudomonas en
general cuentan con dos sistemas de quimiotaxis. Sin embargo, tras la secuenciación del
genoma de F113 (Redondo-Nieto et al., 2013) hemos observado la existencia de tres
sistemas de quimiotaxis en P. fluorescens F113.
MATERIAL Y MÉTODOS
Obtención de mutantes, mediante mutagénesis por inserción.
Obtención de proteína de fusión en el extremo C terminal. Fusión de eCFP al extremo
3’ de flgZ en el plásmido pVLT31.
Análisis de localización mediante microscopio confocal espectral LEICA SP5 con
objetivo 63x y laser de Argón a 458 nm. SIDI-UAM
Complementación de los mutantes con los cósmidos correspondientes aislados de la
librería genómica de F113.
Análisis de movimiento en placas con 0.3% de agar.
Experimentos de anoxia realizado en jarras anaeróbicas
Análisis de competitividad inoculando la misma proporción de estirpe silvestre y
competidor en la base del tallo de la plántula recién germinada. Tras dos semanas de
271
Session IV
SIV-CO-2
colonización se recogen las rizobacterias del ápice de la raíz y se analizan las UFC en
medio selectivo.
Dos diguanilatociclasas y una fosfodiesterasa regulan la funcionalidad del flagelo.
La molécula señal diGMPc está implicada en la señalización que regula la funcionalidad
del flagelo. Existen dos diguanilatociclasas (SadC y WspR) y una fosfodiesterasa
(BifA) cuyos mutantes están afectados en movilidad y por tanto están implicadas en el
mantenimiento de los niveles de diGMPc en relación con el movimiento flagelar. El
genoma de F113 cuenta con una proteína flagelar FlgZ que tiene un dominio PilZ que
detecta diGMPc y responde interaccionando con la proteína FliG del motor flagelar. Su
expresión está regulada por el activador transcripcional de síntesis del aparato flagelar
de Pseudomonas FleQ y su localización en la bacteria depende de los niveles de
diGMPc celulares. Cuando los niveles de diGMPc son altos como consecuencia de la
acción diguanilatociclasa de SadC y WspR, FlgZ se une al motor flagelar
probablemente frenando el movimiento. Cuando los niveles de diGMPc son bajos como
consecuencia de la acción de la fosfodiesterasa BifA la proteína se encuentra dispersa
por la célula favoreciendo el movimiento.
F113 tiene tres sistemas de quimiotaxis necesarios y no intercambiables.
Los mutantes obtenidos en cada una de las histidín quinasas (CheA) de los tres sistemas
presentan distintos fenotipos de movilidad cuando el análisis se realiza tanto en
aerobiosis como en anaerobiosis. Los sistemas quimiotácticos 1 y 3 tienen una mayor
importancia en la colonización competitiva de la alfalfa por F113. Cabe señalar que los
mutantes en CheA1 y CheA3 son inmóviles cuando los experimentos son llevados a
cabo en anaerobiosis. Creemos que la capacidad para realizar quimiotaxis en presencia
de oxígeno o en anaerobiosis es muy importante para el mantenimiento de la
competitividad por la colonización de un nicho ecológico cambiante como es el
rizosférico.
AGRADECIMIENTOS
Este trabajo ha sido financiado por los proyectos BIO2009-08254, BIO2012-316034 y Microambiente
CM.
BIBLIOGRAFÍA
Barahona, E., et al. (2010). Environ. Microbiol. 12: 3185-3195.
Barahona, E., et al. (2011). Appl. Environ. Microbiol. 77: 5412-5419.
Capdevila, S., et al. (2004). Microbiology SGM 150: 3889-3897.
De Weert, S., et al. (2002). Appl. Environ. Microbiol. 56: 1173-1180.
Martínez-Granero, F., et al.(2005). Microbiology SGM 151: 975-983.
Martínez-Granero, F., et al. (2006). Appl. Environ. Microbiol. 72: 3429-3434.
Martínez-Granero, F., et al.(2012). PLoS ONE Vol 7: e31765.
Navazo et al. (2009). Microbial Biotech. 2: 489-498.
Redondo-Nieto, M., et al.(2013). BMC Genomics 14: 54.
272
Session IV
SIV-CO-3
Morel, M.A.1*, Cagide, C.1, Dardanelli, M.S.2, Castro-Sowinski, S.1, 3
1
Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE),
Av. Italia 3318, Montevideo, Uruguay. 2 Departamento de Biología Molecular, Universidad Nacional de
Río Cuarto, Río Cuarto, Argentina. 3 Sección Bioquímica y Biología Molecular, Facultad de Ciencias,
Universidad de la República, Montevideo, Uruguay.
*
[email protected]
RESUMEN
Durante la co-inoculación de alfalfa con Sinorhizobium meliloti y Delftia sp. se produce
un aumento significativo del crecimiento vegetal, comparada con la inoculación simple,
probablemente debido al aumento en la secreción de flavonoides (especialmente
luteolina) y glucosa. Cambios en la secreción de otros metabolitos pueden también
explicar la promoción del crecimiento de alfalfa. Los mismos serán discutidos en la
sesión de póster.
INTRODUCCIÓN
Muchas veces, la co-inoculación, con dos o más microorganismos promotores del
crecimiento vegetal (MPCV), produce un incremento del rendimiento vegetal de
leguminosas, comparado con la inoculación simple con rizobios. Algunos ejemplos son
la co-inoculación con bacterias de los géneros Bacillus y Azospirillum (Morel et al.,.
2012). Las bacterias del género Delftia son β-Proteobacterias y algunas de ellas,
descritas como MPCV, son capaces de fijar nitrógeno en vida libre, producir ácido
indol-3-acético (AIA) y sideróforos (Han et al., 2005; Morel et al., 2011; Ubalde et al.,
2012). Actualmente, existe al menos un registro comercial de inoculante conteniendo
Delftia acidovorans, formulado para el mejoramiento en la producción de soja y canola
(Brett Young™). Los objetivos de esta propuesta fueron: 1) demostrar la capacidad del
aislamiento nativo Delftia sp. JD2 de promover el crecimiento de alfalfa, en ensayos de
co-inoculación con S. meliloti U143 (inoculante nacional), en condiciones de
invernadero; y 2) analizar la composición química de las rizo-deposiciones, obtenidas
en condiciones de hidroponia, durante la co-inoculación de las plantas.
MATERIAL Y MÉTODOS
Estudios en invernáculo. Se determinó el peso seco de la parte aérea y radicular y el
largo de raíz principal de plantas de alfalfa co-inoculadas con U143 y JD2, y los
controles correspondientes (inoculación simple y sin inoculación), luego de 45 días de
crecimiento en invernáculo en macetas conteniendo una mezcla de
tierra:arena:vermiculita (2:2:1).
Estudios analíticos sobre exudados radicales. Se analizó la concentración por
espectrofotometría y la composición por cromatografía de: flavonoides y AIA (LCMS/MS), azúcares y ácidos grasos (GC-FID) y aminoácidos (GC-MS), en los líquidos
de hidroponia obtenidos luego de 4 dias de crecimiento de plantas de alfalfa coinoculadas con U143 y JD2 (y los controles correspondientes).
Los ensayos se realizaron por triplicado y los datos se analizaron estadísticamente por
ANOVA utilizando PAST software versión 2.16 (Hammer et al., 2001).
La producción de materia aérea de alfalfa se incrementó en un 10% cuando se realizó la
co-inoculación con U143 y JD2, comparado con la inoculación simple con rizobios
(Figura 1). Bajo déficit de nitrógeno, JD2 indujo el aumento del desarrollo del sistema
radicular (Figura 1), probablemente debido a la producción de AIA. Sin embargo, no se
273
Session IV
SIV-CO-3
detectó aumento del tamaño del sistema radical durante la co-inoculación, ni en plantas
fertilizadas con nitrógeno.
500
c
800
b
b
700
b
600
Peso seco (mg)
Peso seco aéreo (mg)
400
d
300
c
a
acd
200
b
d
500
400
a
ad
300
200
100
100
JD2
-N
2
3-JD
U14
JD2
U14
3
Con
trol
JD2
N
U14
3-JD
2
JD2
U14
3
N
Con
trol
N
0
0
Figura 1. Peso seco de parte aérea (izquierda) y raíz (derecha). Control y N: plantas sin nitrogenar y
nitrogenadas, respectivamente, sin inocular; U143 y JD2: plantas inoculadas con U143 y JD2, respectivamente;
U143-JD2: plantas co-inoculadas. Todas las inoculaciones fueron bajo déficit de nitrógeno.
Se detectaron cambios en la composición de azúcares exudados entre los diferentes
tratamientos. Es de destacar que durante la co-inoculación solo se detectó glucosa, el
azúcar con mayor contenido energético, entre los analizados (Tabla 1).
Tabla 1. Composición en azúcares de las rizo-deposiciones.
Azúcar
Ribosa
Galactosa/Celobiosa
Glucosa
U143
15±2
19±3
66±5
JD2
25±4
29±3
46±3
U143+JD2
----100
A excepción de la naringenina (precursora de otros flavonoides más complejos), durante
la co-inoculación se produjo un aumento de la secreción de los flavonoides analizados,
resaltando el aumento en luteolina en un factor de 150 (Tabla 2).
Tabla 2. Composición (µM) en flavonoides, AIA y triptófano (Trp) de las rizo-deposiciones. Apigenina,
Api; Crisina, Cri; Genisteína, Geni; Naringenina, Narg; Luteolina, Luteo; Morina, Mor; Naringina, Nar.
AIA
U143+JD2 JD2
U143
0.009
Trp Api Cri Geni Narg
Mor Nar
Luteo
0.15 18 19 17 0.06 3.07
- 0.3
0.16 0.3 4 0.7 2.2 0.15
- 0.09
0.3 6 10
6
11 0.02 - 0.11
9
Basados en el aumento de secreción de luteolina durante la co-inoculación, se continuó
con la evaluación del efecto de la luteolina y/o de las rizo-deposiciones de alfalfa sobre
las proteínas secretadas por Delftia sp. JD2. Estos resultados y otros serán discutidos y
mostrados en la sección de póster durante el congreso.
AGRADECIMIENTOS
A PEDECIBA, ANII, Amsud-Pasteur.
BICLIOGRAFÍA
Hammer, O., et al. ( 2001). Palaeontología Electrónica 4:9.
Han, J., et al. (2005). Syst Appl Microbiol 28: 66-76.
Morel, M.A., et al. (2011). Arch. Microbiol. 193: 63-68.
Morel, M.A., et al.(2012). Crop Plant, Dr. A. Goyal (Ed.), In Tech, DOI: 10.5772/37413
Ubalde, M.C., et al. (2012). Curr Microbiol 64: 597-603.
274
Session IV
SIV-CO-4
Estrés salino, calidad nutricional y comportamiento postcosecha, en
lechuga inoculada con Azospirillum.
Fasciglione, G. *, Casanovas, M., Yommi, A., Quillehauqui, V., Roura, S., Barassi, C.A.
Unidad Integrada: Facultad de Cs. Agrs. (UNMdP) -EEA Balcarce (INTA). Ruta 226 Km 73,5 CC 276
(7620) Balcarce, Argentina *Becaria CONICET.
*
[email protected]
RESUMEN
La inoculación de semillas de Lactuca sativa, cv. Elisa con A. brasilense Sp245 revirtió
la disminución en la emergencia debida a la salinidad e incrementó la partición a raíces.
A cosecha, la supervivencia, el peso fresco y los contenidos de agua, ascorbato y
clorofila, fueron significativamente mayores en las plantas inoculadas, manteniéndose
este efecto durante la poscosecha. Asimismo, la inoculación redujo el Potencial de
Browning en plantas estresadas, lo cual podría atribuirse a la mayor capacidad
antioxidante de las plantas inoculadas debida, en parte a los mayores niveles de
ascorbato.
INTRODUCCIÓN.
Revertir los efectos negativos de la salinidad y aumentar el valor nutricional de los
productos se encuentran entre las demandas tecnológicas de la producción hortícola
(Fasciglione et al., 2012). A pesar de que se ha avanzado en el estudio de inoculantes a
base de Azospirillum (PGPR, del inglés Plant-Growth Promoting Rhizobacteria) para
cultivos extensivos, poco se sabe de su aplicación en producciones intensivas (Barassi et
al., 2007). El objetivo del presente trabajo fue evaluar si la inoculación con
Azospirillum podría actuar como paliativo del estrés salino, así como mejorar la calidad
nutricional y el comportamiento postcosecha de plantas de lechuga.
MATERIAL Y MÉTODOS
1.Ensayo plántulas. Semillas de lechuga controles (C) e inoculadas con 109 células de A.
brasilense.semilla-1 (I) se sembraron en bandejas plásticas de 66 celdas conteniendo
sustrato comercial. Se regó por capilaridad con soluciones de NaCl: 0, 40, 80 y 120
mM. A los 30 días desde la siembra (dds) se evaluó el porcentaje de emergencia (PE) y
los pesos secos de la parte aérea y de la raíz, calculándose la partición aérea (PA) y a
raíces (PR).
2.Ensayo a cosecha y poscosecha. Plántulas C e I provenientes de los tratamientos 0 y
40 mM de NaCl se trasplantaron a macetas hasta la cosecha (95 dds), momento en el
que se determinó: supervivencia (S), peso fresco aéreo (PFA), contenido de clorofila
(CF), contenido relativo de agua (CRA) e índices representativos de calidad
nutricional: Potencial de Browning (PB), contenido de Ácido Ascórbico (AA) y
capacidad antioxidante total (AAnt). Para el ensayo de poscosecha, las plantas se
envasaron en bolsas de polietileno y se almacenaron durante 20 días a 4°C y 98% HR.
A los 10 y 20 días de poscosecha (dpc) se determinaron: el CRA y los índices
nutricionales ya mencionados.
1.Ensayo plántulas. El PE disminuyó al incrementarse el nivel de salinidad,
independientemente del nivel de inóculo (datos no mostrados). La inoculación revirtió
este efecto negativo de la salinidad, incrementando el PE en un 12%. Las plántulas I
creciendo con 80 y 120 mM de NaCl, evidenciaron una mayor PR (Tabla 1).
2. Ensayo a cosecha y poscosecha. La salinidad redujo el PFA y el CRA e incrementó la
AAnt y el contenido de AA, en ambos niveles de inóculo (datos no mostrados).
Independientemente del nivel de salinidad, la inoculación incrementó tanto la S (15%)
275
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SIV-CO-4
como el PFA (18%), ambos factores determinantes del rendimiento del cultivo. Los
principales componentes que determinan la calidad en lechuga, la turgencia, el color y
la ausencia de pardeamiento, se ven afectados durante el almacenamiento. Al analizar
CF, CRA y AA fueron significativos (p≤0.05) los incrementos asociados al factor
inóculo (Tabla 2). El mayor CRA de las plantas I podría deberse a que la mayor PR
observada en las plántulas incremento el desarrollo radical en etapas posteriores (Tabla
1). Si bien el contenido de CF, CRA y AA disminuyó significativamente en todos los
tratamientos durante la poscosecha, los valores de las plantas I fueron siempre
significativamente superiores a las C (Tabla 2), relacionado con un producto de mayor
calidad.
Tabla 1. Partición a raíces (%) a los 30 dds de plantas de lechuga C ó I creciendo sin ó bajo tres niveles
de estrés salino. Letras diferentes indican diferencias entre los niveles de inóculo para cada nivel de
estrés salino según test LSD (p<0,05).
Partición a Raíces (%)
Inóculo
0
40
Na Cl (mM)
80
120
C
23,81+1,67a 21,14+1,54a 23,13+0,75b 12,61+0,54b
I
23,34+2,03a 22,01+0,79a 27,86+1,47a 19,65+1,05a
La ocurrencia de estreses abióticos induce incrementos en el contenido de fenoles en
tallos de la lechuga, estos se oxidan y generan pardeamiento y manchas marrones,
disminuyendo la calidad comercial. Al analizar el PB fue significativa la interacción
Inóculo y Salinidad (p< 0.05), ya que la inoculación redujo el PB sólo en las plantas
estresadas (Tabla 3). Esta reducción posiblemente se asocie al contenido foliar de AA
que fue superior en las plantas I (Tabla 2). Asimismo, la inoculación incrementó la
AAnt únicamente a cosecha (datos no mostrados).
Tabla 2. Evolución del CRA y de los contenidos de AA y de CF en plantas de lechuga I o C durante la
poscosecha. Los valores son promedio de los obtenidos en condiciones de salinidad 0 y 40 mM de NaCl.
Letras diferentes indican diferencias significativas dentro de cada nivel de inóculo, según test LSD (p<0.05).
CRA
(%)
AA
(mg.100g PS-1)
CF
(mg.100g PS-1)
C
I
C
I
C
I
Días de poscosecha
0
10
20
71,7+2,1b
73,1+1,6b
68,8 +1,0b
75,2+0,9a
76,5+1,1a
73,9+1,6a
178,7+13,2b 138,5+5,7b
71,7+2,1b
286,9+18,1a 205,2+15,9a
75,2+0,9a
769,2+30,9b 630,3+24,7b 562,8+35,4b
869,6+27,1a 733,6+32,3a 679,5+33,3a
Tabla 3: Evolución del PB durante la poscosecha en plantas de lechuga I o C, creciendo a 0 ó 40 mM de
NaCl. Letras diferentes indican diferencias significativas dentro de cada nivel de inóculo, según el test LSD.
Potencial de Browning (Abs 320 nm/g PF)
0 dpc
10 dpc
20 dpc
NaCl
C 0,03+0,003b 0,18+0,004a 0,22+0,02a
0 mM
I
0,04+0,002a 0,18+0,003a 0,19+0,02a
C 0,06+0,003a 0,26+0,003a 0,46+0,01a
40 mM
I
0,04+0,003b 0,17+0,001b 0,25+0,02b
BIBLIOGRAFÍA
Barassi, C.A., et al. (2007). Global Science Books. Dynamic Soil, Dynamic Plant 1: 68-82.
Fasciglione G. et al. (2012). J. Sci. Food Agric. 92: 2518-2523.
276
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Bioprospecção de Bactérias Isoladas de Milho para Promoção de
Crescimento de Plantas.
Ikeda, A.C.1*, Szilagy-Zecchin, V.J.1, Hungria, M.2, Kava-Cordeiro, V.1, Glienke, C.1,
Galli-Terasawa, L.V.1
1
Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brasil. 2 Embrapa Soja, Londrina,
Brasil.
* [email protected]
RESUMO
Isolados bacterianos associados a raízes de milho identificados por sequenciamento
parcial do gene 16S RNAr foram avaliados em testes de promoção de crescimento
vegetal. Também foram conduzidos testes in vitro para a capacidade de produção de
sideróforos, solubilização de fosfato, produção de AIA, FBN e produção de enzimas
líticas. Cinco isolados apresentaram resultados promissores na caracterização
enzimática e nos testes de atividade promotora de crescimento e, portanto, poderão ser
avaliados in vivo quanto a parâmetros de crescimento vegetal em ensaios em casa de
vegetação.
INTRODUÇÃO
Associações entre raízes de gramíneas e bactérias podem trazer benefícios de
incremento na produtividade do milho. Bactérias presentes no solo também podem
promover indiretamente o crescimento vegetal, por inibição de fito-patógenos ao
produzirem enzimas líticas e, de forma direta, atuam em processos de FBN e
solubilização de fosfato, na produção de substâncias análogas a fitormônios, como a
auxina, e compostos queladores de ferro (sideróforos) (Araújo et al., 2010). O
sequenciamento do gene 16S RNAr é uma ferramenta útil para a identificação da
diversidade e estudos filogenéticos de bactérias.
MATERIAL E MÉTODOS
Os 24 isolados bacterianos provenientes de fragmentos radiculares de milho, pertencem
à coleção do LABGEM, UFPR-Curitiba, Brasil. O DNA genômico foi extraído de
acordo com Raeder e Broda (1985). A reação de sequenciamento do gene 16S RNAr foi
realizada de acordo com Weisburg et al. (1991) e a qualidade das sequências foi
avaliada pelo programa Phred com edição no programa BioEdit versão 7.0. As
sequências editadas foram comparadas no GenBank pelo programa BLAST em busca
de alinhamentos significativos. A avaliação da produção das enzimas líticas (celulase,
quitinase e pectinase), a detecção da atividade esterásica, a produção de sideróforos, de
ácido indol acético (AIA), a solubilização de fosfato e o potencial de fixação biológica
de nitrogênio (FBN), foram realizados de acordo com Cattelan (1999).
RESULTADOS E DISCUSSAO
Os resultados dos testes in vitro estão apresentados na Tabela 01. Os isolados que
apresentaram os maiores índices de produção de AIA e melhor desempenho nos testes
qualitativos foram identificados como pertencentes ao gênero Bacillus sp. Isolados
desse gênero são amplamente descrito em estudos com promoção de crescimento em
plantas (Wahyudi et al., 2011). Os isolados LGMB137, LGMB196 e LGMB216
apresentaram resultados positivos em pelo menos cinco dos sete testes conduzidos e
podem ser utilizados em ensaios de casa de vegetação, ainda que a produção de AIA
apresente índices baixos, em comparação com o valor máximo observado (22,77
µg/ml). Araújo e Guerreiro (2010) observaram que a maioria dos isolados de Bacillus
sp. que promoveram crescimento em milho não são os maiores produtores in vitro de
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AIA, uma vez que a adição de auxina microbiana pode alterar o nível ótimo da auxina
endógena, causando inibição do crescimento da planta. Os isolados LGMB189 e
LGMB222 apresentaram o segundo e o terceiro maiores níveis de produção de AIA e
resultado positivo para pelo menos cinco dos sete testes realizados, sendo assim
promissores na promoção de crescimento in vivo.
A capacidade de solubilizar fosfato está associada ao aumento da disponibilidade de
fósforo para a planta e da eficiência de fixação biológica de nitrogênio. Na cultura de
milho, a utilização de bactérias com resultado positivo para FBN e solubilização de
fosfato, colabora para a diminuição do uso de fertilizantes fosfatados e nitrogenados,
reduzindo custos para o produtor e com menor impacto ambiental. Também, a produção
de sideróforos disponibiliza ferro para a planta e inibe, por competição, a colonização
de fito-patógenos. Assim, bactérias com resultado positivo para a produção de enzimas
líticas podem ser promissoras para o biantagonismo, uma vez que essas enzimas podem
quebrar compostos da parede celular de fito patógenos (Hernández, 2004; Singh, 2013).
Concluindo, os isolados LGMB137, LGMB189, LGMB196, LGMB216 e LGMB222 foram
selecionados para avaliação de desempenho em ensaios in vivo por parâmetros de
crescimento vegetal em casa de vegetação.
Tabela 1. Resultados dos testes in vitro de promoção de crescimento e identificação por sequenciamento
parcial do gene 16S RNAr de isolados bacterianos isolados de raízes de milho.
AGRADECIMENTOS
À Semilia-Genética e Melhoramento pelo material genético. O trabalho foi parcialmente financiado pelo
Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq-Microrganismos Facilitadores
(557746/2009-4) e CNPq-Repensa (562008/2010-1).
REFERÊNCIAS
Araújo, W.L., et al. (2010). CALO, 169p.
Araújo, F.F. e Guerreiro, R.T. (2010). Ciên, Agrotec. 34: 837-844.
Cattelan, A.J. (1999). Embrapa Soja, 36p.
Hernández, A. (2004). Ver. Colom. Biotec. 6: 6-13
Raeder, U., and Broda, P. (1985). Lett. Appl. Microbiol. 1: 17-20.
Singh, J.S. (2013). Resonance. 275-281.
Wahyudi, A.T., et al. (2011). J. Microbiol. Antimicrob. 3: 34-40.
278
Session IV
SIV-CP-02
Caracterización de una metilobacteria aislada de la superficie del
grano de arroz.
Gallego Parrilla, J.J., Alías-Villegas, C., Díaz-Olivares, I.M., Gutiérrez Alcántara, R.,
Madinabeitia-Peiró, N., Bellogín, R.A. *, Espuny, M.R.
41012-Sevilla. España.
* [email protected]
RESUMEN.
De granos de arroz que desarrollaban un color rosa en algunas plántulas que crecían en
tubos en condiciones asépticas se aisló una bacteria que crecía en AMS con metanol
(0,5%) como fuente de carbono formando colonias de un color rosa intenso. Esta
bacteria, a la que se denominó RS, tiene características promotoras del crecimiento
vegetal como solubilización de fosfatos y producción de AIA, que se traducen en una
mejora en la germinación y alargamiento de la raíz de plantas de arroz. Además es
productora de acil homoserina lactona y puede crecer hasta en concentraciones 800 mM
de NaCl. La amplificación, secuenciación y análisis del gen que codifica el ARN16S
determinó que esta bacteria presenta un 96,29 % de similitud con Methylobacterium
aquaticum.
INTRODUCCIÓN.
El género Methylobacterium incluye un grupo de bacterias metilotrofas facultativas
pigmentadas de rosa que crecen con compuestos de un solo carbono como formiato,
formaldehído o metanol como única fuente de carbono. Las especies de
Methylobacterium están ampliamente distribuidas en la naturaleza (suelo, agua, polvo,
sedimentos, aire, ambientes hospitalarios, etc.) y son abundantes en la filosfera de las
plantas. Methylobacterium coloniza las partes aéreas y subterráneas de las plantas como
epi- y endófita. Tras la colonización, expresan los genes implicados en la metilotrofía
beneficiándose del metanol producido por las plantas. Se han publicado diversos
artículos donde se describe el papel como promotora del crecimiento vegetal a través de
mecanismos directos o indirectos, como la producción de fitohormonas o enzimas que
modulan el crecimiento vegetal, la secreción de compuestos implicados en biocontrol o
en la supresión de la enfermedad. Curiosamente esta bacteria ha sido aislada de aguas
potables y otros ambientes relacionados con la actividad humana y esto es debido a que
son altamente resistentes a cloro (Hiraishi et al.,1995).
MATERIAL Y MÉTODOS.
Para el crecimiento de RS se utilizaron los medios AMS con metanol o King B. Para el
estudio de las propiedades promotoras del crecimiento se emplearon ensayos de
solubilización de fosfatos en NBRIP (Shekhar, 1999), de producción de ácido indol
acético (AIA) (Patten and Glick, 2002), producción de sideróforos (Alexander and
Zuberer, 1991). El crecimiento a distintas concentraciones de NaCl (100, 300, 600, 800
y 1000 mM) se llevó a cabo en King B líquido midiendo la absorbancia a 600 nm cada
24 h durante 3 días. La producción de acil-homoserina lactonas se realizó usando dos
biosensores, Chromobacterium violaceum CV026 (McClean et al., 1997), y
Agrobacterium tumefaciens NT1 (Cha et al., 1998).
Para estudiar el efecto de esta bacteria sobre las plantas de arroz se usaron semillas
descascarilladas de arroz (Oryza sativa “Puntal”) desinfectadas con hipoclorito sódico.
Las plantas se cultivaron en cámaras iluminadas. El test índice de germinación de arroz
se realizó adaptando el método de Belimov et al. (2001) con algunas modificaciones. Se
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realizaron recuentos del índice de germinación cada 12 horas durante 5 días. El ensayo
de alargamiento de raíces de arroz se llevó a cabo siguiendo el método adaptado de
Poonguzhali et al (2008). Tras 15 días de cultivo en condiciones asépticas en el interior
de un recipiente de cristal cerrado en cámara iluminada, se extrajeron las plantas y se
midieron las raíces.
RESULTADOS Y DISCUSIÓN.
Durante la realización de ensayos que se realizaban para el estudio de las propiedades
como PGPR de algunas bacterias, se observó que algunos granos de arroz tomaban un
color rosa-rojizo al cabo de unas dos semanas, a pesar de que las semillas de arroz
habían sido previamente desinfectadas con un tratamiento de 30 minutos de hipoclorito
sódico. De esos granos se aisló una bacteria que por su coloración podría ser una
metilobacteria facultativa pigmentada de color rosa (PPFM) por lo que se probó su
crecimiento en AMS con metanol como fuente de carbono, comprobándose que crecía
en este medio. Dado que se había aislado de plantas de arroz que presentaban aspecto
saludable se estudió si presentaba algunas propiedades que beneficiaran a la planta y
que pudieran clasificarla como PGPR. De este modo se comprobó que produce AIA,
solubiliza fosfatos, presenta actividad ureasa, siendo, además, productora de AHL
detectadas en los ensayos con los dos biosensores (CV026 y NT1 pZLR4). Sin
embargo, no produce sideróforos y no presenta actividad celulasa. Por otra parte, no
acelera la germinación pero sí promueve el desarrollo de un mayor número de raíces a
corto plazo y un alargamiento radicular a más largo plazo. Se ha descrito que las
especies de Methylobacterium producen considerables cantidades de AIA y de
citoquininas que estimulan la germinación de semillas y el desarrollo de las plantas y
algunas presentan ACC desaminasa (Madhaiyan et al, 2006).
Esta bacteria presenta una identidad del 96.296 % con Methylobacterium aquaticum, lo
que podría significar que se trate de una nueva especie.
AGRADECIMIENTOS.
Este trabajo ha sido financiado por los proyectos AGL2009-13487-C04 del Ministerio de Ciencia y
Tecnología del Gobierno Español y de Excelencia P10-AGR5821 de La Junta de Andalucía.
BIBLIOGRAFÍA.
Alexander, D.B., and Zuberer, D.A. (1991). Biol. Fert. Soils 12: 39-45.
Belimov, A.A., et al. (2001). Can. J. Microbiol. 47: 642-652.
Cha, C., et al. (1998). Mol. Plant-Microbe Interact. 11: 1119-1129.
Hiraishi, A., et al.(1995).Appl. Environm. Microbiol. 61: 2099-2107.
McClean, K.H., et al., (1997). Microbiology 143: 3703–3711.
Madhaiyan M., et al. (2006). Planta 224: 268–278.
Patten, C.L., and Glick, B.R. (2002). Appl. Environm. Microbiol. 68: 3795-3801.
Poonguzhali, S,. et al. (2008). Appl. Microbiol. Biotechnol. 78: 1033–1043.
Shekhar, C. (1999). FEMS Microbiol. Lett. 170: 265-270.
280
Session IV
SIV-CP-03
Resistencia a metales pesados de bacterias aisladas de leguminosas de
las Marismas del Odiel y del entorno del Río Tinto.
Lara-Dampier, V.1, Acera-Mateos, P.1, Alías-Villegas, C.1, Gómez-Cárdenas, A.J.1,
Temprano, F.2, Camacho, M.2, Bellogín, R.A.1, Espuny, M.R.1*
1
41012-Sevilla. España. 2 IFAPA Centro Las Torres-Tomejil. Sevilla. España.
* [email protected]
RESUMEN
Se ha analizado la resistencia a distintos metales pesados (plata, cadmio, cobalto,
cromo, cesio, cobre, níquel, arsénico, zinc y mercurio) a 50 rizobios aislados de nódulos
y a otras bacterias asociadas a los mismos de leguminosas recolectadas de distintas
localizaciones de las Marismas del Odiel y del entorno del Río Tinto. La mayoría de los
aislamientos de la misma zona presentaron valores de tolerancia a metales semejantes, y
en algunos casos alcanzaron los valores considerados como de resistencia. Algunas de
los aislamientos seleccionados crecieron a las siguientes concentraciones milimolares de
metales: As 7-10; Cd 0,5; Co 0,5-1; Cr 0,3-0,5; Cs 40; Cu 2-3; Hg 10-50 (micromolar);
Ni 2; Zn 1-2. La mayoría de los aislamientos presentaron resistencia a varios metales.
Cinco aislamientos del grupo de los no rizobios produjeron sideróforos, apareciendo, en
algunos casos, pigmentos difusibles en los medios con determinados metales.
INTRODUCCIÓN.
Los métodos que emplean plantas para descontaminar suelos afectados por la presencia
de metales pesados (fitorremediación) son menos costosos que los métodos
fisicoquímicos y de menor o nulo impacto ecológico (Dary et al., 2010). La
fitorremediación depende de la capacidad de la planta para solubilizar y/o movilizar
metales pero presenta como inconveniente que consume mucho tiempo. Así, la mayoría
de las plantas, excepto las hiperacumuladoras de metales, no son rentables para
fitorremediación debido a su poco y lento desarrollo en los ambientes contaminados.
Para hacer más rentable la fitorremediación en estas circunstancias habría que utilizar
bacterias promotoras del crecimiento vegetal por cualquier mecanismo, directo o
indirecto, que, además, fueran resistentes a metales, ya que estas bacterias juegan un
papel importante modificando la disponibilidad y toxicidad de los mismos (Rajkumar et
al., 2010; Rajkumar et al., 2012).
MATERIAL Y MÉTODOS.
Los aislamientos se realizaron en medio YMA con Rojo Cong o después de la
desinfección de los nódulos con hipoclorito de sodio. Tras su purificación en el mismo
medio se eliminaron redundancias seleccionando sólo uno de los clones que presentaron
el mismo perfil ERIC (cebadores ERIC1 y ERIC2). Posteriormente se distinguieron dos
grupos de bacterias, aquellas que amplificaron el gen nodC (nodCF2 y nodCI, Laguerre
et al., 2001) y que nodularon en la leguminosa de la que se aisló o en otra similar, y
aquellas bacterias que no lo hicieron, que fueron consideradas como no rizobios. A
todas ellas se les estudió la resistencia a metales en las siguientes formas: HgCl2, CdCl2,
CoCl2, NiCl2, AgNO3, ZnSO4·7H2O, CuSO4·5H2O, K2Cr2O7, Na2HAsO4.7H2O, CsCl.
Se prepararon soluciones concentradas (1 M, 1mM o 0,1 mM según los casos) que se
esterilizaron por filtración. A partir de ellas se prepararon placas de TY o YMA3
añadiendo los volúmenes correspondientes cuando el medio estaba a 50º C. Se
colocaron gotas de 10 microlitros de cultivos en TY o YM3 de los distintos aislamientos
en fase logarítmica tardía. El crecimiento se observó a los 5, 7 y 14 días. El estudio de la
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producción de sideróforos se realizó en placas de microtítulo usando 100 μl de
sobrenadante de 7 días de las bacterias en medio líquido (TY y medio mínimo M9) y
100 μl de reactivo de CAS modificado; siguiendo el método de Alexander y Zuberer
(1991).
Algunas de las áreas de las Marismas del Odiel y del entorno del Río Tinto presenta
contaminación por metales pesados derivada de la actividad minera ancestral que se
realiza en esta zona de la franja pirítica del suroeste de la península Ibérica y por otras
actividades antropogénicas, como los dragados de canales de zonas industriales. Por
ello, estas áreas fueron seleccionadas para aislar rizobios de nódulos de leguminosas y
otras bacterias asociadas a estos nódulos que crecieron en las placas de aislamiento.
Los aislamientos se hicieron de: Ornithopus compressus y Lotus corniculatus, de una
zona recuperada pero contaminada por metales (Zn, Pb, As y Hg) de las Marismas del
Odiel; de Medicago marina y Medicago sp, de una zona arenosa de las Marismas del
Odiel cuyos análisis no manifestaron metales pesados, y de Trifolium sp y Medicago
polymorpha del entorno de Río Tinto, también contaminada con metales pesados (Cu,
Ni y Hg).
Los resultados preliminares mostraron que muchos de los aislamientos pueden crecer a
concentraciones de metales pesados que se consideran de resistencia (1 mM en los casos
de Cu2+, Ni2+, Zn2+ y 0,5 mM en los casos de Co 2+ y Cr3+, Brim et al., 1999; Nieto et al.,
1987) y, en ocasiones, superiores. Cinco aislamientos del grupo de los no rizobios
produjeron sideróforos. Además, estos mismos aislamientos produjeron pigmentos
amarillos difusibles en las placas de algunos metales, lo que sugiere que pueda tratarse
de sideróforos inducidos por dichos metales.
AGRADECIMIENTOS
Este trabajo ha sido financiado por el proyecto de Excelencia P10-AGR5821 de La Junta de Andalucía.
BIBLIOGRAFÍA
Alexander, D.B., and Zuberer, D.A .(1991). Biol. Fertil. Soils 12: 39-45.
Brim, H., et al. (1999). Syst. Appl. Microbiol. 22, 258–268.
Dary, M., et al. (2010). J. Hazard. Mater., 177, 1–3, 323-330
Rajkumar, M, et al. (2010). Trends Biotechnol. 28: 142.
Rajkumar, M, et al. (2012). Biotechnol. Adv. 30: 1562-1574.
282
Session IV
SIV-CP-04
Comparación de las características microbiológicas del suelo en
huertos de aguacate bajo manejos orgánico y convencional.
Bárcenas-Ortega, A.E.1*, Chávez-Bárcenas, A.T.1, García-Saucedo, P.A.1, OlaldePortugal, V.2, Tulais-Alvarado, C.A.1, Zavala-Gómez, A.1
1
Facultad de Agrobiología “Presidente Juárez” de la Universidad Michoacana de San Nicolás de Hidalgo,
México, 2 CINVESTAV Unidad Irapuato, México.
* [email protected]
RESUMEN
Para determinar el impacto del manejo convencional sobre la microbiota del suelo, se
compararon algunas características microbiológicas en huertos de aguacate ‘Hass’ bajo
manejos orgánico y convencional en el municipio de Uruapan, Michoacán, México. En
los huertos bajo manejo orgánico se encontró mayor cantidad de microorganismos del
suelo (bacterias, hongos y actinomicetos) que en los huertos con manejo convencional;
lo mismo ocurrió con el número de esporas de hongos micorrizógenos arbusculares
(HMA), superándolos en un 130%.
INTRODUCCIÓN
El aguacate es el cultivo más importante de Michoacán por su impacto sobre la
economía regional, pero es también el principal responsable de la pérdida de cobertura
forestal debido al cambio de uso de suelo, pérdida de la biodiversidad, modificación
micro-climática y uso indiscriminado e intensivo de agroquímicos. El componente
microbiológico es muy importante en el funcionamiento de los ecosistemas y constituye
un marcador biológico potencialmente útil para evaluar sus perturbaciones (Calvo et al.,
2008). El objetivo de la investigación fue: comparar las características microbiológicas
en árboles de aguacate ‘Hass’ bajo manejos orgánico y convencional en el municipio de
Uruapan, Michoacán, México, para determinar el impacto del manejo convencional
sobre la microbiota del suelo.
MATERIAL Y MÉTODOS
El estudio se realizó en cuatro huertos establecidos por pares, uno con manejo orgánico
frente a otro con manejo convencional. Se efectuaron muestreos de suelo en el área de
goteo del árbol. El número de unidades formadoras de colonias (UFC) de bacterias,
hongos y actinomicetos en los suelos se obtuvo mediante la técnica de dilución seriada
y vaciado en placa con medio selectivo (Alvarez et al., 2000), extracto de sueloglucosa-agar para bacterias, medio Martin (1950) para hongos y para actinomicetos el
medio agar Czapeck-Dox (Merck). Para extraer las esporas de HMA del suelo se utilizó
el protocolo de tamizado húmedo y decantación propuesto por Gerdemann y Nicolson
(1963), seguido de centrifugación en sacarosa (Walker y Mizecw, 1982); las esporas
obtenidas se montaron en preparaciones usando alcohol polivinílico glicerol y se
identificaron y contaron usando microscopio compuesto.
En los huertos bajo manejo orgánico se encontró mayor cantidad de microorganismos
del suelo (bacterias, hongos y actinomicetos) que en los huertos con manejo
convencional (Figura 1A), lo que se traduce en un mayor equilibrio; lo mismo ocurrió
con el número de esporas de hongos micorrizógenos arbusculares, superándolos en un
130% (Figura 1 B).
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Media de N° esporasen 100g ss
MEDIAS DEL No DE UFC • 104/g ss-1
A
80
70
60
50
40
A
30
B
20
10
0
ORG
900
B
800
700
600
500
A
400
300
200
B
100
0
ORGÁNICO
CONV
CONVENCIONAL
MANEJO
MANEJOS
Figura 1. Comparación de medias de: A) UFC de microorganismos (bacterias, hongos y actinomicetos)
y B) número de esporas de HMA, encontradas en huertos de aguacate con dos tipos de manejo en
Uruapan, Mich. Letras distintas indican diferencias estadísticas significativas.
Las diferencias entre los dos tipos de manejo coinciden con Sánchez et al. (2009) que
encontró que la biomasa y actividad microbiana del suelo fueron significativamente
mayores en cultivos con manejo orgánico en comparación con aquellos manejados de
manera convencional. Esto puede deberse a los contenidos de materia orgánica que
fueron más altos en los huertos con manejo orgánico y a la no utilización de
agroquímicos como fertilizantes, herbicidas, plaguicidas y otros insumos de síntesis
química. Acuña et al. (2006) mencionan que los efectos de prácticas agrícolas, así como
los producidos por fertilizantes y sistemas de cultivo, pueden ser evaluados a partir de
las determinaciones de la biomasa microbiana, su actividad metabólica y el conteo de
las poblaciones microbianas más importantes de la microflora del suelo, por su parte
Vandermer (1995) afirma que, la agricultura orgánica trata de cerrar el ciclo de
nutrientes en sus granjas, proteger la calidad del medio ambiente y mejorar las
interacciones biológicas beneficiosas y los procesos, además, puede reducir algunos
efectos negativos atribuidos a la agricultura convencional y tiene beneficios potenciales
en la mejora de la calidad del suelo.
AGRADECIMIENTOS
A CONACYT, México, por financiar el proyecto.
BIBLIOGRAFÍA
Acuña, O., et al. (2006). Memorias XII Reunión ACROBAT, Santa Catarina, Brasil. 222-233.
Álvarez S., et al. (2000). Agrociencia 34: 523-532.
Calvo, P., et al. (2008). Estudio de las poblaciones microbianas de la rizosfera del cultivo de papa
(solanum tuberosum) en zonas altoandinas. Ecología Aplicada. Departamento Académico de Biología.
Universidad Nacional Agraria La Molina, Lima, Perú. Pp 141-148.
Gerdemann, J.W., and Nicolson, T,H. (1963). Trans. Br. Mycol. Soc. 46: 235-244.
Sánchez, M., et al. (2006). Actividad y biomasa microbianas como indicadores de materia orgánica en
sistemas de cultivo de maracuyá (Passiflora edulis) en Toro, Valle del Cauca, Colombia.
Revistas.unal.edu.co
Vandermeer, J. (1995). Ann. Rev. Ecol. Syst. 26: 201-224.
Walker, C., and Mizecw, M.(1982). Can. J. Bot. 60: 2518-2529.
284
Session IV
SIV-CP-05
Efecto de la inoculación foliar y radicular de bacterias PGPR en el
cultivo de Aguaymanto (Physalis peruviana).
Flores, L., Ogata, K. *, Zúñiga, D.
Nacional Agraria La Molina. Lima -Peru. Web: www.lamolina.edu.pe/lmt.
*
[email protected]
RESUMEN
Se utilizaron cuatro cepas aisladas del cultivo de aguaymanto. Dos de las cepas
incrementaron la germinación y el porcentaje de semillas pubescentes in Vitro. Por otro
lado, la cepa Aa25, inoculada radicularmente, incremento significativamente la altura y
el número de hojas de la planta, en conjunto con la inoculación foliar de la cepa
Azo16M2.
INTRODUCCIÓN
El aguaymanto, cultivo nativo del Perú, está teniendo mayor demanda en los mercados
internacionales, por sus propiedades medicinales y alta producción de vitamina C. La
exportación peruana de aguaymanto está en aumento respecto a los años anteriores; del
año 2007 (6.8 mil Kg) al año 2012 (57.2 mil Kg) hay un incremento de más de 800 %
(SIICEX, 2013). Los microorganismos promotores de crecimiento (PGPR) son
ampliamente investigados por su capacidad de inducción de resistencia a patógenos y
estimulación del crecimiento en plantas (Barka et al., 2000). Es por esto, que en esta
investigación se evalúa la respuesta de la planta bajo inoculación foliar y radicular con
estos microorganismos.
MATERIAL Y MÉTODOS
Ensayos de germinación in vitro en semillas de aguaymanto, descrito por Zúñiga
(2012). Se utilizaron 2 inóculos de diazotrofos Da29 y Da 30 y 2 de actinomicetos Aa25
y Aa23.
Preparación del inoculo con cepas diazótrofas en medio LMC a 28°C por 48 h y cepas
de actinomicetos sembradas en medio ISP1 a 28°C por 120 h (Shirling y Gottlieb,
1966).
Instalación del ensayo en campo trasplantando plántulas de aguaymanto (provenientes
de almácigos con una altura de 3-4 cm) al campo, considerando 1m de distancia entre
plantas. Se utilizó un diseño de bloques completos al azar (DBCA). Se añadieron 100 g
de compost a cada hoyo de siembra, siendo luego homogenizado con el suelo. El ensayo
se realizó en el Jardín Botánico “Octavio Velarde Nuñez” en la Universidad Nacional
Agraria La Molina.
Ensayo de germinación in vitro en semillas aguaymanto.
A los 7 días de instalado el ensayo se observó que tres de las cepas (Da29, Aa25 y
Aa23) incrementaron el % de germinación de semillas de aguaymanto respecto al
control sin inocular. También se evaluó la presencia de pubescencia en las radículas de
las semillas germinadas, encontrándose que el 79% y el 82% de las plantas inoculadas
con las cepas Da29 y Aa25 respectivamente presentaron diferencias significativas en
comparación al control sin inocular (48%) (Figura 1). La pubescencia es importante
para el crecimiento de la planta, ya que permite una mayor área de toma de nutrientes.
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De este modo se demostró la capacidad PGPR a nivel in vitro de las cepas
seleccionadas.
Efecto de la inoculación en campo.
Las semillas fueron inoculadas con las cuatros cepas mencionadas anteriormente, de
manera individual y sembradas en almacigo. Al cabo de un mes se realizó la medición
de altura y número de hojas de las plántulas, encontrándose un incremento del
crecimiento con tres de las cepas: Da29 (3.91cm), Aa25 (3.99cm) y Aa23 (4.18cm),
respecto al control (3.82cm). Después de 24 h, las plántulas fueron transplantadas a
campo y reinoculadas a nivel radicular con las mismas cepas. A los 15 días se realizó
una inoculación foliar con la cepa diazótrofa Azo16M2, a todos los tratamientos a
excepción del control, cuyo efecto positivo en el crecimiento de aguaymanto fue
demostrado previamente (Ogata y Zúñiga, 2013). Un mes después se realizó la
medición de la altura y número de hojas de las plántulas, pudiéndose observar que el
tratamiento inoculado con la cepa Aa25 en interacción con Azo16M2 presento un
incremento significativo de estos parámetros, respecto al control sin inocular (Tabla 1).
Por lo tanto la cepa Aa25 resulta ser promisoria en el cultivo de aguaymanto, desde la
germinación hasta el desarrollo del cultivo.
Tabla 1. Ensayo a nivel de campo con las cepas
DA29, Da30, Aa25 y Aa23 como inoculantes
radiculares y la cepa Azo16M2 como inoculante
foliar.
Tratamiento
Altura
N° Hojas
Da29
2.800 a
3.333 a
Da30
4.211 b
4.222 ab
Aa23
4.656 b
4.000 ab
Aa25
9.556
Control
d
6.711 c
7.222
c
4.889 b
* Las letras en minúscula corresponden a los grupos
de homogenización encontrados mediante la prueba de
Rangos Multiples LSD.
Figura 1. Efecto de PGPRs en el % de semillas
pubescentes. Las letras en las barras corresponden
a los grupos de homogenización encontrados
mediante la prueba de Rangos Multiples LSD.
AGRADECIMIENTOS
Proyecto PROCYT-CONCYTEC 325-2011, FDA Biol. 111-UNALM, Lima, Perú.
BIBLIOGRAFÍA
Barka, E., et. al. (2000). FEMS Microb. Let. 186: 91–95.
Ogata, K. y Zúñiga, D. (2013). Informe final. Proyecto Procyt-Concytec 325-2011.
Shirling, E., and Gottlieb, D. (1966). Int. J. Syst. Bacteriol. 16: 313-340.
SIICEX. (2013). Estadística de Biocomercio. Edición No4. www.siicex.gob.pe
Zúñiga D. (2012) Universidad Nacional Agraria La Molina. 1ra Edición. Lima, Perú. pp. 40.
286
Session IV
SIV-CP-06
Bacillus sp. y hongos micorrícicos para el control biológico del
nemátodo Meloidogyne incognita en el cultivo de Aguaymanto (Physalis
peruviana).
Isla, F.1*, Carbonell, E.2, Ogata, K.1, Zúñiga, D.1
1
Laboratorio de Ecología Microbiana y Biotecnología Marino Tabusso. Dpto.de Biología, Universidad
Nacional Agraria La Molina. Lima-Perú. Web: www.lamolina.edu.pe/lmt. 2 Laboratorio de Nematología.
Dpto. de Agronomía, Universidad Nacional Agraria La Molina. Lima-Perú.
* [email protected]
RESUMEN
Se evaluó la dosis de nemátodos para determinar el grado de infestación de las raíces en
aguaymanto; luego se probó el uso de los hongos micorrícicos y una cepa PGPR de
Bacillus sp. como biocontrolador en plantas infectadas. La inoculación de la cepa de
Bacillus sp. y doble inoculación de Bacillus sp. y micorrizas incrementó
significativamente la altura de plantas y mejoró el número de frutos respecto a la planta
infectada con nemátodos.
INTRODUCCIÓN
El cultivo del aguaymanto (Physalis peruviana) planta nativa del Perú, va adquiriendo
importancia en la exportación debido a las propiedades nutricionales y medicinales, en
los últimos años se viene intensificando su cultivo ya que ha aumentado la demanda de
exportación. En el Perú no existen formas adecuadas en el control de nemátodos, siendo
la principal forma de control el uso de plaguicidas químicos costosos, tóxicos para la
salud y el medio ambiente. Las especies de Bacillus sp. se han reportado como
promotoras de crecimiento en un número amplio de plantas (Kokalis-Burelle et al.,
2002); además este género es muy efectivo en el control biológico de nemátodos y se ha
demostrado que reduce la eclosión de huevo hasta en un 90% (Nagesh et al., 2005). Por
otro lado los hongos micorrícicos mejoran la toma de nutrientes del suelo,
principalmente fosfato y protegen a la raíz (Liu et al., 2012).
MATERIAL Y MÉTODOS
Efecto de la poblacional inicial sobre el grado de severidad radicular.
Se sembraron plántulas de aguaymanto en macetas de 2 kg en sustrato estéril 2:1 (suelo:
arena) y se inoculó después de 1 semana con diferentes niveles de población de
nematodos. Los tratamientos fueron: 0,1000, 3000, 5000, 10.000 y 20.000 nemátodos
por kg de sustrato. Luego se determinó el grado de severidad radicular (escala del 0 al
5) según (1985).
Preparación de agentes de biocontrol.
Se extrajeron las esporas de hongos micorríticos nativos de aguaymanto y se inoculó
100 esporas de hongos formadores de micorrizas por plántula. Se regó con solución de
Long Ashton con 15 ppm de P. Luego de una semana se inoculó con Bacillus sp. aislada
previamente de suelos de cultivo de papa en zonas altoandinas del Perú (Calvo et al.,
2010), esta cepa con capacidad PGPR se sembró en LMC e incubó a 35°C por 72 horas.
Se tomo 1 ml de la suspensión (108 UCF/ml).
Inóculo de nemátodos.
Una semana después de haber transplantado las plantas al invernadero se inoculó con
5000 nemátodos por kilo de sustrato. La extracción de nemátodos fue mediante la
técnica de Hussey y Barker (1973).
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Session IV
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Diseño experimental.
El ensayo consistió de 5 tratamientos: (C) Control, (N) M. incognita, (B+N) Bacillus sp.
+ M. incognita, (M+N) Micorrizas + M. incognita, (B+M+N) Bacillus sp. + Micorriza
+ M.incognita y analizado con un diseño completamente al azar (DCA). Se evaluó el
crecimiento de la planta y número de frutos en condiciones de invernadero durante 90
días.
En el primer ensayo, el índice de infección radicular más alto y significativo lo presentó
el tratamiento de 5000 nemátodos por kg de sustrato, con un grado de 5, así también
disminuyó significativamente el peso fresco radicular de la planta (Figure 1). En el
segundo ensayo, se observó diferencias significativas entre los tratamientos,
encontrándose que la inoculación con la cepa de Bacillus sp., micorrizas y la doble
inoculación (B+M) disminuyen el daño causado por los nemátodos entre10-14% (Tabla
1), lo cual se evidencia con el aumento significativo en la altura de planta y número de
frutos en comparación al tratamiento infestado solo con nemátodos (Tabla 1).
Tabla 1. Efecto de la inoculación en el
desarrollo del cultivo de Aguaymanto.
Figura 1. Efecto de la población inicial de M.
incognita en el grado de severidad radicular (GSR) y el
peso fresco de las raíces (PFR). Las letras indican la
significancia del PFR, letras iguales no son
significativamente diferentes (p < 0.05).
Tratamientos
Altura
de las
plantas
N˚
Frutos
(C)
75.93 c
5.88 c
Eficiencia de los
tratamientos
respecto al
control (%)
100
(N)
60.00 a
1.13 a
79
(B+N)
70.25 bc
2.63b
92.5
(M+N)
67.88 b
1.25 ab
89.4
(B+M+N)
70.68 bc
2.13 ab
93.1
Los promedios seguidos por letras iguales no
son significativamente diferentes (p < 0.05)
Con estos resultados se podría aplicar inoculantes como Bacillus sp. y micorrizas en el
cultivo de aguaymanto para controlar la infección por nemátodos (M. incognita) y
mejorar el crecimiento de la planta, dentro de una agricultura sostenible.
AGRADECIMIENTOS
PROYECTO PROCYT-CONCYTEC 325-2011, FDA Biol. 111-UNALM, Lima, Perú.
BIBLIOGRAFÍA
Calvo, P., et al. (2010). Bra. J. Microbiol. 41:899-906.
Fassuliotis, G. (1985). The role of nematologist in the development of resistant 1: 233-240.
Hussey, S., and Barker, R. (1973). Plant Dis. Repor. 57: 1025-1028.
Kokalis-Burell, N., et al. (2002). Plant Soil 238: 257-266.
Liu, R., et al. (2012). Mycorrhiza 22: 289-296.
Nagesh, M., et al. (2005). J. Biol. Control 19: 65-69.
288
Session IV
SIV-CP-07
Identificación de dos bacterias diazótrofas asociadas a una
Brassicaceae (Lepidium meyeni Walp.) de suelos altoandinos del Perú.
Chumpitaz, C., Ogata, K., Santos, R., Zúñiga, D. *
Laboratorio de Ecologia Microbiana y Biotecnologia Marino Tabusso, Dpto. Biología, Universidad
Nacional Agraria La Molina. Lima12, Perú. Web: www.lamolina.edu.pe/lmt.
*
[email protected]
RESUMEN
Se amplificó el gen nifH de la dinitrogenasa reductasa en dos cepas, LMTZ064-109 y
LMTZ064-119, asociadas a la rizósfera de Lepidium meyenii Walp. Los aislados
pertenecen a los géneros Stenotrophomonas y Rahnella, respectivamente, siendo ambas
gram negativas, cocobacilos y oxidasa negativa. La aparición de un producto de
aproximadamente 370 pb perteneciente al gen nifH confirmó la capacidad fijadora de
nitrógeno molecular de ambas cepas.
INTRODUCCIÓN
La maca (L. meyenii Walp.) es una Brassicaceae que habita en regiones altoandinas del
Perú entre los 3700-4500 msnm. Es muy apreciada por sus cualidades nutricionales y
medicinales (Tovar, 2001). Al ser un cultivo muy extractivo
genera un
empobrecimiento de los suelos, disminuyendo los rendimientos (Zúñiga, 2009). Los
diazótrofos, por otro lado, son un grupo de microorganismos capaces de fijar N 2 a
formas más asimilables en diferentes ambientes. El gen nifH de la Fe-proteína de la
Nitrogenasa (enzima responsable del proceso de fijación) es usado como un indicador
determinante de esta capacidad (Zehr et al., 2003). El objetivo de este estudio fue
identificar la capacidad fijadora de N2 de los aislados bacterianos asociados al cultivo
de maca.
MATERIAL Y MÉTODOS
Reactivación y caracterización morfológica y bioquímica de bacterias conservados.
Amplificación BOX PCR de acuerdo a Versalovic et al. (1991).
Amplificación del gen nifH de acuerdo a Barua et al. (2011).
Análisis filogenético por medio del gen ARN ribosomal 16S según Weisburg, et al.
(1991), usando la base de datos del Genbank (NCBI).
Un total de siete bacterias (LMTZ064-21, LMTZ064-30, LMTZ064-94, LMTZ064-95,
LMTZ064-109, LMTZ064-119 y LMTZ064-120) fueron seleccionadas de la
colección de bacterias promotoras de crecimiento vegetal (186 cepas) aisladas de
suelos de cultivo L. meyenni (Garcia, 2011). Estas cepas fueron caracterizadas
morfológicamente de acuerdo a su crecimiento en medio mineral sin nitrógeno (MMN) por 48 horas a 28°C. Las colonias tuvieron formas convexas, incoloras, de borde liso,
mucosas y diámetro entre 0.5-2 mm. Todas resultaron ser gram negativas, cocobacilos
y oxidasa negativa.
De acuerdo al análisis BOX PCR se obtuvieron cinco perfiles distintos (Figura 1A), a
los cuales se les evaluó su capacidad fijadora de nitrógeno por medio de la
amplificación del gen nifH de la dinitrogenasa reductasa (Fe-proteína). Solo dos
aislados (LMTZ064-109 y LMTZ064-119) resultaron ser nifH positivos (Figura 1B).
289
Session IV
SIV-CP-07
A
B
A B C C D E
E
A B
C D
E
nifH
370pb
Figura 1. A) Perfiles BOX A1R, A (21), B (30), C (94,
95), D (109) y E (119, 120). M: marcador 1kb
(fermentas). B) PCR nifH de perfiles BOX diferentes.
Cepas nifH+: LMTZ064-109 y LMTZ064-119. CIAT899
Rizhobium tropici (control+). M: marcador 100 pb
(Axygen).
Figura 2. Árbol filogenético en base al gen del ARNr
16S mostrando la localización de los aislados
LMTZ064-109 y LMTZ064-119.
Se realizó un
análisis Neighbor-joining con un valor bootstrap
calculado para 1,000 réplicas. Escala: 2% de
secuencias divergentes
De acuerdo al análisis filogenético realizado (Figura 2), se encontró que la cepa
LMTZ064-109 está relacionada a S. maltophila ATCC 13637T con 98%.
Stenotrophomonas spp. es un habitante común de suelos y plantas (Ryan et al., 2009),
especies de este género pueden actuar como promotores de crecimiento vegetal
mediante diferentes mecanismos como la fijación de nitrógeno y producción de
fitohormonas (Liba et al., 2006). Por otro lado, la cepa LMTZ064-119 tuvo un 100%
de similitud con Rahnella aquatilis HX2. Esta especie se reportó por primera vez
como una enterobacteria fijadora de nitrógeno presente en rizósferas de cultivos de
maíz y trigo (Berge et al., 1991) y su capacidad como PGPR en rizósfera de cultivos de
soja y papa (Kim et al., 1997; Kohashikawa, 2010) al producir fitohormonas y
solubilizar sales fosfato. En este trabajo se pudo determinar que las dos bacterias
aisladas del cultivo de maca fueron capaces de fijar nitrógeno molecular. Este
parámetro es importante a tener en cuenta para la producción de inoculantes
bacterianos, ya que además de reducir el uso de fertilizantes químicos, promueve el
crecimiento de cultivos de importancia económica dentro de una agricultura
sustentable; mejorando a su vez la calidad del suelo.
AGRADECIMIENTOS
Perú biodiverso GTZ-CONCYTEC, 2009. Procyt 309-2009-CONCYTEC. FDA Biol.111-UNALM.
BIBLIOGRAFÍA
Barua, S., et al. (2012). Microbiol. Res. 167: 95-102.
Berge, O., et al. (1991). Can. J. Microbiol. 37: 195-203.
Garcia, M.M. (2011). Tesis Biología. Efecto de bacterias diazotróficas en el cultivo orgánico de maca
(Lepidium meyenii Walpers) de San Pedro de Cajas-Junín. Universidad Nacional Agraria La Molina. Lima,
Perú.
Kohashikawa, N. (2010). Tesis Biología Comportamiento de diferentes bacterias PGPR sobre el crecimiento
del cultivo de papa (Solanum tuberosum), Universidad Nacional Agraria La Molina. Lima, Perú
Kim, K.Y., et al. (1997). FEMS Microbiol. Lett. 153: 273-277.
Liba, C.M., et al. (2006). J. Appl. Microbiol. 101: 1076–1086.
Ryan, P., et al. (2009). Nature Rev. Microbiol. 7: 514-25
Tovar, O. (2001). Publicación CONCYTEC. Lima 144 pp.
Versalovic, J., et al. (1991). Nucleic Acids Res. 19, 6823-6831.
Weisburg, W.G., et al. (1991). J. Bacteriol.173: 697-703
Zehr, J.P., et al. (2003). Environ. Microbiol. 5: 539-554.
Zúñiga, D. (2009). Perú biodiverso GTZ-Concytec. Lima, Perú.
290
Session IV
SIV-CP-08
Selección de cultivos rizobianos aislados de nódulos de leguminosas de
diferentes regiones del Perú con capacidad de promover el crecimiento
de lechuga, páprika y tomate.
Soriano-Bernilla, B.1*, Prado-Chávarry, G.2, Zavaleta-Verde, D.2, Valdez-Nuñez, R.3
1
Dpto.de Microbiología y Parasitología-Facultad de Ciencias Biológicas, Universidad Nacional de
Trujillo, Perú. 2 Universidad César Vallejo, Piura-Perú. 3 Gloria S.A Tarapoto, San Martín-Perú.
* [email protected]
RESUMEN
La selección y evaluación de los cultivos rizobianos aislados de nódulos de leguminosas
y posibles PGPR se realizó en laboratorio, determinando la altura de las plantas,
longitud de las hojas, peso seco de la parte aérea, raíz y peso seco total de las plántulas
de lechuga y páprika, así como la germinación de las semillas de tomate. Se encontró
que los cultivos rizobianos Rc-455-02, Rf 188-03, Ra112-01 y Ra 014-02 promueven el
crecimiento de no leguminosas considerándose una alternativa biológica frente a
fertilizantes químicos que contaminan nuestro ecosistema.
INTRODUCCIÓN
En años recientes, se ha tomado interés de utilizar rizobacterias promotoras de
crecimiento vegetal (PGPR) en la producción de cultivos agrícolas. Esto aplicado a
semillas, tubérculos o raíz, y son capaces de colonizar las raíces de las plantas y
estimular el crecimiento y rendimiento de cultivo.
Dentro de la flora peruana se encuentran especies útiles para el hombre como la papa,
tomate, pimientos, tabaco y berenjena, entre otras. Una de las hortalizas también de
importancia en el Perú por su producción es la lechuga, se consume cruda, debido a su
aporte de calcio, vitamina A y minerales (Ogata et al., 2008). Investigadores evaluaron
el efecto de 30 cepas bacterianas en la germinación y el crecimiento de lechuga,
obteniendo un incremento en la germinación del 36.5% con respecto al control sin
inocular. Asimismo, nuestro país está orientado a la exportación de páprika y
actualmente se ha convertido en uno de los líderes del mercado mundial. El páprika
presenta un valor nutricional alto en contenido de vitamina C, A, sales minerales,
azúcares y oleorresinas (capsaicinas, que otorga el carotenoide colorante) (Santillana et
al., 2005; Quintero et al.., 2000).
MATERIAL Y MÉTODOS
Colección y transporte de los nódulos a partir de leguminosas (frijol común, caupí,
arveja y frijo palo) de regiones peruanas (Lambayeque, Cajamarca, La Libertad y
Ucayali).
Aislamiento de rizobios a partir de los nódulos según metodología de Somasegaran y
Hobe (1994); caracterización bioquímica y autenticación de cultivos rizobianos.
Preparación y estandarización de los cultivos rizobianos autenticados.
Determinación de la capacidad promotora de crecimiento vegetal por cultivos
rizobianos mediante pruebas bioquímicas.
Tratamiento de las semillas de lechuga, páprika y tomate.
Evaluación de la siembra e inoculación de las semillas de lechuga y páprika en muestras
de suelo agrícola preparada en bolsas de polietileno. Se determinó la longitud del tallo y
raíz, peso seco de la parte aérea y radicular de las plántulas después de 20 días de
inoculadas. Se realizó la inoculación rizobiana de las semillas de tomate colocadas en
una placa Petri y al quinto día se hizo la medida de radícula e hipocotilo.
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Session IV
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De 503 cultivos aislados, 234 se consideraron positivos a las pruebas de purificación y
autenticación determinándose así la presencia de cultivos rizobianos.
Figura 1. Porcentaje de
cultivos rizobianos aislados
Figura 2 Autentificación de
los cultivos rizobianos en
leguminosas
Se seleccionaron, mediante pruebas bioquímicas, a 24 cultivos rizobianos aislados de
frijol por tener actividad promotora del crecimiento vegetal, y se determinó en base a
sus mecanismos de promoción directa o indirecta, en la cual indica que si reaccionan
positivamente a una de las pruebas consideradas como promotoras de crecimiento
vegetal (producción de indol, AIA, sideróforos y solubilización de fosfatos) sean
consideradas como tal y dentro de este grupo se seleccionó a Rc-455-02 y Rf 188-03
para la evaluación en plántulas de lechuga y páprika, después de 20 días de inoculadas.
Figura 3. Plántulas de
lechuga.
Figura 4. Plántulas de páprika.
Figura 5. Plántulas
de tomate.
De los rizobios aislados de nódulos de arveja se seleccionaron a 8 cultivos para la
evaluación de la germinación de semillas de tomate y dentro de éstos a Ra 112-01 y Ra
14-02 por ser positivos al menos de una de las pruebas realizadas y presentar diferencia
significativa entre la longitud de radícula e hipocotilo con respecto al control en
semillas de tomate.
BIBLIOGRAFÍA
Ogata K., et al. (2008). Zonas Áridas; 12: 137-153.
Quintero, I., et al. (2000). Rev. Fac. Agron. (LUZ). 17: 482-491.
Santillana, N., et al. (2005). Ecol. Apl. 4: 47-51.
Somasegaran, P., and Hoben, H. (1994). Handbook for rhizobia: methods en legume-Rhizobium
technology. Springer-Verlag. New York.
292
Session IV
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Promoção de crescimento de tomate (Solanum lycopersicum L.)
estimulado por Bacillus amyloliquefaciens FZB42.
Szilagyi-Zecchin, V.J. *, Ruaro, L., Mógor, A.F.
Departamento de Fitotecnia e Fitossanitarismo, Universidade Federal do Paraná, Brasil.
* [email protected]
RESUMO
A bactéria B. amyloliquefaciens foi inoculada em sementes de tomate, em diferentes
doses, e não alterou o potencial germinativo. Na produção de mudas, a menor dose,
proporcionou significativos incrementos em todos os aspectos da parte aérea da planta.
INTRODUÇÃO
O tomateiro é cultivado no mundo inteiro e possui grande relevância econômica e social
(Faostat, 2011). Os microrganismos vêm despertando atenção devido à necessidade de
reduzir o uso de produtos químicos, visando um sistema de agricultura sustentável,
centrada na proteção ambiental (Vale et al., 2010). Uma das estratégias é explorar os
benefícios da ação destes microrganismos na forma de inoculantes (Lucy et al., 2004).
Objetivou-se neste estudo verificar a capacidade do B. amyloliquefaciens em promover
o crescimento de mudas de tomate.
MATERIAL E MÉTODOS
A solução de B. amyloliquefaciens FZB42- (Omex® Agrifluids do Brasil Ltda) a 1x1011
UFC/mL foi inoculada nas sementes na proporção de 320 uL/g de semente. Os
tratamentos corresponderam a porcentagens em volume aplicado.
A cultivar de tomate utilizada foi Santa Clara I-5300 (Isla®). O teste de germinação foi
realizado segundo a RAS (2009). Aos dez dias, foram avaliados volume e comprimento
das raízes e hipocótilo, no programa Win-Rhizo, acoplado a um scanner LA1600.
A produção de mudas foi conduzida de acordo com Koyama et al., (2013). Aos 30 dias
após plantio as mesmas variáveis acima foram analisadas, além da determinação de área
foliar, massa seca da parte aérea e raiz e altura da planta.
No teste de germinação, verificou-se que nenhum tratamento alterou negativamente o
percentual de germinação. No entanto, também não contribuíram para incrementos na
parte aérea ou raiz das plântulas. O tratamento de bactéria a 80% chegou a inibir o
volume de raiz e hipocótilo, indicando um provável excesso de dose.
Na produção de mudas, a menor dose de bactéria, mostrou-se superior para todos os
itens avaliados (exceto clorofila) da parte aérea (Tabela 1). As demais doses não
diferiram da testemunha. Uma parte aérea maior indica uma melhor taxa fotossintética,
que implica em mais fotoassimilados, que poderão ser translocados para os frutos nos
estádios seguintes (Taiz and Zeiger, 2004). Este aumento da parte aérea pode ter uma
relação características benéficas já constatadas para esta cepa, tais como, presença de
compostos auxínicos (Idris et al., 2004), produção de sideróforos (Chen et al., 2007) e
solubilização de fosfato inorgânico (Cuartas et al., 2010).
No sistema radicular, as maiores doses tiveram influência negativamente. A menor dose
se igualou a testemunha. Neste estádio de desenvolvimento observou-se também um
indicativo de excesso de dose nos tratamentos 3 e 4.
293
Session IV
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Tabela 1. Mudas de tomate inoculadas com B. amyloliquefaciens em diferentes doses, avaliadas aos 30
dias após plantio. (DMS) Diferença mínima significativa, (MG) Média Geral, (CV) Coeficiente de
Variação. Curitiba, Paraná, UFPR, 2013.
Tratamentos
1 - água
2 - bact20
3 - bact40
4 - bact80
MG
CV (%)
Volume
(cm3)
5.33 a
5.11 a
4.85 a
4.61 a
4.98
13.66
MUDAS DE TOMATE 30 DIAS APÓS PLANTIO
Raiz
Parte aérea
Comprimento Massa seca Área
Comprimento Volume
(cm)
(g) *
(cm2)*
(cm) *
(cm3)
2232.39 a
0.1789 a
180.69 b
162.77 c
16.02 b
2156.89 a
0.1734 a
255.16 a
261.32 a
20.02 a
2111.69 a
0.1489 b
202.71 b
201.71 b
16.37 b
2031.23 a
0.1223 c
208.82 b
224.78 b
15.52 b
2133.05
0.15
211.85
212.65
16.99
12.33
9.95
12.80
12.66
18.15
Massa seca
(g) *
0.8893 b
1.1586 a
0.9504 b
0.8111 b
0.95
9.79
Clorofila
total (mg/g)
6.67
a
8.11
a
6.83
a
8.52
a
7.53
27.53
Teste de Duncan a 5% de probabilidade.
* Teste de Duncan a 1% de probabilidade.
A inoculação de tomate com B. amyloliquefaciens promoveu incrementos na parte aérea
da planta, mostrando-se benéfica a utilização da menor dose na produção de mudas.
AGRADECIMENTOS
Agradecemos à Omex® Agrifluids do Brasil Ltda, por ceder a cepa da bactéria e à CAPES (Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior) pelo apoio financeiro.
REFERÊNCIAS
Chen XH. et al. (2007). Nature Biotech. 25: 1007-1014.
Cuartas, CAR. (2010). Faculty of Auburn University. 144p.
FAOSTAT. (2011). Disponível em: <http://www.faostat.fao.org.> Acessado em 12/07/2012.
Idris, EES. et al. (2004). J. Plant Dis. Protect.111: 583-597.
Koyama, R., et al. (2013). Amazon. J. Agric. Environ. Sci. 55: 282-287.
Lucy, M. et al. (2004). Antonie Leewenhoek 86: 1-25.
RAS, Regras para análise de sementes. (2009). 365p.
Taiz, L., and Zieger, E. (2004). Fisiología Vegetal. 719 pp.
Vale, M. et al. (2010). Plant growth and health promoting bacteria. pp. 21-43.
294
Session IV
SIV-CP-10
Efecto de volátiles emitidos por la bacteria bCT34 en la promoción de
crecimiento de Arabidopsis thaliana.
Parada, M.1*, Quiroz, A.2, Parra, L.2 Mendoza, D.1, Fincheira, P.1,
2
1
Centro Biotecnológicos de Estudios Microbianos (CEBEM), Facultad de Ciencias Agropecuarias y
Forestales, Universidad de La Frontera. Chile. 2 Laboratorio de Química Ecológica, Facultad de
Ingeniería, Ciencias y Administración. Universidad de La Frontera. Chile.
*
[email protected]
RESUMEN
Muchos de los microorganismos presentes en el suelo tienen la capacidad de emitir
Compuestos Orgánicos Volátiles (COVs) los que permiten aumentar el crecimiento
vegetal, sin embargo, su modo de acción es poco conocido. En este trabajo se aplicaron
indirectamente 8µl de inoculante de la cepa bacteriana bCT34 a plántulas de
Arabidopsis thaliana en placas Petri divididas y ordenadas en una distribución
completamente al azar. La captura de COVs fue realizada mediante el uso de SPME y la
identificación por medio de cromatografía gaseosa acoplada a espectrometría de masas.
Los resultados indican que plantas de Arabidopsis thaliana inoculadas con bCT34
aumentan su peso fresco total, el número de hojas, la longitud radical y el número de
raíces secundarias (p<0,05). Además, se identificaron COVs tales como oximas,
terpenos, hidrocarburos aromáticos y aldehídos. Se concluye que la bacteria bCT34 solo
a través de la emisión de COVs puede modificar la arquitectura radical en A. thaliana y
aumentar el número de hojas.
INTRODUCCIÓN
Algunos microorganismos de suelo tienen la capacidad de promover el crecimiento
vegetal a través de mecanismos directos (fitohormona) e indirectos (antibióticos)
(Bhattacharyya and Jha, 2012). Últimamente ha surgido un mecanismo asociado a
COVs emitidos por bacterias y definidos como compuestos de bajo peso molecular
(<300g/mol-1) que tienen una alta presión de vapor (Ortiz et al., 2009). Ryu et al.
(2003) demostraron por primera vez que volátiles bacterianos emitidos por Bacillus
subtilis GB03 y Bacillus amyloliquefaciens IN937a son capaces de promover el
crecimiento en Arabidopsis thaliana a través de la emisión de acetoína y 2,3-butanodiol.
Este reporte se enfoca en determinar los efectos morfológicos en las plántulas tras la
inoculación indirecta de la cepa bCT34 y determinar el perfil preliminar de volátiles
involucrados en la relación A. thaliana- bCT34 y sus efectos morfológicos en las
plántulas tras su inoculación indirecta.
MATERIAL Y MÉTODOS
La planta modelo utilizada fue Arabidopsis thaliana ecotipo Col0 cultivada en medio
básico Murashinge y Skoog (MS 0.5 X) suplementado con 1% sacarosa y 0,8% de agar
(pH 5.8). La bacteria bCT34 del cepario del laboratorio del Centro Biotecnológico de
Estudios Microbianos (CEBEM) y propagada en el medio universal Plate Count. Las
semillas a utilizar se desinfectaron con hipoclorito de sodio por 8 minutos y lavadas con
agua destilada estéril. Plántulas con una semana de desarrollo fueron inoculadas
indirectamente en medio MS con 8µl de la bacteria bCT34 disuelta en Caldo Nutritivo
(Densidad Óptica 0,1 a 600 nm) utilizando placas Petri divididas y con un fotoperiodo
16:8 y a 22˚C. Los efectos morfológicos fueron evaluados a las 2 semanas de
inoculadas. La captura e identificación de volátiles fue realizada al mismo tiempo según
la metodología descrita por Schalchli et al. (2011). El análisis estadístico de
comparación de medias fue realizado en SPSS versión 11.5.
295
Session IV
SIV-CP-10
Se observó un aumento significativo del peso fresco total y del número de hojas, así
como también se observó diferencias en la arquitectura radical.
(A)
a
(B)
a
b
b
(C)
(D)
P
P+I
Figura 1. Efecto de la inoculación en el número de raíces laterales, longitud radical y número de hojas
de A.thaliana comparadas con plantas no inoculadas. (A) N˚ de raíces secundarias (B) longitud radical
(C) N˚ de hojas (D) Arquitectura radical. Donde P= A. thaliana y P+I= A. thaliana inoculada con
bCT34. Letras diferentes indican diferencias significativas. (Prueba T- Student, α ≥ 0.5, n = 4).
Preliminarmente se identificaron algunos volátiles emitidos desde la interacción A.
thaliana-bCT34. Los compuestos identificados por medio de una comparación de
espectros de masas de la biblioteca NIST: fueron acetato de etilo, estireno, metoxi fenil
oxima, - y -pineno, limoneno e hidrocarburos aromáticos, entre otros. Probablemente
el efecto de los volátiles activó mecanismos que podrían estar involucrados en producir
fitohormonas del crecimiento, observado por ejemplo, en el incremento de raíces
secundarias, el cual pudiera ser causado por un aumento de la auxina y con ello la
regulación de la división y expansión celular. El análisis preliminar de los perfiles de
COVs no mostró la presencia de acetoína o 2,3-butanodiol, además los COVs
identificados no han sido bioensayados para probar su efecto sobre el crecimiento de A.
thaliana. En una segunda etapa se probarán nuevas metodologías de atrapamiento de
volátiles y se inducirá la respuesta utilizando compuestos puros y sus respectivas
mezclas. El crecimiento de las plantas estimuladas por los COVs de bCT34 podría ser
producto de la activación de citocininas implicadas a través de su acción en la división
celular y regulación de componentes específicos del ciclo celular (Ryu et al, 2003). Sin
embargo, se necesitan más pruebas para establecer el mecanismo de señalización que se
ve activado en las plántulas mediante la emisión de volátiles de bCT34. Por otra parte,
el aumento del número de hojas, puede estar provocado por una mayor disponibilidad
de nutrientes presentes en el medio de cultivo, modificación la arquitectura radical.
AGRADECIMIENTOS
Proyecto Fondo de Investigación para Bosque Nativo, CONAF 059/2011. Proyecto FONDECYT
1100812.
BIBLIOGRAFÍA.
Bhattacharyya, P.N., and Jha, D.K. (2012). World J. Microbiol. Biotechnol. 28: 1327-1350.
Ortiz, R., et al., (2009). Plant Signal Behav. 4: 701-712.
Ryu, C., et al. (2003). Plant Physiol.100: 927-4932.
Schalchli, H., et al. (2011). Chemistry and Ecology. 27: 503-513.
296
Session IV
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Functionality of phosphate solubilizing bacteria isolated from the
rhizosphere of papaya plants under conventional and organic farming
systems.
Melo, J.1*, Azevedo-Junior, R.R. 1, Eutropio, F.J.1, Correira, P.2, Carvalho, L. 2, Meleiro,
A.I.2, Teixeira, M.S.1, Carolino, M.2, Ramos, A.C.1, Cruz, C.2
1
Enviromental Microbiology and Biotecnology Lab, University of Vila Velha (UVV). Street Comissário
José Dantas de Melo 21, 29102-770, Vila Velha, Espirito Santo State, Brazil. 2 Centre for Environmental
Biology, Faculty of Science, University of Lisbon, Ed. C2,1749-016 Lisbon, Portugal.
* [email protected]
ABSTRACT
In order to determine the effects of agricultural practices on soil microbial communities
this study compare phosphate solubilizing bacteria in two different soil managements: a
conventional with manure, synthetic fertilizers and pesticides; and as organic system a
soil amended with a composting product. 12 bacterial strains were isolated from
rhizospheric soil samples of Carica papaya L. from Espírito Santo State – Brasil. All
the strains were positive for inorganic and organic phosphorous solubilization, with
isolates from organic system having higher P solubilizing capacity. We found microbial
functional differences between the rhizosphere of C papaya subjected to distinct
managements, and a higher number of P solubilizing bacteria in organic soil.
INTRODUCTION
Conventional farming systems have been associated with loss of soil fertility, soil
erosion, and ground water pollution (Drinkwater et al., 1995). In addition, some
conventional agricultural practices inhibit the activity and function of soil microbes.
Unlikely, the organic farmers do not use synthetic fertilizers or pesticides and attempt to
have a close nutrient cycle on their farms, protect environmental quality, and enhance
beneficial biological interactions and processes (Vandermer, 1995). Soils contain
enormous numbers of diverse living organisms assembled in complex and varied
communities. These organisms play an essential role in the sustainable function of all
ecosystems, including nutrient recycling and acquisition, plant health by suppression of
undesirable organisms. Currently, phosphate solubilizing microorganisms have attracted
the attention of agriculturists as soil inoculums to improve the plant growth and yield.
The goal of this work was to evaluate the influence of organic farming in comparison
with conventional agriculture on soil microorganism with particular incidence in
phosphate solubilizing bacteria.
Strains Isolation. Bacterial strains were isolated from the rizosphere of Carica papaya
L. according to Nautiyal, (1999).
Viable Phosphate Solubilizing Bacteria. Plate counts of solubilizing bacteria were
determined in solid medium (NBRIP) of serial dilutions of each soil samples.
Phosphate Solubilization Index. Bacterial strains were inoculated in NBRIP medium
using different inorganic and organic phosphate sources (e.g. Phytate, Tricalcicum
phosphate). Results were expressed as a solubilizing index (SI) representing the
diameter of halo solubilization per colony diameter. According to Silva Filho & Vidor
(2000) P solubilization can be classified as low (IS < 2); medium (2< IS < 3) and high
(IS > 3).
Phosphate solubilizing quantification. Bacterial strains were grown in liquids media
(NBRIP) using different inorganic and organic phosphate sources (e.g. Phytate,
297
Session IV
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Tricalcium phosphate). Solubilized phosphate was quantified according to a modified
assay of D’Angelo et al.(2001).
Pairwise interactions. The isolated strains were tested for their pairwise interactions in
Nutrient Broth and their growth rate and P solubilization quantification were
determined.
The colony formation unit did not differ in both systems (data not shown), but
abundance of P solubilizing bacteria was significantly higher (P<0.05) in organic soil
than on conventional agriculture (Figure 1A). All strains are classified as low
solubilizers of tricalcium phosphate as the index was lower than 2 units, nevertheless
strains from organic soils had a higher SI (Figure 1B). All strains are classified as
medium solubilizers of organic phosphate with a SI higher than 2 and the strains
isolated from organic agriculture have the highest solubilization index (Figure 1B). Pi
quantification tend to be higher in organic soil compared with conventional practice
(Figure 1C). Pairwise interactions between the isolated strains have shown that
combined strains from different origin (organic and conventional) can positively interact
and enhanced their growth (data not shown). Although the number of total viable
bacteria is about the same in both soil systems the number and activity of solubilizing
bacteria differ in organic and conventional farming, which led us to conclude that
different agriculture practices influence microbial functionality. We suggest that organic
management or introducing microbial consortia with bacteria isolated from organically
managed fields into conventional fields are potential strategies to improve phosphorus
use efficiency.
A
Solubilization Index
50000
40000
30000
20000
Organic
Pi Quantification
300,00
Tricalcium
250,00
2,0
1,5
200,00
150,00
1,0
100,00
0,5
50,00
0,00
0,0
Conventional
350,00
Phytate
2,5
10000
0
C
B
mg Pi /L
60000
CFU g -1
3,0
Solubilizing Phosphate Bacteria
Solubiliziation Index
70000
Conventional
Organic
Conventional
Organic
Figure 1. Phosphate solubilization bacteria in conventional versus organic agriculture. A. Total of viable
phosphate solubilizing bacteria in NBRIP medium. B. Solubilization index of bacterial strains. C.
Quantification of solubilized phosphate using Ca3PO4 as P source.
ACKNOWLEGMENTS
Acknowledgments: This work is supported by CNPq/475436/2010-5 and FAPES/54687985-2011 grants.
FAPES also conceded fellowships to FJE and JM.
REFERENCES
D’Angelo, E., et al. (2001). J. Environ. Qual. 30:2206-2209.
Drinkwater, L.E., et al. (1995). Ecol. Appl. 5: 1098-1112.
Nautiyal, C.S. (1999). FEMS Microbiol. Lett. 170: 265-270
Silva Filho, G.N., et al. (2000). Rev. Bra. Ciên. Solo 24:311-319.
Vandermer,, J. (1995). Annu. Rev. Ecol. Syst. 26: 201-224.
298
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Plant-growth promoting Sphingomonas sp. associated with annual ryegrass.
Dourado, A.C.1, Castanheira, N.2, 3, Cortés Pallero, A.3, Delgado Rodriguéz, A.I.3,
Alves, P.I.L.1 ,2, Barreto Crespo, M.T.1, 2, Fareleira, P.3*
1
Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal. 2 Instituto de Tecnologia Química e
Biológica/UNL, Oeiras, Portugal. 3 Instituto Nacional de Investigação Agrária e Veterinária, Oeiras,
Portugal.
*
[email protected]
ABSTRACT
Bacterial strains previously isolated from annual ryegrass were found to be closely related to
Sphingomonas paucimobilis and to contain a nifH gene sequence similar to Rhizobium spp.
Although all isolates were able to grow in N-free medium, no acetylene reduction activity could
be detected. Plant inoculation assays showed increases in the biomass of plants grown in either
N-free or N-supplemented medium.
INTRODUCTION
The genus Sphingomonas includes about 80 species of strictly aerobic, chemoheterotrophic αProteobacteria that contain glycosphingolipids as cell envelope components (Takeuchi et al.,
2001). Sphingomonas spp. are widely distributed in natural environments, such as soils, aquatic
systems and plants, but the only species described to date as capable of nitrogen fixation is S.
azotifigens, originally isolated from the rhizosphere and roots of rice (Xie and Yokota, 2006).
Some species, including S. paucimobilis, are opportunistic pathogens and have been isolated
from human clinical specimens and hospital environments (Ryan and Adley, 2010).
Annual ryegrass (Lolium multiflorum Lam.) is a forage crop that is extensively used in poorproductive areas in southern Portugal dedicated to the exploitation of cork and holm oaks
(“montado” or “dehesa”). While searching for diazotrophic bacteria associated with this crop,
we have recently obtained a collection of isolates originated from both the root external
environment and surface-sterilized plant tissues. The collection included a group of
Sphingomonas sp. that was able to grow well in nitrogen-free culture medium (work to be
published). Here we describe the characterization of these isolates and the evaluation of their
ability to promote plant growth.
Twenty-one bacterial isolates from annual ryegrass were used. Near full length 16S
rDNA (1.5-kb) was amplified from genomic DNA and sequenced. A fragment of the
recA gene (468-bp) was obtained according to Chen et al. (2012) and sequenced. A
fragment of the nifH gene (725-bp) was amplified as described by Xie and Yokota
(2006); an inner segment (432-bp) was re-amplified (Rösch et al. 2002) and used for
sequencing. The most similar sequences in GenBank were selected and phylogenetic
trees were constructed by the neighbor-joining method (Saitou and Nei, 1987) using the
Kimura’s two-parameter substitution model (Kimura, 1980) in MEGA version 5.0. The
acetylene reduction activity was assessed by gas chromatography in semi-solid NFb
cultures (Mascarua-Esparza et al., 1988). Auxin production was evaluated according to
Ashgar et al. (2002) in 16h-grown cultures in TY medium supplement with 100 µg/ml
tryptophan. Plant inoculation assays were performed in Evans medium; 3-5 days old
seedlings were inoculated with 108 cells, incubated for 4 weeks in a controlledenvironment growth chamber, and afterwards removed and dried until constant weight.
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A group of 21 bacterial strains previously isolated from annual ryegrass using an N-free
semi-solid medium (NFb) was addressed in this work. Sequence analyses of the 16S
rDNA and the housekeeping gene recA revealed that the isolates were phylogenetically
related to S. paucimobilis (respectively 99.8% and 100% similarity with the type strain
DSM 1098), which is not known as a diazotroph, and more distant from the nitrogenfixer S. azotifigens (Figure 1A). Despite their ability to grow in N-free medium, no
acetylene reduction activity could be detected in any of the isolates. The search for a
nifH gene fragment revealed a distinct sequence from S. azotifigens, and more related to
Rhizobium sp. and Sinorhizobium (now Ensifer) sp. sequences (Figure 1B).
Interestingly, the same sequence was found in the type strain S. paucimobilis DSM
1098, indicating the presence of a nitrogen-fixing gene in this species.
Figure 1. Neighbor-joining phylogenetic trees based on (A) 16S rDNA and (B) nifH gene sequences.
The numbers at the nodes indicate bootstrap values based on 1,000 replicates (>70%). Sequences in
boldface were obtained in this work. GenBank accession numbers are enclosed in parentheses.
Plant inoculation assays revealed that most Sphingomonas isolates could enhance the
growth of annual ryegrass plants in N-free Evans medium, with average increases of
50% and 25% respectively in the roots and shoots biomass, relative to non-inoculated
controls. These results suggested that, regardless the negative results in the ARA assay,
nitrogen fixation could be an important process in the association of these bacteria with
the host plant. However, subsequent inoculation assays with isolate G2Ac10 indicated
that increases in the roots biomass could also occur when nitrogen was present in the
culture medium, suggesting the involvement of mechanisms other than nitrogen
fixation. IAA production among isolates varied between 6.6 and 24.4 µg/ml culture.
Five isolates produced above 20 µg IAA/ml culture, but there was no obvious
relationship with major increases in plants biomass. Preliminary tests with the nonpathogenic S. azotifigens also resulted in enhanced roots biomass, revealing a good
potential as an alternative inoculant for annual ryegrass.
ACKNOWLEGMENTS
Work financed with National Funds by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through
Project PTDC/AGR-AAM/100577/2008 and grant # PEst-OE/EQB/LA0004/2011. A. Cortés Pallero and
A.I. Delgado Rodríguez were granted by Fundecyt, España. N. Castanheira is granted by FCT, Portugal.
A.C. Dourado and N. Castanheira contributed equally to this work.
REFERENCES
Asghar, H.N., et al. (2002). Biol. Fertil. Soils 35: 231-237.
Chen, H. (2012). Dissertation at the Faculty of Biology Ludwig-Maximilians-University Munich.
Kimura, M. (1980). J. Mol. Evol. 16: 111-120.
Mascarua-Esparza, M.A., et al. (1988). Plant Soil 106:91-95.
Rösch, C., et al. (2002). Appl .Environ. Microbiol. 68: 3818-3829.
Ryan, M.P., and Adley, C.C. (2010). J. Hosp. Infect. 75: 153-157.
Saitou, N., and Nei, M. (1987). Mol. Biol. Evol. 4: 406-425.
Takeuchi, M., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 1405-1417.
Xie, C.-H., and Yokota, A. (2006). Int. J. Syst. Evol. Microbiol. 56: 889-893.
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Paenibacillus species isolated from Pisum sativum nodules in Lanzarote
present several in vitro plant growth promotion mechanisms.
Ramírez-Bahena, M.H.1, 2, Flores-Félix, J.D.3, Tejedor, C.3, León-Barrios, M.4, Peix,
A.1, 2*, Velázquez, E.2, 3
1
Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, Salamanca, Spain. 2 Unidad Asociada
Grupo de Interacción Planta-Microorganismo Universidad de Salamanca-IRNASA-CSIC. 3 Departamento
de Microbiología y Genética, Universidad de Salamanca, Spain. 4 Departamento de Microbiología y
Biología Celular. Universidad de la Laguna. Tenerife. Spain.
* [email protected]
ABSTRACT
In the present work we explored the diversity of Gram positive endophytes isolated
from nodules of Pisum sativum growing in three Lanzarote soils through 16S rRNA
gene sequencing. The results showed the presence of six species from genus
Paenibacillus and two putative new species of this genus close to Paenibacillus
xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis and Paenibacillus
taohuashanense. All strains isolated were able to produce siderophores an indole acetic
acid that are included in the in vitro mechanisms involved in plant growth promotion
abilities. The data obtained suggested that these endophytes can play a role in the
formation and functioning of Pisum sativum nodules and then we are carrying out
microcosms experiments in order to analyze their effect in plant when coinoculated with
Rhizobium strains isolated from the same nodules in Lanzarote soils.
INTRODUCTION
Lanzarote is the most northern and oriental island of Canary archipelago included in the
Biosphere Reserves since 1993. The particular characteristics of the soils present in this
volcanic islands make them interesting ecosystems to study the diversity of legume
nodules. In this way we showed that Phaseolus vulgaris is nodulated in Lanzarote soils
by Ensifer meliloti strains that are not able to nodulate Medicago sativa (Zurdo-Piñeiro
et al., 2009). Nevertheless, there are no data about the endosymbionts of other legumes
cultivated in small orchards in this island neither about the endophytes present in these
nodules. In the last years the study of legume nodular endophytes is increasing with
special emphasis on their ability to have in vitro plant growth promotion mechanisms
that has been found in some nodule endophytic strains including sporulated Gram
positive genera.
The most abundant genera of this bacterial group found in legume nodules are Bacillus
and Paenibacillus and some of their species are able to produce indole acetic acid an
siderophores (Selvakumar et al., 2007, Li et al., 2011).
Therefore, the objective of this work was the isolation and identification of the
endophytic species isolated from nodules of Pisum sativum used as trap plant in three
sites of Lanzarote (Canary Islands) as well as the analysis of the in vitro plant growth
promotion mechanisms presented by the endophytes isolated.
The isolation of endophytes and rhizobia was performed following the method of
Vincent (1970). The Pisum sativum plants were harvested at flowering in two soils
subjected to integrated production in Valladolid (Spain).
Amplification and sequencing of the 16S rRNA gene were performed according to
Rivas et al. (2007). The sequences obtained were compared with those from EzTaxon-e
server (Kim et al., 2012).
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The phosphate solubilization, siderophores production, indole acetic acid production
and nitrogen fixation were analyzed as previously described (García-Fraile et al., 2012).
Pisum sativum plants nodulated in the three soil samples analyzed from Guatiza and
Mala, one of them recovered from a volcano soil. The nodules were effective and
typical of rhizobia, nevertheless most of the isolated colonies on YMA plates despite
their mucoid aspect and white colour did not correspond to rhizobia but to Paenibacillus
species. Only a strain from species Rhizobium leguminosarum was recovered from the
Mala soil.
The Paenibacillus species were identified as Paenibacillus validus, Paenibacillus
xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis, Paenibacillus
taohuashanense and Paenibacillus polymyxa with more than 99.5% identity in the 16S
rRNA gene. Some strains belonged to undescribed species from genus Paenibacillus
related to Paenibacillus xinjiangensis, Paenibacillus peoriae, Paenibacillus borealis
and Paenibacillus taohuashanense.
All the strains presented at least one of the four in vitro mechanisms of plant growth
promotion studied. Siderophores were produced by all strains and particularly by the
strains of Paenibacillus validus.
ACKNOWLEDGMENTS
This work was supported by MINECO (AGL2010-17380). MHRB is recipient of a JAE-Doc researcher
contract from CSIC, cofinanced by ERDF.
REFERENCES
García-Fraile, P., et al. (2012). PLoS One. 7: e38122.
Kim, O.S., et al. (2012). Int. J. Syst. Evol. Microbiol. 62: 716-721.
Li, L., et al. (2011). FEMS Microbiol. Ecol. 79: 46-68.
Rivas, R., et al. (2007). Lett. Appl. Microbiol. 44: 181-187.
Selvakumar, G., et al. (2007). Curr. Microbiol. 56: 134-139.
Oxford : Blackwell Scientific Publications.
Zurdo-Piñeiro, J. L., et al. (2009). Appl. Environ. Microbiol. 75: 2354-2359.
302
Session IV
SIV-CP-14
Efecto de rizobacterias en el enraizamiento de miniestacas en dos
clones híbridos de Eucalyptus spp.
González-Candia, P.1*, Sossa, K.2, Rodríguez, F.3, Sanfuentes, E.1
1
Laboratorio de Patología Forestal, Facultad de Ciencias Forestales, Universidad de Concepción.
Concepción, Chile. 2 Laboratorio de Biopelículas y Microbiología Ambiental, Centro de Biotecnología,
Universidad de Concepción. Concepción, Chile. 3 Forestal Mininco S.A.
*
[email protected]
RESUMEN
Mediante tres ensayos se evaluaron rizobacterias en el enraizamiento de miniestacas de
dos clones híbridos de Eucalyptus nitens x Eucalyptus globulus. Los resultados de esta
investigación confirmaron el positivo efecto de las rizobacterias en promover el
enraizamiento, confirmando el potencial medioambiental y de bajo costo que esta
estrategia implicaría para la industria forestal. Además la identificación mostró a
Bacillus y Pseudomonas como las bacterias con mayor efecto.
INTRODUCCIÓN
Eucalyptus globulus y sus híbridos son relevantes para industria forestal en Chile, por lo
que mucho esfuerzo se ha destinado a mejorar la producción de esta especie en
condiciones de vivero. En este sentido la reproducción asexual es promisoria, sin
embargo, los problemas en el enraizamiento de algunos clones puede limitar su
implementación (Teixeira et al., 2007). Para mejorar esto, se ha comenzado a utilizar
rizobacterias en virtud de su positivo efecto en el enraizamiento, de su bajo impacto
ambiental y menor costo (Adesemoye et al., 2009). Estas bacterias captan carbono y
nitrógeno desde los exudados de las raíces (Benizri et al., 2001), influenciado el
enraizamiento de la planta (Compant et al., 2010). A pesar de estas ventajas, el efecto
de estas bacterias ha sido poco estudiado en especies forestales (Chanway, 1997). Este
estudio evaluó el efecto de rizobacterias en el enraizamiento de miniestacas de dos
clones híbridos recalcitrantes de Eucalyptus.
MATERIAL Y MÉTODOS
Se aislaron 106 cepas bacterianas desde la rizósfera de cinco clones de Eucalyptus spp.
(Díaz et al., 2009). Fueron realizados tres ensayos (E1, E2, E3) en vivero de la empresa
Forestal Mininco S.A, en Los Ángeles, Chile. Las rizobacterias fueron aplicadas al
substrato por aspersión, y en la base de las miniestacas de dos clones E. nitens x E.
globulus A y B mediante inmersión (108ufc/ml). En E1 se utilizó solo un clon híbrido
mientras que en E2 y E3 se consideraron los dos clones. Como control se utilizó agua
destilada. El diseño experimental fue en bloques completos al azar, con 20 repeticiones
para E1 y E2, y 30 para E3. Luego de 45 días se evaluó el enraizamiento (%). Los
resultados fueron analizados mediante test paramétricos o no paramétricos en función
de la distribución de los residuos (SAS 9.2). Posteriormente las cepas con mejores
resultados fueron identificadas mediante extracción, purificación y secuenciación de
ADNr 16S.
En el primer ensayo 32 cepas aumentaron significativamente (p>0,05) el enraizamiento
de las miniestacas, con un enraizamiento máximo que fue de 75%; 2,6 veces mayor al
control (28%). Estos resultados implican rendimientos comparables a los obtenidos por
Teixeira et al. (2007), mediante inoculación de cepas de rizobacterias en estacas de E.
grandis. En el segundo ensayo 9 y 7 cepas incrementaron significativamente el
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enraizamiento en los clones A y B, respectivamente. En el tercer ensayo 12 cepas
aumentaron el enraizamiento en ambos clones. Estos resultados permiten concluir que
rizobacterias seleccionadas pueden incrementar el enraizamiento en miniestacas de
clones E. nitens x E. globulus, con diferencias clon-específicas. Los resultados de la
identificación indicaron que las cepas bacterianas pertenecieron principalmente al
género Bacillus sp. y Pseudomonas sp. Otras cepas fueron identificadas como
Musilaginibacter sp., Flavobacterium sp. y Rhodococcus sp, géneros de bacterias
ambientales indicadas como bacterias con efecto en el enraizamiento de plantas
(Belimov et al., 2005, Madhaiyan et al., 2010).
Tabla 1. Enraizamiento de miniestacas en los híbridos E. nitens x E.
globulus clones A y B. Se muestran aquellas cepas con
enraizamiento significativamente mayor al control, que además
estuvieron presenten en los resultados de E1, E2 y E3.
Cepa
Control
14
121
46
63
18
49
43
116
56
59
119
Enraizamiento (%) Clon A
7
29
25
28
32
37
28
25
29
27
27
40
Enraizamiento (%) Clon B
54
74
75
74
79
86
78
78
68
67
68
77
AGRADECIMIENTOS
CONICYT, Forestal Mininco S.A.
BIBLIOGRAFÍA
Adesemoye, A., et al. (2009). Microbiol. Ecol. 58: 921-929.
Belimov, A., et al. ( 2005). Soil Biol. Biochem. 37: 241-250
Benizri, E., et al. (2001). Biocontrol Sci and Techn. 11: 557-574.
Chanway, C., (1997). For. Sci. 43 99-112.
Compant, S., et al. (2010). Soil Biol. Biochem. 42: 669-678.
Díaz, K., et al. (2009). World J. Microbiol. Biotechnol. 25: 867-873.
Madhaiyan, M., et al. (2010).IJSEM. 60: 2451-2457.
Teixeira, D., et al. (2007). Braz. J. Microbiol. 38: 118-123.
304
Session IV
SIV-CP-15
Azoarcus sp. CIB, un nuevo endófito del arroz que degrada
anaeróbicamente hidrocarburos aromáticos.
Fernández, H.1, Prandoni, N.1, Fernández-Pascual, M.2, Morcillo, C.2, Fajardo, S.2,
Díaz, E.1, Carmona, M.1*
1
Departamento Biología Ambiental. Centro de Investigaciones Biológicas-CSIC.
Protección Vegetal. Instituto de Ciencias Agrarias-CSIC.
* [email protected]
2
Departamento de
RESUMEN
En nuestro laboratorio hemos demostrado que Azoarcus sp. CIB, una bacteria modelo
en el estudio del catabolismo anaeróbico de compuestos aromáticos, es capaz de invadir
la raíz del arroz y de vivir como endófito. Dado que Azoarcus sp. CIB degrada diversos
contaminantes, podría ser empleada como herramienta biotecnológica en tareas de
endofitorremediación.
INTRODUCCIÓN
Azoarcus sp. CIB es una β-Proteobacteria desnitrificante con la capacidad de degradar,
tanto aeróbica como anaeróbicamente, un elevado número de compuestos aromáticos,
incluyendo contaminantes como el tolueno y m-xileno (López-Barragán et al., 2004).
Otras bacterias del género Azoarcus, e.g., Azoarcus sp. BH72 y A. communis, son
capaces de invadir plantas y vivir como endófitos (Reinhold-Hurek and Hurek, 2000).
Hasta la fecha, no se había descrito ningún Azoarcus que degrade anaeróbicamente
compuestos aromáticos y que sea endófito, llegándose a sugerir la existencia de un
nuevo género, Aromatoleum, para los Azoarcus no endófitos degradadores (ReinholdHurek and Hurek, 2000). En este trabajo presentamos evidencias experimentales de que
Azoarcus sp. CIB es capaz de invadir la raíz del arroz.
MATERIAL Y MÉTODOS
Recuento del número de bacterias dentro de la raíz del arroz. Las semillas de arroz
fueron desinfectadas e inoculadas con bacterias tal y como se ha descrito anteriormente
(Compant et al., 2005). Tras 5 días de crecimiento se determinó el número de viables en
el interior de la raíz siguiendo protocolos ya establecidos (Compant et al., 2005).
Técnicas microscópicas. Se empleó un microscopio confocal Leica TCS-SP5-AOBS y
un microscopio electrónico (TEM) STEM LEO 910. Las muestras para confocal fueron
preparadas según (Reinhold-Hurek and Hurek, 2011). El procesamiento e
inmunolocalización para TEM se llevo a cabo según de María et al. (2005) utilizando
un anticuerpo anti-GFP comercial.
Identificación de genes necesarios para la endofitosis en el genoma de Azoarcus sp.
CIB.
Análisis genómicos en bacterias endofíticas ha permitido identificar una serie de genes
esenciales para establecer un estilo de vida endófito (Krause et al., 2006; ReinholdHurek and Hurek, 2011). Recientemente hemos secuenciado el genoma de Azoarcus sp.
CIB, y se han identificado genes ortólogos a los relacionados en otras bacterias con la
asociación como endófito: i) genes que codifican el flagelo, ii) genes de fimbrias tipo
IV, iii) genes implicados en la síntesis de exopolisacáridos, iv) genes implicados en la
producción de análogos de hormonas vegetales. Estos resultados nos permitieron sugerir
que Azoarcus sp. CIB podría ser el primer degradador anaeróbico de hidrocarburos
aromáticos que se describe capaz de establecerse también como un endófito de plantas.
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Localización de Azoarcus sp. CIB en el interior de la raíz del arroz.
Dado que el arroz es una planta que colonizan otras bacterias del género Azoarcus
(Reinhold-Hurek and Hurek, 2011), se utilizó como modelo de planta huésped para
estudiar la capacidad endofítica de la cepa CIB. Se inocularon, de forma independiente,
plántulas previamente desinfectadas con Azoarcus sp. CIB, Azoarcus communis (control
positivo) y Escherichia coli (control negativo). El recuento de bacterias presentes en el
interior de las raíces reveló que por gramo de raíz había 10 5 células de A. communis,
5x104 de Azoarcus sp. CIB y ninguna de E. coli. Estos resultados sugieren que Azoarcus
sp. CIB es capaz de colonizar la raíz del arroz tan eficientemente como A. communis.
Observación de Azoarcus sp. CIB en el interior de la raíz del arroz.
Azoarcus sp. CIB-GFP, una cepa que expresa la “green fluorescence protein”, fue
detectada en el interior de estructuras de la raíz del arroz tanto mediante observación
con microscopía de fluorescencia (Figura 1A), como con microscopía confocal (Figura
1B). La microscopía electrónica permitió corroborar estos resultados, localizando a
Azoarcus sp. CIB en los espacios intercelulares de la exodermis radicular (Figura 1C).
B
A
C
PC
Az
EI
Az
Az
10 m
50m
0,2µm
Figura 1. Observación microscópica de la interacción de Azoarcus sp. CIB con la raíz del arroz.
(A) Microscopía de fluorescencia de un pelo radicular donde se observa un grupo de Azoarcus sp. CIBGFP. (B) Microscopía confocal en la que se observan agregados de Azoarcus sp. CIB-GFP en las zonas
internas de la raíz. (C) Inmunolocalización con anti GFP de Azoarcus sp. CIB-GFP en un espacio
intercelular de la exodermis radicular de arroz. PC, pared celular; EI, espacio intercelular, Az,Azoarcus.
Este trabajo constituye la primera descripción de un organismo capaz de degradar
anaeróbicamente compuestos aromáticos y de vivir como endófito en plantas, abriendo
la posibilidad de utilizar endofitorrediación de ambientes contaminados con
hidrocarburos aromáticos.
AGRADECIMIENTOS
Este trabajo ha sido financiado por los proyectos BIO2009-10438 y BIO2012-39501 del Ministerio de
Economía y Competitividad y la Comunidad de Castilla-La Mancha proyecto POII10-0211-5015.
BIBLIOGRAFÍA
Compant, S., et al. (2005). Appl. Environ. Microbiol. 71: 1685-1693.
de María, N. et al. (2005). Plant Physiol. Biochem. 43: 985-996.
Krause, A., et al., (2006). Nat. Biotech. 24: 1385-1391.
López-Barragán, M.J., et al. (2004). J. Bacteriol. 186: 5762-5774.
Reinhold-Hurek, B., and Hurek, T. (2000). Int. J. Syst. Evol. Microbiol. 50: 649-659.
Reinhold-Hurek, B. and Hurek, T. (2011). Curr. Op. Plant Biol. 14: 435-443.
306
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SIV-CP-16
Impacts of rice-bean intercropping on soil microbial activity and the
development of rice plants.
Razakatiana, A.T.E.1, 2*, Henintsoa, M.3 Becquer, T.4, Ramanankierana, H.2, Baohanta,
H.R.2, Raherimandimby, M.1, Rabeharisoa, L.3, Duponnois, R.5
1
Laboratoire de Biotechnologie et de Microbiologie, Faculté des Sciences, Université d’Antananarivo, BP
906, Antananarivo 101, Madagascar. 2Laboratoire de Microbiologie de l’Environnement (LME), Centre
National de Recherches sur l’Environnement (CNRE), BP 1739, Antananarivo 101, Madagascar.
3
Laboratoire des Radioisotopes (LRI), Université d’Antananarivo, BP 3383, Antananarivo 101,
Madagascar. 4IRD, UMR 210 Eco&Sols, CIRAD – INRA – SupAgro, 2 place Viala, Bâtiment 12, 34060
Montpellier Cedex , France. 5Laboratoire des Symbioses Tropicales Méditerranéennes LSTM UMR
CIRAD/ IRD /SupAgro/UM2 USC INRA TA A-82/ J Campus International de Baillarguet 34398
Montpellier Cedex 5, France.
* [email protected]
ABSTRACT
The impact of the intercropping system with common bean and rice under different
treatments such as organic fertilizer, mineral fertiliser, cropping system with no-tillage
or conventional tillage on plant development and on microbial activity was assessed.
INTRODUCTION
Soil tillage, organic amendment type or amount of phosphorus (P) fertilizer could
influence both plant development, production yield and soil microbial activities
(Asensio et al, 2010). The main objective of the study was to evaluate the effects of
different cropping system on plant development and soil microbial activities. This will
provide new way to reduce the use of chemical fertilizer in farming system.
The study site is a field experiment located in Lazaina, a rural area in the suburb of
Antananarivo, Madagascar (18°46’53.56’’South and 47°32’05.03’’East). Different ricebean intercropping system corresponding to a combination of two soil tillage
(conventional (CT) and no-tillage (NT)), two organic treatments corresponding to an
input of 20 kg P ha-1 as manure (M20) and as green manure of stylosanthes (GM20)
and two levels of, triple superphosphate (TSP) chemical fertilizer (20 (TSP20) and 50
(TSP50) kg P ha-1), were compared to a control (CT without organic and mineral
fertilizers).
The shoot production and the grain yields of rice and bean were determined. Soil
microbial enzymatic activities (rhizospheric soils of rice and bean) were assessed with
an emphasis on phosphatasic activities and FDA (Fluorescein diacetate) hydrolysing
activities. Phosphatasic activities were conducted in acid and alkaline media. The paraNitrophenyl Phosphate (pNPP) substrate in contact with the soil was hydrolyzed to pNitrophenol by phosphatasic enzymes (Feller et al.,1994) and FDA in contact with the
soil was hydrolyzed by different enzymes producing there fluoresceine (Gaël et al.,
2002; Alef K.,1998; Schnurer, J. and Rosswall. T.,1982).
Compared to the control, the organic manure mixed with mineral fertilizer in moderate
doses promotes plant growth and yield of both rice and beans (Fig 1). However, the
green manure promoted a decrease of the shoot biomass productions and grain yields,
probably due to the poor nitrogen content of the stylosanthes shoots that we used.
307
Session IV
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Quantity of pnitrophenol (µg/h/g of
soil)
(a) (a)
(ab) (ab)
(ab)
Production yield
(t.ha-1)
200
3
(a)
(b)
(a) (a) (ab) (a)
0
50
40
30
20
10
0
(b)
(a) (a) (a) (a)
(a)
(a) (a) (a)
c
(abc) (bc) (abc)(a)
(abc) (ab)
(ab) (abc)
(abc)
(a)
Acid phosphatase
Quantity of p-nitrophenol
(µg/h/g of soil)
Production
yield(kg.ha-)
400
50
40
30
20
10
0
2
(b)
(b) (b) (b)
(ab) (ab) (ab)
(a)
1
(a)
(ab)
(a)
0
c
(a)(a)
(ab) (ab)
(ab)
(b)
Acid phosphatase
Alcaline phosphatase
(d
)
(b)
(a) (b)
(ab)
(ab) (ab)
(a) (ab)
(ab) (ab)
(ab) (ab)
(ab)
Alcaline phosphatase
Figure1. Comparison of yield of bean (a), yield of rice (b) and soil phosphatase activity of rice soil (c)
and bean soil (d) on the field expériment in Lazaina
Table 1. Global microbial activity
Treatment
CT-Co
CT-TSP20
CT-TSP50
CT-M20
CT-TSP20-M20
CT-TSP50-M20
CT-TSP20-GM20
CT-TSP50-GM20
NT-TSP20-M20
NT-TSP20-GM20
Fluorecsein diacetat (mg/h/g) of rice soil
5,53 (abc)
2,55 (a)
3,61 (abc)
2,32 (a)
3,03 (ab)
2,63 (abc)
7,05 (bcd)
9,80 (d)
4,20 (abc)
6,95 (cd)
Fluorecsein diacetat (mg/h/g) of bean
soil
3,92(a)
2,21 (a)
4,18(a)
2 (a)
6,68 (a)
4,04 (a)
11,76 (b)
10,78 (b)
3,29 (a)
2,61 (a)
Using stylosanthes as green manure significantly stimulated the global microbial
activity of soil microorganisms evaluated by the fluorescein diacetate hydrolysis assay,
in rice and bean soils (Table1), as observed also by Acosta-Martinez et al. (2007) and
Gaël et al. (2002). Soil phosphatase activity texted by the p-nitrophenyl phosphate
(pNPP) hydrolysis assay (Feller et al.,1994) was also significantly higher in tillage
treatments using manure alone or mixed with mineral fertilizer. Thus, our results
showed that organic fertilizers on rice-bean intercropping system positively influence
soil microbial activities.
ACKNOWLEGMENTS
This work was supported by the project Fabatropimed supported by Agropolis Foundation under the
REFERENCES
Acosta-Martínez, V., et al. (2007). Appl. Soil. Ecol. 37: 41–52.
Alef, K. (1998). Methods in applied soil microbiology and biochemestry. Academic Press, London, pp.
232-233.
Asensio, R. (2010). Les Cahiers du PRAM 8: 25-29.
Feller, C., et al. (1994). Can. J. Soil Sci. 73: 121-129.
Gaël, A., et al. (2002). Activités biologiques et fertilité des sols. ITAB, Paris. 27p.
Schnurer, J., and Rosswall, T. (1982). Appl. Environ. Microbiol. 43:1256-1261.
308
Session IV
SIV-CP-17
Efeito de bactérias PRPG com indução de ácido salicílico em plântulas
de duas cultivares de palma forrageira (Opuntia e Nopalea).
Lyra, M.C.C.P.1*, Pérez-Montaño, F.2, Jiménez-Guerrero, I.2, Madinabeitia, N.2, DiazOlivares, I.M.2, Gutiérrez-Alcántara, R.2, Ollero, F.J.2
1
Agronômico de Pernambuco-IPA. Laboratório de Genoma-Avenida General San Martin, 1371, Bongi,
CEP. 52761000 Recife-Pernambuco. Brasil. 2 Departamento de Microbiología. Facultad de Biología.
Universidad de Sevilla. Avda. Reina Mercedes, 6. 41012 Sevilla-España.
*
[email protected]
ABSTRACT
Prickly Pear Cactus has great economic interest in Brazil due to its ability to withstand
long periods of drought. The use of PRPG bacteria induced with AS can have an effect
on plant growth. This study aimed to evaluate this interaction. We observed that the
Nopalea did most amount of AS was detected by luminescence. The AM07 isolated was
inhibited by the presence of AS. The growth parameters of the plants only showed
significant differences in the variety Miúda due to its homogeneity of germination and
consequently the same physiological stage.
INTRODUÇÃO
A palma forrageira é uma cultura muito utilizada no nordeste brasileiro por apresentar
uma grande capacidade de adaptação às condições de seca. Os mecanismos pelo qual as
rizobactérias promotoras de crescimento de plantas (PGPR) atuam podem ser diretos ou
indiretos e estes mecanismos podem ser ativados em diferentes estágios do crescimento
da planta. Algumas enzimas e compostos fenólicos estão envolvidas nestes diferentes
mecanismos de defesa na planta quando inoculadas com bactérias PGPR (Figueiredo et
al, 2010). Um destes compostos é o acido salicílico (AS) que deriva do aminoácido
fenilalanina. A importância do SA como regulador do crescimento em plantas está
reduzida a poucos processos mas sua presença afetam a síntese de outros reguladores de
crescimento. O AS reduz a síntese de etileno e em algumas espécies pode causar atraso
na senescência de flores ou indução da floração. Ryals et al. (1996) observaram que os
níveis de SA aumenta no tecido da planta quando estas sofrem algum ataque de
patógeno e a aplicação de SA exógeno aumenta a resistência da planta para grande
gama de patógenos.Este trabalho teve como objetivo observar o efeito de dois isolados
endofíticos com e sem indução de acido salicílico em plântulas de palma forrageira .
MATERIAL E MÉTODOS
As sementes de Opuntia fícus indica (var. Orelha de Elefante Africana) e de Nopalea
Cochenillifera (var. Miúda) da família Cactaceae foram oriundas do Agreste
Pernambucano (Banco de Germoplasma de Arcoverde-Estação Experimental do
Instituto Agronômico de Pernambuco-IPA-Brasil). Oito tratamentos foram realizados:
T1: Controle, T2: G. diazotrophicus (AM07), T3: H. seropedicae (AM37), T4: AM07 +
1 mM (10-4M) de ácido salicílico (AS), T5: AM37 + 1 mM AS, T6: AM07 + AM37, T7:
AM07+ AM37 + 1mM AS, e T8: 1 mM AS. Foram estudados: Peso Fresco da Planta
(PFP, g), Comprimento da planta (CP, cm) e Largura do Cladódio (LC, cm – realizada
exatamente na metade da folha). O delineamento foi inteiramente ao acaso, com três
repetições. A inoculação com os isolados foi realizada cinco dias após o transplante
onde as mesmas foram cultivadas em meio DYGs por 24 hs. O SA foi dissolvido em
água segundo Bordiec et al. (2011). A extração e determinação do AS foi realizada de
acordo com Huang et al. (2005) com algumas modificações, onde macerou-se 0,2 g do
tecido da planta, sonicação e centrifugação resultando em um extrato cru contendo AS.
O extrato cru foi misturado com Acinetobacter sp. strain ADP1 (Metzgar et al, 2004) e
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colocada em uma placa e incubada a 37°C por 1 h em três réplicas. A quantificação do
AS (Luminescência) foi realizado no equipamento Synergy HT Biotek com Programa
Gen51.11.
A variedade Orelha de Elefante Africana (Opuntia fícus-indica), não apresentou
diferenças significativas para os parâmetros estudados talvez em função do alto
coeficiente de variação (CV%=40,53). O tratamento T6 com os isolados AM07 e AM37
foi quem obteve o maior percentual em relação ao controle absoluto para a PFP (129%),
vindo logo após os tratamentos T2 (93%) e T3 (81%). Ao adicionar o AS juntamente
com os isolados observamos uma redução da PFP de 17% negativo em relação ao
controle negativo e de 35% menos em relação ao T6. O mesmo comportamento foi
observado para as outras duas variáveis (CP e LC). Resultado não observado para o
parâmetro LC, que foi negativo. Resultados contrários, foram observado por Ryals et al,
(1996) onde observaram que os níveis de SA aumentava no tecido da planta quando
estas sofriam algum ataque de patógeno. Na variedade N. Cochenillifera (var. Miúda)
observamos diferenças significativas em que o melhor tratamento foi T2 seguido dos
tratamentos T3, T7, T5 e T4, respectivamente. Resultados semelhantes foram
observados com o uso simultâneo do AS com os isolados influenciando negativamente
nos tratamentos em que o T8 foi quem apresentou os piores resultados chegando a obter
um percentual de -17% (PFP), 10% (CP) e 9% (LC). O maior valor de luminescência
usando a bactéria Acinetobacter sp. ADP1, foi observado com a Nopalea var. Miúda
nos tratamentos T8 (59,87 ng) e T5 (31,2 ng), os demais tratamentos variaram entre 9 e
12,3 ng. No caso da planta Opuntia var. Orelha de Elefante, o maior valor de AS foi
obtido no T8 (29,92 ng). 100% a mais de AS na planta Nopalea. Se analisarmos estes
dados comparando com as variáveis estudadas de PFP, CP e LC, podemos afirmar que
quanto mais AS na planta mais afeta o crescimento da mesma. O isolado AM07 foi o
mais afetado com a adição de AS nas duas variedades. Podemos afirmar que como a
palma forrageira possui um metabolismo fotossintético do tipo CAM, que o caracteriza
por terem um crescimento lento, estas plantas competem de forma desvantajosa com
outras espécies de plantas quando as condições ambientais são extremas. O uso de AS
inoculado com PRPG em cactáceas deve ser melhor estudado.
CONCLUSÕES
A variedade Nopalea foi quem mais quantidade de AS foi detectada por luminescência.
A bactéria AM07 foi inibida pela presença de AS. Os parâmetros de crescimento das
plantas apresentaram diferenças significativas apenas na variedade Miúda devido a sua
homogeneidade na germinação e consequentemente um mesmo estagio fisiológico. Por
serem de metabolismo lento, resultados satisfatórios poderiam ser observados em um
período de tempo maior, bem como, determinar a melhor dose de AS para estas plantas.
REFERÊNCIAS
Bordiec, S., et al. (2011). J. Exp. Bot. 62: 595-603.
Figueiredo, M.V.B.F., et al. (2010). Plant Growth Promoting Rhizobacteria: Fundamentals and
Applications.
Huang, W.E., et al. (2005). Environ. Microbiol. 7: 1339-1348.
Metzgar, M.L., et al. (2004). Nucleic Acids Res.32: 1792-1797
Ryals, J.A., et al (1996). Plant Cell 8: 1809-1819.
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Análisis cualitativo y cuantitativo de vitamina C en fresas, en presencia
de microorganismos endófitos.
Aranda-Alonso, C.1,
Serrano, A.M.1
2*
, Rodríguez-Carvajal, M.A.1, Ollero, F.J.2, Megías, M.3, Gil-
1
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla. Sevilla, España. 2
Departamento de Microbiología Vegetal y Biología Molecular, Facultad de Biología, Universidad de
Sevilla. Sevilla, España. 3 Departamento de Microbiología y Parasitología, Facultad de Farmacia,
Universidad de Sevilla. Sevilla, España.
* [email protected]
RESUMEN
El contenido en ácido L-ascórbico o vitamina C en fresas ha sido determinado mediante
HPLC-PDA en fase reversa. Fueron analizadas ocho tesis inoculadas con diferentes
microorganismos endófitos y en tres de ellas el aumento en ácido L-ascórbico fue muy
significativo, siendo la cepa Pantoea vagans LMG 24199 (T) (NTABE-P310) +
Micorriza la que presentó un efecto más acusado como inductor de la biosíntesis de
vitamina C.
INTRODUCCIÓN
La vitamina C está formada en un 90% por ácido L-ascórbico (AA), que es la forma
predominantemente activa, si bien también se puede encontrar en la forma oxidada:
ácido L-deshidroascórbico (DHA), que también presenta actividad biológica (Campos et
al., 2009). Aunque para cuantificar de forma precisa el contenido en vitamina C de la
matriz biológica en cuestión es importante determinar el contenido de ambos, se puede
considerar únicamente el porcentaje de ácido L-ascórbico en una determinación rápida y
aproximada.
En este trabajo se presenta un método de análisis HPLC-PDA en fase reversa, que no
compromete la integridad del ácido ascórbico y que a la vez proporciona unos
resultados fiables, siendo rápido y de bajo coste.
MATERIAL Y MÉTODOS
Preparación de las muestras y extracción del ácido ascórbico.
Se parte de unos 10 g de fresas trituradas. La rotura mecánica de las células se lleva a
cabo con nitrógeno líquido en mortero y, a continuación, se liofiliza la muestra. El
liofilizado se trata con 10 ml de una disolución de H3PO4 (3%) y AcOH (8%) (Frenich
et al., 2005), se centrifuga a 15000 rpm durante 10 min a 5 °C y se recoge el
sobrenadante, que se diluye con agua hasta 50 ml. El extracto resultante se pasa a través
de filtros de nylon de 0,45 µm para su posterior inyección en el HPLC-PAD (Pallauf et
al., 2012).
Condiciones cromatográficas.
Se ha utilizado un sistema de HPLC Waters-Alliance 2695 acoplado a un detector PDA
Waters 2996 (Photo Diode Array), con una pre-columna SecurityGuard Cartridge C18
4 x 3,0 mm ID y una columna Hyperclone ODS C18 - 5µm x 4,0 mm x 250 mm
(Phenomenex, EEUU). La fase móvil empleada ha sido H 2SO4 0,01% (Burini, 2007). El
volumen de inyección para cada ensayo fue de 100 µl y cada cromatograma se ha
registrado durante 30 minutos.
Debido a la inestabilidad térmica observada en las muestras y en los patrones, éstos se
prepararon frescos para cada análisis y manteniendo en hielo los viales justo antes de
introducirlos en el carrusel del equipo.
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Antes de establecer el método de cuantificación, se hicieron ensayos para detectar el
posible efecto matriz intrínseco en muestras biológicas. La variación en las respuestas
instrumentales fue considerable, por lo que se corroboró la presencia del efecto matriz y
se decidió aplicar la técnica de las adiciones estándar (Miller, 2002) para subsanar este
efecto en la cuantificación.
Los resultados de dicha cuantificación fueron los siguientes:
El incremento que se produce a causa de los microrganismos endófitos es más que
notable.
Este resultado plantea la necesidad de realizar ensayos futuros más exhaustivos, para
confirmar estas bacterias y hongos como agentes inductores de la biosíntesis de
vitamina C.
AGRADECIMIENTOS
Agradecemos la financiación al Ministerio de Educación y Ciencia (AGL2009-13487-C04-02).
BIBLIOGRAFÍA
Burini, G., et al. (2007). J. Chromatogr. 1154: 97-102.
Campos, F. M., et al. (2009) Quim. Nova 32: 87-91.
Frenich, A. G., et al. (2005). J. Agric. Food Chem. 53: 7371-7376.
Miller, J., (2002). Estadística y Quimiometría para Química Analítica. p 296.
Pallauf, K., et al. (2012). J. Food Compos. Anal. 21: 273-281.
312
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SIV-CP-19
Occurrence of rhizobacteria with PGPR activities in different
ecosystems and agro-ecosystems of northern, central and southern
Chile.
Barra, P., Jorquera, M., Acuña, J. *, Lagos, L., Marileo, L., Inostroza, N., Mora, M.L.
*
[email protected]
ABSTRACT
The occurrence of rhizobacteria with PGPR activities in different ecosystems and agroecosystems of northern, central and southern Chile was studied. Around 200 isolates
showed the ability to growth in culture media supplemented with phytate and 1aminocyclopropane-1-carboxylate (ACC) as sole source of phosphorus and carbonnitrogen, respectively. Genes involved in phytate (β-propeller phytase gene) and ACC
(acdS gene) degradation were detected by PCR in isolates from both ecosystems and
agro-ecosystems. Indole acetic acid (IAA) production (9-67 μg mL-1) was also observed
in selected isolates.
INTRODUCTION
Soil bacteria play an essential role in nutrient cycling and plant productivity. In
terrestrial ecosystems, bacterial communities of the plant rhizosphere carry out
functions that are essential to plant growth. Thus, plant-growth promoting rhizobacteria
(PGPR) have been isolated from agro-ecosystems and commonly proposed as
inoculants to contribute to nutrition (e.g. nitrogen and phosphorus) and stress tolerance
of plants. However, all of our knowledge of rhizosphere bacterial communities for
different plants so far has come from studies on agricultural crops and forest tree
species, and almost nothing is known yet about microbial communities in soil under
natural vegetation. In comparison to managed ecosystems, the long term selection and
co-evolution of microbial communities under natural vegetation provides a unique
opportunity to examine how certain rhizobacteria may help plants acquire nutrients and
survive in poor soils. Under this scenario, molecular tools, such polymerase chain
reaction (PCR), now offer great promise for exploring the genetic diversity of
rhizobacteria and understanding how bacterial communities function to support plant
growth and tolerance to environmental stress. The main goal of this study was to
explore the occurrence of rhizobacteria with PGPR activities in different ecosystems
and agro-ecosystems of northern, central and southern Chile. The PGPR activities and
related genes evaluated were: phytate-degrading activity (β-propeller phytase [BPP]
gene); 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (acdS gene) and
indole acetic acid (IAA) production.
Rhizosphere samples were collected from plants grown in different ecosystems
(Atacama Desert [22°S; 68°W], Conguillio National Park [38°S; 71°W], Patagonia
[53°S; 70°W]) and agro-ecosystems (avocado orchards [32°S; 70°W], cereal crops and
pastures [38°S; 72°W]) from northern, central and southern Chile. Rhizosphere samples
were enriched in diverse minimal culture media supplemented with phytate (PSM
media) and ACC (DF media) as sole source of phosphorus and carbon-nitrogen,
respectively. Isolates grown in PSM and DF media were screened by PCR for putative
genes involved in phytate (BPP gene) and ACC (acdS gene) degrading activities as
described by Jorquera et al. (2012). Production of phytohormone, indole acetic acid
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(IAA), was also evaluated in 25 isolates showing PCR positive isolates for BPP and
acdS genes.
Count of culturable bacteria revealed higher bacterial loads rhizosphere soils of
Conguillio National Park, cereal crops and pastures (1-10×106 CFU g-1) compared with
Atacama Desert, avocado orchards and Patagonia (1-5×105 CFU g-1). Around 220 from
800 isolates grown in media enriched with phytate and ACC were selected for PCR
screening based on different phenotypes. Amplification of BPP and acdS genes was
observed in both ecosystems and agro-ecosystems. By using PSM media, 7-29% and 222% of isolates showed positive PCR amplification for BBP gene in ecosystems and
agro-ecosystems, respectively. In relation to acdS gene, 9-27% and 12-32% of isolates
showed positive PCR amplification in ecosystems and agro-ecosystems, respectively.
By using DF media, 0-26% and 0-18% of isolates showed positive PCR amplification
for BBP gene in ecosystems and agro-ecosystems, respectively. Respect to acdS gene,
21-29% and 6-28% of isolates showed positive PCR amplification in ecosystems and
agro-ecosystems, respectively. Our results also showed higher IAA productions in
isolates from agro-ecosystems (23-67 μg mL-1) compared with ecosystems (9-35 μg mL1
). This study revealed the occurrence of rhizobacteria with PGR activities in diverse
managed and undisturbed ecosystems. Rhizobacteria with PGR activities obtained from
natural vegetation may be taken in account to help plants to acquire nutrients in agroecosystems with lower rates of fertilization and improve the tolerance of plants to
stressful conditions (e.g. drought, salinity or frost).
ACKNOWLEGMENTS
This work was supported by the CONICYT Doctoral Scholarships 21110473 and FONDECYT Project
no. 1120505.
REFERENCES
Jorquera, M.A., et al. (2012). Microbial Ecol. 64: 1008-1017.
314
Session IV
SIV-CP-20
Efecto de plaguicidas sobre la actividad biológica de la cubierta vegetal
(L. perenne) de un sistema de biopurificación denominado lecho
biológico.
Diez, M.C.1*, Gallardo, F.2
1
Departamento. de Ingeniería Química, Universidad de La Frontera, Temuco, Chile. 2 Departamento de
Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Temuco, Chile.
*
[email protected], [email protected]
RESUMEN
Se evaluó el efecto de repetidas aplicaciones de una mezcla de plaguicidas (atrazina,
clorpirifos e iprodiona) sobre la actividad biológica en la cubierta vegetal de L. perenne
de un sistema de biopurificación denominado lecho biológico. En general, las
actividades biológicas en la biomezcla no variaron significativamente entre el control y
los tratamientos en relación a la concentración de plaguicida adicionado. El ácido
succínico se encontró en los exudados radicales en la mayor concentración, mientras
que el menor encontrado fue el ácido oxálico.
INTRODUCCIÓN
El sistema de biopurificación denominado lecho biológico está diseñado para retener y
degradar plaguicidas en elevadas concentraciones. La adsorción y degradación suceden
en una biomezcla conformada por suelo, paja y turba en proporción volumétrica de
1:2:1. El suelo aporta microorganismos y capacidad de adsorción, la paja propicia el
crecimiento de hongos con actividad ligninolítica y la turba adsorbe contaminantes y
mantiene la humedad necesaria para la actividad biológica en la biomezcla. Sobre esta
biomezcla, se instala una cubierta vegetal de césped que propicia una rizósfera que
colabora con la degradación de los contaminantes a través de los exudados radicales y
por la absorción de contaminantes; además de favorecer la evapotranspiración en el
sistema de biopurificación (Castillo et al., 2008).
MATERIAL Y MÉTODOS
Para evaluar el efecto de los plaguicidas sobre la actividad biológica de la cubierta
vegetal de L. perenne (ballica) de un lecho biológico, se instaló un ensayo en modalidad
destructiva durante 90 días. Se utilizaron vasos de plumavit (300 ml) con 100 g de
biomezcla c/u y cuya característica se presenta en la Tabla 1. Se germinaron las semillas
por dos semanas bajo cámara de crecimiento con temperatura de 20 ±1 ºC y un foto
período de 17 h/día. Luego se raleó dejando 6 plántulas con características fenotípicas
semejantes por vaso. Luego al día 15 se contaminan los vasos con 4 niveles de una
mezcla de atrazina (ATZ), clorpirifos (CHL) e iprodiona (IPR) (0, 1, 5, 10 mg/l)
dejando 3 vasos sin contaminar (blancos) para cada tiempo de muestreo. Al día 16 se
tomaran 12 vasos de los cuales 3 son blanco (vasos sin contaminar) y 9 contaminados
con la respectiva concentración de los plaguicidas y se realizan los análisis
correspondientes. Se re-contaminan luego cada 15 días y se realiza toma de muestra
antes y después de cada contaminación. Antes y después de cada aplicación, se realizan
los análisis de fluoresceína diacetato (FDA), ureasa, peroxidasa, fosfatasa ácida,
plaguicida residual y ácidos orgánicos exudados.
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Tabla 1. Características fisicoquímicas de la biomezcla preparada con suelo trumao (Andisol):
Biomezcla
pH
CO
(%)
NTK
(%)
C/N
4,76
19,6
0,60
32,7
CO: carbono orgánico; NTK: nitrógeno total Kjeldahl
La actividad ureasa no presentó diferencias significativas entre el control y las dosis de
plaguicidas adicionados. Luego de la primera adición de los plaguicidas, la actividad
ureasa disminuyó drásticamente, incrementando luego su actividad a partir del día 31 y
hasta el día 59, luego de lo cual disminuyó nuevamente en todos los tratamientos
utilizados. La actividad fosfatasa ácida no presentó diferencias significativas entre el
control y las dosis de plaguicidas adicionados. La fosfatasa duplicó su actividad entre la
primera y segunda contaminación, manteniéndose prácticamente sin modificar hasta el
final del ensayo con valores entre 15 y 20 µg PNP/g, a pesar de las sucesivas
contaminaciones con los plaguicidas. Respecto de la actividad de la FDA, ésta presentó
un comportamiento oscilatorio en el tiempo, sin mostrar una directa con la
concentración ni con la degradación de los plaguicidas y similar al observado en
estudios anteriores (Tortella et al., 2012) lo cual puede deberse a que la FDA incluye un
grupo de enzimas hidrolíticas las cuales son producidas por varios tipos de
microorganismos. La actividad peroxidasa se mantiene similar hasta el día 44 del
ensayo en valores de 0,4 a 0,6 U/g y si mostrar diferencias significativas entre los
tratamientos luego de lo cual incrementa hasta valores de 1 a 1,2 U/g, debido a la mayor
actividad de los hongos que se desarrollan favorecidos por la presencia de la paja.
El ácido orgánico que se encontró en mayor concentración en la biomezcla corresponde
a ácido succínico (5,689 mg/l), seguido de ácido cítrico (1,624 mg/l). Los ácidos málico
y oxálico se encontraron en menor concentración (0,536 y 0,197, respectivamente).
A modo general podemos señalar que los pesticidas extraídos en los exudados radicales
incrementan durante el tiempo debido a las re-contaminaciones a las cuales fueron
siendo sometidas las plantas y a la acumulación de pesticidas en la raíz de las plantas.
En general, la iprodiona en las 3 concentraciones adicionadas (1, 5 y 10 mg/l) se
presenta en mayor concentración en los exudados indicando su menor degradación.
Estos resultados son concordantes con los resultados obtenidos de la degradación de
iprodiona en la biomezcla sin la cubierta vegetal (Diez et al., 2013). Por otro lado,
atrazina y clorpirifos presentan un nivel elevado de remoción.
AGRADECIMIENTOS
Este trabajo ha sido financiado por el proyecto Fondecyt 1120963.
BIBLIOGRAFÍA
Castillo M.d.P. et al. (2008). J. Agric. Food Chem. 56: 6206-6219.
Diez, M.C. et al. (2013). J. Biobased Mat. and Bioenergy (en prensa).
Tortella, G.R. et al. (2012). Biodegradation DOI 10.1007/s10532-013-9619-4.
316
Session V
Phisiology and biochemistry of beneficial
microorganisms and associated plants
Session V
SV-P-1
Fijación de nitrógeno en leguminosas en entornos variables: el
bacteroide, el nódulo y la planta.
Arrese Igor, C.*
* [email protected]
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leguminosarum.
Palacios, J.M.1*, Rubio-Sanz, L.2, Prieto, R.I.1, Menéndez-Cerón, M.1, Albareda, M.1,
Clavijo, C.1, Imperial, J.1, 3, Mandrand-Berthelot, M.A.4, Rodrigue, A.4, Cacho, C.5, RuizArgüeso, T.1, Brito, B.1
1
CBGP and Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, Universidad Politécnica de
Madrid, Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain. 2 Departamento de
Química y Análisis Agrícola, E.U. Ingeniería Técnica Agrícola, Ciudad Universitaria s/n 28040. 3 C.S.I.C.
Madrid. 4 Université Lyon-INSA-CNRS UMR5240, Lyon, France. 5 IHCP JCR, Ispra, Italy.
*
[email protected]
INTRODUCTION
Transition metals such as Fe, Cu, Mn, Ni, or Co are essential nutrients, as they are
constitutive elements of a significant fraction of cell proteins. Such metals are present in
the active site of many enzymes, and also participate as structural elements in different
proteins. From a chemical point of view, metals have a defined order of affinity for
binding, designated as the Irving-Williams series (Irving and Williams, 1948)
Mg2+ < Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+>Zn2+
Since cells contain a high number of different proteins harbouring different metal ions, a
simplistic model in which proteins are synthesized and metals imported into a
“cytoplasmic soup” cannot explain the final product that we find in the cell. Instead we
need to envisage a complex model in which specific ligands are present in definite
amounts to leave the right amounts of available metals and protein binding sites, so
specific pairs can bind appropriately. A critical control on the amount of ligands and
metal present is exerted through specific metal-responsive regulators able to induce the
synthesis of the right amount of ligands (essentially metal binding proteins), import and
efflux proteins. These systems are adapted to establish the metal-protein equilibria
compatible with the formation of the right metalloprotein complexes. Understanding
this complex network of interactions is central to the understanding of metal
metabolism for the synthesis of metalloenzymes, a key topic in the Rhizobium-legume
symbiosis. In the case of the Rhizobium leguminosarum bv viciae (Rlv) UPM791 Pisum sativum symbiotic system, the concentration of nickel in the plant nutrient
solution is a limiting factor for hydrogenase expression, and provision of high amounts
of this element to the plant nutrient solution is required to ensure optimal levels of
enzyme synthesis (Brito et al., 1994).
Usually, transition metals are present at low levels in natural habitats, and specific, high
affinity transport systems have been developed by bacteria to import them from the soil
solution. At the same time, these elements are toxic even at moderate concentrations,
since they can catalyze the production of free radicals, or compete with other metals for
the active site of metalloenzymes. In this talk we will present our recent work on the
mechanisms involved in the homeostasis of transition metals nickel and cobalt in free
living and symbiotic cells of the endosymbiotic bacterium Rhizobium leguminosarum.
319
Session V
SV-P-2
Bacteroid
Ni
HupE
Ni-Malate
Ni
Ni
HupE2
Ni
Ni-Citrate
Ni
?
NiFe Hydrogenase
Ni
Ni
Ni-X
Ni
Ni
dmeRF
?
?
Ni
Ni
Ni
Figure 1. Nickel uptake and efflux systems involved in nickel
homeostasis in the Rhizobium leguminosarum-Pisum sativum symbiosis.
Nickel complexes in the nodule cytosol.
A first stage to be considered in the supply of metals to the bacteroid in legume nodules
is the chemical species present in the nodule cytosol surrounding the symbiosome.
Analysis of the chemical nature of nickel complexes has been made possible through
the development of a new analytical methodology for the direct identification and
quantification of nickel complexes in extracts from nodule cytosol (Cacho et al., 2010),
a useful tool to study the mechanism involved in the nickel provision for endosymbiotic
bacterial cells. The use of this methodology has revealed that nickel species vary in
different legume plants. Pea nodules contain similar levels of nickel malate and nickel
citrate, whereas most nickel is chelated with citrate in lentil nodules. Other legumes
such as bean or soybean contain different balances of nickel chelates.
The binding affinity of the metal ions for the different organic ligands might influence
the capacity of the metal to be detached from the chelate, or to be transported as a
chelate across the different membranes. Nickel complexes found in plant cytoplasm has
been used as substrates for experiments of hydrogenase induction of wild-type and
hupE/hupE2 mutants to ascertain the actual role of such compounds in the provision of
this metal for metalloenzyme synthesis under controlled conditions. At this point,
further characterization of potential metal- or chelate- transporters through the plantderived peribacteroid membrane is required to ascertain the actual roles of metal
complexes in the provision of nickel to the bacteroid.
Nickel transport through bacterial outer membrane.
Once inside the symbiosome, and before crossing the cytoplasmic membrane, metals
must cross the bacterial outer membrane (OM). Whereas small molecules (<600 Da)
can cross this membrane by passive diffusion through porins when they are moderately
concentrated in the environment, larger molecules and molecules present at low
concentration require energy-dependent systems. The OM does not maintain a proton
gradient nor ATP synthesis, and these energy requirements can be fulfilled by TonBdependent transport systems, in which a protein complex (TonB-ExbB-ExbD) spanning
the periplasmic space is able to transduce proton-motive-force from cytoplasmic
membrane to power up transport in the outer membrane (Postle and Larsen, 2007).
Although most TonB-dependent transporter (TBDT)-like proteins are predicted to
transport iron compounds (siderophores or heme) or cobalamine, recent reports indicate
that this kind of receptors are also involved in the uptake of other metals, such as nickel.
In the case of endosymbiotic bacteria, a TonB-dependent transporter (TBDT) involved
in nickel uptake has been found adjacent to hydrogenase genes in the genome of B.
320
Session V
SV-P-2
japonicum (Schauer et al., 2008). This gene has been cloned and transferred into R.
leguminosarum, and its role on the import of nickel ions for hydrogenase synthesis in
this bacterial species is being analyzed.
Nickel uptake through cytoplasmic membrane.
Once in the periplasm, metal ions must cross the cytoplasmic membrane, the main
permeability barrier for metal uptake. Two proteins (HupE and HupE2) of the
HupE/UreJ family are the main nickel transporters through cytoplasmic membrane in
Rlv UPM791 (Brito et al., 2010). These transporters are integral membrane proteins
with six predicted transmembrane domains that work as single-component permeases.
Mutant analysis demonstrated the essentiality of these proteins for the synthesis of
active R. leguminosarum NiFe hydrogenase under nickel limitation both in free-living
culture and in symbiosis (Brito et al. 2010). Hydrogenase activity determinations and
Ni63 transport assays demonstrated that HupE is a nickel transporter that provides this
metal for the synthesis of hydrogenase in Rlv UPM791 free-living cells and lentil
bacteroids. Site directed mutagenesis has allowed the identification of two histidine-rich
domains essential for Ni2+ transport. The characterization of kinetic and functional
properties of Rlv UPM791 HupE is being carried out through heterologous expression
of HupE in a nik-deficient Escherichia coli background. The specificity of this
transporter for Ni2+ is being determined in competition assays with other divalent metal
ions. A HupE variant lacking the N-terminal periplasmic region of the mature protein
has been constructed to ascertain the potential role of this domain in ion scavenging and
concentration around the transporter. The relevance of other residues conserved in
members of the HupE/UreJ family is being tested through the analysis of the
corresponding site-specific mutants. The results of the on-going work will be presented
and discussed.
Figure 2. Topological model proposed for
the R. leguminosarum nickel transporter
HupE. Histidine residues are highlighted,
and histidine-rich motifs are indicated on
the top (adapted from Brito et al., 2010).
Metal efflux systems.
The commonest mechanism for metal resistance in living cells is metal efflux. Efflux
systems described in different bacteria include members of the Resistance-Nodulationcell Division (RND) protein family, P-type ATPases, cation diffussion facilitators, and
other resistance factors (Nies, 2003). In order to have an idea of the metal resistance
systems present in endosymbiotic bacteria we have carried out g enomic and
physiological analyses of R. leguminosarum strains isolated from ultramafic soils.
321
Session V
SV-P-2
Transposon mutagenesis of the highly resistant R. leguminosarum strain UPM1137 has
led to the identification of potential metal efflux determinants conferring resistance to
high metal levels under free-living and symbiotic conditions. A metal-inducible
resistance mechanism involving a member of the cation diffusion facilitator (CDF)
family has been studied in detail. Phenotypic characterization of a deletion mutant has
shown that this protein acts as a key component for cobalt detoxification in R.
leguminosarum. The gene encoding this CDF forms an operon with a nickel-and cobaltresponsive metalloregulator of the RcnR-type. Highly similar operons have been
identified in other endosymbiotic bacteria, suggesting that metal-inducible, RcnRregulated CDF systems constitute a widespread strategy for transition metal
detoxification in this group of bacteria. The characterization of such resistance systems
will allow a better understanding on how endosymbiotic bacteria regulate intracellular
metal levels, a central aspect of metal homeostasis in this relevant group of bacteria.
ACKNOWLEDGEMENTS
This work has been funded by Spain’s MICINN (project BIO2010-15301 to J.P.), Comunidad Autónoma
de Madrid (Microambiente-CM S2009/AMB-1511 to T.R.A), Universidad Politécnica de Madrid (AL11P(I+D)-4 to B.B.), and by France’s PICS project (SYMBIONI n°5992 to A.R. and MA. M-B.).
REFERENCES
Brito, B., et al. (1994). J. Bacteriol. 176: 5297-5303.
Brito, B., et al. (2010). J. Bacteriol. 192: 925-935.
Cacho, C., et al. (2010). Talanta 83: 78-83.
Irving, H., and Williams, R.J.P. (1948). Nature 162: 746-747.
Nies, D.H. (2003). FEMS Microbiol. Rev. 27: 313-339.
Postle, K., and Larsen, R.A. (2007). Biometals 20:453-465.
Schauer, K., et al. (2008). Trends Biochem. Sci. 33: 330-338.
322
Session V
SV-CO-1
Engineering a hydrogen biosensor in the nitrogen-fixing strain
Rhodobacter capsulatus.
Rubio, L.M.
*
[email protected]
ABSTRACT
R.capsulatus has a system to regulate the expression of its uptake hydrogenase in
response to H2. We are studying this system and using some of its elements to generate
a H2 biosensor: a R. capsulatus strain able to detect presence of H2 yielding a response
of our choice.
INTRODUCTION
Numerous microorganisms can produce H2 by reactions related to their metabolism and
most of them are also able to consume it. Hydrogenase enzymes (metalloproteins that
contain iron-sulfur clusters and Fe-Ni or Fe-Fe binuclear active sites) catalyze the
reaction: 2H+ + 2e- ↔ H2. [NiFe]-H2ases have been thoroughly studied in Bacteria and,
in some cases, their expression is regulated by the so-called soluble sensor
hydrogenases. In addition, nitrogenase enzymes produce H 2 when reducing N2 into
NH3. The nitrogen-fixing bacterium R. capsulatus carries nitrogenase and hydrogenase
enzymes able to produce H2. It also carries a system to detect H2 that is composed of
three proteins: a H2-sensor hydrogenase (HupUV), a histidine kinase (HupT) and a
response regulator (NtrC-like transcription factor, HupR) (Vignais et al., 2005). In the
presence of H2, this sensor triggers expression of hydrogenase structural and
biosynthetic genes. The aim of this work is to construct a tool relying on this complex
regulatory signal cascade that will transduce H2 levels into a signal of our choice. A
reporter gene has been introduced in a wild-type strain of R. capsulatus, so that it is
expressed in presence of H2. This biosensor will allow us to screen or select for
randomly generated nitrogenase mutants with increased H2-producing activity.
The strain used in this study is R. capsulatus SB1003. This strain was cultured under
aerobic (synthetic air) or anaerobic conditions (N 2) and in the presence or absence of
6.5% H2 in the gas phase. Chromosomal mutations were generated by simple or double
recombination using pVIK112 (Kalogeraki and Winans, 1997) or pK18mobsac
plasmids (Kirchner and Tauch, 2003) -galactosidase activity assays were carried out
as described by Miller (1972). RNA from cultures of exponentially growing cells (OD
of 0.8) was extracted by using TRizol and chloroform. cDNA was obtained using High
Capacity cDNA Reverse Transcription kit using above RNA as template (Applied
Biosystem). Expression analyses were performed on cDNA samples by RT-qPCR.
RESULTS AND DICUSSION
Detection and analysis of hupS promoter.
The expression of structural genes for the uptake hydrogenase is activated when the
phosphorylated form of the transcriptional regulator HupR binds to the promoter of
hupS (small subunit of uptake hydrogenase). A putative promoter sequence upstream of
hupS was cloned into the lacZ-containing vector pMP220 (Spaink et al., 1987), and this
construct was introduced into R. capsulatus to generate R. capsulatus pMP220::PhupS
323
Session V
SV-CO-1
strain. Both strains, R. capsulatus pMP220 (wild type) and R.
capsulatuspMP220::PhupS were cultured in PY medium under aerobic conditions and
in absence or the presence of 6.5 % H2. In absence of externally-added H2, βgalactosidase activity was higher in R. capsulatus pMP220::PhupS than in wild type
(800 vs. 17.5 Miller units, respectively). Furthermore, in presence of 6.5 % H 2 strain
pMP220::PhupS exhibited 1450 Miller units compared to 56 units in the wild-type
strain. This result shows that the PhupS region is indeed a promoter and that it is
activated by H2.
Constructing and testing the “hydrogen sensor”.
The regulatory effect of H2 in the expression of hydrogenase structural genes was
analyzed by RT-qPCR. R. capsulatus was cultured in the absence or the presence of 6.5
% H2. Total RNA was extracted, and hupR and hupS expression was analyzed by RTqPCR. hupR and hupS expression was ca. 10 times higher in the presence of H 2 than in
its absence.
The lacZ expressing H2 sensor was constructed into the chromosome of R. capsulatus.
A recombinant suicide plasmid, fusing PhupS to the lacZ reporter
(pVIK112::PhupS::lacZ), was constructed and introduced into R. capsulatus wild-type
strain by biparental matting to generate strain PhupS::lacZ, which contains the lacZ
reporter gen inserted into the chromosome. R. capsulatus wild-type and PhupS::lacZ
strains were cultured under the same conditions for 7 hours and their β-galactosidase
activities determined. In the PhupS::lacZ strain activities were found to be higher in the
presence of H2, confirming functionality of our H2 sensor tool in a stable chromosomal
location.
The H2 sensor strain will be used to screen or select clones expressing randomly
generated nitrogenase variants able to produce high levels of H 2 into the cell (see
Barahona E. et al. communication: Towards the optimization of hydrogen production by
nitrogenase: In vitro directed evolution of Rhodobacter capsulatus molybdenum
nitrogenase).
REFERENCES
Adams, M.W.W., et al. (1981). Biochim. Biophys. Acta. 594:105-176.
Vignais, P.M., et al. (2005). Biochem. Soc. Trans. 33: 28-32
Kalogeraki, V.S., and Winans, S.C. (1997). Gene. 188: 69-75.
Kirchner, O., and Tauch, A. (2003). J. Biotechnol. 104: 287-299
Miller, J.H. (1972). Cold Spring Harbor, N.Y.
Spaink, H.P., et al. (1987). Plant Mol. Biol. 9: 27-39.
324
Session V
SV-CO-2
MtNramp1 mediates iron import in rhizobia-infected Medicago
truncatula cells.
Rodríguez-Haas, B.1, Finney, L.2, Kryvoruchko, I.3, González-Melendi, P.1, Udvardi,
M.3, Imperial, J.1, 4, González-Guerrero, M.1*
1
Centro de Biotecnología y Genómica de Plantas (CBGP) Universidad Politécnica de Madrid. Campus de
Montegancedo, Pozuelo de Alarcón, 28223 Madrid, España. 2 Biosciences Division. Advanced Photon
Source, Argonne National Laboratory Argonne, Illinois, Estados Unidos de América. 3 The Samuel
Roberts Noble Foundation, Plant Biology Division, Ardmore, Oklahoma, 73401, USA. 4 Consejo
Superior de Investigaciones Científicas (CSIC). Madrid, España.
* [email protected]
ABSTRACT
Symbiotic nitrogen fixation is a process that requires relatively high quantities of iron
provided by the host legume. Using synchrotron-based X-ray fluorescence, we have
determined that this iron is released from the vasculature into the apoplast of zone II of
M. truncatula nodules. This overlaps with the distribution of MtNramp1, a plasma
membrane iron importer. The importance of MtNramp1 in iron transport for nitrogen
fixation is indicated by the 60% reduction of nitrogenase activity observed in knockdown lines, most likely due to deficient incorporation of this essential metal cofactor at
the necessary levels.
INTRODUCTION
Iron is an essential growth-limiting micronutrient for plants (Grotz and Guerinot, 2002).
Most of the proteins (nitrogenase, leghemoglobin, etc…) involved in symbiotic nitrogen
fixation require iron cofactors (Udvardi and Day, 1997). In spite of this very little is
known about how iron reaches the nodule and how it is incorporated by rhizobiacontaining cells. Here we show that iron is released in the infection/maturation areas of
indeterminate nodules, and that a Nramp transporter introduces it into the infected cells.
Synchrotron-based X-ray fluorescence
X-ray fluorescence assays were performed as described by Rodríguez-Haas et al (2013).
Plant transformation.
M. truncatula plants were transformed with Agrobacterium rhizogenes as indicated
(Boisson-Dernier et al., 2001).
MtNramp1 localization.
Promoter:GUS and confocal immunofluorescence studies were performed as described
(Limpens et al., 2009).
Generation of knock-down plants
The first 450 bp of MtNramp1 cDNA sequence were cloned in pENTR1A and
transferred using Gateway Technology to pFRN.
Nitrogenase activity measurements.
Nitrogenase activity was measured as described (Ruíz-Argüeso et al., 1979).
There are three possible mechanisms for iron transfer to the nodule: i) increased uptake
from the epidermis, ii) use of accumulated stores from the nodule primordia, and iii)
long-distance transport from the vasculature. These possibilities would present different
iron distribution patterns, and these could be determined by means of synchrotron-based
325
Session V
SV-CO-2
X-ray fluorescence. The data showed that iron is transported by the vasculature and
accumulates in the apoplast of the zone II of the nodule (Figure 1A). This also indicates
that plasma membrane iron importers must be present in these cells to introduce
apoplastic iron into the cytosol.
In a previous microarray analysis we identified MtNramp1 as a metal transporter gene
induced more than 7-fold by nodulation. This result was verified by qPCR. MtNramp1
was able to complement the phenotype of the fet3/fet4 yeast mutant that has a reduced
capability of incorporating iron from the medium. Promoter:GUS studies of MtNramp
indicated that it is expressed in the apical region of the nodule, coincidental with zone
II, where iron is being released into the apoplast. This was verified with
immunolocalization of HA-tagged MtNramp1 using confocal microscopy (Figure 1B).
It appeared that MtNramp1 is localized in the plasma membrane of cells of nodule zone
II. All these results suggest that MtNramp1 could be the transporter that introduces
apoplastic iron into the infected cells that would be used for nitrogen fixation. This was
tested by obtaining knock-down lines and determining the nitrogenase activity of their
nodules, since nitrogenase is one of the most abundant iron proteins in the nodule. On
average, a 90 % reduction of expression levels was achieved that caused a 60 %
reduction of nitrogenase activity (Figure 1C), supporting a role of MtNramp1 in
symbiotic iron transport.
B
Zone III
C 100
% Nitrogenase activity
A
Zone II
Zone I
80
60
40
20
WT
Knock-down
Figure 1. Iron transport in M. truncatula nodules. (A) Elemental distribution in Zone II. Iron is in green,
zinc in blue and calcium in red. (B) Immunolocalization of MtNramp1. Blue is DAPI stained DNA, green
indicates GFP expressing rhizobia and red is MtNramp1-HA labelled with anti-HA mouse antibody
conjugated to Alexa594. (C) Nitrogenase activity in wild type and MtNramp1 knock-down nodules.
ACKNOWLEDGMENTS
This work was supported by the Ramón y Cajal Fellowship RYC-2010-06363, and the Marie Curie
International Reintegration Grant MENOMED (all to MGG). Use of the Advanced Photon Source was
supported by the U.S. Department of Energy under contract number DE-AC02-06CH11357.
REFERENCES
Grotz, N., and Guerinot, M.L. (2002). Curr. Opin. Plant Biol. 5: 158-163.
Udvardi, M.K., and Day, D.A. (1997). Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 493-523.
Rodríguez-Haas, B., et al. (2013). Metallomics doi 10.1039/c3mt00060e
Boisson-Dernier, A., et al. (2001). Mol. Plant Microbe Interact. 14: 695-700.
Limpens, E., et al. (2009). Plant Cell 21: 2811-2828.
Ruíz-Argüeso, T., et al. (1979). Biochem. Biophys. Res. Comm. 86: 259-264.
326
Session V
SV-CO-3
Selección de líneas de garbanzo tolerantes a la salinidad empleando
caracteres de producción de biomasa y funcionamiento nodular como
indicadores.
Gómez, L.A.1*, Vadez, V.2, Vaillhe, H.3, Pernot, C.3, Drevon, J.J.3
1
Instituto de Suelos. Autopista, Costa-Costa y Antigua Carretera de Vento, Capdevila, Boyeros. La
Habana, Cuba. 2 Instituto Internacional de Investigaciones de Cultivo para el Trópico Semiárido.
Patancheru, Hyderabad 502 324, Andhra Pradesh, India. 3 Instituto Nacional de Investigaciones
Agronómicas, INRA, Place Viala F-34060, Montpellier, France.
*
[email protected]
RESUMEN
Se estudió la tolerancia a la salinidad de 30 líneas cultivadas en solución nutritiva, evaluando
nodulación, conductancia nodular al O2 y biomasa. En condiciones no salinas las plantas
mostraron alta nodulación y crecimiento, mientras que la salinidad (25 mM NaCl), disminuyó
significativamente estos parámetros. La sensibilidad a la sal determinada como crecimiento
foliar, y nodular relativo y eficiencia de uso de la simbiosis con Mesorhizobium mostró
importantes diferencias entre líneas, las que se agruparon en 4 clases. Se concluyó que la
tolerancia a la salinidad está relacionada con alto potencial simbiótico y elevada y estable
conductancia nodular al O2.
INTRODUCCIÓN.
La salinidad afecta cerca de 80 millones de ha de suelos cultivable en todo el mundo un factor
que ejerce un impacto negativo sobre la fijación simbiótica del nitrógeno (FSN) y el
rendimiento de grano de plantas de garbanzo (Cicer arietinum L.) en interacción con
Mesorhizobium, dado la particular sensibilidad de esta simbiosis a la salinidad (Flowers et al.,
2010). Una alternativa para resolver este problema pudiera ser la selección de líneas resistentes.
El presente trabajo evalúa la tolerancia a la salinidad de 30 líneas inoculadas con la cepa de
Mesorhizobium ciceri UPMCa 7.
MATERIAL Y MÉTODOS
Se evaluaron 30 líneas (ICCV2; ICCV10; ICC67; ICC867; ICC1431; ICC1915; ICC2580;
ICC3946; ICC4495; ICC4593; ICC5003; ICC5337; ICC6263; ICC6306; ICC8058; ICC8522;
ICC8950; ICC9942; ICC10885; ICC11121; ICC12155; ICC13357; ICC15518; ICC15610;
ICC96029; JG11; CSG8962; ICC4973 (L550); INRAT 93.1 y Amdoun, Vadez et al., 2007),
cultivadas en solución nutritiva (Drevon et al., 1988), a dos niveles de salinidad (0 y 25 mM
NaCl). Se evaluó la conductancia nodular al O2 (CNO), biomasa foliar (BF), número de nódulos
(NN), peso individual de los nódulos (PIN), biomasa de nódulos (BN), relaciones entre órganos,
Crecimiento Relativo foliar y nodular relativo y Eficiencia en la Utilización de la Simbiosis con
Mesorhizobium (EUSM), según L’Taief et al., 2007. Los datos se procesaron para componentes
principales (ACP) y dendograma (XLSTAT, Addinsoft SARL, Paris, Francia, 2010).
En condiciones no salinas las plantas exhibieron alta nodulación y crecimiento (Tabla
1), mientras bajo salinidad se afecto la BN (11 – 89 %); el NN (12 – 83 %), y la BF (2589 %), las raíces se afectaron solo en el 50 % de las líneas. El ACP en el tratamiento no
salino evidencio que F1, F2 y F3 fueron vectores de “distribución de biomasa”,
“nodulación y crecimiento” y “crecimiento individual de los nódulos” respectivamente,
en cambio en condiciones salinas F1, F2 y F3 fueron considerados vectores de “EUSM”,
“nodulación” y “crecimiento Foliar y Nodular relativo” respectivamente.
Los índices CFR, CNR y EUSM variaron considerablemente entre genotipos los que se
agruparon en 4 clases y 8 sub-clases de acuerdo a su tolerancia a la salinidad (Tabla 1).
327
Session V
SV-CO-3
A 0 mM NaCl las líneas representativas de las clases 1, 2, 3 y 4 fueron ICC4593,
ICC13352, ICC10885 e ICC8522 respectivamente, las clases 3 y 4 agruparon las líneas
con más alta nodulación y crecimiento (Tabla 1). En condiciones salinas las líneas
representativas de las clases/ sub-clases 1A, 1B, 1C, 2A, 2B, 2C, 3 y 4 fueron CSG
8962, ICC6306, ICC12155, ICC15610, ICC3946, INRAT 93-1, ICC8950 y Amdoun
respectivamente, la clase 3 agrupó las líneas más tolerantes (Tabla 1).
Las líneas ICC8950, ICC1431, ICC4495, Amdoun, ICC4973 e ICC5337 fueron
consideradas tolerantes, mientras ICCV10, ICC13357 e ICC1915 sensibles.
Tabla 1. Clases/sub-clases resultado del análisis de dendograma para disimilitudes fenotípicas
líneas cultivadas a 0 and 25 mM de NaCl.
Clases
BF
BN
NN
PIN
BR
CFR CNR
/sub-classes
g planta-1 g planta-1 Numero x planta-1 mg nodulo-1 g planta-1
0 mM
1
3.60
0.355
212
1.86
0.46
2
3.20
0.606
458
1.44
0.51
3
5.85
0.529
646
0.82
0.81
4
7.37
0.785
984
0.79
0.91
25 mM NaCl
1A
0.62
0.058
56
1.04
0.14
30.1 39.7
1B
1.55
0.186
132
1.51
0.34
36.4 43.6
1C
1.24
0.120
108
1.38
0.20
92.4 48.0
2A
2.11
0.239
212
1.21
0.45
59.6 72.7
2B
2.62
0.210
225
0.93
0.52
89.4 34.4
2C
1.81
0.154
196
0.85
0.36
37.0 26.4
3
2.03
0.201
138
1.50
0.27
91.9 122.0
4
3.36
0.366
322
1.17
0.56
54.9 49.5
en 30
EUSM
3.12
5.74
6.7
7.1
11.4
8.7
8.3
8.3
La pO2 crítica asociada al metabolismo nodular mostró en el tratamiento no salino un
valor medio de 40 % y no varió por influencia de la sal. La pendiente de la relación
consumo de O2 y pO2 multiplicado por el área nodular (cm2 g-1 nódulo) que representa
la conductancia nodular al O2 (CN0) incremento significativamente (p<0.001) solo en
las líneas sensibles, por lo tanto el incremento en la CNO pudiera ser un mecanismo de
adaptación en respuesta a la demanda de N de la planta y en compensación a la
disminución de la nodulación por estrés salino, pero también por el alto costo de la
respiración de la FSN (Jebara y Drevon, 2001). Se concluyó que la tolerancia del
garbanzo a la salinidad está relacionada en parte a la capacidad de mantener alto
potencial simbiótico bajo condiciones salinas, el cultivar ideal parece ser el que exhibe
un gran número de pequeños nódulos y una elevada y estable CNO.
BIBLIOGRAFÍA
Drevon, J.J., et al. (1988). Plant Physiol. Bioch. 26: 73-78.
Flowers, T.J., et al. (2010). Plant. Cell. Environ. 33: 490-509.
L´Taief, B., et al. (2007). J. Plant Physiol. 164: 1028-1036.
Vadez, V., et al. (2007). Field. Crop. Res. 104: 123-129.
328
Session V
SV-CO-4
Estrés abitotico induce cambios en las señales de comunicación y en la
interacción temprana entre maní y rizobacterias.
Departamento de Biología Molecular, Facultad de Ciencias Exactas, Físico-Químicas y Naturales,
Universidad Nacional de Río Cuarto, Argentina.
*
[email protected]
ABSTRACT
En este trabajo mostramos como el estrés hídrico (PEG6000-0,28MPa) es capaz de
inducir cambios en la composición de lípidos de rizobacterias y en rizodeposiciones de
maní, importantes para la señalización rizosférica, en la movilidad bacteriana y de
relevancia en los eventos tempranos de interacción.
INTRODUCCIÓN
La diversidad estructural de moléculas producidas por las leguminosas al igual que las
rizobacterias con capacidad de asociarse con ellas, es vasta. Proponemos examinar
cómo el perfil de lípidos de rizobacterias y de rizodeposiciones de maní (señal
rizosférica), así como la movilidad de rizobacterias (evento temprano de interacción)
que se asocian a maní, cambia frente estrés hídrico (PEG6000 -0,28MPa). Es relevante
el papel que proteínas, lípidos, flavonoides, ácidos orgánicos, entre otros, juegan en la
tolerancia a condiciones de estrés abiótico y cambios ambientales pueden comprometer
la eficacia del diálogo molecular entre ambos socios, alterándose consecuentemente el
desarrollo de diversos eventos como por ejemplo la movilidad, la adhesión, la
colonización y la nodulación (Dardanelli et al., 2009; Guasch-Vidal et al., 2013).
MATERIAL Y MÉTODOS
Recolección y análisis de rizodeposiciones: plantas de 7 días de crecimiento en solución
Hoagland (PEG6000 -0,28MPa) fueron cosechadas y las rizodeposiciones fueron
empleadas en los estudios (Dardanelli et al., 2008).
Composición de ácidos grasos (AG) de rizodeposiciones y de rizobacterias: el análisis
de los esteres metílico se realizó a partir de los lípidos totales, por cromatografia
gaseosa (GC) según Paulucci et al. (2013).
Identificación y cuantificación de fosfolípidos de rizobacterias: mediante TLC,
empleando Acetato de sodio [1-14C], agregado al medio de cultivo al momento de la
inoculación se realizó el estudio de estas moléculas (Paulucci et al., 2013).
Estudios de movilidad swarming y swimming de rizobacterias: fue llevado a cabo tal
como se describe en Albareda et al. (2006) y Medeot et al. (2010).
Efecto del estrés hídrico sobre lípidos de rizodeposiciones de Arachis hypogaea
Las plantas de maní fueron sometidas a un gradiente de PEG6000, determinando que el
efecto significativo ocurre a un potencial de -0,28MPa (estrés moderado). La
composición de AG de las rizodeposiciones muestra que existen cambios con
disminución de ácidos palmítico, palmitoleico y esteárico, mientras que se observó un
incremento de oleico y otras moléculas no identificadas (NI) de más de 20 carbonos.
Efecto del estrés hídrico sobre lípidos de las rizobacterias asociadas a Arachis
hypogaea
Estudios realizados con PEG6000 sobre las rizobacterias Azospirillum brasilense AZ39
y Bradyrhizobium sp. SEMIA6144 indicaron una mayor tolerancia por parte de los
329
Session V
SV-CO-4
microorganismos en comparación con el vegetal. Sin embargo dentro de las bacterias
empleadas, AZ39 mostró ser mayor tolerante al efecto de PEG6000 que SEMIA6144.
En la condición de estrés inducido con PEG6000 -0,28MPa (15mM), AZ39 presentó
una disminución de la biomasa húmeda en un 34% con respecto al control cuando
alcanza la fase estacionaria, mientras que SEMIA 6144 disminuyó un 48%. El perfil de
AG de AZ39, se modificó mostrando incrementos significativos a nivel de palmítico,
esteárico y otros NI de mas de 20C, y una disminución del AG oleico, mientras que
SEMIA6144 incrementó el AG cis-vaccenico, y disminuyó el palmítico, palmitoleico y
esteárico frente al estrés hídrico. Estudios de TLC revelaron que los principales
fosfolípidos en AZ 39 son fosfatidilcolina (PC), fosfatidiletanolamina (PE) y
fosfatidilglicerol (PG) y cardiolipina (Cd) y en estrés se incrementó PC y disminuyó PE
y Cd.
Efecto del estrés hídrico sobre la movilidad de rizobacterias que se asocian a maní
Con respecto a la movilidad, se evaluó swarming y swimming de bacterias crecidas en
condiciones de estrés hídrico, observando un incremento de los dos tipos de movilidad
cuando las bacterias estuvieron sometidas a dicha condición experimental (Figura 1
paneles A y B). Estos son los primeros reportes de movilidad en estas condiciones
experimentales para rizobacterias.
A
B
Figura 1. Swarming (Agar-agua 0.5%) y Swimming (Agar-agua 0.3%) en condición control y estrés
PEG6000 -0,28 MPa (15mM). Paneles A AZ39 y B SEMIA 6144.
AGRADECIMIENTOS
Este trabajo fue financiado por Proyecto PIP CONICET y SECyT UNRC.
BIBLIOGRAFÍA
Albareda, M., et al. (2006). FEMS Microbiol. Lett. 259: 67-73.
Dardanelli, M., et al. (2008). Soil Biol. Biochem. 40: 2713-2721.
Dardanelli, M., et al. (2009). Symbiosis 40: 175-180.
Guasch-Vidal, B,. et al. (2013). Mol. Plant Microbe. Interact. 26: 451-460.
Medeot, D., et al. (2010). FEMS Microbiol. Lett. 303: 123-131.
Pauluci, N., et al. (2013). J Appl. Microbiol. 114: 1457-1467.
330
Session V
SV-CP-01
Movilidad en cepas de rizobios nodulantes de maní.
Vicario, J.C.*, Dardanelli, M.S., Giordano, W.F.
Departamento de Biología Molecular, FCEFQyN, Universidad de Río Cuarto. Córdoba. Argentina.
*
[email protected]
RESUMEN
La movilidad es una característica de los rizobios que puede contribuir en el éxito de la
competitividad simbiótica de estas bacterias (Ames et al., 1981). En este trabajo se
evaluó la capacidad de movilidad de cepas nodulantes de maní en diferentes
condiciones y se realizaron estudios de morfología por SEM. El comportamiento mostró
no ser homogéneo en las condiciones ensayadas y se observó un aumento significativo
en el largo de las células swarmer en comparación con las vegetativas.
INTRODUCCIÓN
Se han identificado en bacterias seis categorías de movilidad: swimming, swarming,
twitching, gliding, sliding y darting (Henrichsen, 1972). Los movimientos de mayor
importancia en el suelo son los de tipo swimming, el cual puede también ocurrir en
medio de cultivo con baja concentración de agar (0,2-0,4%). En contraste al
movimiento bacteriano individual que ocurre en el swimming, el swarming es
considerado un comportamiento bacteriano grupal asociado con migración en superficie
semisólidas (Williams y Schwarzhoff, 1972). Un mayor número de flagelos son
requeridos para el swarming, lo cual puede deberse a una mayor fricción de la colonia
con el medio (Harshey, 2003). Cuando las células son inoculadas en la superficie de un
medio agar, se forman colonias regulares en el punto de inoculación. Luego las células
inician un proceso de diferenciación en células largas multinucleadas e hiper flageladas
llamadas células swarm.
OBJETIVO
Evaluar la movilidad bacteriana de cepas simbiontes de maní, determinándose si la
misma puede ser el condicionante para una respuesta efectiva cuando la bacteria se
inocula en la semilla.
MATERIAL Y MÉTODOS
Se realizaron ensayos de movilidad para las cepas recomendadas de Bradyrhizobium
C145, SEMIA6144, USDA4438 y las nativas 15A y Pc34 aisladas de nódulos radicales
de maní cultivados en el sur de Córdoba (Bogino et al., 2010) La concentración de agar
para todos los ensayos de swimming fue de 0,3% y para todos los ensayos de swarming
fue de 0,5%. La movilidad fue determinada midiendo el diámetro del halo. Los ensayos
anteriormente descriptos, fueron modificados para realizar un estudio de movilidad en
un gradiente de agar, fuentes de carbono y cambios de temperatura de ensayo.
Se realizaron estudios de microscopía electrónica de barrido (SEM) según lo descripto
por Cheng et al. (2009).
La concentración de agar en la cual se obtuvieron los mayores diámetros de movilidad
para las cepas de estudio fue, 0,3% seguida por 0,5%. Estas concentraciones se
seleccionaron para realizar los estudios de swimming y swarming respectivamente. Las
5 cepas presentaron los mayores valores de movilidad cuando la fuente de carbono fue
Arabinosa. El comportamiento con las demás fuentes fue variable. A temperatura de
incubación inferior a la optima para el crecimiento de rizobios (28 ºC), disminuye la
331
Session V
SV-CP-01
movilidad tipo swimming y swarming de las bacterias simbiontes de maní ensayadas.
Todas las cepas con excepción de C145 presentaron un aumento significativo en el
largo de las células swarmer comparándolas con las vegetativas (Figura 1), mientras que
el ancho de las células swarmer no se vio aumentado en ningún caso.
A
B
Figura 1. Microscopía electrónica de barrido de células vegetativas y swarmer de Bradyrhizobium. (A)
SEMIA6144 células vegetativas; (B) SEMIA6144 células swarmer tomadas del borde del frente de
swarm. Escala = 1µm.
AGRADECIMIENTOS
BIBLIOGRAFÍA
Ames, P., et al. (1981). J. Bacteriol. 148: 728-729.
Bogino, P,. et al. (2010). J. Basic Microbiol. 50: 274-279.
Chang, H., et al. (1999) Annu. Rev. Microbiol. 57: 249-273.
Henrichsen, J. (1972). Bacteriol. Rev. 36: 478-503.
Sharma, M., et al. (2002). Curr. Sci. India 83: 707-715.
Vincent, J. (1970). Handbook N° 15. Blackwel Scientific Publication, Oxford, UK.
Williams, F.D., et al. (1978). Annu. Rev. Microbiol. 32: 101-22.
332
Session V
SV-CP-02
Effects of phosphorus and nitrogen level fertility on growth
parameters of Faba bean inoculated with rhizobia.
Maazaoui, H.1, 2, 3*, Sifi, B.1, Drevon, J.J.3
1
Laboratory of Agronomic Sciences and Techniques, National Institute of Agronomic Research of
Tunisia (INRAT) Hédi Karray stree,t 2080 Ariana, Tunisae. 2 Faculty of Sciences of Bizerte, Tunisia. 3
INRA, UMR Eco&Sols, 1 Place Pierre Viala, F34060, Montpellier, France.
* [email protected]
ABSTRACT
In the aim to study the influence of phosphorus and nitrogen fertilization and rhizobia
inoculation on growth parameters of Faba bean, experiments were conducted in
glasshouse. Isolated rhizobia from nodulated Faba bean roots were cultivated in a
selective growth media. Our work suggests that nodulation may be affected by the
concentration level of phosphorus and nitrogen and it is less important when the
rhizosphere is deficient in phosphorus. The results indicated that phosphorus apply in
combination with rhizobia inoculation increased nodules and biomass production.
Highest dry weights were recorded at 125 µM and 175 µM of phosphorus and 0.5 mM
and 2 mM of nitrogen. We conclude that Faba bean crop should be grown preferably
with 175 µM of phosphorus and 0.5 mM of nitrogen concentration along with rhizobia
inoculation under glasshouse conditions.
INTRODUCTION
Phosphorus (P) is one of the major essential macronutrients for plant growth and
development ( E hr l ic h, 1990). Its concentration levels in soil were variable between
400-1,200 mg kg −1. Phosphorus is present in soil in two forms, as organic and
inorganic phosphates. To convert insoluble phosphates both organic and inorganic
compounds in accessible form to the plant, is an important trait for a PGPR
in increasing plant yields (Igual et al., 2001; Rodríguez et al., 2006). The
concentration of soluble P in soil is usually very low, normally at levels of 1 ppm or less
(Goldstein, 1994). The plant takes up several P forms but major part is absorbed in the
forms of HPO4. The phenomenon of P fixation and precipitation in s o i l is g e ne r a l l y
h ig h l y d e p e nd e nt o n p H a nd s o i l t yp e . Several reports have documented
microbial P release from organic P sources (McGrath et al., 1995; Ohtake et al., 1996;
McGrath et al., 1998; Rodríguez and Fraga, 1999).
Experiments were conducted in glass house at INRA Montpellier to evaluate the
response of faba bean (Vicia fabae) to nitrogen and phosphorus fertilizer combined to
inoculation with Fb1 Rhizobium strain. For this purpose, hydro aeroponic culture into 1
L pots was conducted using the commercial faba bean (Castel variety) and Fb1 as the
only selected rhizobia. Five levels of phosphorus (P) (25, 75, 125, 175 and 225 µM) and
three levels of nitrogen (N) (0, 5, 2, and 6 mM) with three replications were used in the
work. The full doses of P and N were applied at sowing. Plants of three replicates were
potted 60 days old after sowing to evaluate nodules and shoots dry weights.
Plant nodules were significantly affected at 6 mM N applies independently on P applies.
It is quite possible that the excess of N had inhibited faba bean nodulation since the
available carbohydrates seeping from the shoot and the root system would be low
333
Session V
SV-CP-02
causing feeble rhizobial growth resulting in lower nitrogen. These results are in
accordance with those obtained by Alexander (1978), who demonstrated that excess of
nitrogen reduced nodulation, root hair production and leghaemoglobin synthesis. We
noticed that nodulation dry weight increased significantly at 175 µm P and 0.5 mM and
2 mM N concentration. At these two nitrogen levels and 225 µM P Nodulation dry
weights was significantly decreased.
80
0,5N
0,5N
Shoot dry weight (mg)
Nodule dry weight (mg)
6
2N
4
6N
2
0
2N
60
6N
40
20
0
25
75
125
175
225
25
P (µm)
75
125
175
225
P (µm)
Figure 1. Effect of P and N concentration on
nodule dry weight.
There was significant response of faba bean biomass improvement at the combination
of 175 µM P level with rhizobia inoculation. The data indicated that the highest shoot
dry weight was registered with both 0,5 and 2 mM N levels combined to 175 µM P.
Faba bean growth was decreased at the three nitrogen levels and 225 µM P (Figure 2).
ACKNOWLEGMENTS
under the reference ID 1001-009 FABATROPIMED scholarship programme provided by the EU for the
stay of Houda Maazaoui in Montpellier .
REFERENCES
Alexander, M., (1978). Introduction to soil microbiology. John Wiley & Sons, New York and London.
Brady, N.C., and Weil, R.R. (1999). Agron. J. 53:464-465.
Ehrlich, H.L. (1990) Geomicrobiology (2nd edn.). Dekker, New York, p 646.
Goldstein, A.H. (1994) . T orriani-Gorni, A. et al. (eds) ASM Press, Washington, pp 197-203.
Igual, J.M., et al. (2001). Agronomie 21: 561 -568.
McGrath, J.W. (1998). Appl. Environ. Microbiol. 64: 3 56-35.
O h t a k e , H., et al. (1996). Res. Conserv. Recycl. 18: 125-134.
Rodríguez, H,. (2006). Plant Soil 287: 15-21.
Rodríguez; H., and Fraga, R. (1999). Biotechnol. Adv. 17: 319-339.
334
Figure 2. E
Session V
SV-CP-03
Phytate mineralising bacteria increase in the rhizosphere of nodulated
common bean (Phaseolus vulgaris) under P deficiency.
Maougal, R.T.1, 2*, Brauman, A.3, Plassard, C.1, Abadi, J.1, Djakoun, A.2, Drevon, J.J.1
1
INRA, Eco&Sols, 1 Place Pierre Viala, F34060, Montpellier, France. 2 Laboratoire de Génétique,
Biochimie et Biotechnologies Végétales, Département de Biologie et d’Ecologie, Université Constantine
1, Route de Ain el Bey, Constantine, Algeria. 3 IRD, Eco&Sols, Land Department developement,
Bangkok, Thailande.
*
[email protected]
ABSTRACT
To characterize the functional microbial community involved in the mineralization of
phytate in the rhizosphere of Phaseolus vulgaris, putative phytate hydrolysing bacteria
were isolated from nodulated roots of Phaseolus vulgaris cultivated in two contrasted
soil in terms of P availability. Their ability of these rhizosphere isolates to degrade
phytate was confirmed enzymatically. Our work demonstrates that phytate-mineralizing
bacteria represent a tiny but significant part (1%) of the rhizospheric population of
Phaseolus vulgaris and this community is stimulated in P-deficient soil.
INTRODUCTION
Low phosphorus availability is a primary constraint to common bean (Phaseolus
vulgaris L.) production since approximately 60% of beans in Latin America and 44 %
of beans in Africa (Wortmann and Allen, 1994) are grown on severely P-deficient soils.
However, the organic P pool may account for 29-65% of total soil P (Patel et al., 2010).
Among rhizosphere bacteria that may exert a beneficial effect on plant growth, phytatemineralizing bacteria (PMB) are able to use phytases to hydrolyse phytate to phosphate
(Vohra and Satyanarayana, 2003; Patel et al., 2010). Our study hypothesis is that the
density of this functional bacterial community will be more important in the plant
rhizosphere of Phaseolus vulgaris L. in P deficient conditions than in P sufficient one.
To confirm this hypothesis, we numerated and characterized the phytate-mineralizing
bacteria in the rhizosphere of Phaseolus vulgaris cultivated in a low-P soil with P
sufficient versus P deficient supply.
The common bean recombinant inbred lines 147 (Drevon et al., 2011) was used as
model plant and cultivated under glasshouse, into 1 L pots filled with a low-P soil from
a cultivated field of the experimental station of Institut National de la Recherche
Agronomique at Maugio, Montpellier, France. The soil was added with a deficient
(P15) and a sufficient (P50) P supply.
Serial dilutions of the soil suspension were plated on LB medium and on Angle et al.
(1991) medium after modification by Richardson and Hadobas (1997). All isolates with
visible ability to mineralize phytate were counted and isolated, and subsequently
inoculated in a liquid phytate-specific media where bacterial growth and medium Pi
were measured at regular time intervals.Their phytase activity was measured on
bacterial pellet and supernatant after a 72 h incubation period according to Shimizu
(1992) with modifications.
The bacteria able to grow on the modified Angle medium represented 1% of total
number of bactaria that were abble to grow on the rich medium (LB). Under P15, the
density of phytate-utilizing bacteria on Angle medium was 10 fold higher in
335
Session V
SV-CP-03
rhizospheric soil than in bulk soil, whereas under P50 there was no significant
difference between both soils (data not shown). This suggests a selection of phytatemineralizing bacteria in the rhizosphere of P-deficient bean.The growth curves of
phytate-mineralizing bacteria in liquid medium with phytate as sole source of P could
be grouped in 3 types shown in Figure 1A significant accumulation of Pi in the culture
medium was observed for 26 isolates (data not shown).
200
0,4
100
0
0,0
B
34
500
OD (630nm)
1,2
400
300
0,8
200
0,4
100
0
0,0
C
OD (630nm)
500
44
1,2
400
300
0,8
200
0,4
0,0
100
0
24
48
72
96
120
144
0
168
Phytase activity (nmol Pi)
0,8
A
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
47
48
52
I
2
Phytase activity (nmol Pi)
300
Pi concentration (µM)
400
OD (630 nm)
500
Bacillus subtilis 111b
2
A
1,2
31
44 43 59 40
II
38
42 36 34
III
B
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
1
Time (h)
22
20
I
18
17
23
3
2
13
II
9 14
12
15 16
III
Figure 1. Kinetics of growth ( ) and concentration of Pi ( ) in the growth medium for
rhizobacterial isolates with Na-IHP as sole source of P. for A, Bacillus subtilis 111b as
representative of group II; B, isolate 34 as representative of group II; C, isolate 44 as representative
of group III. Data are means ± sd of 3 replicated cultures.
Figure 2. Extracellular (left) and Cell-associated phytase activity (right) of rhizobacterial isolates. A:
on P 50, B: on P15.
The phytase activity varied considerably among isolates of the 3 above groups of curves
(Figure 2). A majority of isolates produced extra-cellular phytase in agreement with the
results by Hussin et al. (2007) for maize. However, additional or exclusive cellassociated phytase was found for some isolates. This suggests that phytate could be
mineralized either externally or within the periplasm of the bacteria; these two
conditions seem to involve different phytases.
Overall, our results indicate that nodulated-bean plants can increase in case of P
deficiency, the phytase activities of a diverse community of phytase hydrolysing
bacteria within their rhizosphere.
ACKNOWLEGMENTS
under the reference ID 1001-009 and AVERROES scholarship programme provided by the EU for the
stay of Rim Tinhinen Maougal in Montpellier.
REFERENCES
Angle, J.S., et al. (1991). Appl. Environ. Microbiol. 57: 3674-3676.
Drevon, J.J., et al. (2011). Proc. Environ. Sci. 9: 40-46.
Hussin, A.S.M., et al. (2007). World J Microbiol Biotech. 23: 1653-1660.
Patel, K.J., et al. (2010). Appl. Soil Ecol. 44: 252-261.
Richardson, A.E., and Hadobas P.A. (1997). Can. J. Microbiol. 43: 509-516.
Vohra, A., and Satyanarayana, T. (2003). Crit. Rev. Biotech. 23: 29-60.
Wortmann, C. S., and Allen, D. J. (1994). CIAT Occasional Publication Ser. 11.
336
Session V
SV-CP-04
Is the contrast between nodulated recombinant imbred lines of
common bean (Phaseolus vulgaris L.) linked with available phosphorus
in soil? A multi-local field test in Mediterranean conditions.
Alkama, N.1, 2, 3*, Jaillard, B.1, Ounane, S.M.2, Ounane, G.2, Drevon, J.J.1
1
INRA, UMR Eco&Sols, 1 Place Pierre Viala, 34060 Montpellier, France. 2 Ecole Nationale Supérieure
Agronomique (ENSA), Département de phytotechnie. Avenue Hassan Badi, El Harrach 16200 Alger,
Algérie. 3 Université Mouloud Mammeri, Faculté des Sciences biologiques et agronomiques, Département
d’Agronomie, Tizi Ouzou, Algeria.
* [email protected]
ABSTRACT
In order to assess the relation between symbiotic nitrogen fixation and soil phosphorus,
a multi-local test was proposed to producers of Tizi Ouzou area in Algeria, without
modification of their cultivation practises. The nodulation and growth of the cultivar
traditionally used by farmers, was studied with seven potentially interesting
recombinant inbred lines selected among the crossing of BAT 477 and DOR 364 in
addition. The sampling was performed at the flowering stage. The major finding in this
work is that nodule biomasss were positively correlated with Olsen-P. Although, the
curvilinear regressions of nodule biomass and shoot biomass as a function of Olsen-P
suggest the existence of 2 ranges of Olsen-P among studied sites that are separated by
critical P values. It is concluded that the low nodulation of the RILs was partly
compensated by increasing the efficiency in use of the rhizobial symbiosis.
INTRODUCTION
Among legumes, common bean (Phaseolus vulgaris L.) is the most cultivated and
appreciated in the world. Indeed, the bean constitutes a staple food for 500 million
humans for its high content in proteins which can substitute the animal proteins which
are often inaccessible for many people (Pujola et al., 2007). Unfortunately, common
bean is often cultivated in marginal lands, where more than 50% of the soils are
deficient in phosphorus. Like for all legumes, phosphorus is a major limiting factor for
common bean. Vadez and Drevon (2001) demonstrated the existence of a genetic
variation for the phosphorus use efficiency (PUE) for N 2-dependent growth of common
bean. These authors selected recombinant inbred lines (RILs) from the crossing between
BAT 477 and DOR 364, that were contrasting in their PUE for their symbiotic nitrogen
fixation and were adapted to the Mediterranean conditions (Trabelsi, 2001). The
objective of this work was consequently to focus on the relationship between Olsen-P
and RILs cultivated in farm conditions in a Mediterranean agro-ecosystem.
The experimental sites were located in Tizi-Ouzou district, Algeria, at 100 km east of
Algiers. This area was selected for its importance in bean production. Fifteen sites were
initially selected, representing the diversity of agro-ecological conditions where beans
are produced. Soils of the 15 sites were characterized by a standard sampling using a
drill at a 30 cm depth, before sowing the bean culture. Seven Recombinant Inbred Lines
(RILs) were tested. They result from crossing between BAT 477 and DOR 364 carried
out by Ribet and Drevon (1996) within the framework of a co-operation CIAT-INRA.
“El Djadida” was used as a local reference for comparison with the RILs since this
cultivar is traditionally used by the farmers in this area. The culture tests were carried
out randomly in each sites divided into lines, each line carrying a genotype among the
Dj canopy. At the stage of full flowering, i.e. 45-46 days after emergence, 10 plants for
337
Session V
SV-CP-05
each genotype were excavated within 20 cm deep and around the root. Shoots, nodules
and roots of the harvested plants were separated, oven dried and weighted.
The question was to determine if the availability of P had an effect on the nodulation of
roots, and thus on shoot biomass production by common bean. Figures shows mean
nodule (Figure 1) and shoot (Figure 2) biomasses of the 8 lines of common bean as a
function of Olsen-P concentration in soil of the 8 experimental sites. We observed a
highly significant relation between biomasses and Olsen-P concentration in soil. The
best fits were obtained with a curvilinear equation. For nodule and shoot biomasses, the
optima were observed for a Olsen-P concentration of about 40 and 35 mg kg -1 of OlsenP in soil, respectively : behind this value, nodule and shoot biomasses increased with
Olsen-P concentration, since above this value nodule and shoot biomasses decreased
with Olsen-P concentration in soil. Moreover, the relation which linked nodule biomass
and shoot biomass of the 8 lines of common bean to Olsen-P concentration in soils
presented systematically a best fit with a curvilinear regression, by increasing from 0 to
about 40 mg of P kg-1 soil, and by being quite constant or decreasing beyond this value
of P. P would limit nodulation whereas above 40 mg of P kg -1 soil, the P availability
would be higher than the nodule requirement for most RILs.
30
y = 4.04x - 22.8 - 0.052x2
R=0.55 (P<0.001)
Shoot biomass (g plant-1)
Nodule biomass (mg plant-1)
130
90
50
10
0
10
20
30
40
Concentration of Olsen-P in soil (mg
50
y = 1.05x + 3.8 - 0.015x2
R=0.50 (P<0.001)
25
20
15
10
60
0
kg-1)
10
20
30
40
50
60
Concentration of Olsen-P in soil (mg kg-1)
Figure 2. Shoot biomasses, versus
concentration of Olsen-P in soil, of the 8
genotypes grown on the experimental
sites retained. Data are means and SD of
10 replicates collected at flowering stage.
Figure 1. Nodule biomasses, versus
concentration of Olsen-P in soil, of the 8
genotypes grown on the experimental
sites retained. Data are means and SD of
10 replicates collected at flowering stage.
ACKNOWLEDGMENTS
This work was supported by Aquarhiz project of the EU INCOMED program and Tassili PHC of the
French ministry of foreign affairs. The authors are grateful to the local farmers and the students of
Mouloud Mammeri University for their excellent contribution.
REFERENCES
Pujola, M., et al. (2007). Food Chem. 102: 1034-1041.
Vadez, V., and Drevon, J.J. (2001). Agronomie 21: 691-699.
Trabelsi, M. (2001). In: Fixation symbiotique de l’azote et développement durable dans le Bassin
méditerranéen. Ed INRA, Paris 2003. Les colloques, N° 100, pp. 45-58.
Ribet, J., and Drevon, J.J. (1996). New Phytol. 132: 383-390.
338
Session V
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Phosphoenol pyruvate phosphatase transcript in nodule cortex of
Phaseolus vulgaris.
Bargaz, A.1*, Lazali, M.2, Ghoulam, C.3, Drevon, J.J.2
1
Swedish University of Agricultural Sciences, Department of Biosystems and Technology, PO Box 103,
2
3
SE-230 53 Alnarp, Sweden. INRA, UMR Eco&Sols, 1 Place Viala, F34060, Montpellier, France.
Equipe de Biotechnologie Végétale et Agrophysiologie des Symbioses, Faculté des Sciences et
Techniques Guéliz, BP 549, 40000, Marrakech, Maroc.
*
[email protected]
ABSTRACT
Increases of acid phosphatases (APases) enzymes are among mechanisms which lead to
increase efficiencies both of N2 fixation and nodule respiration under P deficiency. Our
findings have revealed that activities and differential expression of phosphoenol
pyruvate phosphatase (PEPase) transcripts were positively correlated with increases
both of the rhizobial symbiosis efficiency (EURS) and nodule O2 permeability. The
induced enzyme activity and the marked transcript localization of this APase in nodule
cortex would control nodule respiration and contribute to adaption of nodulated legumes
to low P availability.
INTRODUCTION
In nodules, expression of large number of Glycine max APase “GmPAP” genes was
mainly detected under P-deficiency (Li et al., 2011). This organ was reported to be
strongly enriched in highly tissue-specific genes (Libault et al., 2010; Severin et al.,
2010). Overall, most APases are nonspecific hydrolyzing Pi from a broad spectrum of
Pi mono-esters and may have different functions as described for a soybean APase gene
GmPAP3 whose expression alleviates oxidative stress caused by salinity and osmotic
constraint (Li et al., 2008). The involvement of APases in tolerance to various abiotic
and biotic constraints, led us to assume that PEPase may have a multiplicity of functions
as well as those related to phosphate metabolism and should provide new insights into
adaptation to P-deficiency. Thus, the present work hypothesizes that differential
expression in the nodule cortex, of PEPase may contribute to low P adaptation and may
be involved in the regulation of nodule respiration.
Nodules of about 3 mm diameter of each RIL corresponding to 50 ± 05 mg of nodule
fresh weight (FW) were carefully detached at 42 days after transplanting (DAT) and
immediately frozen in liquid nitrogen and stored at -80 °C until use for PEPase activity
assay. Sample preparation and fixation for in situ reverse transcription polymerase chain
reaction (RT-PCR) were set according to the protocol described by Molina et al. (2011).
The method involves in situ amplification of specific nucleic acid sequences on nodule
sections, followed by fluorescence detection of the localized PCR product via epifluorescence microscopy.
To our knowledge, this study is the first to reveal that the PEPase transcription was
induced by P-deficiency with differential expression among nodule tissues. The
differential localization of transcripts encoded for PEPase in the outer cortex and
infected zone under P-deficiency (Figure 1) opens new insights into understanding the
physiology of N2-fixing legumes as well as requirements for N2 fixation and regulation
of nodule permeability to O2 diffusion. Under P-deficiency, the correlation between
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Session V
SV-CP-05
PEPase enzyme activity or transcript localization in infected cells and EURS or N2
fixation (Figure 2) suggest that this APase is involved in nodule metabolism that is
linked to N2 fixation and the overall N2-dependent growth of the legume in
hydroaeroponics. The marked increase in PEPase transcripts in the nodule cortex of Pdeficient nodules not only suggests an increase in intracellular Pi scavenging but also
opens up a challenge to understand whether such sub-localization is involved in the
scavenging of Pi from extracellular organophosphates.
Figure 1. In situ localization of PEPase transcripts
(green spots) in nodules of common bean RIL115
and RIL147 grown under a sufficient (250 P)
versus a deficient (75 P) P supply. InC, infected
cell; IC, inner cortex; IZ, infected zone; OC, outer
cortex; UC, uninfected cell; VT, vascular trace
parenchyma.
Figure 2. Efficiency of use of rhizobial symbiosis
of common bean RIL115 and RIL147 inoculated
with R. tropici CIAT899 and grown under a
sufficient (open circles) versus a deficient (filled
circles) P supply. Data represent individual values
of 14 replicates harvested at 42 DAT. Ndw,
Nodule dry weight.
ACKNOWLEGMENTS.
This work was supported by the FABATROPIMED project financed by Agropolis Fondation under the
REFERENCES
Li, W.Y.F., et al. (2008). New Phytol. 178: 80-91.
Li, C., et al. (2011). Ann Bot. 109: 275-285.
Libault, M., et al. (2010). Plant J. 63: 86-99.
Severin, A.G., et al. (2010). BMC Plant Biol. 10: 160.
340
Session V
SV-CP-06
Phytate-mineralizing rhizobia from Vicia faba symbiosis in an agroecosystem of south of France.
Domergue, O.1, 2*, Chouayekh, H.3, Abadie, J.1, Amenc, L.1, Pernot, C.1, de Lajudie, P.4,
Galiana, A.5, Drevon, J.J.1
1
INRA, Eco&Sols, 1 Place Viala, 34060 Montpellier, France. 2 INRA, Campus de Baillarguet TA A82/J
34398 Montpellier. 3 Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de
Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP “1177” 3018 Sfax, Tunisie. 4 IRD, LSTM,
Campus de Baillarguet TA A82/J 34398 Montpellier. 5 CIRAD, LSTM, Campus de Baillarguet TA A82/J
34398 Montpellier.
*
[email protected]
ABSTRACT
Functional diversity of rhizobia involved in phytate mineralization was determined in
Vicia faba symbiosis in an agro-ecosystem of south of France. Among 60 isolates from
V. faba nodules, 27 were able to hydrolyze phytate on selective medium. Amplified
rhizobial phytase mRNA signal has been localized in V. faba nodules by using in situ
RT-PCR with β-propeller phytases specific primers. Our results suggest that phytatemineralizing rhizobia identified as R. leguminosarum bv. viciae could be found in V.
faba symbiosis.
INTRODUCTION
Via its ability to fix atmospheric N2, V. faba - rhizobia symbiosis can offer important
ecosystem services including renewable N inputs into crops and soil, and in a
diversification of cropping systems. However, symbiotic nitrogen fixation (SNF) can be
limited by such abiotic factors as N excess and phosphorus (P) deficiency (Graham and
Vance, 2003).
With their ability to hydrolyse phytate to myo-inositol and inorganic phosphate (Pi)
phytases increase P bio-availability for plants growth and development (Mullaney and
Ullah 2003). Despite that these enzymes are widely distributed among microbial cells
(Ullah and Gibson, 1987), those belonging to the class of β-propeller phytases (BPPs)
have been characterized mainly from the Bacillus genus (Kerovuo et al., 2000a).
Because of potential value of phytase producing bacteria in restoring soil health by
mobilizing phytate-P and making it available for plant use, selecting indigenous
rhizobia harbouring phytase genes could provide sustainable tools for increasing SNF in
low-P soils. Identifying phytate-mineralizing rhizobia in V. faba symbiosis from south
of France agro-ecosystem is the aim of our study.
Vicia faba - rhizobia isolates were sampled from multi-local areas of an agro-ecosystem
in the south of France. Rhizobial isolates were selected for their ability to hydrolyze
phytate on Angle et al. (1991) medium modified by Richardson and Hadobas (1997).
Identifying phytase-encoding genes in phytate-mineralizing isolates was carried out by
PCR detection, using BPPs specific primers. Phylogenetic diversity of phytate
mineralizing isolates was assessed by recA gene sequencing (Gaunt et al., 2001). V.
Faba - rhizobia symbiosis was performed by inoculation and hydro-aeroponic culture.
Nodules preparation and fixation, for in situ reverse transcription polymerase chain
(RT-PCR) were carried out according to Bargaz et al. (2011) protocola.
341
Session V
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Screening for phytate-hydrolyzing isolates from V. faba nodules originating from south
of France agro-ecosystem multi-local areas showed that 27 of the 60 rhizobial isolates
screened were able to mineralize phytate on selective medium (Figure 1). These phytase
producers were identified as Rhizobium leguminosarum bv. Viciae strains by recA
sequencing which is in agreement with the results shown by Abd-Alla (1994).
Using BPPs specific primers, phytase genes were amplified from the V. faba - rhizobial
isolates and in situ V. faba nodules transcription signals were localized. The results
show that the transcripts corresponding to BPPs were mainly localized in nodule cortex
and infected zones (Figure 2).
Figure 1. Peripheral halo zone induced by phytatemineralizing rhizobia on Angle et al. (1991)
medium modified by Richardson and Hadobas
(1997).
Figure 2. In situ β-propeller phytases (BPPs)
transcripts localization in Vicia faba nodule, using
BPPs specific primers according to Molina et al.
(2011) protocol. BPPs transcripts were mainly
localized in nodule cortex and infected zones.
To our knowledge, this study is the first to reveal BPPs transcription in V. faba nodule
tissues. The differential localization of BPPs transcripts opens new field into
understanding the use of organic P by N2-fixing legumes as well as P requirements for
N2 fixation. However, further experiments to confirm in situ localization of BPPs
transcripts into V. faba nodules are needed.
ACKNOWLEGMENTS.
This work was supported by the Great Federative Project FABATROPIMED financed by Agropolis
Fondation under the reference ID 1001-009.
REFERENCES
Abd-Alla, M.H. (1994). Lett. Appl. Microbiol. 18: 294-296.
Angle, J.S., et al. (1991). App. Environ. Microbiol. 57: 3674-3676
Bargaz, A., et al. (2011). J. Exp. Bot. 63: 4723-4730.
Gaunt, M.W., et al. (2001). Int. J. Syst. Evol. Microbiol. 51: 2037-2048.
Graham, P.H., and Vance, C. P. 2003. Plant Physiol. 131:872–877.
Kerovuo, J., et al. (2000a). Biochem. Biophys. Res. Com. 268: 365-369.
Mullaney, E.J., and Ullah, H.J. (2003). Biochem. Biophys. Res. Com. 312: 179-184.
Richardson, A.E., and Hadobas, P.A. (1997). Can. J. Microbiol. 43: 509-516.
Ullah, A.H., and Gibson, D.M. (1987). Prep. Biochem. 17: 63-91.
342
Session V
SV-CP-07
Daily simulation of plant and microbial transfers of carbon between
the soil and the atmosphere under wheat-faba bean intercropping.
Ibrahim, H.1*, Hatira, A.1, Blavet, D.2, Drevon, J.J.2, Pansu, M.2
1
U.R. 04/UR/10-02 Pedology, Department of Geology, Faculty of Sciences of Tunis, El Manar
University, 2092 Tunis, Tunisia. 2 IRD, UMR Eco&Sols, 1 Place Pierre Viala, F34060, Montpellier,
France.
*
[email protected]
ABSTRACT
The objective of this work was to collect the data necessary to apply a Model of the
transformations of Organic matter by Micro-Organisms of the Soil (MOMOS) to one
cultural cycle of a legume-cereal agrosystem in the Mediterranean region. This study
has demonstrated that the MOMOS equation system is able to predict the daily
exchanges of labile and stable C between the soil and the atmosphere. This fills a gap
concerning modelling of direct microbial control on C evolutions and emerges as a new
tool for agro-ecology and global change.
INTRODUCTION
This work aims to present an agro-ecological application of the MOMOS model (Pansu
et al., 2004, 2010) which is centred on microbial functioning and appears very sensitive
to meteorological, edaphic and biological conditions. In contrast with other published
propositions which need long term comparisons to quantify significant C transfer
between soils and atmosphere, our experimental work aimed to answer to 2 questions:
(i) could MOMOS predict the daily evolution at short term of leaving and dead forms of
organic carbon in complex systems, (ii) could we couple the equations of OC
decomposition with different equations of OC production and propose a new theory for
agro-ecology and global change.
The soil water content (θ) was predicted using the SAHEL model (Penning de Vries et
al., 1989). TAO (Thuriès et al., 2002) we use Van Soest method for the determination
of biochemical compositions of plant material. For MOMOS (Pansu et al., 2004; 2009;
2010; Bottner et al., 2006) we compare predicted and measured values for carbon and
nitrogen content in plant, soil and microbial biomass
Figure 1a reproduces the measured and daily predicted values of microbial OC during
one year of cereal-legume intercropping. It illustrates the growth of microorganisms
associated to plant growth and OC brought to soil from the part lost from
photosynthesis.
Figure 1b shows that the sum of predicted C from the mortality of plant roots and
shoots, which provide the C substrates for microbial growth, was greater than the daily
total CO2-C respired by microorganisms and plant roots over the whole cultivation
period.
The total C input increased again at harvest where 80% of leaf and stem material
wasmodelled as falling to become litter, in addition to decomposition of the remaining
shoots and roots by natural mortality. This showed that the intercropping was a sink of
OC during all the cultivation period and became a source of OC about two months after
the harvest, but during a period of all processes were slowed by the winter conditions.
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Session V
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The increase of measured and predicted total OC during the intercropping season was
not significant, as in other modelling studies which need lon

environment interactions (ibempa)

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