Proceedings Undergraduate Research Experiences

Transcripción

Proceedings Undergraduate Research Experiences
2015, Vol. 1:1
Resident Instruction for Insular Areas Educational Grant Program
2015
Resident Instruction Grants Program for Institutions of Higher Education in Insular Areas
Undergraduate Research Experiences
Participating Students and Faculty
Student
Mentor
Area of Expertise
Andréa Paola Ríos
[email protected]
Abner A. Rodríguez
[email protected]
Ruminant Nutrition
Forage Conservation
Natalie Vázquez
[email protected]
Esbal Jiménez
[email protected]
Animal Reproduction
Miltón González Serrano
[email protected]
Leyda Ponce de León
[email protected]
Dairy Products
Camille Cordero
[email protected]
Jaime Curbelo
[email protected]
Dairy Cattle
Mastitis
Nicolle Irizarry Larriuz
[email protected]
Paul F. Randel
[email protected]
Animal Nutrition
Abdiel Santana Rivera
[email protected]
David Sotomayor
[email protected]
Soil and Water Quality
Soil Fertility
Jennifer Rivera San Antonio
[email protected]
Roberto Vargas
[email protected]
Nematology
Mibet Cordero
[email protected]
Maria del Carmen Librán
[email protected]
Horticulture
Alexandra Ramírez Irizarry
[email protected]
Pablo Morales Payan
[email protected]
Organic Agriculture
Veronica Rivera
[email protected]
Lydia Rivera
[email protected]
Tropical Plant
Pathology/Mycology
Abner A. Rodríguez Carías, Ph.D.
Professor
Department of Animal Science
Project Coordinator
Table of Content
Intake, growth performance, and carcass yield of meat-type rabbits fed with different levels of
corn stalk silage.
1
Andrea P. Ríos, Abner A. Rodríguez and Luis C. Solórzano
Expression of prolactin receptor in the skin of dry Holstein cows with phenotypic
differences in hair coat.
6
Natalie M. Vázquez and Esbal Jiménez
Use of exopolysaccharides producing lactic acid bacteria in the manufacture of low fat
Gouda cheese. Milton S. González and Leyda Ponce de León
10
The effect of scrotal temperature on semen quality parameters in Holstein Friesian and
Jersey bulls.
16
Camille Cordero-Aponte and Jaime E. Curbelo-Rodríguez
The retained placenta malady in a Puerto Rican dairy cattle herd: prevalence, treatment
and effects on animal performance.
23
Nicole M. Irizarry, Paul F. Randel, Héctor Santana and Ricardo Montañez
Soil nitrogen mineralization in soils cropped to maize (Zea Mays) of the southern
semiarid zone of Puerto Rico.
29
Abdiel Santana, Johannies Rivera-Zayas and David Sotomayor-Ramírez
Comparative Study of Organic and Conventional Farms in Puerto Rico Using Nematode
Communities as Environmental Indicators.
37
Jennifer M. Rivera San Antonio and Roberto Vargas
Growth responses of lettuce plants (Lactuca sativa L., cv. “Black Seeded Simpson”)
grown under a hydroponic system (NFT) receiving inorganic and organic nutrient
solution.
42
Mibet M. Cordero Ruiz and María del Carmen Librán
Effects of Exogenous Biostimulants on Tahiti Lime (Citrus latifolia) Trees Affected by
the Citrus Greening Disease in Lajas, Puerto Rico.
51
Alexandra I. Ramírez-Irizarry and J. Pablo Morales-Payan
Rhytismatales and other pathogenic fungal species of native trees of Puerto Rico
Verónica M. Rivera Vega and Lydia. I. Rivera Vargas
58
Intake, growth performance, and carcass yield of meat-type rabbits fed with different
levels of corn stalk silage
Andrea P. Ríos, Abner A. Rodríguez and Luis C. Solórzano
Abstract
Among the most important problems facing rabbit producers in Puerto Rico is the high cost of
commercial feed (CF), therefore, alternatives to its use need to be evaluate. Rabbits are nonruminant herbivores capable of degrading fiber, thus inclusion of fermented forages might
represent a useful alternative. An experiment was conducted to evaluate the inclusion of corn
stalk silage (CS; 28% DM) in diets for growing rabbits based on voluntary intake, growth
performance, and carcass yield. Thirty New Zealand white rabbits (averaging 5 weeks of age and
700g live weight) were distributed by initial weight into 30 elevated cages and randomly
assigned to three treatments; control (T1, 100% CF), and inclusion of CS at 25% (T2) and 50%
(T3) of the total dry matter offered. Diets were offered at 5% of rabbit body weigh daily on a dry
matter basis during 59 days. Data collected on feed intake, weight gain, feed conversion, and
carcass dressing percentage were statistically analyzed using a completely randomized design
with 10 replicates per treatment and Bonferroni-test for mean separation. Total intake was higher
(P<0.05) in rabbits fed the 100% CF diet (59.03g/d), than in those offered 50% CS (40.74g/d),
but not significantly different from those fed 25% CS (49.53g/d). Control rabbits showed higher
(P<0.05) daily gain (T1=17.58 g, T2=12.46 g, T3=6.60 g) and lower feed conversion ratio than
animals that received CS in the diet (T1=3.38, T2=4.01, T3=6.17). Carcass yield percentage was
lower (P<0.05) in animals fed diets containing 50% CS than in those fed 0 and 25% CS. In
summary, corn stalk silage at 25% and 50% inclusion rates can be used in growing rabbit diets
but with resulting lower productive performance than that obtained by animals fed 100% CF.
Inclusion of CS in the diet at the 25% level did not affect rabbit carcass yield.
Keywords: Rabbits, Corn Silage, Performance
Resumen
Entre los principales problemas que enfrentan los productores de conejos en Puerto Rico se
encuentra el alto costo del alimento concentrado (AC), por esta razón las alternativas de
alimentación deben ser evaluadas. Los conejos son herbívoros no rumiantes con capacidad de
degradar fibra, por lo que la inclusión de forrajes conservados en su alimentación podría
representar una alternativa útil. Se realizó un experimento para evaluar la inclusión de ensilaje de
materia vegetativa de maíz (EM; 28% MS) en dietas para conejos destinados a la producción de
carne, basado en los criterios de ingesta voluntaria, ganancia en peso y rendimiento de la canal.
Treinta conejos de la raza Nueva Zelandia blanco (promedio de 5 semanas de edad y 700 g de
peso vivo) fueron distribuidos, según su peso inicial, en 30 jaulas elevadas y asignados
aleatoriamente a tres tratamientos; control (T1, 100% AC), y la inclusión de EM a 25 (T2) y 50%
(T3) del total ofrecido en materia seca. Las dietas se ofrecieron diariamente al 5% del peso vivo
del animal en base seca durante 59 días. Se recolectaron datos de consumo de alimento, ganancia
o pérdida de peso, conversión alimenticia y rendimiento de la canal. Los datos se analizaron
según un diseño completamente al azar con 10 repeticiones por tratamiento. La prueba de
Bonferroni se utilizó para la separación de medias. El consumo total fue mayor (P <0.05) en los
1
conejos alimentados con la dieta AC 100% (59.03 g/d), que en los que se les ofrecieron 50% EM
(40.74 g/d) o 25% EM (49.53 g/d). Los conejos de control presentaron mayor (P <0.05)
ganancia diaria en peso (T1= 17,58 g, T2= 12,46 g, T3= 6,60 g) y una menor (P<0.05)
conversión alimenticia que los animales que recibieron EM en la dieta (T1= 3.38, T2= 4.01, T3=
6.17). El porcentaje de rendimiento de la canal fue menor (P <0,05) en los animales alimentados
con dietas que contenían 50% EM que en los alimentados con 0 y 25% EM. En resumen, el
incluir ensilaje de maíz en 25 y 50% de la materia seca dietética puede ser utilizado para
alimentar conejos, pero este presenta un menor rendimiento productivo comparado con el
obtenido por los animales alimentados con 100% AC. Inclusión de EM en la dieta a un nivel de
25% no afectó el rendimiento de la canal.
Palabras Claves: Conejos, Ensilaje de Maíz, Rendimiento
Introduction
The last agricultural census in Puerto Rico reports that commercial rabbit farms and animal
inventory have been declining. In 2007, there were 119 commercial farms and 38,519 rabbits,
numbers that decreased in 2012 to 98 farms and 33,982 animals (USDA/NASS, 2014). Feeding
cost is among the most important problems that affect local rabbit producers. Currently, rabbit
diets are based exclusively of commercial feeds, which are disadvantageous due to: high price,
low energy density, and inadequate fiber content. Rabbits are non–ruminant herbivores that
practice hindgut fermentation and are capable of degrading fiber. Thus, they can utilize forages
and fibrous by – product feeds as a major dietary component (Tonson et al., 1999). Inclusion of
fermented forages in rabbit diets could represent a useful option to reduce feed costs for
commercial rabbit producers. Corn silage might be a candidate for this purpose because of its
high yield, good nutritive value and easy handling. This study was designed, to evaluate the
inclusion of corn stalk silage as an integral part of the diet for growing meat–type rabbits based
on the criteria intake, growth performance and carcass yield.
Material and Methods
The study was conducted in a pavilion specialized for rabbit production located in the Alzamora
Farm at the University of Puerto Rico, Mayagüez Campus (Figure 1). Thirty New Zealand White
rabbits (averaging 5 weeks of age and 700g live weight) were obtained from the UPRM
Agricultural Experiment Station Small Animal Research Farm in Lajas. Animals were
distributed by initial weight into thirty elevated cages (Figure 2) and randomly assigned to three
treatments; control (T1, 100% CF), and addition of corn stalk silage (CS) at 25% (T2) and 50%
(T3) levels. The silage was prepared from a Mayorbela variety, harvested at 90 d. of growth
forage. The was chopped in to pieces of 2 -3 cm length and ensiled in 8-gal plastic silos for a
minimum of 21 d. Diets were offered daily at 5% of rabbit live weight on dry matter a basis
during 59 days (Figure 3). Nutrient composition of both feedstuffs was determined using
standard procedures (AOAC, 1990; Van Soest et al., 1991), while pH and concentrations of
fermentation products of CS were determined in a commercial laboratory. The offered feed and
orts were weighted daily and total intake was calculated. Rabbit body weight was recorded
weekly (Figure 4) and used to determine total and average daily gain and feed to gain
conversation ratio. After 60 days of experimentation the animals were sacrificed in the
slaughterhouse of the Small Animal facilities in Lajas (Figure 5).
2
Figure 1. Pavilion for Rabbit Production
Figure 2. Elevated Rabbit Cages
Figure 3. Diet Daily Offering
At slaughter hot carcass weight was recorded (Figure 6) to determine carcass-dressing
percentage. Liver, kidney and stomach weighs were also recorded and expressed as percentage
of final weight (Figure 7). Data collected on feed intake, weight gain, feed conversion, carcass
dressing percentage, and organ weights were statically analyzed using a completely randomized
design with 10 replicates per treatment and Bonferroni-test for mean separation.
Figure 4. Rabbit Body Weight Measurement
Figure 5. Slaughtered Animals
Figure 6. Hot Carcass Weight Measurement
Figure 7. Organ Weight Measurement
3
Results and Discussion
The nutrient profile of the CF utilized in this experiment is in agreement with the manufacture’s
label, while the CS had a chemical composition and ensiling characteristics typical of those
observed for corn stalks ensiled in a tropical environment (Table 1). Obviously the starch content
of only 0.4% is much lower than that of CS including the grain. Total intake was higher (P<0.05)
in rabbits fed the 100% CF diet (59.0g/d), than in those offered 50% CS (40.7g/d), but not
significantly different from those fed 25% CS (49.53g/d). Control rabbits showed higher
(P<0.05) daily gain (T1=17.5 g, T2=12.4 g, T3=6.60 g) and more efficient feed conversion than
animals receiving either level of CS in the diet (T1= 3.38, T2= 4.01, T3= 6.1). Carcass yield
percentage was lower (P<0.05) in animals fed the diet containing 50% CS than in those fed 0 and
25% CS. Similar organ weights as a percentage of final rabbit weight were observed in the three
treatments. In this experiment inclusion of both levels of CS resulted in lower intake and
productive performance than that obtained by rabbits fed 100% CF. The CS - containing diets
had lower crude protein and starch contents than the 100% CF, which likely affected animal
performance. Therefore, to accomplish desirable growing rates in rabbits fed CS, ensiling of
vegetative material with higher starch content in combination with the use of protein
supplementation would seem to be necessary. Under the conditions of the present experiment,
inclusion of CS in the diet at the 25% level did not significantly affect rabbit carcass yield, but
lower yield was observed when CS was included at 50%, which is probably a reflection of lower
nutrient density of the diet.
Table 1. Chemical composition and fermentation profile of commercial feed and corn silage
Chemical Composition (%)
Item
CF
CS
CS
DM
93.1
26.4
pH
3.92
OM1
93.5
97.6
Lactic Acid
6.611
ASH1
6.5
2.4
CP1
Starch1 NDF1
ADF1 Lignin1
17.6
17.8
37.1
22.1
6.0
9.5
0.4
63.5
41.1
5.9
Ensiling Characteristics (%)
Acetic
N-H3/N
Acid
L/A Butyric Acid Propionic Acid
Total
1.961
3.371,2
.171
.041
10.1
1
Dry matter basis, 2Lactic acid/acetic acid ratio
Conclusion
Corn silage at 25 and 50% inclusion rates can be use in growing rabbit diets but with lower
productive performance than that obtained by animals fed 100% CF especially at the higher level
of inclusion. Inclusion of CS in the diet at the 25% level resulted in only a
small not significant effect on rabbit carcass yield.
4
Table 2. Intake, performance, and carcass yield of meat – type rabbits fed with different
proportions of commercial feed and corn stalk silage
Component
Intake (g/d)
CC
CS
Total
100:0
Proportion CF:CS
75:25
59.03a
---59.03a
38.9b
10.5b
48.5ab
22.4c
18.3a
40.7b
.01
.01
.01
Body weight (g)
Initial
Final
695.0
1732.1a
699.4
1434.7b
688.1
1077.6c
.97
.01
Gain (g)
Daily
Total
17.5a
1037.3a
12.4b
735.27b
6.6c
389.4c
.01
.01
Feed Conversion Ratio
3.38b
4.01b
6.17a
.01
Hot carcass yield (%)
49.2a
47.7a
43.8b
.01
Organ weigh as % final
weight
Liver
Stomach
Kidney
5.91
14.0
1.3
5.93
13.2
1.3
5.02
12.4
1.3
.12
.39
.93
50:50
P
a,b
Means with unlike superscripts in the same row differ p<0.05
Cited Literature
AOAC. 1990. Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists.
Arlington, VA.
Tonson, M.A.; S.A. Abd El-Lateif and M.R.M. Ibrahim 1999. Growth performance of NZW
rabbits fed diets containing cassava leaves and stems meal as a substitution of clover hay meal.
Egyptian J.Nutr. and Feeds, 2: 541. USDA/NASS. National Agricultural Statistical Services. Nass.usda.gov. 2014
http://agcensus.usda.gov/Publications/2012/Full_Report/Puerto_Rico/st72_1_014.pdf
Van Soest, P. J., Robertson J. B. and Lewis, B. A. 1991. Methods for dietary fiber, neutral
detergent fiber, and non-starch polysaccarides in relation to animal nutrition. J. Dairy Sci. 74:
3583 – 3597.
5
Expression of prolactin receptor in the skin of dry Holstein cows with phenotypic
differences in hair coat
Natalie M. Vázquez and Esbal Jiménez
Abstract
The development of technologies to ameliorate the effects of heat stress in dairy cattle to
maintain high milk production has been a long-term goal of researchers. These technologies in
many cases augment the costs of producing milk. Recently, phenotypic differences (animals with
shorter hair) were discovered in dairy cattle which may be an adaptation to heat stress. Increased
secretion of prolactin has been observed in animals under heat stress. This hormone, which acts
by activating the prolactin receptor (PRLR), also has been implicated with the growth of hair
follicles in many mammals. Consequently, a study was performed to investigate if differences
exist in the expression of PRLR between short-haired (SH) and normal-haired (NH) cows. Skin
biopsies were obtained from 12 Holstein non-pregnant, dry cows; six classified as SH and six
NH. RNA was extracted from the skin biopsies for cDNA synthesis. Further amplification and
determination of relative expression of PRLR was determined by using specific primers and Real
time PCR analysis. The relative expression of PRLR in SH and NH cows was similar (P=0.17).
Numerically, the relative expression of PRLR in SH cows was 1.09 times greater than NH cows.
Further investigations should test if structural differences exist in PRLR of SH and NH cows,
which might increase or decrease the actions of prolactin in the skin of these animals.
Keywords: short hair, prolactin receptor expression, dairy cattle
Resumen
El desarrollo de tecnologías para disminuir los efectos del estrés por calor en el ganado lechero,
para mantener una alta producción de leche, ha sido un objetivo por muchos años de los
investigadores. El problema con estas tecnologías es que aumentan los costos de producción de
leche. Recientemente, diferencias fenotípicas (animales con pelo más corto) en la capa del pelo
fueron descubiertos en el ganado lechero, lo que puede ser una adaptación al estrés por calor. Se
ha observado que la secreción de prolactina aumenta en animales bajo estrés por calor. Esta
hormona, que actúa mediante la activación del receptor de prolactina (PRLR), también ha sido
implicada con el crecimiento de los folículos pilosos en muchos mamíferos. Por lo tanto, se
realizó un estudio para investigar si existen diferencias en la expresión de PRLR entre vacas con
pelo corto (SH) y pelo normal (NH). Se obtuvieron biopsias de la piel de 12 vacas Holstein no
preñadas y horras; seis clasificadas como SH y seis NH. Se extrajo ARN de las biopsias de piel
para la síntesis de cDNA. Luego se amplificó y determinó la expresión relativa de PRLR
mediante el uso de cebadores específicos y análisis de PCR en tiempo real. La expresión relativa
de PRLR en vacas SH y NH fue similar (P = 0.17). Numéricamente, la expresión relativa de
PRLR en vacas SH fue 1.09 veces mayor que las vacas NH. Estudios adicionales deben
realizarse para investigar si existen diferencias estructurales en PRLR de vacas SH y NH, que
puedan aumentar o disminuir la acción de prolactina en la piel de estos animales.
Palabras Claves: pelo corto, la expresión del receptor de prolactina, ganado lechero
6
Introduction
The dairy industry suffers an economic crisis due to increases in production costs such as food,
water, electricity and labor. It is therefore very important to find methods to improve
productivity efficiency in dairy farms. Phenotypic differences in hair coat were discovered in
dairy cattle possibly as an adaptation to heat stress. Specifically, the slick hair (SH) cow is the
animal with shorter, thinner, and shinier hair (Olson, 2006). There have been several studies that
compare milk production between both groups that found that SH cows have a higher milk
production, lower vaginal temperature and lower respiration rate compared to NH (Olson, 2006).
Prolactin (PRL), known primarily as a lactogenic hormone, has been observed to increased in
animals under heat stress. This hormones, which acts by activating the prolactin receptor (PRLR)
has also been implicated in the process of angiogenesis, osmoregulation, immune response, and
hair growth and development; it also has a direct relationship to the environment’s temperature
(Alamer, 2011). The PRLR are found mainly in the hair follicle, skin and around the sweat
glands (Nixon et al., 2002). SH cows produce more PRL to keep equilibrium and maintain
thermoregulation compared to NH cows (Collier et al., 2008). Stressed cows will experience
increases in the production of PRL which in turn will increase the amounts of the receptors.
Hence, it is important to understand PRLR expression in the skin of animals with different hair
coat.
Materials and Methods
After shaving of the rump, skin biopsies (5mm) from dry Holstein cows (n=6 per treatment; SH
and NH cows) were obtained and immediately stored in liquid nitrogen for subsequent RNA
extraction. Skin samples were then homogenized using a Polytron and RNAzol (Molecular
Research Center, Cincinnati, OH). Total RNA was isolated according to the manufacturer's
instructions. RNA concentration and quality was measured using a nanospectrophotometer.
Reverse transcription and Real Time PCR amplification were performed by using SYBR FAST
kit KAPPA ONE-STEP qRT-PCR in a final volume of 20 ul with total RNA of 40ng. Reaction
cycles consisted of the cDNA synthesis at 42 ° C for 5 minutes followed by inactivation of the
RTMix at 95 ° C for 10 minutes. Further amplification was performed with 45 cycles of a
denaturation step for 4 seconds at 95 ° C and an alignment and extension step at 60 ° C for 30
seconds. PRLR and Glycerolaldehyde-3-phosphate-dehydrogenase (GAPDH) were designed
using PrimerQuest (Integrated DNA Technology, IDT). The PRLR primers were 5’GGAAGGAGCCAACATGAA-3’ and 5’-TCAGCGTGTAATTGGTAGG-3’ for forward and
reverse, respectively. The GAPDH primers were 5’-CAGCGACACTCACTCTTCTAC-3’ and
5’-GGAAGTCAGGAGATTCTCAGT-3’ for forward and reverse, respectively.
The relative expression of PRLR was similar (P=0.17) between SH and NH cows. Numerically,
the relative expression of PRLR in SH cows was 1.09 times greater than NH cows (Figure 1).
The skin is one of the organs with the lowest expression of PRLR, and perhaps a greater number
of animals were needed to detect statistical differences. Evidence has been found that supports
7
the existence of a major gene responsible for producing short, thin and slick hair; it is known to
be a mutation in the chromosome 20, specifically in the PRLR (Olson et al., 2003). Since no
differences in the expression of the gene itself were detected, maybe it is because it affects the
function of the PRLR instead of its expression in the animal. Collier et al. (2008) compared
production of PRLR during heat stress between SH and NH and concluded that SH produce
more PRL than NH to maintain their thermoregulation. Another research studied PRLR mRNA
expression and PRL concentration in the plasma between SH and NH which affected the hair
cycle acting directly at cellular level where they used PRL inhibitors found out that as expression
levels of total PRLR mRNA gradually decreased in skin, PRL concentration in plasma also
decreased and fiber growth rates increased at the same time (Chunwang et al., 2010). In 2011,
Alamer et al. determined that a direct relationship exists between PRL and the environment’s
temperature; whereas the environmental temperature increased so did the concentration of PRL
in the blood. A study where PRL was increased to induce follicle cycles during which PRLR
expression changes were determined by ribonuclease assays suggested that the cycle-related
patterns of PRLR expression were regulated by PRL, thereby modulating hormonal
responsiveness of seasonally growing hair follicles (Nixon et al.,2002).
Figure 1. Relative expression of prolactin receptor in Holstein cows with different hair coat
Conclusion
The expression of the prolactin receptor in the skin between slick hair cows and normal hair
cows was similar (P<0.05). Further investigations should test if structural differences exist in
8
PRLR of SH and NH cows, which might increase the actions of prolactin in the skin of these
animals.
Cited Literature
Alamer, M. (2011). The Role of Prolactin in Thermoregulation and Water Balance During Heat
Stress in Domestic Ruminants. Asian Journal of Animal and Veterinary Advances, 6(12), 1153–
1169.
Chunwang, Y., Lixin, D., Wei, Z., Xiaoping, K., & ZHIHAI, J. (2010). Expression of Prolactin
Receptor mRNA after Melatonin Manipulated in Cashmere Goats Skin during Cashmere
Growth. Asian- Australasian Jorurnal ofAnimal Science, 23(10), 1291–1298.
Collier, R. J., Collier, J. L., Rhoads, R. P., & Baumgard, L. H. (2008). Invited review: genes
involved in the bovine heat stress response. Journal of dairy science, 91(2), 445–454.
Nixon, A. J., Ford, C. A., Wildermoth, J. E., Craven, A. J., Ashby, M. G., & Pearson, A. J.
(2002). Regulation of prolactin receptor expression in ovine skin in relation to circulating
prolactin and wool follicle growth status. The Journal of endocrinology, 172(3), 605–614.
Olson, T. (2006). Evaluation and utilization of the Slick hair gene in Florida and Caribbean
Dairies. USDA NIFA.
Olson, T., Lucena, C., Chase, C. C., & Hammond, A. C. (2003). Evidence of a major gene
influencing hair length and heat tolerance in cattle. Journal of Animal Science, 81(1), 80–90.
9
Use of exopolysaccharides producing lactic acid bacteria in the manufacture of low fat
Gouda cheese
Milton S. González and Leyda Ponce de León
Abstract
In different shapes, textures and flavors, dairy products has been the joy and the primary source
of calcium, and other nutrients such as potassium and vitamin D in almost everyone’s daily diet.
Among them all, cheese demand and consumption has increased all over America in the last
decades. Particularly, Gouda cheese is one of the most popular cheeses in the world, by its dense
texture and creamy flavor. Although, it is recommended to consume low-fat or fat-free dairy
products, most of the regular products like Gouda cheese, contain many saturated fats and LDL
(Low-Density Lipoprotein) cholesterols. If not eaten in the right portions can increase the risk for
coronary heart diseases and diabetes. Also, the majority of low fat or fat free cheeses tend to be
dryer, harder, and have a “rubbery” texture, which overshadows the possibility of becoming part
of the consumer’s preference. Therefore, lactic acid bacteria are implemented as natural
thickeners to improve the texture. The Streptococcus thermophilus ssp. strain is a high efficient
thermophile organism that produces an exopolysaccharide secretion (EPS), an extracellular
enzyme that works as a biological defense mechanism against environmental threats to the cell,
especially against dehydration. The EPSs produced by S. thermophilus form a barrier that
increase the viscosity and moisture, by preventing the cheese from draining much of the water
that is lost in the molding and pressing process. Preliminary data shows that EPS cheeses have a
greater compressibility in comparison with Non-EPS ones. Consequently, more desirable traits
are found in the EPS low fat Gouda cheese, like a smoother, moister, and creamer texture than
regular manufactured low fat Gouda cheese. Therefore, the EPS treatment is a potential
alternative to improve the organoleptic qualities such as texture and mouthfeel in low fat Gouda
cheese.
Keywords: Low Fat Gouda Cheese, EPS, Texture Improvement
Resumen
En diferentes formas, texturas y sabores, los productos lácteos han sido el encanto y la fuente
primaria de calcio, y otros nutrientes como potasio y vitamina D en la dieta diaria. Entre todos
ellos, la demanda y el consumo de queso en Estados Unidos ha aumentado en las últimas
décadas. Particularmente, el queso Gouda es uno de los quesos populares, por su textura densa y
sabor cremoso. Aunque, se recomienda consumir productos lácteos reducidos en grasa o
descremados, la mayoría de los productos regulares como el queso Gouda, contienen grasas
saturadas y el colesterol LBD (Lipoproteína de Baja-Densidad). Si no se consumen en las
porciones correctas puede aumentar el riesgo de enfermedades coronarias del corazón y diabetes.
Además, la mayoría de los quesos bajos en grasa o descremados tienden a ser más secos, más
duros, y tienen una textura "gomosa", que reduce la posibilidad de formar parte de la preferencia
del consumidor. Por lo tanto, se implementan bacterias productoras de ácido láctico como
espesantes naturales para mejorar la textura. La cepa de Streptococcus thermophilus ssp. es un
organismo termófilo de alta eficiencia, el cual produce una secreción de exopolisacáridos (EPS),
sustancia extracelular que funciona como un mecanismo de defensa biológico contra amenazas
ambientales para la célula, especialmente contra la deshidratación. El EPS producido por S.
10
thermophilus forma una barrera que aumenta la viscosidad y humedad, previniendo así que el
queso drene la mayor parte del agua que se pierde en el proceso de moldeado y prensado. Los
datos preliminares muestran que quesos con EPS tienen una mayor capacidad de compresión en
comparación con los quesos Sin-EPS. Consecuentemente, se encuentran más rasgos deseables en
el queso Gouda bajo en grasa con EPS, como mayor humedad, y una textura más suave y
cremosa que el queso Gouda bajo en grasa fabricado tradicionalmente.
Palabras Claves: Queso Gouda Bajo en Grasa, EPS, Mejoramiento de Textura
Introduction
Lactic acid bacteria (LAB) have become an important component of dairy products industry, by
its efficient fermentation qualities (Cogan et al., 2007). Two of the most common LAB used as
starter cultures in cheese production are Lactobacillus delbrueckii spp. and Streptococcus
thermophilus spp., both work effectively at a temperature range of 36-45°C. Despite their
facultative differences, S. thermophilus spp. being aerobic, and L. delbrueckii spp. being
anaerobic, they form a suitable combination for long term fermentation as they complement each
other in the different stages of cheese manufacture. Moreover, S. thermophilus spp. is capable of
synthesize exopolysaccharides (EPS), an extracellular polymers that may be assembled as
capsular polysaccharide, or may form a slime layer loosely attached to the cell surface or
secreted into the environment. (Madigan et al., 1997). EPS are theorized to protect bacteria
against detrimental environmental conditions, also it has been shown to protect the cells against
phage infection. (Kang and Cottrell, 1979). In addition, it has been thought that EPS produced by
LAB have beneficial impact on human health such as cholesterol-lowering ability (Pigeon et al.
2002), immunomodulation, and antitumoral activities (Kitazawa et al., 1998; Chabot et al.,
2001), and prebiotic effects. (DalBello et al., 2001; Korakli et al., 2002). One of the most
important EPS attribute is its water-binding properties, which can be applied to reduced fat
cheeses. It increases the moisture in the non-fat portion, interfere with protein-protein
interactions and reduce the rigidity of the protein network, while increasing the viscosity of the
serum phase (Hassan, 2008). Reduced-fat cheeses tend to be drier, and due to high casein content
imparts a firm and rubbery body and texture (Emmons et al., 198; Mistry et al., 1993; Metzger
and Mistry, 1995; Mistry, 2001). Thanks to the advances in biotechnology industry and food
science, this EPS custom can be used as a substitute to the fat properties, providing consistency,
moisture and lowering synergies (Ruas-Madiedo et al., 2005). This represents a reduction in the
saturated fats and LDL (Low-Density Lipoprotein) content in cheese, while potentially lowering
the risk to suffer coronary heart diseases, obesity and diabetes (USDA, 2011).
The project was conducted in a Milk and Dairy Products and Food Microbiology laboratories
located in the Alfredo Ramírez de Arellano y Rosell building at the University of Puerto Rico,
Mayagüez Campus.
11
Cheese Making
Approximately three hundred and sixty liters of whole milk were obtained from UPRM
Agricultural Experiment Station Dairy Farm in Lajas. The milk was divided into two portions of
nearly one hundred and eighty liters and the fat percent was determined by Babcock method.
After the fat percent determination to a 3.0%, one hundred and eighty liters are skimmed (figure
1), divided into two portions of ninety liters of skimmed milk, and standardize up to a 1.5% by
calculating and adding 4.4 pounds of warmed skimmed cream to each portion by the Pearson
Square method. First two 1.5% fat milk portions were pasteurized in two tanks at 64°C for thirty
minutes, and let cool. At 42°C the Sacco® Lyofast commercial starter cultures were added
respectfully to each tank: one with Non Exopolysaccharide-Producing Streptococcus
thermophilus spp., and Lactobacillus delbrueckii spp. bulgaricus; and another with
Exopolysaccharide-Producing Streptococcus thermophilus spp., Lactobacilus delbrueckii spp.
bulgaricus, and Lactobacillus delbrueckii spp. lactis. Titulable Acidity tests were performed at
this point to verify if the cultures were producing lactic acids. The animal rennet was applied to
the milk, and let them set for about thirty minutes until it forms the clot. The cheese curds were
cut with knives and cooked in the whey for thirty minutes at 42°C. Whey was drained from the
tank and the cheese curds were directly salted. The cheese curds are placed in a rectangular mold
with a cheese cloth, and were compressed using a hydraulic press for eighteen hours. The cheese
blocks were vacuum packed and placed in a cava at 15°C for three months. These procedures
were repeated with the 3.0% fat milk portions, excluding the skimming and standardizing
procedures. Through ripening process, every fourteen days samples were collected and tested
from each cheese block.
Texture Analysis
The texture firmness analysis consisted in cutting cheese in a wheel shape with about the same
shape and sizes. These samples were kept at 25°C, were compress with a Pasta Adhesive
Aluminum probe with measures of 50mm X 37mm with 1Kg of force, by a TX-XT2 Texture
Analyzer that registered the peak positive distance that progress on the cheese sample surface,
determining the hardness of the cheese.
Moisture Analysis
The moisture percent in cheese sample was determined by a Microwave Humidity/Solid
Analyzer System by placing 2-3 grams of shredded cheese in fiberglass pads into the CEM
machine. The cheese samples were dried and the differences in weight were determined.
Microbial Enumeration
In addition, a LAB Enumeration test was performed by homogenizing 11 grams of cheese
sample in a 20% solution of sodium citrate. Serial dilutions were made using peptone water at
1%, and then were served in petri dishes using the pour plate technique. LAB were enumerated
using MRS agar and incubation for 48 hours at 36°C in a anaerobic jar (Wehr, 2004).
12
This study investigated the contributions of the EPS produced by Streptococcus thermophilus
spp. and its properties to retain moisture and improve the texture of the low fat Gouda cheese.
Analysis on 1.5% and 3.0% milk fat cheeses made with EPS-producers commercial starter
cultures showed to increase the moisture levels through the ripening process, even though they
lose some humidity through time. According to Figure 2, the EPS cheeses had the higher
moisture values due to its water-binding property. Although, unexpectedly the 1.5% Milk Fat
Non-EPS cheese had a sudden increment in its moisture level, perhaps this could result from a
change in the environment humidity levels or an error from the CEM analyzer. Also a texture
firmness analysis was performed by applying 1 Kg force over the cheese sample surface. As
Figure 1 shows, 3.0% milk fat cheeses had a bigger peak positive distance through the first three
weeks, and the they decrease to 4.296 mm and 2.279 mm by 56 days due to the available
moisture and cream in cheese samples, while being EPS one the higher value. On the other hand,
1.5% milk fat cheeses in the first two weeks they remain constant, but from that point the NonEPS decreased its peak positive distance to a 0.706 mm by 56 days, while the EPS cheese
increased its value to 1.716 mm, meaning a softer texture. Furthermore, LAB enumeration was
made to ensure if the populations where in the specific range to carry the metabolic activities in
the cheese. The LAB populations shown by table 1 presents bigger numbers of colonies for the
3.0% milk fat cheeses than 1.5% milk fat, while the EPS cheeses than Non-EPS ones. Thus to
the higher levels of moisture and viscosity in the cheese from the EPS, a higher number of LAB
may be found.
Table 1. Lactic Acid Bacteria Enumeration in dilutionsof 10-7
Cheeses
LAB Population
3.0% Milk Fat Non-EPS
1.28 x109
3.0% Milk Fat EPS
1.29 x109
1.5% Milk Fat Non-EPS
8.0 x108
1.5% Milk Fat EPS
8.39 x108
Figure 1. Gouda Cheese Texture Development
13
Figure 2. Humidity percentage in cheese samples
Conclusions
EPS-Producing LAB such as Streptococcus thermophilus spp. could be implemented to increase
the moisture retention in low fat Gouda cheese, since also they were in the specific range of
population to perform the metabolic activity. In Addition, the EPS helps to decrease the low fat
Gouda cheese firmness. Thus EPS properties embodies positive traits for the dry and rubbery
texture of the low fat Gouda cheese.
14
Cited Literature
Chabot, S., H. L. Yu, L. De Léséleuc, D. Cloutier, M. R. van Calsteren, M. Lessard, D. Roy, M. Lacroix,
and D. Oth. 2001. Exopolysaccharide from Lactobacillus rhamnosus RW-9595M stimulate TNF, IL-6
and IL- 12 in human and mouse cultured immunocompetent cells, and IFN-g in mouse splenocytes. Lait
81: 683-697.
Cogan, T., Beresford, T., Steele, J., Broadbent, J., Shah, N., & Ustunol, Z. (2007). Invited Review:
Advances in Starter Cultures and Cultured Foods. J. of Dairy Science, 90, 4005-4021.
Dal Bello, F. D., J. Walter, C. Hertel, and W. P. Hammes. 2001. Invitro Studyof prebiotic properties of
levan-type exopolysaccharides from lactobacilli and non-digestive carbohydrates using denaturing
gradient gel electrophoresis. Syst. Appl. Microbiol. 91: 232-237.
Emmons, D. B., M. Kalab, E. Larmond, and R. J. Lowrie. 1980. Milk gel structure X. Texture and
microstructure in Cheddar cheese made from whole milk and from homogenized low fat milk. J. Texture
Stud. 11: 15-34.
Hassan, A. 2008. ASDA Foundation Scholar Award: Possibilities and Challenges of ExopolysaccharideProducing Lactic Cultures in Dairy Food. J. Dairy Sci. 91(4): 1282-1296.
Kang, K. S., and I. W. Cottrell. 1979. Polysaccharide, p. 417-481. in H. J. Peppler and D. Perlman
nd
(ed.),Microbial technology: Microbial processes, 2 ed., Vol 1. Academic Press, Inc., New York.
Kitazawa, H., T. Harata, J. Uemura, T. Saito, T. Kaneko, and T. Itoh. 1998. Phosphate group requirement
for mitogenic activation of lymphocytes by an extracellular phosphopolysaccharide from Lactobacillus
delbrueckii spp. bulgaricus. Int. J. Food Microbiol. 40: 169-175.
Korakli, M., M. G. Gänzle, and R. F. Vogel. 2002. Metabolism by bifidobacteria and lactic acid bacteria
of polysaccharide from wheat and rye, and exopolysaccharides produced by Lactobacillus
sanfranciscensis. J. Appl. Microbiol. 92:958-965.
th
Madigan, M. T., J. M. Martinko, and J. Parker. 1997. Brock Biology of Microorganisms. 8 ed. Prentice
Hall International Ltd., London, UK.
Metzger, L. E., and V. V. Mistry. 1995. A new approach using homogenization of cream in the
manufacture of reduce fat Cheddar cheese. 2. Microstructure, fat globule distribution, and free oil. J.
Dairy Sci. 78: 1883- 1895.
Mistry, V. V. 2001. Los fat cheese technology. Int. Dairy J. 11: 413-422.
Mistry, V. V., and D. L. Anderson. 1993. Composition in microstructure of commercial full-fat and lowfat cheeses. Food Struct. 12: 259-266.
Pigeon, R. M., E. P. Cuesta, and S. E. Gilliland. 2002. Binding of free bile acids by cells of yogurt starter
culture bacteria. J. Dairy Sci. 85: 2705-2710.
Ruas-Madiedo, P., and De los Reyes-Gavilán, C. 2005. Invited Review: Methods for the Screening,
Isolation and Characterization of Exopolysaccharide Produced by Lactic Acid Bacteria. J. Dairy Sci.,
88(3), 843-843.
USDA MyPlate Dairy Group -- All about the Dairy Group. (n.d.). Retrieved March 27, 2015, from
http://www.choosemyplate.gov/food-groups/dairy.html
Wehr, H., & Frank, J. (2004). Standard Methods for the Examination of Dairy Products (17th ed.).
Washington DC: American Public Health Association.
15
The effect of scrotal temperature on semen quality parameters in Holstein Friesian and
Jersey bulls
Camille Cordero-Aponte and Jaime E. Curbelo-Rodríguez
Abstract
In Puerto Rico, the use of exotic bovine dairy breeds to increase milk production has been
commonly implemented for decades. High temperatures and humidity, characteristic of
our island, have been reported to adversely affect the reproductive performance of bulls.
This study was conducted to evaluate the effect of scrotal temperature on semen quality
parameters in Jersey and Holstein-Friesian bulls. Jersey and Holstein bulls (n=3/breed)
were evaluated. Scrotal surface temperature was determined in different anatomical
locations of the scrotum [i.e., proximal (P), medial (M) and distal (D)] using infrared
thermography. Several semen quality parameters were evaluated in each bull using the
computer-assisted sperm analysis system (HT CASA II). No significant differences
between semen quality parameters among bull breeds where observed except for
proximal (p=0.0153) and distal droplet (p=0.0405), which were superior in Jersey bulls.
Scrotal temperatures of Jersey bulls were significantly lower than Hosltein bulls with
Mmin of 32.7± 0.07°C vs 34.35 ± 0.30 °C (p=0.005) and Davg 32. 72 ± 0.47 vs 34.51±
0.42 °C (p= 0.0547), respectively. The results found in this study suggest that Jersey bulls
posses superior reproductive capacity than Holstein bulls, in part by their superior scrotal
thermoregulatory capacity.
Keywords: Semen quality, scrotal surface temperature, thermography imaging
Resumen
En Puerto Rico, el uso de razas lecheras bovinas exóticas, empleadas para incrementar la
producción de leche, ha sido implementado durante décadas. Altas temperaturas y
humedad, características de nuestra isla, tienen impacto adverso sobre la capacidad
reproductiva de los toros. Este estudio se realizó para evaluar el efecto de la temperatura
escrotal sobre los parámetros de calidad del semen en toros lecheros. Se evaluaron toros
de la raza Jersey y Holstein (n = 3 / raza). Se determinó la temperatura escrotal en
distintas localidades anatómicas del escroto [i.e., proximal (P), medial (M) y distal (D)]
utilizando termografía infrarroja. Varios parámetros de calidad de semen se evaluaron en
cada toro utilizando el sistema de análisis de semen (HT CASA II). En resumen, no se
observaron diferencias significativas en parámetros de calidad del semen entre razas de
toros, a excepción de la gota citoplásmica proximal (p = 0.0153) y la gota citoplásmica
distal (p = 0.0405), las cuales fueron superiores en los toros Jersey. Temperaturas
escrotales de toros Jersey fueron significativamente menores que la de toros Holstein, con
un Mmin de 32.7 ± 0.07 °C vs 34.35 ± 0.30°C (p = 0.005) y Davg 32. 72 ± 0.47 vs 34.51 ±
0.42 °C (p = 0.0547), respectivamente. Los resultados encontrados en este estudio
sugieren que los toros Jersey poseen capacidad reproductiva superior a toros Holstein, en
parte por su mayor capacidad termoregulatoria escrotal.
Palabras Claves: Calidad de Semem, temperatura de superficie escrtola, termografia
16
Introduction
The geographical position of Puerto Rico is described as tropical climate governed by
high temperatures and humidity throughout the year. The dairy industry of our island is
the most important economically. Dairy farmers have implemented different genetic
strategies such as the use of exotic dairy breeds from temperate regions (e.g., Europe) to
increase milk production. It has been reported that high ambient temperatures have a
negative effect on the productive performance of cattle, whether a limitation on milk
production, pregnancy rate, growth rate, and in the case of dairy bulls, their reproductive
capacity and semen quality. Research studies have found a relationship between heat
stress and semen quality parameters (Rios et al. 2013). In Puerto Rico there is insufficient
or no information on semen quality parameters and reproductive capacity of local dairy
bulls. This information is of relevant importance to increase reproductive efficiency in
local dairy farms Lunstra and Coulter (1993) found that infrared thermography of scrotal
surface is a useful tool for determining the reproductive potential of diary bulls and that
there was a clear association between the estimation of scrotal thermoregulation and the
quality of semen and fertility in bulls. Similarly, it has been found that higher
temperatures cause testicular degeneration (Rios et al. 2013). That is why, it is expected
that high temperatures in which bulls are constantly exposed will cause a negative effect
on semen quality parameters and therefore a decrease in their reproductive capacity.
Therefore, the aim of this study was to evaluate the effect of scrotal temperature on
semen quality parameters in Jersey and Holstein Friesian bulls.
Two dairy bull breed from two commercial dairy farms were used. The dairy "Tai South
Farm" provided 3 Jersey bulls and the dairy "Enriqueta" provided 3 Holstein Friesian
bulls. Both dairies are located in Lajas, Puerto Rico. Each bull was restricted individually
in chute (Fig. 1 panel A). Once restrained, infrared thermography of the scrotal surface
was performed on each bull (Fig. 1 panel B). The thermal camera (T8 FLIR) was
positioned 3ft from the rear of each bull. Also, certain observations were made and in
case of any presence of blood or wounds annotations were made. Then, using an
electroejaculator, a semen sample was obtained from each bull (Fig. 1 panel C). Once
each sample was collected, an extender formula, previously prepared in the Reproduction
Laboratory, was applied in a 1:1 ratio, and time of collection and application of extender
was annotated. Then, a questionnaire was provided to complete information including
animal management practices and animal characteristics (data not shown). The samples
were then transferred to the Molecular Genetics and Musculoskeletal Biology laboratory
at the Department of Animal Science. Each sample was diluted using a micropipette in a
1:10 ratio with a buffer solution. Three microliters from the sample dilution was obtained
17
and placed in a Leja 4 Chamber 20 Micron Slide. Finally an analysis of semen quality
parameters was performed using the HT CASA II system.
Figure 1. Restrainment of bulls (panel A), infrarred thermography imaging of scrotal surface (Panel B), and
semen collection (Panel C) of bulls included in the study.
Sperm motility parameters
The Static parameter represents the non-moving sperm cells in a semen sample. No
significant differences (p0.4365) were observed between breeds (Fig. 4). A possible
explanation for this is the high variability observed (SE ± 25.0549 and 8.6845) for
Holstein and Jersey bulls respectively. The Progressive parameter considers sperm
moving with straightness and an average path velocity. No significant differences were
detected (p=0.6939). This could also be explained by the variability observed between
Holstein bulls (SE ±7.5810) and Jersey bulls (SE ± 4.3552) (Fig. 4). The Motile
parameter denotes a sperm that moves more than its head length from its original position
due to acquisition. In this case as well, there were no significant differences (p=0.4365).
High variability observed in Holstein and Jersey bulls (SE± 25.0549 and 8.6845,
respectively) could be a possible cause for no observable significant differences (Fig. 4).
Lastly, the slow motility parameter represents a sperm moving with less than the
minimum required for a curvilinear velocity or average path velocity. No significant
differences were observed (p=0.6596) among breeds.
Sperm morphology parameters
Bent tail represents any sperm with an exceeding bending rate. No significant differences
were observed (p=0.8176) between Holstein and Jersey bulls. Sperm are categorized as
having Coiled Tail if the tail bends 180º or more over its length. No significant
differences were observed (p=0.6432) between breeds. The DMR (Distal Midpiece
Reflex) indicates if the tail of the sperm is wrapped around a distal cytoplasmic droplet.
In this study, no significant differences were observed (p=0.5994). The Distal Droplet
indicates the presence of a cytoplasmic droplet further down the tail from the base of the
head while the Proximal Droplet represents a cytoplasmic droplet that is attached to the
midpiece or base of the head of the sperm. In both cases, these are considered as defects
and immaturity in sperm. Significant differences were observed (p= 0.0405 and 0.0153)
18
in the Distal and proximal droplet parameters respectively, between both breeds. These
significant differences might be associated with abnormal scrotal temperatures exhibited
in bulls.
Normal Sperm fraction and sperm concentration
The Normal fraction values show significant differences between both breeds (p=
0.0153). This value might be associated to the differences observed in scrotal
temperatures (lower in Jersey). The Motile concentration is derived from motile cells
only and the Total concentration includes the concentration of all parameters. In this
study no significant differences were observed in both cases with motile concentration
having p= 0.1051 and total concentration having p= 0.1961. In the case of total
concentration a high variability can be observed between Jersey (SE± 343.6) and
Holstein (SE± 16.0307) bulls (Fig. 6).
Table 1- Semen quality parameters percentages of motility for Jersey and
Holstein bulls individually and their average percentages
Breed
Parameters Percentage (%)
Jersey 1
Static
28.0
Progressive
11.8
Motile
72.0
Slow
11.5
Jersey 2
2.2
26.7
97.8
2.2
Jersey 3
Average
1.7
10.63
21.3
19.93
98.3
89.36
2.8
5.5
Holstein 1
Holstein 2
10.3
6.7
20.5
26.7
89.7
93.3
2.6
6.7
Holstein 3
83.6
1.5
16.4
13.4
Average
35.53
16.23
66.46
7.56
Table-2 Semen quality parameters percentages of morphology for Jersey and
Holstein bulls individually and their average percentages
Breed
Jersey 1
Jersey 2
Jersey 3
Average
Holstein 1
Holstein 2
Holstein 3
Average
Parameters Percentage (%)
Bent Tail
Coiled Tail
DMR
Distal Droplet
Proximal Droplet
13.5
0.0
2.6
5.36
1.3
6.7
11.9
6.63
5.1
0.0
0.3
1.8
2.6
0.0
0.0
0.86
0.7
2.2
0.0
0.96
0.0
0.0
1.5
0.5
4.5
6.7
6.4
5.86
3.7
0.0
2.4
2.03
9.1
2.2
15.3
8.86
29.5
46.7
31.3
35.83
19
Infrared thermography imaging of scrotal surfaces
Scrotal temperature was measured on three different anatomical locations: proximal,
medial and distal. Three different values, the maximum, minimum and average
temperature were obtained for each section of the scrotal surface of the bulls. For the
proximal variables, the Pmax (p=0.4847), Pmin (p=0.3642 ) and Pavg (p=0.3642 ) show no
significant differences between the breeds evaluated. Medial temperatures shown no
significant differences were observed for the Mmax (p= 0.6184). Significant differences
were observed for the Mmin (p=0.055), but no significant differences were observed for
Mavg (p=0.1739). For distal variables, the Dmax (p=0.1734) and the Dmin (p=0.1245)
demonstrated no signficiant differences between breeds, but in the case of Davg
(p=0.0547) significant diffrences were observed. These values in whicha significant
difference can be noticed suggest that Jersey bulls have thermoregulatory advantage over
Holstein bulls.
Percentages (%)
120
100
80
60
40
Jersey
20
Holstein
0
Semen Quality Parameters of Motility
Figure 2: Sperm motility parameters between Holstein (purple) and Jersey (gray) bulls. Data for each
parameter expressed in % ± SE. Significant difference in quality parameters between breeds expressed in
* p=values <0.05.
Percentages(%)
45
40
35
30
25
20
15
10
5
0
*
*
*
Jersey
Holstein
Semen Quality Parameters of Morphology
Figure 3: Sperm morphology parameters between Holstein (purple) and Jersey (gray) bulls. Data for each
* p=values <0.05.
20
1400
Concedntrartion M/ml
1200
1000
800
Jersey
600
Holstein
400
200
0
Concentration
Figure 4: Sperm concentration parameters between Holstein (purple) and Jersey (gray) bulls. Data for each
* p=values <0.05.
Maximum Infrared
Temperature (C±°)
38
37
36
35
34
33
32
31
30
Jersey
Holstein
Proximal
Medial
Distal
Anatomic Scrotal Locality
Figure 5: Anatomic scrotal locality maximum temperature readings between Holstein (purple) and Jersey
(gray) bulls. Data for each parameter expressed in % ± SE. Significant difference in quality parameters
between breeds expressed in * p=values <0.05.
Minimun Infrared
Temperature (Cº)
37
36
35
34
33
32
31
30
29
28
Jersey
Holstein
Proximal
Medial
Distal
Figure 6: Anatomic scrotal locality minimum temperature readings between Holstein (purple) and Jersey
21
*
Average Infrared Temperature
(C°)
38
37
36
35
34
*
33
32
Holstein
31
30
29
Jersey
Proximal
Medial
Distal
Figure 7: Anatomic scrotal locality average temperature readings between Holstein (purple) and Jersey
Conclusion
In this preliminary study, there was an evidently high varibility in sperm quality
paramters between Holstein-Friesian and Jersey bulls. The results confirm an association
between scrotal temperatures in bulls and semen quality parameters. Higher scrotal
temperatures in bulls suggest a larger parcentage of abnormalities in sperm such as, bent
tail, coiled tail, DMR, and proximal and distal droplets. Specifically there were
significant differences in distal and proximal droplet parameters between these two
breeds. In addition, Jersey bulls show numerically larger sperm concentration than
Holstein bulls. Despite the fact that both breeds’ reproductive capacity could be adversely
affected by high scrotal temperatures, the differences in abnormalities could imply that
Jersey bulls might be a more suitable breed for reproduction in tropical and subtropicl
regions in comparison to the Holstein Friesian breed.
Cited Literature
Fiaz, M., Usmani, R. H., Abdullah, M., Ahmad, T. 2009. Evaluation of semen quality ofHolstein
Friesian and Jersey bulls maintained under subtropical environment. PakVet J, 30(2):75-78
Lunstra, D. D., Coulter, G. H., 1993. Scrotal Thermography as a tool for predicting semen quality
and natural-mating fertility in young beef bulls.
Ríos, V. C., Ortiz, N. M., Valencia, A. F., Orjuela, J. A., 2013. Estrés calórico y ysu relación
con variables reproductivas en machos bovinos en la Amazonia Colombiana. Revista electrónica
de Veterinaria. Retrieved from
http://www.veterinaria.org/revistas/redvet/n040413/041309.pdf
22
The retained placenta malady in a Puerto Rican dairy cattle herd: prevalence, treatment
and effects on animal performance
Nicole M. Irizarry, Paul F. Randel, Héctor Santana and Ricardo Montañez
Abstract
Retained placenta (RP) is a reproductive dysfunction universally present in dairy cattle
worldwide. In Puerto Rico it has received rather little research attention. The present work,
conducted in the dairy herd of the University of Puerto Rico at Mayaguez, involves one phase of
experimentation, in which two medical procedures for treating RP are being compared: (T1)
hormones (oxytocin and prostaglandin) to expedite expulsion of the placenta plus antibiotic and
(T2) hormones only; with the object of determining if the antibiotic is really necessary to treat
routine cases of RP or if its use is not cost effective. Of the 68 parturitions that took place in the
herd from the start of experimentation, in Sept. 2014, until the present writing, in mid Jan. 2015,
only 6 cows retained the placenta for 24 hours or more (8.82% of incidence) of which 3 received
T1 and 3 received T2. Data collection will continue throughout the current academic semester.
The second phase of the work will involve a search of the herd records, covering the years 2012
to 2014 and part of 2015, to determine the overall prevalence of RP and possible differences in
prevalence due to the independent variables: parturition number (first vs. second or later), season
of the year (hotter vs. cooler months), and perhaps breed group (pure or high grade Holstein vs.
crossbred). Also of interest, is the effect of RP on subsequent reproductive performance as
judged by criteria such as interval until the next parturition, interval from calving to the next
service and number of inseminations to achieve pregnancy.
Keywords: Retained placenta, Dairy cattle, Antibiotic treatment
Resumen
La retención de placenta (RP) es un problema reproductivo comúnmente presente en el ganado
lechero a nivel mundial. En Puerto Rico este tema ha recibido muy poca atención en el ámbito
científico. Este trabajo, llevado a cabo en el hato lechero de la Universidad de Puerto Rico en
Mayagüez, envuelve una fase experimental, en donde dos tratamientos veterinarios para tratar la
RP están siendo comparados: hormonas oxitocina y prostaglandina para acelerar la expulsión de
la placenta con adición de antibiótico (T1) y hormonas exclusivamente (T2); con el objetivo de
determinar si el antibiótico es realmente necesario para tratar casos rutinarios de RP o si su uso
es o no costo efectivo. De los 68 partos ocurridos desde el inicio del experimento en septiembre
2014 hasta el presente en el mes de enero 2015, 6 vacas retuvieron la placenta por 24 hrs o más
(8.82% de incidencia) de las cuales 3 recibieron T1 y 3 recibieron T2. La recolección de datos
continuará por lo que resta de este semestre académico. La segunda fase del experimento
envuelve una búsqueda en los récords de la vaquería desde el 2012 hasta el 2014 y parte del 2015
para determinar la incidencia total de RP y diferencias en estos casos debido a variables
independientes, tales como: número de partos (primer parto vs. partos múltiples), época del año
(meses calientes vs. meses frescos), y la raza (Holstein puras vs. cruce de razas). También se
desea conocer el efecto de RP en el futuro desempeño reproductivo de la vaca evaluando el
intervalo entre partos, intervalo entre un parto y el siguiente servicio, y el número de
inseminaciones requeridas para lograr la preñez.
Palabras Claves: Retención de placenta, Ganado lechero, Antibióticos
23
Introduction
After calving, the cow usually expels the placenta in the first 24 hours by the action of hormones
naturally released in the organism to degrade the placenta and stimulate uterine contractions. If
the placenta is not expelled properly, leaving remanents inside the system for a long time, an
infection could develope. The retained placenta makes the perfect medium for bacteria to grow,
causing the bacteria present in the organism combined with the bacteria that enter the system at
the moment of calving to develop a massive infection. These infections are designated as metritis
or endometritis at varying levels of severity, which are different types of uterine infections. Some
signs of infection are: strong stench, uterine discharges with a brownish red color, poor appetite,
decrease production, absence of striations due to the low rhythm of uterine contractions and
unstable temperature or fever in severe cases. Chronic or continuous cases of metritis affect the
fertility of the cow; in detriment to the production and eventually, the farm income. One of the
causes associated with the RP problem is the diet during the dry period. The nutritional factor
during this period is vital, providing the cow with the necessary nutriments for the development
of the embryo. In the south of Puerto Rico, pastures are usually of low quality in certain periods
of the year, especially in the dry seasons, which may be a causal factor. Different methods exist
to treat animals with retained placenta: uterine lavage, antibiotic or hormone administration, or
both; the administration of hormones being the normal protocol followed in cases of RP in the
studied herd. The usage of antibiotics is limited because they are expensive and bacteria can turn
resistant to them if its usage is constant. Based on this fact, it was expected that the addition of
the antibiotic in this research would result in no marked impact on the recovery and prognosis of
a cow with retained placenta, which justifies reducing the usage of antibiotics in routine cases of
RP.
Antibiotic treatment versus Hormones only treatment
The study was conducted in the dairy operation at the Agricultural Research Station of the
University of Puerto Rico, Mayaguez Campus, at Lajas, Puerto Rico. Cows and heifers about to
calf were kept under regular management for pregnant dairy females. After calving, if the animal
expelled the placenta in the first 24 hours it joined the reference group with no treatment. If in 24
hours the cow had not expelled the placenta, it was assigned to the antibiotic treatment or the
control group (hormones only). Data were collected during weekly visits (Figure. 2) and the
attending veterinarians did the post-partum examinations every two weeks.
Individuals under the first treatment (T1) received an intramuscular injection twice daily for a 5day period of 5.00 ml of oxytocin and 5.00 ml of prostaglandin F2α with an addition of a
subcutaneous injection of antibiotic (Excede). Individuals under the second treatment (T2) or
control group received only oxytocin and prostaglandin in the same dosages.
The parturitions considered for inclusion in the research were all that occurred in the months of
September through January.
24
Figure 1. Expelled Placenta
This image shows a placenta on the ground expelled normally.
Figure 2. Field Data Collection
Body and behavior observations were taken as part of the weekly data collection
25
Incidence from 2012 to 2014
Data were collected from the PC-Dart and Madero Dairy System (AFI Milk) programs, in the
digital records of the farm for the 2012 to 2014 years interval.
Treatment with antibiotic vs. control treatment
Data from the cows that retained placenta during the experiment and were assigned either to T1
or T2. Of the 68 parturitions at the moment of summarizing, 8.82% of the cows retained the
placenta.
Table 1. Cows under T1 and T2.
Cow ID
T1 or T2
Calving
Date
(m/d/yr)
Post-calving
Live Weight
(lb)
Calf
Survival
Days
Retaining
Placenta
Post-partum Examination
209
T2
09/10/14
879
L
2
273
T1
09/15/14
1.039
L
9
302
T2
10/08/14
-
D
11
Mastitis possibly associated
with placental retention
Retained remanents of the
placenta discovered several
days later
Metritis and mastitis
61
T2
12/06/14
1,215
L
6
134
T1
12/07/14
1,084
D
5
298
T1
1/20/15
950
D
7
Acute metritis; 50 days of
infection.
No complications after freemartin and twin male birth
Post-partum weakness
following dystocia
Figure 3. Cow Weakened by metritis. The backbone of this very
lean animal can be appreciated easily. The severe infection
reduced her appetite and production
26
Figure 4. Infected cow. This is a dystocic
cow straining to expel the placenta, that
several days later, developed a chronic
infection.
Figure 5. Uterine discharge. Cow that calved
normally with uterine discharge several hours later.
2012-2014 Incidence
Table 2. Percentage of retained placenta cases from 2012-2014
Year
# of Calvings
# of RP cases
RP %
2012
26
7
26.92
2013
2014
84
127
11
21
13.10
16.54
Total
237
39
16.46
Most of the cases from 2012 were treated with antibiotics. In 2013, the standardized protocols or
RP cases at this farm changed to the usage of hormones, which it is still the norm.
No dairy herd is completely free of RP problems, however good reproductive management and
optimal feeding help to minimize this malady. “Metritis affects as many as 25% of the roughly 9
million dairy cows in the United States, costing nearly $400 per case in lost productivity and
27
treatment costs.” (Cornell University, 2011). The overall incidence found in the present research
from 2012 to January 2015 was 16.33%. Comparing our results with the overall mean for the
United States, the incidence found is proportionally higher for a small herd.
A similar experiment was conducted in the State of Aguascalientes, Mexico (Wonchee, Z. et.al.,
2002) based on the observation of 668 parturitions, from which 243 had RP and were treated
with different combinations of prostaglandin, gonadotropins and oxytetracycline. Although the
present data are too limited for firm conclusions, the Wonchee research concluded that antibiotic
treatment has no significant advantage in RP cases; furthermore these authors also found no
advantage from use of the hormones either.
Conclusion
Further data are needed to confirm conclusions of this study, but tentatively it appears that
antibiotic use in routine cases (with no other complications) of retained placenta can be
eliminated with a saving in costs. The incidence of retained placenta in the research herd has
slightly increased in the past two years, as the herd size has doubled, being 16.46% for the
overall 2012-2014 period. This means that further attention and improved management is needed
to reduce the incidence of retained placenta to as low a level as possible.
Cited Literature
Cornell researchers create first metritis vaccine to protect dairy cows. 25/10/2014, of Cornell
University, College of Veterinary Medicine.
Web: http://www.vet.cornell.edu/news/metritis.cfm
Wonchee, Z., Lozano, R., González, E., (2002). Evaluation of different treatments used in the
early postpartum in dairy cows with metritis. Téc Pecu Méx, 40 (1), 105-117.
28
Soil nitrogen mineralization in soils cropped to maize (Zea Mays) of the southern semiarid
zone of Puerto Rico
Abdiel Santana, Johannies Rivera-Zayas and David Sotomayor-Ramírez
Department of Crops and Agro-environmental Sciences
Abstract
Quantified soil nitrogen (N) mineralization rates under controlled conditions can be used to
estimate rate constants and potentially mineralizable N pools. This information can be used to
guide fertilizer-N management in crops and improve agroecosystem sustainability. Soils from the
Jacaguas series (Fluventic Haplustolls) and Guamaní series (Torrifluventic Haplusteps) were
sampled from plots of experimental fields evaluating the effects of fertilizer-N on maize yield.
The Jacaguas soil was under a three-year maize maize-cowpea (Vigna unguiculata) rotation and
Guamaní was under a fallow-maize cropping system. Soils were sampled from plots historically
fertilized with 120 kg N/ha under fallow-maize and additional samples were gathered from
Jacaguas soil under a cowpea rotation. Soils were air-dried and sieved to pass a 2-mm sieve and
packed to selected bulk density in mesocosms. Fresh cowpea residue at a rate of (125 kg N/ha)
was added to soils. The Guamaní and Jacaguas soils were incubated with (AM) and without (FA)
cowpea amendment and the Jacaguas soil under the cowpea rotation was incubated without
cowpea amendment to evaluate the residual soil N mineralization (CC). A 20-week laboratory
incubation leaching experiment was performed. Controlled leachings were made at 0, 1, 2, 3, 4, 6,
8, 12, 16 and 20 weeks, and analyzed for inorganic N (NH4+-N and NO3-N). We report on results
of data gathered up to 6 weeks. Soils amended with cowpea (AM) or with a history of cowpea
rotation (CC) had a higher N accumulation of inorganic N (Nmin) at 6 weeks than unamended
soils. The Jacaguas soil with A or CC treatments had higher rates of N mineralization during the
early stages (0 to 3 weeks) but lower cumulative Nmin at 6 weeks than Guamaní soil.
Cumulative Nmin at 6 weeks was in the order of Guamaní (AM) > Guamaní (FA) > Jacaguas
(AM) = Jacaguas CC > Jacaguas (FA), with values of 58, 48.6, 39.2, 35.2, 25.3, mg N/kg,
respectively. The results gathered to date suggest that amending soils with a covercrop residue
such as cowpea is an efficient way to provide soil N to a crop and optimize the use of N
fertilizers.
Keywords: Soil Nitrogen, Nitrogen Mineralization, Nitrogen Management
Resumen
La cuantificación de las tasas de mineralización de nitrógeno (N) en el suelo bajo condiciones
controladas puede ser usada para estimar las constantes de las tasas de mineralización y las
reservas de N potencialmente mineralizables. Esta información puede ser usada como guía para
el manejo de fertilizante nitrogenado en cultivos and y mejorar la sustentabilidad agro-ecológica.
Suelos de la serie Jacaguas (Fluventic Haplustolls) y serie Guamaní (Torrifluventic Haplusteps)
fueron muestreados en parcelas de campos experimentales donde se evalua los efectos de
fertilizante N en rendimientos de maíz. El suelo Jacaguas estaba bajo un sistema de rotación
maíz maíz-caupi (Vigna unguiculata) por tres años y el suelo Guamaní bajo un sistema de
siembra-barbecho con maíz. Las muestras de suelos fueron tomadas de parcelas con historial de
fertilización de 120kg/ha bajo sistema maíz-barbecho, además se tomaron muestras adicionales
del suelo Jacaguas bajo sistema de rotación con cobertor caupi. Las muestras tomadas fueron
29
secadas al aire y luego cernidas con un tamiz de 2mm para ser empacadas a una densidad
aparente seleccionada en mesocosmos. Material fresco de caupi fue agregado a los suelos a una
tasa (125 kg N/ha). Los suelos Guamaní y Jacaguas fueron incubados con una enmienda de caupi
(AM) y sin enmienda (FA) y el suelo Jacaguas bajo historial de rotación con caupi (CC) fue
incubado sin enmienda para de este modo evaluar la mineralización del N residual. Se llevó a
cabo un procedimiento de incubación y extracción de 20 semanas en el laboratorio. Extracciones
controladas fueron llevadas a cabo en 0, 1, 2, 3, 4, 6, 8, 12, 16 y 20 semanas, y estas fueron
analizadas para N inorgánico (NH4+-N y NO3-N). Se reportan resultados de data colectada hasta
la sexta semana. Suelos enmendados con caupi (AM) o con historial de rotación de caupi (CC)
tuvieron mayor acumulación de N inorgánico (Nmin) a las 6 semanas, en comparación a los
suelos sin enmendar. El suelo Jacaguas con tratamiento A o CC tuvo mayores tasas de
mineralización durante etapas tempranas (0 a 3 semanas) pero menor Nmin acumulado a las 6
semanas que el suelo Guamaní. Nmin acumulado a las 6 semanas fue en orden Guamaní (AM) >
Guamaní (FA) > Jacaguas (AM) = Jacaguas CC > Jacaguas (FA), con valores de 58, 48.6, 39.2,
35.2, 25.3, mg N/kg, respectivamente. La data colectada hasta la fecha sugiere que enmendar
suelos con residuos de un cobertor como caupi es una manera eficiente de proveer N del suelo al
cultivo y optimizar el uso de fertilizante nitrogenado.
Palabras Claves: Nitrógeno del Suelo, Mineralización de Nitrógeno, Manejo de Nitrógeno
Introduction
Soil N mineralization is a process in which organic matter is degraded into its mineral
constituents, therefore making soil inorganic N available to plants. Mineralization occurs
continuously under natural soil conditions (temperature, humidity, pH, aeration), but the two
most influencing factors are temperature and soil water content (Cabrera, 2007). The
quantification of soil N mineralization rates under controlled conditions can be used to estimate
rate constants and potentially mineralizable N pools. Quantified mineralization rates can help
understand how and when N is becoming available, since N becomes available gradually from
organic matter we can measure how it becomes available from mineralization processes
throughout time. By quantifying cumulative soil N we can also estimate mineralizable N pools,
taking in consideration factors like the soils organic matter content, temperature, water content
and other less influential but important characteristics.
This information can be used to guide fertilizer-N management in crops, by determining the soils
capacity to provide inorganic N and minimizing the amount of N fertilizer applied. By
identifying the moment in which soil N is available and the moment of critical nutrient need in
the plant, it is possible to synchronize the stated and have a better use of the N pool available
from the soil to the crop. Determination of N available from the soil can also help better
understand the N Cycle. This knowledge can lead to better N management to avoid
contamination by excessive application of fertilizer-N, while also enhancing the soils
microbiology, therefore improving agro-ecosystem sustainability.
The latest years various multinational biotechnology companies dedicated to production of seeds
have established in the southern semi-arid zone of Puerto Rico. This area is one of the major
agricultural lands in the country. This zone has highly fertile land and it is cropped to a variety of
30
crops including but not limited to maize. An optimum management of this lands soil can result in
better crop production and a more sustainable and eco-friendly agriculture.
A 20-week soil incubation leaching experiment was performed, under controlled laboratory
conditions. Soils from the Jacaguas series (Fluventic Haplustolls) and Guamaní series
(Torrifluventic Haplusteps) were sampled from plots of experimental fields evaluating the effects
of fertilizer-N on maize yield on DOW Agroscience facilities at Guayama and Santa Isabel, PR.
The Jacaguas soil was under a three-year maize maize-cowpea (Vigna unguiculata) rotation and
Guamaní was under a fallow-maize cropping system. Soils were sampled on both soils from
plots historically fertilized with 120 kg N/ha under fallow-maize and additional samples were
gathered from Jacaguas soil under a cowpea rotation. Soils were prepared by first being air-dried
and then sieved to pass a 2-mm sieve. Samples were then packed to selected bulk density in
mesocosms. Fresh cowpea residue at a rate of (125 kg N/ha) was added to both soils. The
Guamaní and Jacaguas soils were incubated with (AM) and without (FA) cowpea amendment
and the Jacaguas soil under the cowpea rotation was incubated without cowpea amendment to
evaluate the residual soil N mineralization (CC).
Objectives
Quantify soil N mineralization to estimate rate constants and potentially mineralizable N pools.
Understand cover crop rotation and cover crop amendment role in soil N mineralization
processes.
Sampling depth was from 0 to 20 cm over each sub-plot and taken to the University of Puerto
Rico at Mayagüez. Samples were air-dried and sieved through a 6mm mesh to then be mixed and
homogenized. Soil pH with a 2:1 water ratio, soil moisture by the gravimetric method and soil
texture (Kettler, 2001) were determined for each soil.
Samples were packed using 60 ml syringes as incubation vessels. Syringes were weighted and a
filter composed of 0.4g of celite and a 3.0cm Whatman® micro-fiber filter was added before
weighting again. An amount of 40g of the prepared soils were added to each syringe, by
treatment, and packed to selected bulk density in mesocosms, finally then total weigh was taken.
Table 1. Experimental Treatments
Location
Soil management
Guayama
Fallow-maize
Santa Isabel
Treatment
Descriptor
None
FA
Fallow-maize
Cowpea amendment
AM
Fallow-maize
None
FA
Fallow-maize
Cowpea amendment
AM
Cowpea-maize
None
FA
Cowpea (+ Cowpea) amendment equivalent to 125kg/ha
Control sample to measure N provided by the environment or any other external agent
31
Controlled leachings were made at 0, 1, 2, 3, 4, 6, 8, 12, 16 and 20 weeks based on Stanford &
Smith (1972) and Cabrera & Kissel (1988) with modifications. Inorganic N extractions were
made using 80mL CaCl2 0.01M, leaching for 3 hours on the extraction system (Zero-Max®
Drive Power Block). A digital scale was used to weight samples after each step of the process.
Leached solutions were poured through funnels with filter paper 25mL of each solution sample
were poured into 25mL bottles. For each sample 2 drops of concentrated sulfuric acid were
added and then they were refrigerated until analyzed and for inorganic N (NH4+-N and NO3-N.
A 10mL of N free solution (340 mg CaSO4, 120 mg MgSO4, 15.04 mg K2SO4, 2.19 mg KH2PO4,
1L H2O) were added using the leaching system for 30 minutes to replace exchangeable ions in
the soil sample. The samples were placed in a suction system for 4 hours applying suction at 0.33 kPa to get and appropriate water filled pore space (WFPS), leaving the soils around field
capacity. Soil samples were then incubated for the next leaching. Optimum mineralization
temperature was monitored and kept at 35°C (Kirschbaum, 1994) and water content was
manipulated and kept around field capacity (Paul et al., 2000).
Results up to six weeks are reported. Pre-plant soil fertility parameters (Table 2 & 3) show both
soils have comparable inorganic nutrient availability as organic matter (OM) content. OM,
around 2%, indicates availability of mineralizable soil N. It is evident when comparing these
parameters (P ppm & K ppm) to initial extraction values (T0) that the Jacaguas (SI) soil has had
a longer N fertilizer application than the Guamani (G) soil.
Table 2. General soil fertility pre-plant parameters of Guayama
pH
OM
Bray 1
Ca
Mg
%
7.65
K
Na
CEC
48
25.5
-----P ppm----
2.7
9
3521
867
166
Table 3. General soil fertility pre-plant parameters of Santa Isabel
Treatment
pH
Bray 1
Olsen
-----P ppm---71.43
36.18
Ca
7.65
OM
%
2.08
3445
Mg
K
Na
CEC
----------------------meq/100g-------------620
260.5
89.75 23.43
Fallow
Cover Crop
7.65
1.88
49.68
3873
613.25
29.09
192.25
78
25.33
Extraction values for T0 reveal a substantial difference between Nmin at both locations, for the
G soil values are around 6 mg N/kg soil while at the SI soil which has a longer history of N
fertilizer values start at 48.7 and reach up to 140.6 mg N/kg soil. (Compare with Chart 1 & 2).
Nmin results on chart 2 were expected, values are in the order of SI- CC, SI- AM, and SI-FA.
This shows that CC rotation applied to this soil in combination with fertilizer N have led to the
32
highest amounts of accumulated Nmin, its was followed by AM which had higher values than FA
due to N afforded by the amendment, but lower than CC due to N not being immediately
available from the OM applied. This is supported by the approximate 4 year history of N
fertilizing.
Chart 1. Initial Extraction Guayama
T 0 GUAYAMA
Inorganic N (mg/kg soil)
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
6.6
6.2
0.0
Guayama FA
Guayama AM
Chart 2. Initial Extraction Santa Isabel
T 0 SANTA ISABEL
Inorganic N (mg/kg soil)
160.0
140.6
140.0
120.0
100.0
72.5
80.0
60.0
48.7
40.0
20.0
0.0
Santa Isabel FA
Santa Isabel AM
Santa Isabel CC
33
The Jacaguas soil had higher rates of N mineralization during the first 3 weeks when compared
to the Guamani soil. Rates of Mineralization on the G soil started rising at the third week,
surpassing the SI soil, but have not yet stabilized at six weeks. SI rates seems to be reaching
Vmax values, but further data needs to be gathered form the following weeks to confirm this. N
Mineralization rates are expected to reach Vmax by the 8th week of incubation. As observed in
chart 2.1, Km values for the SI soil are higher than those at the G soil. CC and A treatments for
SI had higher rates of N mineralization compared to FA, this values are also reflected in the
treatments cumulative N values, making soils with any residual OM content have more available
soil N. N Mineralization rates tend to be higher in soils with better aeration and clay content ( ),
this supports the fact of SI rates being higher at earlier weeks due to having a loamy texture. G
rates start rising on the 3rd week of incubation, rising from 15.8 mg N/kg soil to 24.1 mg N/kg
soil, this show that up to date the G soil is capable of providing higher amounts of soil N
compared to the Si soil starting at week 3. The AM treatment had a higher rates of N
mineralization compared to FA, FA has values comparable to the SI values at week 4 but then
surpasses SI rates. Data shows the cowpea amendment starts being mineralized the third week
post amendment making it available as soil Nmin. This is also seen in the SI soil in which the
AM treatment starts surpassing the CC treatment at a lower rate at week 3. Over all rates until
now show the AM treatment as an efficient way to provide soil Nmin, which starts being
available for the third week onward.
Chart 3. N Mineralization Rates for Guayama and Santa Isabel
45
Nmin (mg N/kg)
40
35
G-F
G-A
SI-F
SI-A
SI-CC
30
25
20
15
10
5
0
0
1
2
3
4
5
Time (weeks)
34
6
7
The FA treatment was the less efficient in affording soil Nmin for both of the treated soils. In
contrast the AM treatment was the most efficient Nmin provider for both soils. Accumulated N
goes in order G-AM, G-FA, SI-AM, SI-CC, and SI-FA each with values of 58, 48.6, 39.2, 35.2,
25.3, mg N/kg, respectively. The G soil is able to provide more soil N in comparison to SI soil,
this could be due to having a higher content of clay, therefore creating more desirable conditions
for mineralization. In the G soil, A had around 10 mg N/kg soil more than FA revealing AM as a
more efficient treatment. When referring to SI, AM provided around 4 mg N/kg soil more than
CC and 14 mg N/kg soil more than Fa. CC rates showed a good input of Nmin at early stages
making it a fair soil N provider, but cumulative values are lower than AM. It is expected for
cumulative values on the AM treatment to keep being the highest among all treatments for both
soils, followed by CC in SI and FA.
Chart 4. Cumulative Inorganic Soil N
70.0
58.018
N Total (mg/kg soil)
60.0
50.0
48.606
39.214
40.0
30.0
Guayama FA
Guayama AM
Santa Isabel FA
Santa Isabel AM
Santa Isabel CC
35.279
25.329
20.0
10.0
0.0
Treatments
Conclusions
Soils amended with cowpea (AM) or with a history of cowpea rotation (CC) had a higher N
accumulation of inorganic N (Nmin) at 6 weeks than unamended soils. The Jacaguas (Santa
Isabel) soil with AM or CC treatments had higher rates of N mineralization during the early
stages (0 to 3 weeks) but lower cumulative Nmin at 6 weeks than Guamaní (Guayama) soil.
Cumulative Nmin at 6 weeks was in the order of Guamaní (AM) > Guamaní (F) > Jacaguas (AM)
= Jacaguas CC > Jacaguas (F), with values of 58, 48.6, 39.2, 35.2, 25.3, mg N/kg, respectively.
The results gathered to date suggest that amending soils with a covercrop residue such as cowpea
is an efficient way to provide soil N to a crop and optimize the use of N fertilizers. It is expected
for the AM treatment to keep having the highest M rate constant up to the 20 week and the
higher cumulative N. Therefore confirming a CC amendment is the most efficient treatment for
providing soil N.
35
Cited Literature
Cabrera, M. L. (2007). Mineralización y Nitrificación: Procesos Claves en el Ciclo del Nitrógeno.
Informaciones Agrónomicas de Cono Sur, #34, 1-9
Cabrera, M. L., Kissel, D.E., & Virgil M.F. (2005). Nitrogen mineralization from organic
residues: Research opportunities. Journal of Enviromental Quality, #34, 75-79
Celaya-Michael, H., & Castellanos, A. E. (2011). Mineralización de Nitrógeno en el Suelo de
Zonas Aridas y Semiáridas. Terra Latinoamericana, #29, 343-356
Dou, Z., Toth, J.D., Jabro, J.D., Fox, R.H., & Fritton, D.D. Soil Nitrogen Mineralization During
Laboratory Incubation: Dynamics and Model Fitting. Soil Biology and Biochemistry, # 28, 625632
Garcia, F.O. (1992). Carnon and nitrogen dynamics and microbial ecology intallgrass prairie.
(unedited Doctoral Tesis). Kansas State University, US.
Kettler, T. A., Doran, J. W., & Gilbert, T. L. (2001). Simplified Method for Soil Particle-Size
Determination to Accompany Soil-Quality Analyses. Soil Science of America, #65, 849-852
Kirschbaum M.U. 1994. The temperature dependence of soil organic matter decomposition, and
the effect of global warming on soil organic storage. Soil Biol. Biochem. 27:753-760.
Lacasta, C., Benitez, M., Maire, N., Meco, R. (2006). Efecto de la textura del suelo sobre
diferentes parametros bioquímicos.VII Congreso SEAE Zaragoza 2006. #110
McDonald, N.T., Watson, C.J., Lalor, S.T.J., Laughlin, R.J., & Wall, D.P. (2013). Evaluation of
soil tests for predicting nitrogen mineralization in temperate grassland soils. Soil Science Society
of America Journal, #78, 1051-1064
Stanford, G., Carter, J.N., & Smith, S.J. (1974). Estimates of Potentialy Mineralizable Soil
Nitrogen Based on Short-Term Incubations. Soil Science of America Proceedings, #38, 99-102
Stanford, G., Smith, S.J. (1972). Nitrogen mineralization potentials in soils. Soil Science of
America Journal, #36, 465-472
36
Comparative Study of Organic and Conventional Farms in Puerto Rico Using Nematode
Communities as Environmental Indicators
Jennifer M. Rivera San Antonio and Roberto Vargas
Department of Crops and Agro-Environmental Sciences
Abstract
The nematode communities provide us with valuable information that can be used as a mechanism to
quantifying soil condition. By sorting nematode communities in conventional and organic farms we
can determine the "environmental health" of the soil in both agricultural systems. The objective is to
compare these systems in order to have a standard for “soil health” for specific crops and observe the
effect of both agricultural systems in the soil nematode community. Two certified Organic farms in
Puerto Rico (UPR Experimental Station, Lajas P.R. & Bananera Fabre, Sabana Grande P.R.) and one
Conventional farm (Bananera Fabre, Sabana Grande P.R.) were selected for this study. Samples of
0.6kg, approximately, consisting of three subsamples, were isolated from each plot site and prepared
for nematode extraction using the Christie & Perry Nematode extraction protocol with modifications
and the Baermann technique. The Nematodes were classified according to their trophic level:
bacterial feeding (BF), fungal feeding (FF), plant feeding (PF), predator (P) and omnivore (O).
Results showed that nematode population in an Organic Farming System had higher diversity of
the trophic group (BF, FF, O and P) and presented a lower percentage of Plant Feeding
nematodes when compared to a Conventional farming System. In a Conventional Farming
System we observed a higher population of (PF) nematodes and a low diversity of the trophic
community (BF, FF, O and P). Apparently, high population of Plant Feeding nematodes
suppresses other trophic groups in soil.
Keywords: Nematodes, Organic versus Conventional Farms, Trophic level, Environment, Soil
Resumen
Las comunidades de nematodos proveen información valiosa que puede ser utilizada como
mecanismos para clasificar la condición o calidad del suelo. Al clasificar las comunidades de
nematodos en las fincas convencionales y orgánicas podemos determinar la "salud ambiental"
del suelo en ambos sistemas agrícolas. El objetivo es comparar estos sistemas mediante un
análisis de nematodos del suelo con el fin de obtener un estándar para la "salud del suelo" para
cultivos específicos y observar el impacto de los sistemas agrícolas en la comunidad del suelo.
Dos fincas Orgánicas certificadas en Puerto Rico (Estación Experimental de la UPR, Lajas PR y
Bananera Fabre, Sabana Grande PR) y una finca convencional (Bananera Fabre, Sabana Grande
PR) se seleccionaron para este estudio. Muestras de aproximadamente 0.6 kg, compuestas de tres
sub-muestras, se colectaron y prepararon para la extracción de nematodos utilizando el protocolo
de Christie y Perry modificado y la técnica de Baermann. Los nematodos fueron clasificados de
acuerdo a los niveles tróficos: Bacteriófagos (BF), Fungívoros (FF), Fito-parasíticos (PF),
Depredadores (P) y Omnívoros (O). La población de nematodos en un sistema de agricultura
orgánica mostró una mayor diversidad en los niveles tróficos (BF, FF, O y P) y presentó un
menor porcentaje de nematodos Fito-parasíticos. Aparentemente, una alta población de
nematodos Fito-parasíticos suprimieron los restantes niveles tróficos.
Palabras Claves: Nematodos, Finca Orgánica versus Convencional, Nivel trófico, Suelo
37
Introduction
The origin of the word nematode comes from the Greek Laguage nema- meaning “thread” and ode meaning “like”, thread like. Nematodes are amongst the most abundant group of animals to
exist. They range from 50μm diameter and 1mm in length. Nematode abundance in soils in
managed and unmanaged (soils) ranges from 1–10 million individuals/m2 (Peterson and Luxton,
1982; Lavelle and Spain, 2001). The taxonomic classification of nematode constitute as the
following; Kingdom: Animalia, Clade: Nematoida, Filum: Nematoda. Nematodes are recognized
as a major consumer group in soils, generally grouped into four to five trophic categories based
on the nature of their food, the structure of the stoma (mouth) and esophagus, and the method of
feeding (Yeattes and Coleman, 1982). These groups are classified as bacterial feeders, fungal
feeders, predatory feeders, omnivores, and plant feeders. These five feeding categories are based
primarily on the stoma (Figure1).
Figure 1. Nematodes can be classified into different feeding groups based on the structure of
their mouthparts. (a) bacterial feeder, (b) fungal feeder, (c) plant feeder, (d) predator, (e)
omnivore. Figure credit: Ed Zaborski, University of Illinois
The nematode communities provide useful information that can be used as a soil quantifying
mechanism (Berkelmans, et al., 2003). Nematodes live in a wide range of environments, while
others live in a more restricted area. Scientists had been primarily based their studies of
nematodes on the damage to crops but they also are essential organisms of the soils nutrient
cycle and fertility. It has been proven that some nematodes are more responsive to resource
enrichment than other organisms. (Ferris and Bongers, 2006). Bacterial-feeding nematodes
contribute to the supply of nitrogen to the soil. By incorporating manure, compost or cover crops
there it can stimulate bacterial growth and trigger an effect of the abundance of bacterial-feeding
nematodes. This interaction can make nitrogen available for the plant intake.
38
The study was conducted in a selection of three farms based upon their agricultural systems
practices, one conventional farm and two organic farms. The Conventional Farm (Fabre) located
in Sabana Grande, Puerto Rico produced bananas. The organic farms (Fabre) is also located in
Sabana Grande, Puerto Rico and produced organic bananas in two stages: one 7 month stage and
a 3 year stage project. Finally, the Organic farm of the Experimental Station of the University of
Puerto Rico at Lajas which produced sweet peppers, pumpkin and eggplant. In all the farms the
soil was classified as a Vertisol, Fraternity series. The Fraternity series are classified as fine,
smectitic, isohyperthermic Typic Haplusters. These soils are moderately well drained with a
mean annual temperature of 75 °F and a mean annual precipitation of 45 inches. These soils
present a slow permeability. The soil samples consisted of a three sub-samples of approximately
0.6 kg. The Sub-samples were selected at a distance of (±1 feet) of the tree. The sample was
placed in a plastic bag and kept in a cooler until processed. (Figure 2)
Sample Collection at farms
A total of 13 samples were collected from Organic Farm (5) and 8 samples of the same
Conventional Farm due to the extended size of the farm. However, only the average value of the
sample was taken under consideration as a representation of the effect of the conventional
farming system. The nematodes were extracted using the Christie & Perry Nematode Extraction
(Christie & Perry, 1951) with an adjustment in the process of breaking down the soil. A selection
of 250cc of the soil and 250cc of water were placed in a blender in order to break down the soil
aggregates. The blender was pulsed 2 to 4 times in order to obtain finer soil particles. This
solution was transferred into a coarse sieve (#60) that contains about 20 to 24 meshes per inch to
eliminate debris and a fine sieve (#360) to retain the nematodes. The samples were cleaned with
water thoroughly until the water would pass through the sieve and would look clear. The
suspension retained in the #360 sieve was transferred to a filter that was placed on a grid on top
of a Baermann funnel. The funnels were placed in a rack and incubated for a period of 48 hrs
39
until collected in a vial. An aliquot of the sample was placed under the microscope for
classification. The first 50 observed nematodes were taken under consideration and classified
according to the trophic level.
Results
The nematode population in an Organic Farming System showed a higher diversity of the trophic
group and presented a lower percentage of Plant Feeding nematodes when compared to a
Conventional farming System. In the Organic Farming System (Bananas at a stage of a 7-month
growth) we could observed the absence of Bacterial Feeding and Predator nematodes, whereas
5% of the observed nematodes were Fungal feeders, 32% Plant feeding nematodes and a 14%
Omnivorous nematodes. In the same Organic Farm (Bananas at a stage of 3-Years) we could
observed that the Plant Feeding nematode population decreased by 16% and the Omnivorous
nematodes increased by a 35%. However we observed that the Predator nematode population
disappeared. The Organic Farming System (5-Year Project) that produced Pumpkin, Eggplant
and Sweet pepper showed a great diversity in the nematode population. We observed a decrease
(67%) of Plant Feeding in soil influenced by Sweet peppers, compared to Pumpkin. In these
samples, we also detected an increase in the population of Bacterial Feeding nematodes by 80%,
Predator nematodes by 47% and Omnivorous nematodes by 40%. In these samples, the Fungal
Feeding nematode population decreased by 37%. This effect can reflect the possible nematode
population interaction that could be present once the Plant Feeding nematodes have decreased by
more than a 50% of the original index. In the Conventional Farming System there was a
population of 3% Bacterial Feeding nematodes, 1% of Fungal feeding nematodes, 36% of Plant
feeding nematodes, 2% of Predator nematodes and an 8% of Omnivorous nematodes. By
comparing both systems we observed that in the Organic Farming Systems the Plant Feeding
nematode population decreased by a 22%. As an effect we observed an increase in Bacterial
Feeding nematodes by a 33%, the Fungal Feeding nematodes by a 66%, the Predator nematodes
by a 56% and the Omnivorous nematodes by 18%.
Table 1. Results obtained form soil nematode extraction at Organic and Conventional Farming
Systems.
Bacterial
Feeding
Fungal
Feeding
Organic Banana 7-Month
0
0
Organic Banana 3-Year
0
Organic Pumpkin 5-Year
Farm/Years Practice
Trophic Level
Plant
Feeding
Predator
Omnivore
38
3
9
5
32
0
14
3
4
30
4
8
Organic Eggplant 5 Year
8
3
23
10
6
Organic Sweet Pepper 5-Year
16
3
10
8
13
Conventional Banana 12-Year
3
1
36
2
8
Population based on 50 nematodes counted on samples, Values were adjusted by 50.
40
Conclusion
The impact of organic amendments to the soil decreases the plant feeding nematode population,
allowing other trophic groups to prosper. In a Conventional Farming System of 12-years of
practice the Plant Feeding nematodes suppress other trophic groups. By comparing a
Conventional Farming System (12-years of practice) with an Organic Farming System (5-years
of practice) a significant reduction of 22% in Plant Feeding nematodes could be reach as well as
a more diversity in other nematode species. In a long-term organic system (3-years), it can
promote the development of Omnivorous and Fungal Feeding nematodes. As a result, in an
organic farming system we could support higher and more balanced diversity of the trophic
groups.
Cited Literature
Berkelmans, R., Ferris, H., Tenuta, M. & van Bruggen, A.H.C. (2003). Effects of long-term crop
management on nematode throphic levels other than plant feeders disappear after 1 year of
disruptive soil management. Applied Soil ecology 23, 223-235.
Bongers, T. (1990). The maturity index, an ecological measure of environmental disturbance
based on nematode species composition. Oecologia 83, 14-19.
Bongers, T. & Bongers, M. (1998). Functional diversity of nematodes. Appl. Soil Ecol. 10, 239251.
Christie, J.R. & Perry, V.G. (1951). Removing nematodes from soil. Procedings of the
Helminthological Society of Washington. 18, 106–108.
Peterson, H., & Luxton, M. (1982). A comparative analysis of soil fauna populations and their
role in decomposition processes. Oikos 39: 287–388.
Ugarte, C. & Zaborski, E. (2014). Soil Nematodes in Organic Farming Systems
Wilson, J.B. (1999). Guilds, functional types and ecological groups. Oikos. (86) 507-522.
41
Growth responses of lettuce plants (Lactuca sativa L., cv. “Black Seeded Simpson”) grown
under a hydroponic system (NFT) receiving inorganic and organic nutrient solution.
Mibet M. Cordero Ruiz and María del Carmen Librán
Department of Crops and Agro Environmental Sciences
Abstract
There is a great concern and skepticism of people for the content of chemicals in foods
consumed. Most of this concern came from people who already have a particular disease such as
cancer, among others. Demand for crops grown under organic production systems began in
Europe and has spread to, United States, Puerto Rico and other countries. For these reasons it is
imperative to conduct research dealing with this issues. Today, lettuce crops (Lactuca sativa L.),
is one of the most consumed and demanded horticultural crops in Puerto Rico. There is a need
to conduct
studies, to determine growth responses under an organic nutrient system as
compared with an inorganic nutrient production system. The objectives of this research were; to
determine the effectiveness of an organic fertilizer on lettuce plants grown in an NFT (Nutrient
Film Technique) hydroponic system and to evaluate and compare the growth responses of these
plants grown in an inorganic and organic nutrient solution. The lettuce cultivar to be evaluated is
“Black Seeded Simpson”. During six months, two studies were conducted. In each study, two
treatments were evaluated and replicated five times. Growth parameters evaluated were, number
of leaves, height of the plant, and fresh weight. Results shown an optimum growth parameters on plants
grown under an inorganic nutrient system as compared with plants grown in an organic solution
nutrient system. Further studies should be conducted to evaluate the responses of crops grown
under different organic fertilizers in hydroponic systems.
Keywords: lettuce, NFT, organic fertilizer, inorganic fertilizer
Resumen
Existe una gran preocupación y escepticismo de las personas por el contenido de químicos en los
alimentos que consumen. Mucha de las preocupaciones proviene de personas que ya tienen una
enfermedad particular así como el cáncer, entre otras. La demanda por productos producidos
bajo sistemas de producción orgánica comenzó en Europa y se ha extendido hacia los Estados
Unidos, Puerto Rico y otros países. Por estas razones, es imperativo realizar investigación
relacionada con esta problemática. Hoy día, el cultivo de la lechuga (Lactuca sativa L.), es uno
de los cultivos hortícolas más consumidos y con mayor demanda en Puerto Rico. Existe una
necesidad de realizar estudios, para determinar las respuestas de crecimiento bajo un sistema de
producción nutritivo orgánico y compararlo con un sistema de producción nutritivo inorgánico.
Los objetivos de esta investigación fueron: determinar la efectividad de un fertilizante orgánico
en plantas de lechuga crecidas en un sistema hidropónico NFT (Nutrient Film Technique) y
evaluar y comparar las respuestas de crecimiento de esas plantas crecidas en una solución
mineral orgánica e inorgánica. El cultivar evaluado es la “Black Seeded Simpson”. Durante seis
meses, dos estudios fueron realizados. En cada estudio, se evaluaron dos tratamientos con cinco
réplicas cada uno. Los parámetros de crecimiento evaluados fueron, número de hojas, altura de
42
la planta, y peso fresco. Los resultados mostraron parámetros de crecimiento y rendimiento
óptimo en las plantas crecidas en solución inorgánica al ser comparadas con las plantas crecidas
bajo un sistema de solución nutritiva orgánica. Futuros estudios deben ser dirigidos a evaluar las
respuestas de crecimiento de cultivos crecidos bajo diferentes fertilizantes orgánicos en un
sistema hidropónico.
Palabras claves: lechuga, NFT, abono orgánico, abono inorgánico
Introduction
In the last years the research results demonstrated that the demand for organic products in
United States as well worldwide has been increasing significantly (Kortbech, 2002). Greens and
herbs production is more through recirculating culture. Today, growers interest to provide crop
nutrients from organic sources has increased (Kuack, 2013). There are many reasons of why the
consumption and demand for organic products is increasing in different countries: United States
40.6%, Germany 12.7%, Japan 12.7%, France 6.3%, Italy 5.6%, Great Britain 4.6%, other
Europe 16.2% and the Rest of the World 1.4%, these percentages represent the demand for
organic vegetables over the inorganic vegetables (Willer, 2001). Parents, prefer the organic food
for their children rather than food produced by conventional agriculture, in this manner they can
prevent some diseases related with the chemicals used during farming. For this study organic
fertilizer selected were, Seaweed Creme and Humega both manufactured by Bioflora®.
Organic crops grown with organic solutions will not be added inorganic fertilizers or other
chemical or synthetic substances that harms human health. Lettuce produced in this research are
grown in a closed system NFT (Nutrient Film Technique) indicates that the plants are in contact
with a sheet of mineral solution to a time interval. Crops grown in inorganic solutions are
produced in a conventional hydroponic system, in which fertilizers and agrochemicals are used
artificially synthesized.
This study was conducted in Finca Alzamora and Piñero Greenhouse, at University of Puerto
Rico, Mayaguez Campus (Figure 1). Two studies were conducted. The first study were from
September to December 2014 and the second study were from January to March 2015. Each
study had two treatments replicated five times (Figure 2). In the first study plants were grown;
T1- Inorganic nutrient solution with a Peters® soluble fertilizer 20-10-20/125 ppm N, T2Organic nutrient solution Seaweed Creme® fertilizer/soluble emulsion manufactured with
marine algae. A second study was conducted; T1- Inorganic nutrient solution 20-10-20/150 ppm
N, and T2- Organic nutrient solution Humega® (8% Humic Acid) (Figure 3).
Lettuce seeds from cultivar Black Seeded Simpson were planted in a Clean Start Oasis®
growing medium for hydroponics and placed into a germination tray (Figure 4). Plants were
transplanted to an NFT hydroponic system with two inches height (Figure 5). Once they were
transplanted to the four treatments electrical conductivity and pH, were monitored everyday by
using an EC and pH meter (Figure 6). A daily inspection of lettuce plants watching the growth
43
and development of plants was performed. Once harvested lettuce we take data from growth
parameters which are set numbers of leaves, plant height, pH, electrical conductivity and fresh
weight (Figure 7). Once these data are taken we proceed to evaluate the results.
Figure 1. Piñero Greenhouse
Figure 2. First study (left) and second study (right). T1 inorganic (left) and T2 Organic (right)
Figure 3. Organic (Seaweed Crème) and Inorganic fertilizer
44
Figure 4. Lettuce planted in a Clear Star Oasis
Figure 5. Lettuce ready to be transplanted
Figure 6. Electrical conductivity and pH meter
Figure 7. Harvest time
First Study: September –December 2014
The first study was conducted at Finca Alzamora, Mayaguez Campus. Growth parameters results
from the two treatments shown, that the average numbers of leaves grown in the organic
treatment was 5 lower than plants as compared with the inorganic treatment which were 22
(Figure 8). In the organic treatment the lettuce plants did not have an acceptable growth and
development as the plants in the inorganic treatment. The height of the lettuce plants in the
organic treatment was 2 inches and in the inorganic treatment was 7.93 inches (Figure 9). The
pH in the organic treatment was 7 and in the inorganic treatment were 6.6 (Figure 10). The
electrical conductivity of the organic treatment was 0.35 mmhos and in the inorganic treatment
45
was 1.9 mmhos (Figure 11). This relevant difference in the electrical conductivity could be the
reason of the poor growth and development of the lettuce plants grown in the organic treatment.
The poor growth responses could be an effect of the low absorption by the plant from the
nutrient in the organic fertilizer. According with Kuack, (2013) organic fertilizer could not be
absorbed by the lettuce plants because were not degraded by the Nitrosomas bacteria and could
not be absorbed easily.
Figure 8 and 9. Number of leaves and plant height
46
Figure 10 and 11. pH and electrical conductivity
Second Study: January – March 2015
The second study was conducted in Piñero greenhouse at Mayaguez Campus. The results of the
two treatment, shown that in the organic treatment the average number of leaves were 50
meanwhile in the inorganic treatment were 5,000 (Figure 12). There is a considerable differences
between the first study and the second study. Results shown that lettuce plants grown in 150 ppm
of Nitrogen (20-10-20) shown higher growth parameters as compared with the first study. The
reason should be due to an increase of the concentration of N, which affected on growth and
development of the plants. The average height of the lettuce plants grown in the organic
treatment was 2 inches and in the inorganic treatment was 11 inches (Figure 13). In the first
study the results shown of the height of the lettuce plants in the organic treatment was 2 inches,
showing equal results in this growth parameter. There are no considerable difference between
both studies. The average fresh weight of the organic treatment was 8g as compared with the
inorganic treatment which was 292g (Figure 14). The average pH solution of the organic
treatment was 7.5 and in the inorganic treatment were 7.2 (Figure 15). The pH was not a factor
47
that affected the development of the lettuce plants in the organic treatment because the optimal
pH in hydroponic nutrient solution is between 7.0 – 7.5 and the organic nutrient solution was on
the range. The electrical conductivity of the organic treatment was 3 mmhos and in the inorganic
treatment was 1.2 mmhos (Figure 16). This difference in the electrical conductivity could be the
reason of the poor growth development of the lettuce plants in the organic treatment. The
highest values of Electrical Conductivity could be the cause of leaves burnt of the plants in the
organic nutrient solution. The values of EC (Electrical Conductivity) for a nutrient solution in
hydroponic should be between 1.5 to 2.00 mmhos.
Figures 12 and 13. Number of leaves and plant height
48
Figures 14, 15 and 16. pH in solution, electrical conductivity and fresh weight
49
Conclusion
These results demonstrated in both studies that the organic nutrient solution containing organic
fertilizers such as Seaweed Creme® and Humega®, did not stimulate an optimum growth and
development in lettuce plants. Both studies shown similar results of growth parameters in
treatments containing organic nutrient solution. Poor growth and development were observed.
The lettuce plants from organic nutrient solution did not shown the expected development and
quality as compared with the lettuce plants grown in the inorganic nutrient solution. Further
studies should be conducted in manner to characterize and evaluate the chemical composition
and plant responses produced with organic fertilizers used in hydroponic systems.
Cited Literature
Kuack, D. 2013. Using organic fertilizers for hydroponic lettuce production. University of
Kansas. 2015.
Willer, Helga and Y. Minov. 2001. Articulo elaborado con base en información extractada de
“Organic Agriculture Worldwide”. 2015
50
Effects of Exogenous Biostimulants on Tahiti Lime (Citrus latifolia) Trees Affected by the
Citrus Greening Disease in Lajas, Puerto Rico
Alexandra I. Ramírez-Irizarry and J. Pablo Morales-Payan
Department of Crops and Agro-Environmental Sciences
Abstract
Citrus greening is a relatively new disease in Puerto Rico, associated with
the bacterium Candidatus Liberibacter asiaticus. The pathogen blocks the tree phloem, resulting
general weakening of the trees, staggered death of branches, declining fruit yield, reduced fruit
quality, and eventual death of the tree. It is currently considered the most devastating disease in
citrus. There is no known cure for this disease, and its management relies on practices to contain
its spread and to keep the tree alive and productive for as long as possible. The use of
biostimulants (exogenous organic substances that accelerate growth and may enhance some
physiological processes) may have potential in citrus greening management. Research was
conducted in 2014-2015 in Lajas, Puerto Rico, to determine the effects of selected biostimulants
on adult trees of Tahiti lime (Citrus latifolia) affected by the citrus greening disease. At the
beginning of the experiment, plants were defoliated by approximately 75% and showed little
vigor. Every 2 weeks we applied aqueous solutions of six biostimulants [(a) an extract of the
alga Ascophyllum nodosum, (b) a formulation based on soluble silicon, (c) a blend of vitamins
and enzymes, (d) gibberellin 4/7, (e) an extract of the plant Reynoutria sachalinensis, and (f) an
extract of plants rich in brassinosteroids, spraying the leaves to run-off. All the biostimulants
were applied with an organic surfactant. Thus, a group of plants was sprayed only with the
surfactant dissolved in water to account for its potential effects on the trees. Also, plants without
application of biostimulant or surfactant were evaluated as checks. There were eight trees per
treatment, in a completely randomized design. The results of trees treated with the surfactant
were the same as those of check trees. Chlorophyll concentration in the leaves was not affected
by the biostimulants. As compared to check trees, Tahiti lime trees treated with gibberellin 4/7
tended to have larger crown diameters and were more vigorous than check plants. After four
applications of the biostimulants, trees treated with the silicon biostimulant had a greater survival
rate than those of other treatments. At the time of writing this abstract, this field research is still
ongoing.
Keywords: Citrus, Biostimulants, Citrus greening
Resumen
El citrus greening en una enfermedad relativamente nueva en Puerto Rico, asociada a la bacteria
Candidatus Liberibacter asiaticus. El patógeno bloquea el floema del árbol, provocando su
deterioro progresivo, reducción de su productividad y eventualmente su muerte. Actualmente se
considera la enfermedad más devastadora de los cítricos, ya que no se conoce cura para esta
enfermedad. Las prácticas usadas en huertos enfermos se enfocan en prolongar la vida de los
árboles el mayor tiempo posible. El uso de bioestimulantes (sustancias orgánicas exógenas que
aceleran el crecimiento y pueden mejorar algunos procesos fisiológicos) es una de las prácticas
con potencial para retardar el deterioro de plantas afectadas por citrus greening. Cada 2 semanas
se aplicaron soluciones acuosas de seis bioestimulantes [(a) un extracto de alga Ascophyllum
nodosum, (b) una formulación basada en silicio soluble, (c) una mezcla de vitaminas y enzimas,
51
(d) geberelina 4/7 (e) extracto de la planta Reynoutria sachalinensis y (f) extracto de plantas
ricas en brasinoesteroides (con un agente surfactante para ayudar a los bioestimulantes a penetrar
a las hojas pulverizadas hasta el escurrimiento. Además, un grupo de plantas se pulverizadas solo
con el surfactante disuelto en agua para saber sus posibles efectos en los arboles. También, las
plantas sin aplicación de bioestimulantes o surfactante se evaluaron como controles. Hubo 8
arboles por tratamiento con un orden completamente al azar. Los resultados muestran que el
surfactante por si mismo no afecto a los árboles tratados. No se encontró efecto significativo de
los tratamientos en la concentración de clorofila. Los arboles tratados giberelina 4/7 tendieron a
tener coronas (sistemas de ramas) de mayor diámetro y a ser más vigorosos que los árboles sin
bioestimulante. Luego de cuatro aplicaciones de los tratamientos, los arboles tratados con la
formulación de silicio tuvieron una tasa de supervivencia 25% mayor que los arboles sin
bioestimulante. Al momento de redactar este resumen, la investigación continúa.
Palabras Claves: Bioestimulantes, limón Tahití, citrus greening
Introduction
Citrus in Puerto Rico are traditionally fruits of economic importance, and they generally were
among the top four fruits in terms of annual farm gate value. In Puerto Rico, common limes
(Citrus aurantifolia) and Tahiti or Persian limes (Citrus latifolia) are among the favorite citrus,
with more than 90% of the limes marketed in the island are imported. The number of new lime
orchards planted in the first decade of the 21st century was on the rise in Puerto Rico, when a
new disease, the Citrus greening, was detected in the island in 2009. It has been estimated that
from 2009 and 2014 approximately 80% of the orchards have been affected by the disease,
reducing their productivity and longevity.
The Citrus greening disease has been associated with the bacterium Candidatus Liberobacter
asiaticus, transmitted by the citrus psyllid Diaphoria citri. The psyllid causes direct damage by
sucking sap from the tree, but this is considered minor or tolerable damage as compared to the
indirect and permanent damage caused by its transmitting the pathogen that causes the Citrus
greening into the tree while feeding from it. The transmitted bacterium stays in the phloem,
eventually blocking it, which results in loss of vigor, poor growth, chlorotic mosaic (green and
yellow) with irregular patterns in the leaves, branches with sparse foliage, branch die back, and
loss of leaves, flowers and fruits. Fruits that do not abort may have abnormal coloration, are
generally small, have low juice content and poor juice quality. In Puerto Rico, losses due to this
disease vary, with some orchards being completely lost, while in other orchards 50% of the lime
trees are known to have died between 2011 and 2014. In Florida, USA, the estimated loss to
Citrus greening was $3,600 million between 2006 and 2012 (Evans et al., 2014).
All the widely grown citrus varieties are susceptible to the disease, and there is no known cure
for the Citrus greening. Until new resistant varieties with commercial value are developed, the
disease is managed with practices to retard the infection and development of the disease, and to
prolong the commercial life of the trees. Management practices tried in other countries include
intercropping with guava (Psidium guajava) (which is said to repel the vector but has not been
effective in most places), growing transplants in enclosed structures to ensure new plantings are
52
done with healthy trees, intensive programs of foliar fertilization and insecticides, and removing
symptomatic citrus trees (Ichinose et al., 2012; Grafton-Cardwelll et al., 2013). Those practices
have had varying degrees of success, but there is need for more research to explore other
alternatives that may prolong the commercial life of infected trees.
Biostimulants are organic substances that in relatively small concentrations may influence the
physiology of plants, resulting in accelerated growth, increased uptake and transport of essential
nutrients, enhanced photosynthetic activity, and increased tolerance to biotic and/or abiotic
stress, which may lead to reduced flower and fruit abortion, and higher yield and quality (De la
Cruz, 2013). A number of biostimulants are approved for organic systems, offering a means for
conventional and organic growers to enhance crop performance with little environmental impact
(Flores-Torres, 2013).
The effects of biostimulants in trees affected by the Citrus greening are unknown. Infected trees
will eventually die, but it is possible that because of their effects on growth and vigor
bioestimulants may help prolong the commercial life or trees with the citrus greening. The use of
organic biostimulants in lime trees affected by the citrus greening may be a promising practice,
whose research we are pioneering in Puerto Rico. The objective of this research was to determine
the effects of selected foliar-applied biostimulants on the growth and survival of organicallygrown Tahiti lime trees affected by the citrus greening in Puerto Rico.
This field experiment took place in the Agricultural Experiment Station in Lajas, Puerto Rico
(Figure 1). The trees used are Tahiti lime grafted onto in ‘Cleopatra’ tangerine (Citrus
reticulata), planted in 2006. The orchard has been grown organically since 2007. Trees started to
show symptoms of greening in late 2012.
Figure 1. View of the experimental orchard from above. Lajas, Puerto Rico.
53
We tested six biostimulants (Table 1), which included a seaweed (Ascophyllum nodosum)
extract; a formulation of soluble silicon; a blend of brassins, enzymes and vitamins; a plant
extract rich in natural brassinosteroids; an extract of the plant Reynoutria sachalinensis; and
gibberellin 4/7. All of these biostimulants have been known to enhance citrus performance in the
absence of citrus greening (Diaz-Candelas, 2013), and are approved for use in organic systems.
The rates used (Table 1) were those recommended by the manufacturers for use in citrus. All the
bioestimulants were applied with an organic surfactant (an extract of yucca and garlic), and a
treatment with surfactant alone in water was used to test potential effects of the surfactant on the
plant. An untreated check (no surfactant, no biostimulant) was also included in the experiment.
The biostimulants were applied in aqueous solutions, spraying all the foliage to run-off
(approximately 500 ml per tree), at a frequency of 14 days.
Table 1. Description of treatments tested on citrus greening-affected Tahiti lime trees in Lajas,
Puerto Rico, 2014-2015.
Treatment
Active ingredients
Commercial
Formulation
Rate of
commercial
formulation per
liter of water
Surfactant
Yucca spp. and garlic
(Allium sativum) extract
BioLink ®
1 ml
Ascophyllum nodosum
seaweed extract
Citokininins, gibberellin,
oligosaccharides, amino
acids and betaine
Stimplex®
5 ml
Natural extract
Vitamins, brassins and
enzymes
Organic
Vitazyme®
5 ml
Blend of organic acid
and nutrients
Blend of silicon with humic
and fulvic acids and
micronutrients
QuickSol®
2.5 ml
Gibberella fujikuroi
fungi derived
Giberelins 4/7
ProVide®
50 mg of active
ingredient
Plant extract
Brassins
Comcat®
1g
Reynoutria
sachalinensis plant
extract
Undisclosed
Regalia®
5 ml
Control
54
Mentioning commercial names does not imply a special endorsement from the authors or from
the University to that specific formulation or manufacturer; it is done solely to specify the
formulation used in the experiment as it was available at the time of conducting the research.
We used a completely randomized design, with eight trees per treatment. Canopy height and
diameter, number of symptomatic and asymptomatic branches, SPAD values in expanded leaves
(associated with chlorophyll concentration), vigor (a 0-5 visual scale modified from Inch (2014)
where 0 is dead and 5 is an asymptomatic tree), and tree survival rate were evaluated. The results
were submitted to analysis of variance and separation of means as appropriate.
% Increase above check tress
There was no effect of surfactant alone on plant growth and survival. None of the treatments
significantly affected the SPAD values of expanded leaves, indicating that on average leaf
coloration (a reflection of chlorophyll concentration) was not statistically different (data not
shown). It is possible that the disease affected the availability of nutrients necessary to synthesize
and/or maintain chlorophyll molecules, therefore preventing biostimulants from increasing
chlorophyll concentration as they have been known to do in healthy fruit crop trees. Flowering
and fruit set varied considerably between trees, and yield will be determined at harvest.
As compared to check trees, Tahiti lime trees treated with gibberellin 4/7 tended to have larger
crown height and diameter (Figure 2); this result may be explained by the effect of gibberellins
on promotion of vegetative elongation, particularly on stems and branches. Tree vigor was also
greater in plants treated with gibberellin 4/7 (an average value of 2.4 in the 0-5 scale) than in
check trees (an average value of 1.33 in the 0-5 scale). Asymptomatic new branches tended to be
more abundant in trees treated with biostimulants than in check trees.
25
Gibberellin
15
11
Vitamin/enzyme/brassin
Silicon
0
Reynoutria extract
Figure 2. Effect of selected biostimulants on crown diameter (% above check plants) in Tahiti
lime trees affected by citrus greening. Lajas, 2015.
55
After four applications of the biostimulants, tree survival was 100% in trees treated with
the silicon formulation, as compared to approximately 75% survival in check plants and
those treated with the Reynoutria sachalinensis formulation, and approximately 85% in
all the other treatments (Figure 3). These results may be attributed to the growth
promoting properties of the active ingredients (gibberellins, citokinins, amino acids,
brassins, vitamins, enzymes) in some of the biostimulants tested in this research, which
may have allowed the plants to continue growing sufficiently to survive despite the
disease.
Figure 3. Survival of Tahiti lime trees with citrus greening as affected by selected biostimulants.
Conclusions
As of March 2015, the experiment is still being conducted. So far, some biostimulants appear to
have an effect on tree survival and growth. While fruit yield has not been evaluated, effects on
growth and survival may be an indicative that some of the treatments tested in this research may
be helpful in prolonging the life of citrus greening-affected Tahiti lime trees grown organically.
Cited Literature
Evans, E. A., Ballen, F. H., & Crane, J. H. 2014. Economic Potential of Producing Tahiti Limes
in Southern Florida in the Presence of Citrus Canker and Citrus Greening. HortTechnology,
24:99-106.
56
De La Cruz-Rodríguez, L. F. 2013. Evaluación de dos bioestimulantes agrícolas en naranja
dulce (Citrus sinensis) ‘Valencia Tardía’ en la región montañosa centro-norte de Puerto Rico.
Tesis de Maestría en Horticultura. Universidad de Puerto Rico, Recinto de Mayaguez.p.4-13.
Díaz-Candelas, C. C. 2013. Effect of exogenous bio-regulators on organically-managed Tahiti
lime (Citrus latifolia Tanaka) fruit and essential oil productivity and quality. Tesis de Maestría
en Horticultura. Universidad de Puerto Rico, Recinto de Mayaguez.p.3-17.
Grafton-Cardwell, E. E., Stelinski, L. L., & Stansly, P. A. 2013. Biology and management of
Asian citrus psyllid, vector of the huanglongbing pathogens. Annual review of entomology
58:413-432.
Flores-Torres, C. R. 2013. Efectos de bioestimulantes para sistemas orgánicos y sustentables en
limón ‘Tahití’ (Citrus latifolia Tanaka). Tesis de Maestría en Horticultura. Universidad de Puerto
Rico, Recinto de Mayaguez.p.5-19.
Ichinose,
K., Hoa, N. V., Bang, D. V., Tuan, D. H., & Dien, L. Q. 2012. Limited efficacy of
guava interplanting on citrus greening disease: Effectiveness of protection against disease
invasion breaks down after one year. Crop Protection, 34, 119-126.
Inch, S. 2014. Evaluation of Commercial Citrus Cultivars for Field Tolerance/Resistance to
Huanglongbing in East Central Florida. Abstracts of the American Society for horticulture
Meeting, Orlando, Florida. July 2014.
57
Rhytismatales and other pathogenic fungal species of native trees of Puerto Rico
Verónica M. Rivera Vega1 and Lydia. I. Rivera Vargas2
1
Undergraduate student, General Agriculture Program, 2Professor, Department of Crops
and Agro-Environmental Sciences, University of Puerto Rico-Mayaguez Campus,
Mayaguez, PR 00681 e-mail: [email protected]; [email protected]
Abstract
Most forests in Puerto Rico are devoted to the preservation of natural areas and watersheds,
to the conservation of biodiversity, and as such, as a source of passive recreation. Puerto
Rico’s flora includes 3,243 plant species, and at close to 1,050 are trees, estimating
endemism to be around 13.6%. Currently both biotic and abiotic factors are identified as
clear and present threats to this biological treasure. Among them, the advent of biological
invasions by exotic pests and diseases. Our research objective was to identify and
characterize microorganisms affecting native tree species. Samples were collected from tree
species showing diseased tissues from different locations and ecosystems around the island.
Locations sampled were: the University of Puerto Rico-Mayaguez Campus, the
“Arboretum Metropolitano del Parque Doña Inés” in Tujillo Alto, and coastal areas at
Joyuda, Cabo Rojo, Puerto Rico. Free-hand sections of plant tissue containing fungal
reproductive structures were examined by light and scanning electron microscopy. To
stimulate sporulation of pathogenic fungi, moist chambers containing infected plant tissues
were prepared. In addition, fungi were isolated from tissue sections after placing them in
culture media. For some specimens, DNA was extracted, sequenced and analyzed. Five
different species of fungi were identified on five endemic and native trees of Puerto Rico. A
fungal specimen belonging to the Order Rhytismatales was identified on the leaves of
Cocoloba nervosa collected at the “Arboretrum Metropolitano del Parque Doña Ines”.
Another fungus, Meliola spp. was identified on leaves of Crescentia cujete collected at the
same location. Through the use of molecular techniques, we confirmed the phytopathogenic
fungus, Hilberina caudata affecting the leaves of Cordia sebestena at the coastal areas of
Joyuda, Cabo Rojo, Puerto Rico. As part of this project we also identified the parasitic
green algae, Cephaleuros virescens in Simarouba talae tree. Puerto Rico is an island with
high density population and fragile ecosystems. Thus understanding fungal diseases
affecting our flora will allow us to develop an effective plan for the conservation of native
species of trees in Puerto Rico and the Caribbean.
Key words: Rhytismatales, phytopathogenic fungi, native trees.
Resumen
La mayoría de los bosques en Puerto Rico están dedicados a la preservación de los espacios
naturales y cuencas hidrográficas, y también para la conservación de la biodiversidad, y
como tal, como una fuente de recreación pasiva. La flora de Puerto Rico incluye 3,243
especies de plantas, y cerca de 1,050 árboles, la estimación de endemismo es alrededor de
un 13.6%. Actualmente tanto los factores bióticos y abióticos se identifican como amenazas
claras y presentes a este tesoro biológico. Entre ellos, la llegada de invasiones biológicas de
plagas exóticas y enfermedades. El objetivo de nuestra investigación fue identificar y
caracterizar los microorganismos que afectan a las especies de árboles nativos. Se
58
recogieron muestras de las especies de árboles que mostraban tejidos enfermos de
diferentes lugares y ecosistemas de la isla. Las ubicaciones donde se recolectaron las
muestreas fueron: la Universidad de Puerto Rico Recinto de Mayagüez, el Arboretum
Metropolitano del Parque Doña Inés en Tujillo Alto y las zonas costeras en Joyuda, Cabo
Rojo, Puerto Rico. Cortes histológicos a mano libre del tejido vegetal, que contenían
estructuras reproductivas de los hongos, fueron examinadas por microscopía de luz y
electrónica de rastreo. Para estimular la esporulación de hongos patógenos, se prepararon
cámaras húmedas con tejidos de las plantas infectadas. En adicción, los hongos fueron
aislados de secciones de tejido vegetal luego de, haber sido colocados a crecer en medios de
cultivo. Para algunas muestras, se extrajo ADN y este fue secuenciado y analizado. Se
identificaron cinco especies diferentes de hongos en cinco árboles endémicos y nativos de
Puerto Rico. Una muestra de hongos pertenecientes a la Orden Rhytismatales fue
identificado en las hojas de Cocoloba nervosa recogidos en el Arboretrum Metropolitano
del Parque Doña Inés. Otro hongo, Meliola spp. fue identificado en las hojas de Crescentia
cujete recogido en el mismo lugar. A través del uso de técnicas moleculares, se confirmó
que el hongo fitopatógeno, Hilberina caudata afecta a las hojas de Cordia sebestena en las
zonas costeras de Joyuda, Cabo Rojo, Puerto Rico. Como parte de este proyecto también
identificamos las algas verdes parasitaria, Cephaleuros virescens en el árbol Simarouba
talae. Puerto Rico es una isla con gran densidad de población y frágiles ecosistemas. Por
ende, la comprensión de las enfermedades fúngicas que afectan a nuestra flora nos
permitirá desarrollar un plan efectivo para la conservación de las especies nativas de
árboles en Puerto Rico y el Caribe.
Palabras claves: Rhytismatales, hongos fitopatógenos, arboles nativos
Introduction
Since ancient times trees have been important sources of income and fuel. At the present,
there is no organized production of hardwoods in Puerto Rico, with limited economic
activity in the form of artisanal charcoal and precious wood production. Most forests in
Puerto Rico are devoted to the preservation of natural areas and watersheds, to the
conservation of biodiversity, and as such, as a source of passive recreation. According to
Acevedo-Rodríguez and Strong (2012) the flora of Puerto Rico includes 3,243 plant
species, and at close to 1,050 are trees. These authors also estimate endemism to be around
13.6%. Currently both biotic and abiotic factors are identified as clear and present threats
to this biological treasure. Among them are the encroachment by human settlements, and
lately, the advent of biological invasions by exotic pests and diseases.
The study of diseases affecting tree species in Puerto Rico is almost not existent. Almost
plant pathology scientific work has been done for agricultural plants, so a great knowledge
void exists on threats to forest species, especially those affecting native and endemic plants.
Thus, the principal objective of this investigation was to begin a process of initial
identification of fungal microorganisms that affect native and endemic species of trees in
Puerto Rico.
59
Material and Methods
Leaf and bark samples collection: Leaf and bark tissue samples from endemic tree species
were collected at: the University of Puerto Rico-Mayaguez Campus (UPRM), the
“Arboretum Metropolitano del Parque Doña Inés” in Tujillo Alto, and coastal areas at
Joyuda, Cabo Rojo, Puerto Rico. Six species of trees were selected: Cocoloba nervosa,
Cordia sebestena, Crescentia cujete, Juglans jamaicensis, Simarouba talae and an
unknown species. These species showed symptoms of infection characterized by: wilting,
presence of necrotic tissues, and tar spots or green islands on leaves. Bark was collected
from Juglans jamaicensis and from a fallen unknown tree at the UPRM who’s trunk and
bark showed signs of putrescence. Samples were placed in individual plastic bags in a
portable cooler and transported to the Plant Pathology Lab at UPR-Mayaguez. Samples
were kept at 10°C before processing.
Microorganism Identification: For microorganism’s identification, reproductive
structures were mounted on microscope slides using a drop of lactophenol and examined
with a light microscope (Olympus, Ontario, Canada). For fungal or oomycetes specimens,
tissue sections of about 4mm were dissected from an area close to signs or spots. Dissected
sections were transferred to two culture media: potato dextrose agar (i.e. PDA) for fungal
isolation and PARPH agar for oomycetes isolation. PARPH Agar contained cornmeal and
antibiotics such as: pimaricin (5mg), rifampicin (10mg), ampicillin (250mg), hymezaxol
(50mg) and pentachloronitrobenzene (100mg). Petri plates were incubated at 26̊C. Once
growth was observed on Petri plates, colonies were purified for identification. Taxonomic
keys were used for fungal identification (Barnett and Hunter, 1982; Hanlin, 1990). In
addition, to achieve fungal sporulation, moist chambers were prepared from plant tissue and
incubated at room temperature. Direct isolation from fungal reproductive structures was
used for further purification and identification.
Scanning electron microscopy (SEM) was used to further examined samples from leaves of
C. nervosa and C. sebestena. Tissue sections (approx. 7 mm) were dehydrated and
processed previous to examination following the methodology stipulated by Bonilla (2012).
For fungal DNA analysis, DNA was extracted using DNeasy® Plant Mini Kit (Qiagen,
Alemania), following manufacturer’s instructions. DNA samples were amplified by PCR in
a thermocycler (Eppendorf, Hauppauge, NY). Reaction mix consisted of: 10µL of
AmpliTaq Gold® Fast PCR master mix (Life Technologies, Carlsbad, CA), 1.5µL of each
primer, 3µL of molecular water and 4µL from sample DNA. Primer sets used were
commonly employed to identify fungi belonging within the Order Rhytismatales. Primer
sets were: 5.8SR/LR6, mrSSU1/mrSSU3R, LROR/LR6 and LR3/LR6 (Lantz et al., 2011;
Vilgalys and Hester, 1990). In addition primer set ITS1/ITS4 were used (White et al.,
1990). Thirty PCR cycles were performed using the following parameters: 94°C
denaturation step (1 min), 50°C annealing step (45 s), 50 to 72°C ramp (1 min), and primer
extension at 72°C (1 min). A final 7-min incubation step at 72°C was added after the final
cycle. PCR products were visualized in 1.2% agarose gel electrophoresis containing TAE
buffer and 2.5µL of ethidium bromide. Molecular marker or ladder used was100bp (DNA
60
Ladder, BioLabs, New England). PCR product was purified by QI Aquick® Gel Extraction
Kit, (Qiagen, Germantown, MD) following manufactures instructions. DNA was sequenced
at commercial facilities (Macrogen Inc., Rockvillle, MD). Nucleotide sequences were
analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Three phytopathogenic fungi were identified on leaves of C. nervosa, C. sebestena and C,
cujete. Two saprophytic fungi were identified from bark of J. jamaicensis and from an
unknown fallen tree (Table 1). Table 1 summarized tree species and tissue sampled, and
microorganisms identified. On leaves of C. cujete, we observed pigmented mycelia,
hyphopodia and ascospores of the phytopathogenic fungus, Meliola sp. using light
microscopy (Figure 1). On leaves of C. nervosa, we observed reproductive structures of
Melasmia sp., the imperfect state a fungus belonging to the Order Rhytismatales, which is
also a phytopathogenic fungus (Figure 2). On leaves of C. sebestena we observed
ascospores of Hilberina caudata.using light microscopy (Figure 3). Through molecular
characterization of the LSS region, we were able to confirm the identity of the
pythopathogenic fungus as H. caudata (Figure 4).
Table 1. Trees species and tissue sampled, location and microorganism identified.
Tree
Location
Coccoloba
nervosa
Parque
Doña Inés
Cordia
sebestena
Crescentia
cujete
Juglans
jamaicensis
Simarouba
talae
Unknown
#1
Joyuda ,
Cabo Rojo
Parque
Doña Inés
Parque
Doña Inés
Parque
Doña Inés
UPRM
Tissue
Sample
Leaf
Type of
Microorganism
Phytopathogenic Fungi
Leaf
Microorganism
Identified
Melasmia sp.
Imperfect state,
Order Rhytismatales
Hilberina caudata
Leaf
Meliola sp.
Bark
Order Hysteriales
Saprophytic Fungi
Leaf
Cephaleuros
virescens
Trichoderma sp.
Plant parasitic green
algae
Saprophytic Fungi
Bark
Only samples from C. nervosa and C. sebestena were submitted to DNA analysis. We
were unable to obtained PCR products using primer sets ITS1/ITS4 and 5.8R/LR6. A 250
bp PCR product were obtained using a primer set of a mitochondrial region:
mrSSU1/mrSSU3R but DNA sequences were too short to make final conclusions (data not
shown). Successful PCR amplification was obtained using primer set LROR/LR6 for C.
61
sebestena. A final product of 1,165pb was amplified (Figure 4). The sequence obtained
showed 87% of homology to H. caudata when analyzed by BLAST.
A saprophytic fungus, Trichoderma sp. was isolated from bark of a fallen unknown tree.
Another saprophytic fungus was identified by direct examination of bark tissue of J.
jamaicensis, the fungus belongs to the Order Hysteriales. No oomycete were isolated from
vegetative tissue. Thus, PDA was better isolation media than PARPH. As part of this
project we also identified the parasitic green algae, Cephaleuros virescens in Simarouba
talae tree (Figure 5).
A
B
p
h
Figure 1. Crescentia cujete: A. Sooty mold growing on leaf; B. Reproductive structures of the
phytopatogenic fungus, Meliola sp. examined by light microscopy (40x). Mycelium, perithecia, (p) and
mucronate hyphopodia (h) were observed. Insert: Multicellular ascospores.
A
B
D
C
E
F
Figure 2: Cocoloba nervosa: A: Leaf showing tar spot and chlorosis. B: Group of pycnidia causing spots on
leaf. C: Conidias of Melasmia sp. (40X). D: Unknown structures observed on imperfect state, Melasmia sp.
(40X). E. Conidiophores (40X). F: Mycelial growth protruding through leaf stomata observed by Scanning
Electron Microscopy.
62
A
A
B
C
Figure 3. Cordia sebestena: A. Green islands on leaf (arrow). Photo by Alejandro Segarra; B. Ascospores of
Hilberina caudata. (40X); C. Mycelial growth on leaf observed by Scanning Electron Microscopy.
C
63
L
1
2
3
4
1
2
3
4
C
1,165pb
Figure 4: Agarose gel of PCR products using primer set LR3/LR6 (black numbers) and LROR/LR6 (whire
numbers) for samples of C. sebestena (1 and 2) and C. nervosa (3 and 4; arrow). L= ladder; C = Control
water.
A
B
Figure 5. Simarouba talae A. Leaf showing green algae spots. Photo by Victor Gonzalez. B. Cepahleuros
virescens (Photo from APS Press)
64
Symptoms observed on leaves of C. nervosa were similar to tar spots. A free-hand
histological section of leaf tissue and direct examination by light microcopy allowed the
identification of Melasmia sp., the imperfect stage of a fungus belonging to the order
Rhytismatales (Figure 2). We observed reproductive structures such as pycnidia (Figure
2D), and superficial mycelium invading leaves and protruding through stomata (Figure 2F),
conidiophores were simple or branched (Fig.2E) and conidias hyaline and allantoid
(Fig.2C) (Barnett and Hunter, 1998).
Green islands were observed on leaves of C. sebestena. The fungus invaded the stomata.
Ascospores were clavate, unicellular and hyaline, with the basal end bent (Figure 3B)
(Miller et al., 2014). Through molecular analysis we obtained and identification with 87%
of homology with the phytopathogenic fungus Hilberina caudata (Ascomycota). On leaves
of Crescentia cujete, we identified the fungus Meliola sp.. This genus is abundant in the
tropics and is known for the production of abundant superficial dark brown septate
mycelium. Mucronate hyphopodia are commonly observed associated with mycelium.
Globose, ostiolate perithecia are produced superficially on leaves containing multicellular
dark brown ascospores (Fig.1) (Kenneth, 2008). Trichoderma sp., a saprophytic fungus,
was recollected from an unknown fallen tree that was at the stage of decay. This fungus is
commonly found in different climate zones, having numerous means of survival and
proliferate (Harman, G and Kubicek, 2005). Juglans jamaicensis was also found at the
stage of decay at moment of the recollection, identifying the saprophytic fungus from Order
Hysteriales. We were unable to identify the pathogenic organism that caused its death.
Simarouba talae is a perennial tree, native from P.R that grows on the mountains of the
island. On leaves of S. talae we identified C. virescens a green algae that affect mostly trees
of tropical and subtropical zones. This algae is commonly found affecting mango leaves
(Fig.2) (Litz, 2009). This algae requires high relative humidity to caused infections.
Initially it produces a spot of about 5 to 8mm of diameter, with a red to orange coloration
turning to a necrotic lesion with age (Litz, 2009).
Conclusion
In conclusion, we identified five different species of fungi in five endemic and native trees
of Puerto Rico. Three of them, H. caudata, Meliola sp. and Melasmia sp. are known
phytopathogenic species. Trichoderma sp. and a fungus from the Order Hysteriales are
saprophytic species. A green algae, C. virescens was identified on leaves of a native tree
of P.R. Puerto Rico is an island with high density population and fragile ecosystems. Thus
understanding fungal diseases affecting our flora will allow us to develop an effective plan
for the conservation of natives and endemic species of trees in Puerto Rico and the
Caribbean.
Cited Literature
Acevedo-Rodríguez, P., Strong, M. T. 2012. Catalogue of seed plants of the West Indies.
Smithsonian Contributions to Botany. 98: 1-1192.
65
Barnett, H.L. and Hunter, B.B. 1998. Illustrated genera of imperfect fungi, 4th edition.
American Phytophathological Society Press, MN, USA, 174pp
Bonilla Avilés, L.L. 2012. Identificación de especies anamórficas de añublos polvorientos
(Ascomycota: Orden Erysiphales) presentes en plantas ornamentales y aromáticas en Puerto
Rico Tesis. M.S Depto. Cultivos y Ciencias Agroambientales, UPR. Mayagüez, P.R.133pp
Cooke, M.C. 1871. Handbook of British fungi, Vol.II. Macmillan and CO. London, UK,
757pp
Hanlin, R.T. 1990. Illustrated genera of Ascomycetes,Vol.I. American Phytopathological
Society Press, MN, USA, 194pp
Harman, G.E and Kubicek, C.P. 2005. Trichoderma & Gliocladium: Enzymes, biological
control and applications, Vol.II. Taylor & Francis Ltd. PA, USA
Kenneth, R. 2008. Westcott’s plant disease handbook, 7th edition. Springer-Verlag Berlin
Heidelberg, NY, USA
Lantz, H., Jonhston, P.R, and Minter, D.W. 2011. Molecular phylogeny reveals a core clade
of Rhytismatales. The Mycological Society of America, KS, USA
Litz, R.E. 2009. The Mango: Botany, production and uses, 2nd edition. CAB International.
MA, USA, 232-233pp.
Miller, A.M., S.M. Huhndorf, and J. Fournier. 2014. Phylogenetic relationships of five
uncommon species of Lasiosphaeria and three new species in the Helminthosphaeriaceae
(Sordariomycetes). Mycologia, 106(3), 2014, pp. 505–524
Vilgalys R. and Hester M. 1990. Rapid genetic identification and mapping of enzymatically
amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246.
Acknowledgements
This project was supported by CARIPAC program and Agriculture and Food Research Initiative Competitive
Grant no. 2012-01871 from the USDA National Institute of Food and Agriculture, Hispanic Serving
Institution.
66
“This project was supported by the Resident Instruction
in Insular Areas Program Competitive Grant no. 201370004-21018 and 2014-70004-22033 from the USDA
National Institute of Food and Agriculture.”

Proceedings Undergraduate Research Experiences

Transcripción

Documentos relacionados

historic background - Government Development Bank for Puerto Rico

Caribe - New York University

our NAI Peru Overview

Modern Myths Concerning Life on Mars