Rhynchophorus ferrugineus

MICROSCOPY RESEARCH AND TECHNIQUE 00:000–000 (2010)
Infection of the Red Palm Weevil (Rhynchophorus ferrugineus)
by the Entomopathogenic Fungus Beauveria bassiana:
A SEM Study
BERENICE GÜERRI-AGULLÓ,1* SONIA GÓMEZ-VIDAL,1 LETICIA ASENSIO,1 PABLO BARRANCO,2
1
AND LUIS V. LOPEZ-LLORCA
1
Department of Marine Sciences and Applied Biology, Laboratory of Plant Pathology, Multidisciplinary Institute for Environmental
Studies (MIES) Ramon Margalef, University of Alicante, Ap. 99, 03080 Alicante, Spain
2
Department of Applied Biology, CITE II-B, E.P.S., University of Almeria, 04120 Almerı́a, Spain
KEY WORDS
biological control; fungal morphogenesis; appressoria; insect cuticle degradation
ABSTRACT
The Red Palm Weevil (Rhynchophorus ferrugineus) is a devastating pest of palms
in the Mediterranean, Middle East, and Eastern countries. No effective control measures are available. R. ferrugineus has been found naturally infected by the entomopathogenic fungus Beauveria
bassiana, but its infection process in this host is unknown. We have studied the infection of R. ferrugineus larvae and adults by B. bassiana using dry conidia and conidia suspensions using scanning electron microscopy (SEM). In early stages, SEM revealed acquisition of B. bassiana conidia
by cuticle ornamentation in legs, antennae, and elytra of R. ferrugineus adults. Subsequently, conidia germinated and frequent episodes of hyphal/conidial fusion were found. Appressoria, signs of
adhesion and cuticle degradation led to penetration (even direct) and colonization of R. ferrugineus
hosts by the fungus. B. bassiana conidiophores were found in a R. ferrugineus cuticle, which indicate the completion of the life cycle of the fungus in the insect host. SEM has proven that dry conidia of B. bassiana is an adequate inoculum for R. ferrugineus infection. SEM revealed that conidia
of B. bassiana attached to the cuticle of R. ferrugineus can germinate and differentiate appressoria.
Microsc. Res. Tech. 00:000–000, 2010. V 2009 Wiley-Liss, Inc.
C
INTRODUCTION
The red palm weevil Rhyncophorus ferrugineus is a
devastating palm pest. This weevil affects more than
20 palm species (Barranco et al., 2000) including the
date palm (Phoenix dactylifera L.), Canary Islands
date palm (P. canariensis Hort), coconut (Cocos nucifera L.), and African oil palm (Elaeis guineensis Jacq.).
Rhynchophorus ferrugineus was introduced in Spain
mainland in 1995 (Barranco et al., 1996a, 1996b) and
then spread to all palm growing areas in the Mediterranean and recently also to the Canary Islands. The
pest has caused large economic losses in date palms
worldwide for the last 30 yr (Faleiro, 2006; Murphy
and Briscoe, 1999), and still there are no effective control measures.
Entomopathogenic fungi infect a wide range of insect
pests of socioeconomic importance (Bidochka et al.,
2000). Beauveria spp. are important coleopteran pathogens (Tanada and Kaya, 1993). Entomopathogenic
fungi are, therefore, being put forward as biological
control agents in Integrated Pest Management (IPM)
programs to control R. ferrugineus current outbreaks
(Faleiro, 2006; Murphy and Briscoe, 1999).
Scanning electron microscopy (SEM) has frequently
been used to evaluate the infection process of entomopathogenic fungi versus their insect hosts (Asensio
et al., 2005; Kirkland et al., 2004; Neves and Alves,
2004; Pedrini et al., 2007; St. Leger et al., 1991; Talaeihassanloui et al., 2007). SEM has also been applied to
study morphological features of R. ferrugineus (Salama
C
V
2009 WILEY-LISS, INC.
and Abdel Aziz, 2001) and those of the close relative
species R. palmarum (Saı̈d et al., 2003).
R. ferrugineus has been found naturally infected by
the entomopathogenic fungus Beauveria bassiana
(Güerri-Agulló et al., 2007, unpublished), but its infection process in this host is unknown. We have studied
the infection of R. ferrugineus larvae and adults by
B. bassiana using dry conidia and conidia suspensions
using SEM.
In this study, we present a time-course of the infection process of R. ferrugineus by two B. bassiana isolates obtained from naturally infected insects. We have
used SEM to study the infection process of B. bassiana
in R. ferrugineus cuticle and whole insects. We have
used R. ferrugineus adults because they can be readily
infected with B. bassiana outside the palm tissues.
Infected adults can deliver the fungus to uninfected
insects and to palm tissues. Our results indicate that
R. ferrugineus cuticle captures abundant inoculum of
*Correspondence to: Berenice Güerri-Agulló, Department of Marine Sciences
and Applied Biology, Laboratory of Plant Pathology, Multidisciplinary Institute
for Environmental Studies (MIES) Ramon Margalef, University of Alicante,
Apdo. 99, 03080 Alicante, Spain. E-mail: [email protected]
Received 9 July 2009; accepted in revised form 28 October 2009
Contract grant sponsor: Spanish Ministry of Science and Innovation; Contract
grant numbers: INIA (TRT2006-0016-C07-02) and PETRI (PET2007-0469-01);
Contract grant sponsor: Generalitat Valenciana; Contract grant numbers:
BFPI06/493 and GVPRE/2008/038.
DOI 10.1002/jemt.20812
Published online in Wiley InterScience (www.interscience.wiley.com).
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GÜERRI-AGULLÓ ET AL.
Fig. 1. SEM of 7- to 10-day old Rhynchophorus ferrugineus uninfected (control) larvae. (a) General
(dorsal) view. Bar 5 2 mm. (b) Close-up of head region. Bar 5 500 lm.
Fig. 2. Light and SEM of uninfected (control) Rhynchophorus ferrugineus adult. (a) General view. Bar 5 1 cm. (b) SEM of rostrum and
antenna organs. Parts: scape (sc), pedicel (pe), flagellum (fl), club (cl),
and flagellomeres (fla). Bar 5 2.5 mm. (c) SEM of elytra showing long
furrows. Bar 5 1 mm. (d) SEM of R. ferrugineus leg. Parts: femur (fe),
tibia (ti), and tarsus (ta). Bar 5 2.5 mm. (e) Close-up of femur, showing long and thin setae and short and wide thorns. Bar 5 500 lm.
[Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
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Fig. 3. SEM of young (7- to 10-day old) Rhynchophorus ferrugineus larvae infected with Beauveria
bassiana. (a) Detail of insertion of mouthpart with hyphae on the cuticle 2 days after inoculation. Bar 5
100 lm. (b) Close-up showing two intercalary appressoria (arrows). Bar 5 20 lm.
Fig. 4. SEM of femur and tibia of Rhynchophorus ferrugineus
adults infected with Beauveria bassiana. (a) Femur and tibia joint
area showing hyphae and conidia 6 days after inoculation (DAI). Note
abundant spines or coeloconic sensilla (cuticle ornamentations).
Bar 5 500 lm. (b) Close-up of the tibia showing a hyphal network
with conidia and cuticle spines. Bar 5 100 lm. (c) Detail of the former
showing hyphae and conidia in the spine socket. Bar 5 50 lm.
the fungus that leads to insect death. With the information obtained, we are developing a mycoinsecticide
based on B. bassiana for biocontrol of R. ferrugineus.
cell counter polystyrene vials with cap (VWR International, Barcelona, Spain) containing 15 mL of artificial
food substrate. Fifteen days later, larvae were then
transferred to new VWRblood cell counter polystyrene
vials with fresh food substrate. After 30 days, larvae
were transferred to 150 cm3 containers containing 30
mL of artificial diet. Food substrate was replaced every
15 days for three months. Larvae were then introduced
in 150 cm3 containers with 15 g of palm fiber as substrate for pupation.
B. bassiana isolates (203 and 193) were obtained
from naturally infected R. ferrugineus adults in E and
SE Spain. All fungi belong to our laboratory collection
and were kept at 48C in the dark on corn meal agar
(CMA; BBL Sparks, MD).
MATERIALS AND METHODS
Insects and Fungi
R. ferrugineus was reared in the laboratory of Entomology at the University of Almeria (Spain) in a
SANYO MLR-351 incubator at 25 6 0.58C in the dark.
Adults were bred in pairs using sugarcane (Saccharum
officinarum L.) as both food and oviposition substrate.
Eggs were collected from sugarcane and were placed in
sterile 9-cm diameter plastic Petri dishes with an artificial food substrate (Alarcon et al., 2002). Emerged larvae were individually transferred to 20-mL VWRblood
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GÜERRI-AGULLÓ ET AL.
and larvae (7- to 10- or 30-d old) were inoculated with
B. bassiana as follows. Insects of the same stage were
placed in pairs in 9-cm Petri dishes containing 24-dayold CMA colonies of a given B. bassiana isolate for 15
min. Controls consisted of insects placed on Petri
dishes containing CMA only.
Insects were then placed in moist chambers at 258C
for 12 h. Afterward, insects were transferred to sterile
Petri dishes to avoid insect reinoculation by the lost
inoculum around the chamber. Samples were collected
at 1–2 d for 7- to 10-d-old larvae, 3–4 days for 30-d-old
larvae, and 1, 2, 4, 5, 6, 7, and 9 days for adults.
R. ferrugineus Excised Cuticle
Thirty-six R. ferrugineus cuticle fragments (ca. 5 3
5 mm) were excised axenically from several parts of
the insect body. Eighteen fragments were inoculated
with a given B. bassiana isolate (see Insects and Fungi
section), by rubbing them on a CMA plate with a 24-dold colony of the fungus under assay. Eighteen control
fragments were treated with Petri dishes containing
uninoculated CMA only. Eighteen cuticle fragments
per isolate were alternatively inoculated with B. bassiana conidia suspensions as follows. Each fragment was
inoculated with 20 lL of a 1 3 106 conidia!ml21 suspension in 0.02% Tween-20 (Sigma) on R. ferrugineus cuticle. Controls consisted of fragments treated with 20 lL
of 0.02% Tween-20 only. After treatment, cuticle fragments were incubated as already explained for 1, 2, 3,
and 4 days. At each time, six cuticle fragments were
collected and stored as for whole insects (see earlier)
until processed for SEM.
Scanning Electron Microscopy
Frozen adults and larvae from bioassays were
fragmented before processing for SEM. The legs, elytra, abdomen, and head were excised from R. ferrugineus adults, whereas 30-d-old larvae were fragmented in two pieces (head and body). Insect pieces
and cuticle fragments were fixed overnight at 48C in
4% glutaraldehyde in 0.05 M phosphate buffer (pH
7.3) and rinsed three times (10 min each) in phosphate buffer. Samples were then dehydrated in an
ethanol series and critical point dried. Some cuticle
fragments were cut with a fresh scalpel blade before
further processing. Specimens were sputter-coated
with gold, observed, and photographed in a HITACHI S-3000 N SEM at 16.5 6 4.6 kV (Asensio et al.,
2005; Lopez-Llorca et al., 1999).
Fig. 5. SEM of legs of Rhynchophorus ferrugineus adults infected
with Beauveria bassiana. (a) Extensive growth in the insertion of first
and second tarsomere 9 DAI. Bar 5 100 lm. (b) Close-up showing possible appressorium. Bar 5 20 lm. (c) Claws at the end of last tarsal
segment 9 DAI showing hyphal growth. Bar 5 250 lm. (d) Close-up of
claw insertion area with abundant hyphal growth. Bar 5 100 lm.
Inoculation of R. ferrugineus With
Entomopathogenic Fungi
R. ferrugineus Larvae and Adults. R. ferrugineus
adults (up to one month after emergence from pupae)
RESULTS
Larvae of R. ferrugineus had a typical appearance in
the SEM showing a soft body and hard sclerotized head
region (Fig. 1a). A close-up of the head of a 7-10-dayold larvae showed a smooth surface except for the presence of scarce long (up to ca. 400 lm) and thin sensilla
(Fig. 1b). Older larvae due to their large size and softness were nonamenable to fragmentation for conventional SEM processing and are, therefore, not included
in this study.
R. ferrugineus adults had the typical general structure of a coleopteran (Fig. 2a). The clavate antenna
showed a long scape (sc; Fig. 2b) typical for this species.
The scape was followed by a pedicel (pe, Fig. 2b) and 6
flagellomeres (fla; Fig. 2b). The apical flagellomere is
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SEM OF RPW INFECTION BY BEAUVERIA BASSIANA
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Fig. 6. SEM of antennae of Rhynchophorus ferrugineus adults
infected with Beauveria bassiana. (a) Close-up of area between two
flagellomeres 2 DAI showing ungerminated conidia. Bar 5 50 lm. (b)
Detail of a conidium (arrow head) with bipolar germination 2 DAI.
Bar 5 10 lm. (c) Flagellum 7 DAI showing hyphae and conidia. Bar 5
100 lm. (d) Close-up of accumulation of B. bassiana inoculum
between two flagellomeres 9 DAI. Bar 5 20 lm. (e) Close-up of sensory surface of club 9 DAI showing abundant fungal growing over the
sensilla. Bar 5 100 lm. (f) Detail showing hyphae growing over sensory surface. Bar 5 20 lm.
the club (cl, Fig. 2b). The adjacent part, the pronotum,
had a smooth appearance with characteristic spots.
The remainder of the thorax and the abdomen was protected by two elytra masked with dark stripes (narrow
furrows) (Fig. 2c). A SEM of a leg (Fig. 2d) clearly
showed three regions: the femur (fe, Fig 2d), the tibia
(ti, Fig. 2d), and the tarsus. The tarsus is divided into
five tarsomeres. The distal tarsomere shows two tarsal
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GÜERRI-AGULLÓ ET AL.
Fig. 7. SEM of elytron of Rhynchophorus ferrugineus adults inoculated with Beauveria bassiana (dry conidia) 1 day after inoculation.
(a) General view. Bar 5 1 mm. (b) Detail of elytral furrow showing
abundant conidia. Bar 5 100 lm. (c) Germinated conidia
(‘‘germlings’’) of B. bassiana on a R. ferrugineus cuticle zone with cuticle ornamentaions. Bar 5 20 lm. (d) Close-up of B. bassiana germ-
lings. Bar 5 10 lm. (e) B. bassiana germlings displaying bidirectional
germ tubes. Bar 5 20 lm. (f) Detail of germling on a microtopographic
grove. Bar 5 10 lm. (g) Germlings adhereing (arrow head) to R. ferrugineus cuticle. Bar 5 10 lm. (h) Close-up of appressorium with signs
of adhesion/degradation to R. ferrugineus cuticle. Bar 5 1 lm.
claws. A close-up of the femur showed details of the
abundant long and thin (ca. 100 lm) sharp spines present in one side of the femur plus short and wide thorns
(Fig. 2e).
When larvae of R. ferrugineus were inoculated
with dry conidia of the entomopathogenic fungus B.
bassiana, they showed signs of infection within 3–5
d (data not shown). The head region in the mouthMicroscopy Research and Technique
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Fig. 8. SEM of elytron of Rhynchophorus ferrugineus adult inoculated with Beauveria bassiana (dry conidia) 2 days after inoculation.
(a) Extensive hyphal development. Bar 5 50 lm. (b) Detail showing
early anastomosis of two germ tubes. Bar 5 5 lm. (c) Close-up showing early conidial fusion. Bar 5 5 lm. (d) B. bassiana hyphal fusion
(arrow head) and attempted hyphal anastomosis (circle) on R. ferrugineus cuticle. Bar 5 20 lm. (e) B. bassiana germ tubes entering R. ferrugineus hair socket. Bar 5 20 lm. (f) Hyphal network with conidial
fusion (arrow head). Bar 5 20 lm. (g) Intercalary appressorium with
mucilage. Bar 5 5 lm.
part insertion showed abundant hyphae of the fungus 2 days after inoculation (DAI) (Fig. 3a). By that
time, B. bassiana had started to differentiate penetration structures such as laterally or intercalary
appressoria produced by mycelial hyphae (Fig. 3b,
arrowheads).
When R. ferrugineus adults were inoculated in the
same way they acquired B. bassiana inocula in several
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GÜERRI-AGULLÓ ET AL.
Fig. 9. SEM of elytron of Rhynchophorus ferrugineus adult inoculated with Beauveria bassiana (dry conidia) 3 days after inoculation.
(a) General view of cuticle showing two furrows with extensive mycelial development. Bar 5 500 lm. (b) Detail of hyphae and germinated
conidia. Bar 5 50 lm. (c) Close-up of germlings showing conidial
fusion (arrowhead), conidium-hypha fusion (solid square), and
appressorium adhering to cuticle (solid circle). Bar 5 20 lm. (d)
Close-up of mature appressorium (septate) with mucilage (arrowhead). Bar 5 5 lm.
regions of their bodies. R. ferrugineus legs closer to the
fungal colony captured inoculum of the fungus. The femur and tibia joint area contained numerous spines—
coeloconic sensilla—which showed abundant B. bassiana conidia (Fig. 4a and 4b). Coeloconic sensilla (Fig.
4c) showed abundant conidia and hyphae of B. bassiana. Six DAI a hyphal network was present (Fig. 5a)
on the tarsomeres. There were abundant hyphae
(9 DAI) between the first and the second tarsal segment (Fig. 5b). Similar features were found in the
insect claws (Fig. 5c) and their joint areas (Fig. 5d).
A time-course of the fungus development on R. ferrugineus antennal cuticle is shown in Figure 6. Early after inoculation (2 d), the area between two flagellomeres showed ungerminated conidia (Fig. 6a). Some
conidia showed germination signs, including bipolar
germ tubes (Fig. 6b). Five to seven days later, hyphal
growth was found in these zones (Fig. 6c and 6d). The
sensory surface of the club with abundant sensillae
captured copious conidia, which developed a dense mycelium (Fig. 6e and 6f) in late infection steps.
Elytra fragments from R. ferrugineus adults exposed
to B. bassiana dry conidia were also observed with
SEM. Their large surfaces also showed abundant B.
bassiana inocula (Fig. 7a and 7b). On R. ferrugineus
elytral cuticle, early (1 DAI) morphogenetic events of
B. bassiana were easily found (Fig. 7c–7h). Cuticle surface had either densely spinal ornamentations (Figs. 7c
and 7d, 7g and 7h) or microfolder structures (Fig. 7f).
In the former, areas spines had abundant B. bassiana
conidia in coeloconic sensilla (Fig. 7c) just as for R. ferrugineus legs. Bipolar germ tube formation was very
frequent (Figs. 7c and 7e, arrowheads). Even short (ca:
5 lm long) germ tubes started to differentiate appressoria (Fig. 7f). These were formed in microfolder structures (Fig. 7f, arrowhead) or spinal areas (Fig. 7g,
arrowhead) of R. ferrugineus cuticle. These early
formed appressoria showed signs of mucilage production or degradation of R. ferrugineus cuticle (Fig. 7h).
In the areas of elytra fragments with more hyphal
development (Fig. 8a, 2 DAI), frequent episodes of
hyphal/conidial fusion were found. These included
germ tube anastomosis (Fig. 8b), germ tube apex fusion
(Fig. 8d, arrowhead), and directional growth (Figs. 8d,
circle). Conidial fusion possibly through conidial anastomosis tubes (CATs) was also detected (Fig. 8c and 8f,
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Fig. 10. SEM of elytron of Rhynchophorus ferrugineus adult inoculated with dry conidia (a, b) or conidia suspensions in water (c, d)
of Beauveria bassiana 4 days after inoculation. (a) Differenciation of
sympodial (zig-zag) typical B. bassiana conidiophores (arrowhead) on
R. ferrugineus cuticle. Bar 5 20 lm. (b) Hyphae penetrating (arrow-
heads) R. ferrugineus cuticle. Bar 5 20 lm. (c) Signs of cuticle degradation (arrowhead) surrounding B. bassiana germling. Bar 5 10 lm.
(d) Extensive cuticle degradation surrounding B. bassiana hyphae
(arrowheads). Bar 5 20 lm.
arrowhead). Fully developed hyphae were found entering coeloconic sensilla (Fig. 8e). Some of these developed intercalary appressoria (Fig. 8g). Mucilage formation was also associated with these structures (Fig. 8g,
arrowhead). Further fungus development was particularly obvious in elytral furrows (Fig. 9a and 9b). CATs
were frequently found (Fig. 9c, arrowhead). Tubes fusing conidia-hyphae (Fig. 9c, solid square) were also
found. Mature appressoria (with septae) (Fig. 9c, solid
circle; 9d arrowhead) were found. These produced large
amounts of mucilage.
Formation of typical sympodial (zig-zag) B. bassiana
conidiophores in elytra fragments was also found
(Fig. 10a, arrowhead). Signs of direct cuticle penetration were found in spinal areas (Fig. 10b, arrowheads).
In the surroundings of well-developed appressoria
signs of extensive cuticle degradation or masking cuticle ornamentations by mucilage production were particularly obvious (Fig. 10c, arrowhead). In the vicinity
of hyphae, these signs were also found (Fig. 10d, arrowheads).
Lateral views of cuticle fragments revealed the
elytral furrows (Fig. 11a). These were areas of thinner
cuticle (Fig. 11a, arrowhead). A close-up view showed
the multilayer structure of R. ferrugineus cuticle
(Fig. 11b). An unequivocal sign of cuticle penetration is
shown in Figures 11c and 11d.
Microscopy Research and Technique
DISCUSSION
Entomopathogenic fungi are among the most relevant biological agents suggested to control R. ferrugineus (Faleiro, 2006). Particularly, we have found
B. bassiana infecting naturally R. ferrugineus (GüerriAgulló et al., 2007, unpublished). In this study, we
have studied the infection of R. ferrugineus larvae, and
adults by B. bassiana isolates from naturally infected
insects. We have used dry conidia and conidia suspensions to describe the infection process of R. ferrugineus
by the entomopathogenic fungus B. bassiana using
SEM. SEM has already been used to study the infection process of entomopathogenic fungi as palm endophytes (Gomez-Vidal et al., 2006), colonizing palm
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GÜERRI-AGULLÓ ET AL.
Fig. 11. SEM of Rhynchophorus ferrugineus cuticle fragments inoculated with Beauveria bassiana. (a) General view of elytral fragment
showing a furrow (arrowhead) sideways 3 DAI. Bar 5 200 lm. (b)
Lateral view of a cuticle fragment from R. ferrugineus thorax 1 DAI.
Bar 5 100 lm. (c) Lateralclose-up of elytron showing a hypha (arrowhead) penetrating the cuticle 1 DAI. Bar 5 25 lm. (d) Detail of penetration point. Bar 5 2 lm.
waste (Lopez-Llorca and others, 1999) and infecting
palm pests other than R. ferrugineus (Asensio et al.,
2005). To the best of our knowledge, there are only two
SEM studies available on Rhynchophorus spp. limited
to the sensory organs (Saı̈d et al., 2003; Sharaby and
Al-Dosary, 2007).
In this study, R. ferrugineus larvae inoculated with
B. bassiana showed fungus growth between the mouth
and frons regions. Because these larvae in our study
remained alive for several days (results not shown),
they could spread the fungus when feeding on palm tissues. Presence of intercalary appressoria on the head
region would suggest attempts of fungus penetration.
Similar structures have also been found in fungal parasites of nematode eggs (Lopez-Llorca and Claugher,
1990).
R. ferrugineus larvae under natural conditions
(infecting palms) are a difficult target for entomopathogenic fungi due to their endophytic behavior (Al-Manie
and Alkanhal, 2004; Wattanapongsiri, 1966). Therefore, we focused our study on R. ferrugineus adults the
likely target for B. bassiana under field conditions.
Infected adults can remain alive for a few days after
infection. Some of them can even bear fungal inoculum
outside body while insects are still alive (data not
shown). These insects could infect healthy R. ferrugineus insects. R. ferrugineus adult cuticle showed under
SEM microstructures (spines, microfolders, and sensilla) able to acquire B. bassiana conidia. Sensilla were
also conducive to inoculum activation and differentiation, especially in the insect legs and ellytrae. Host
surface microstructure is relevant for differentiation of
infective structures by fungal pathogens (Apoga et al.,
2004; Hoch et al., 1987; Kolattukudy et al., 1995;
Madelin et al., 1967).
B. bassiana conidia germinated on R. ferrugineus cuticle and often showed bipolar germ tubes in excised
cuticle. Hyphae and conidia fusion episodes (e.g.,
hyphae-hyphae, conidium-conidium and hyphae-conidium) were often observed. We found structures resembling CATs in B. bassiana. These structures were
recently described in Neurospora crassa (Roca et al.,
2005) using SEM, and their function or role is still a
matter of discussion. CAT formation has never been
reported before in entomopathogenic fungi. The high
frequency of CATs like structures of B. bassiana on R.
ferrugineus cuticle suggests that they may play a role
in the infection process. The true nature of CATs like
structures and their possible role in insect pathogenicity should be analyzed experimentally.
Appressoria were formed terminally on germ tubes
and laterally or intercalary in hyphae. These structures are well known from fungal invertebrate pathogens (nematophagous and entomopathogenic fungi) as
penetrating structures (Lopez-Llorca and Claugher,
1990; Lopez-Llorca et al., 2002; Nahar et al., 2008; St.
Leger et al., 1989, 1996). The high frequency of B.
bassiana appressoria differentiated on R. ferrugineus
cuticle suggests that this structure provides the signals
required for the triggering of this key event in fungus
penetration of the host. Adhesives and cuticle degrading enzymes are formed by fungus pathogen when
invading their hosts (Donatti et al., 2008; Zhang et al.,
2008). Appressoria and mucilaginous substances have
been detected for most entomopathogenic fungi including Metarhizium anisopliae and B. bassiana (Holder
and Keyhani, 2005; Wang and St. Leger, 2007).
This inoculum activation and differentiation led to
host infection. SEM of R. ferrugineus cuticle fragments
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SEM OF RPW INFECTION BY BEAUVERIA BASSIANA
has shown—for instance—that elytral furrows are
zones with thinner cuticle underneath. They are,
therefore, together with sensilla and sensilla sockets
body areas especially prone to B. bassiana infection.
This could be important for the establishment of epizootics or massive infections of R. ferrugineus with B.
bassiana when the fungus is applied under natural
conditions. Late stages of B. bassiana pathogenesis to
R. ferrugineus were characterized by extensive mucilage production and possible degradation of cuticle
ornamentations by appressoria. Cuticle penetration
and B. bassiana conidiophores indicated the completion of the life cycle of the fungus in the insect host.
SEM has helped to study the germination, adhesion,
and differentiation of a B. bassiana isolate from R. ferrugineus on its host cuticle. We have confirmed acquisition of inocula, differentiation of the fungus, adhesion,
colonization, penetration, and esporulation. Wang and
St. Leger (2005) reported that full infection process
(adhesion, germination, and appressoria differentiation) was found in M. anisopliae on its host cuticle.
However, only parts of this developmental process
were displayed on nonhost cuticles. In this way, SEM is
a good tool to evaluate the formulations of entomopathogenic fungi. Such formulations are being developed
in our laboratory to control R. ferrugineus in palms
under natural conditions. Hyphal fusion and adhesion
could be analyzed and correlated with the pathogenicity of the fungus measured in bioassays both in lab and
under semifield—controlled conditions—including
palms. These studies are also in progress in our
laboratory.
ACKNOWLEDGMENTS
The authors thank Dr. J.M. Llorens (Conselleria
d’Agricultura, Pesca i Alimentació, Generalitat
Valenciana) for kind supply of natural infected Rhynchophorus ferrugineus insects for Beauveria bassiana
isolation and Mr. Andrés Amorós (SSTTI, Universidad
de Alicante) for technical support with SEM.
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