Beauveria Bassiana As Endophyte in Cocoa

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Artículo Científico Posada, F.J.; Chaves, F.C.; Gianfagna, T.J.; Pava-Ripoll, M.; Hebbar, P.: Beauveria bassiana as endophyte in cocoa

ESTABLISHMENT OF THE FUNGAL ENTOMOPATHOGEN Beauveria bassiana AS AN ENDOPHYTE IN COCOA PODS (Theobroma cacao L.)

ESTABLECIMIENTO DEL HONGO ENTOMOPATÓGENO Beauveria bassiana COMO ENDÓFITO EN FRUTOS DE CACAO (Theobroma cacao L.)

Francisco J. Posada1, Fabio C. Chaves2, Thomas J. Gianfagna3, Monica Pava-Ripoll4, Prakash Hebbar5

1 I.A. Ph.D. Programa de Ingeniería Agronómica. Universidad de Ciencias Aplicadas y Ambientales U.D.C.A, Calle 222 No. 54-37, Bogotá, D.C., Colombia. [email protected] (corresponding author); 2 Ph.D. Department of Plant Biology and Plant Pathology, Rutgers, The State University of New Jersey, 59 Dudley road, New Brunswick, NJ 08901 USA. 3 Biologist, Ph.D. Department of Plant Biology and Plant Pathology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901 USA. 4 Bacteriologist, Ph.D. Department of Entomology, University of Maryland, College Park, MD 20742, USA. 5 Microbiologist, Ph.D. US Department of Agriculture-APHIS-PPQ, Emergency & Domestic Programs, 4700 River Road # 5C-0340. Riverdale, Maryland, USA, 20737.

Rev. U.D.C.A Act. & Div. Cient. 13 (2): 71-78, 2010

ABSTRACT RESUMEN

The entomopathogenic fungus Beauveria bassiana was El hongo entomopatógeno Beauveria bassiana fue re-aislado re-isolated from surface sterilized pieces of the cocoa pod de superficies desinfectadas de fragmentos de la parte externa outer section and peduncle, two and three months after y del pedúnculo del fruto de cacao luego de dos y tres meses flowers of greenhouse cocoa plants were hand-pollinated de que las flores de plantas criadas en invernadero fueran and sprayed with a 1x107 mL-1 suspension of B. bassiana polinizadas manualmente y asperjadas con una suspensión conidia. The colonization frequency of B. bassiana on cocoa de 1x107 mL-1 esporas de B. bassiana. La frecuencia de pods was 0.8% and 1.3% two and three months post-spraying colonización de B. bassiana en el fruto de cacao fue de 0,8% y of conidial suspension, respectively. These results indicate 1,3% luego de dos y tres meses de la aspersión de las esporas, that this entomopathogenic fungus can be established as respectivamente. Estos resultados indican que este hongo an endophyte on cocoa pods if flowers are sprayed. Other entomopatógeno puede ser establecido como hongo endófito naturally occurring endophytic fungi such as Acremonium de frutos de cacao luego de la aspersión de las flores de caco spp., Aspergillus spp., Cladosporium spp. and Penicillium con esporas del hongo. Durante el estudio, también se aislaron spp. as well as seven morphospecies of bacteria were also otros hongos endófitos del fruto de cacao como Acremonium isolated from cocoa pods. These findings suggest that cocoa spp., Aspergillus spp., Cladosporium spp. y Penicillium spp, pods are conducive for endophytic fungal growth of both y siete morfoespecies de bacterias, demostrando su presencia naturally occurring and inoculated B. bassiana, and could natural como endófitos de esta planta. Estos resultados possibly lead to an innovative methodology to manage insect sugieren que los frutos de cacao contribuyen al crecimiento pests in biocontrol programs. tanto de hongos endófitos naturales como del hongo B. bassiana inoculado, y esto podría conllevar al establecimiento Key words: Ascomycota, hypocreales, biological control, de una metodología novedosa para el manejo de insectos endophytes, entomopathogens, Theobroma cacao. perforadores de frutos de cacao en programas de control biológico de insectos plaga.

Palabras clave: Ascomycota, hypocreales, control biológico, endófitos, entomopatógenos, Theobroma cacao.

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INTRODUCTION Snellen (Lepidoptera: Gracillariidae) in Southeast Asia (Flood et al. 2004). Monalonium spp sucks the cocoa fruits and C. Fungal endophytes live naturally in plants without causing cramerella borers the pods, both causing significant economic any apparent infection (Maheshwari, 2006). A vast number losses. C. cramerella adults lay their eggs on the cocoa pod of naturally occurring fungal endophytes have been identified and larvae enter the pod, feeding on the seed endosperm. The and these include several entomopathogenic fungi such larvae eventually exit the pod and pupate in the leaf litter, with as Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: subsequent adult emergence. Larval feeding causes petrifaction Hypocreales) (Vega et al. 2008). This fungus has also been and clumping of cocoa beans and premature pod ripening. artificially established as an endophyte to control insect pests Several reports indicate that both insect pests are susceptible on different plant species such as banana (Musa sp.) (Akello et to B. bassiana (Lim et al. 1988; Montealegre & Rodríguez, al. 2009), maize (Zea mays) (Wagner & Lewis, 2000; Cherry et 1989; Wiryadiputra, 2001). al. 2004), date palm (Phoenix dactylifera) (Gómez-Vidal et al. 2006), opium poppy (Papaver somniferum Vault) (Quesada- The objective of this experiment was to explore the flower Moraga et al. 2006), coffee (Coffea arabica L.) (Posada et al. inoculation methodology to establish B. bassiana as an 2007; Posada & Vega 2006) and cocoa (Theobroma cacao L.) endophyte in cocoa pods. After conidial inoculation on flowers, (Posada & Vega, 2005). several pods were sampled monthly for a period of five months to evaluate the establishment of this fungus as an endophyte Artificial inoculations of plants with conidial suspension of B. on cocoa pods. This study also reports the naturally occurring bassiana have been applied using several methods such as, fungal and bacterial endophytes present on different parts of the seed soaking, soil drenching, leaf spraying, stem injection, and cocoa pod over a period of time. This would be the first attempt ridicule inoculation (Posada & Vega, 2005; 2006; Posada et al. to determine whether the flower inoculation methodology can 2007). However, the establishment of this fungus on other be used to establish the entomopathogenic fungus B. bassiana plant parts, such as flowers or fruits, has not been fully explored. as an endophyte of cocoa pods. Spraying flowers with a conidial suspension of B. bassiana may provide protection against economically important insect pests MATERIAL AND METHODS that feed upon fruits and pods. Yet, to use this methodology, it is important to consider that in most plants, the numbers of The isolate of B. bassiana used to spray the cocoa flowers flowers usually exceeds the numbers of mature fruits since both was Bb04005 and was obtained from Sustainable Perennial internal and external factors induce the abortion of flowers and Crops Laboratory, US Department of Agriculture in Beltsville, immature fruits (Stephenson, 1981). MD. This isolate was originally isolated from a yellow jacket wasp (Vespula sp.; Hymenoptera: Vespidae), thus ensuring The flowers also attract beneficial insects that have been used to its entomopathogenicity. Before running the experiment, we inoculate fungal conidias of biological insecticides. For instance, determined the percentage of germination of this B. bassiana insect pollinators such as bees and bumblebees have vectored strain. For this, 15µL of the conidial suspension were plated per conidia of fungal agents such as Trichoderma harzianum triplicate, on 2.5% Noble agar (Becton Dickinson, Sparks, MD) Rifai and Gliocladium roseum Bainier (= Clonostachys rosea and incubated at 25±2ºC for 24 hours. After the incubation (Link: Fries) Schroers, Samuels, Seifert and Gams) to control time, three subsets of agar pieces (1cm2) were sampled per Botrytis cinerea on strawberries (Peng et al. 1992; Yu & Sutton, plate, slide mounted and observed under the microscope. One 1997; Kovach et al. 2000; Shafir et al. 2006). Similarly, the hundred conidia were observed per subsample and assessed dissemination of entomopathogenic fungi by insects has been for the presence of the germ tube. studied in many agricultural ecosystems (Vega et al. 2007) and bees have also been used to disseminate Metarhizium The experiment used eight 15-year old “Amelonado” cocoa trees anisopliae (Metchnikoff) Sorokin on oilseed rape flowers to growing in greenhouses at Rutgers University (New Brunswick, reduce populations of the pollen beetle, Meligethes aeneus NJ). The flowers of four trees (60 flowers per tree) were sprayed Fabricius (Butt et al. 1998). However, none of these insect- with 1x107 B. bassiana conidia suspended in 0.1% Triton mediated dissemination studies focused on the establishment X-100 (Sigma-Aldrich Co., St. Louis, MO) (+Bb). The flowers of B. bassiana as a plant endophyte. of the remaining trees (60 flowers per tree) were sprayed with autoclaved 0.1% Triton X-100 (control) (-Bb). The treatments The establishment of B. bassiana as an endophyte in cocoa were applied after the cocoa flowers were hand-pollinated. Spray plants may be used to control insect pests that attack cocoa applications were performed using a hand-held Jet-Pak spray pods through the world, including the cocoa bug, Monalonium system (Sprayon Products, Inc., Cleveland, OH). After spraying, spp. (Hemiptera: Miridae) in America (Montealegre & Rodríguez, flowers were individually covered with plastic bags to increase 1989) and the cocoa pod borer, Conopomorpha cramerella humidity content and promote germination of fungal conidia.

72 Artículo Científico Posada, F.J.; Chaves, F.C.; Gianfagna, T.J.; Pava-Ripoll, M.; Hebbar, P.: Beauveria bassiana as endophyte in cocoa

Flowers set fruits and cocoa pods were collected each month previously by Posada & Vega (2005). Thus a total of 60 pieces after spraying for a period of five months. Pods were evaluated were evaluated per pod: 24 from the outer part of the pod, for the presence of fungal and bacterial endophytes and for 24 from the inner part, and 12 from the peduncle (Figure 1). the colonization frequency of B. bassiana within the pod. Each Plates were incubated at 25 ± 2°C in a 12:12 photoperiod month, two to five pods from each treatment were harvested. and observed for fungal development for two months. Fungal Individual pods were cut open into four transversal sections colonies that were developing from cocoa pieces were counted plus the peduncle (Figure 1). The husk of each transversal and then individually isolated on fresh YMA plates, incubated as section was divided in two parts: outer and inner parts (Figure described before and identified using various taxonomic keys 1 #1-4 and #5-8, respectively), and the peduncle was divided (St. Germain & Summerbell, 1996; Barnett & Hunter, 1998; into two parts: one attached to the pod and the other attached Watanabe, 2002). Bacterial colonies growing on YMA plates to the tree (Figure 1 #9 and #10, respectively). The pieces were counted and grouped as morphospecies according to were surface sterilized by submerging them in 0.5% sodium colony characteristics: morphology, color and shape. hypochlorite for two minutes, 70% ethanol for two minutes, and then rinsed in sterile water (Arnold et al. 2001). They were The number of colonies of endophytic bacteria and fungi subsequently dried with sterile tissue paper and the brownish present on pod pieces over time were recorded. The edges were removed using a sterile scalpel. Each piece was colonization frequency of B. bassiana was expressed using the subsequently cut in fragments of ca. 3x4 mm and six fragments formula of Fisher and Petrini (1987) [colonization frequency= were placed on Petri dishes containing yeast malt agar (YMA; 100 x (pieces colonized by a single endophyte / total number Sigma-Aldrich Co., St Louis, MO) plus antibiotics as described of pieces evaluated)].

Figure 1. Cocoa pod sections sampled for presence of naturally occurring endophytic fungi and bacteria and for Beauveria bassiana after flowers were sprayed with a conidial suspension (+Bb) or with sterile water plus 0.1% Triton X-100 (control) (-Bb). For each section (labeled 1 to 10 in the figure), six pod pieces were plated on yeast malt agar (YMA).

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To assess the quality of the surface sterilization method, 15µL four fungi were identified to morphospecies. Aspergillus sp., of the water used to rinse the pieces after they were surface Cladosporium sp. and Penicillium sp. were recovered as sterilized were plated on YMA plus antibiotics (Posada & Vega, endophytes at each evaluation time (Table 1). The number of 2005). Plates were evaluated for the presence of endophytic colonies of Penicillium sp. was predominant in both pod and microorganisms four days after inoculation peduncle pieces and was followed by Aspergillus sp. Rubini et al. (2005) reported the diversity of endophytic fungi on branches RESULTS AND DISCUSSION of cocoa trees in Brazil; Acremonium sp. and Cladosporium sp. were recovered among those ones, but Penicillium sp. and This is the first report of the establishment of the entomopathogen Aspergillus sp. were not, thus indicating that the presence of fungus B. bassiana as an endophyte in fruits (cocoa pods) these last two fungi is probably limited to the cocoa pods. after the flowers were sprayed with a conidial suspension. The germination of B. bassiana Bb04005 conidia was >90%. Lower numbers of endophytic bacteria colonies were found The evaluation of surface sterilized water did not yield any on cocoa pod pieces over time. The total number of colonies microorganisms, thus assuring that the external surface of the of endophytic bacteria found one, two, three, four and five pods and peduncles were properly surface sterilized. months post-spraying were 0, 27, 10, 0 and 1, respectively, on (+Bb) treatments and 1, 7, 5, 7 and 0, respectively, on The cacao plant produced a high number of flowers, however (-Bb) treatments. Bacterial colonies recovered from cocoa pod the abortion rate of flowers and immature fruits is very high, pieces belong to seven different bacteria that were identified and generally no more than 5% of the flowers develop into as morphospecies. Posada & Vega (2005) also reported five mature fruits (Bos et al. 2007). Hence, it is not surprising that morphospecies of bacteria from different parts of cocoa from 480 flowers that were hand pollinated and sprayed, only seedlings inoculated with B. bassiana (seedling inoculation 40 set fruits (8.3%) and 30 developed to maturity (6.3%): 16 method) and also from the control treatments. However, their of them were sprayed with B. bassiana (+Bb) and 14 were associations with fungal endophytes are yet to be determined. controls (–Bb). Two and four pods were sample per treatment in the first and second month post-spraying, respectively. Five B. bassiana is reported as a natural endophyte in C. arabica pods were sampled from (+Bb) treatments whereas three (Vega et al. 2010) and Theobroma gileri (Evans et al. 2003). pods were sampled from (-Bb) plants in the third month. In However, B. bassiana was never recovered from the control the fourth and fifth month, three and two pods were sampled pods (-Bb) in our study. This fungus was neither found as an from (+Bb) treatments, respectively, whereas two and three endophyte on branches from cocoa trees (Rubini et al. 2005) pods were sampled from (-Bb) treatments, respectively. The or cocoa seedlings (Posada & Vega, 2005), thus indicating that total number of pod pieces evaluated were 960 in the (+Bb) B. bassiana may not be naturally present as an endophyte treatment and 840 in the (-Bb) treatment. on T. cacao. However, B. bassiana was recovered from outer section of (+Bb) pods, two and three months post-spraying Because the symbiotic or antagonistic effects of the ecological (Table 1). At two months post-spraying, 240 cocoa pod associations of endophytic microorganism are not clear in pieces, belonging to four pods, were evaluated and 92 fungal most of the host plants, it is important to have as much colonies were recovered from those pieces (Table 1). Two of knowledge as possible of the endophytic microbial community those fungal colonies (2.2%) were positively identified as B. present in a particular plant. This will help to establish possible bassiana (one from the third quarter of the pod and another ecological associations among them and ensure the successful from the peduncle of the same pod) (Table 1). Thus, at two establishment of fungal entomopathogens. Results from this months post-spraying, one out of four pods evaluated (25%) study show that several fungal and bacterial endophytes were was positive for B. bassiana and the colonization frequency recovered from different parts of cocoa pods and peduncle was 0.8% (2/240*100). At three months post-spraying with B. in both (+Bb) and (-Bb) sprayed flowers, at each month of bassiana, 300 cocoa pod pieces, belonging to five pods, were evaluation. The total number of colonies of endophytic fungi evaluated and 67 fungal colonies were recovered from those found one, two, three, four and five months post-spraying were pieces. Four of those fungal colonies (5.9 %) were identified 51, 92, 67, 62 and 27, respectively, on (+Bb) treatments and as B. bassiana (all from the fourth quarter of the same pod) 133, 103, 28, 57 and 34, respectively, on (-Bb) treatments. (Table 1). Thus, at three months post-spraying, one out of five Fungal colonies recovered from cocoa pod pieces belong to pods evaluated (20%) was positive for B. bassiana and the nine endophytic fungi that were identified using morphological colonization frequency was 1.3% (4/300*100). traits. One fungus was identified to species level: B. bassiana; four fungi were identified to genus level: Acremonium sp., Although the colonization frequency of B. bassiana in cocoa Aspergillus sp., Cladosporium sp. and Penicillium sp.; and pods was at low levels (0.8% and 1.3% two and three months

74 Artículo Científico Posada, F.J.; Chaves, F.C.; Gianfagna, T.J.; Pava-Ripoll, M.; Hebbar, P.: Beauveria bassiana as endophyte in cocoa 0.0 0.0 5.3 0.0 1.9 1.0 0.0 0.0 0.0 0.0 0.0 0.0 5.3 0.0 1.8 0.0 5.9 0.0 11.8 17.3 42.9 34.6 65.0 18.4 13.6 75.0 25.0 15.8 59.6 17.5 82.4 0 7 0 2 1 0 0 0 7 0 0 0 9 3 0 1 0 4 2 0 % 23 57 46 67 19 14 21 34 10 28 Pod+Ped (%) % 0.0 1.5 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 3.5 1.8 0.0 0.0 0.0 0.0 2.9 0.0 11.7 12.0 28.6 24.8 44.7 71.4 10.7 22.8 10.5 44.1 0 2 0 0 0 3 0 0 0 3 0 0 0 2 6 1 0 0 0 0 1 0 N 16 38 33 46 12 20 13 15 Peduncle (%) % 0.0 5.3 7.5 3.8 0.0 1.0 3.9 1.0 1.0 1.0 0.0 0.0 3.6 0.0 0.0 0.0 0.0 7.0 3.5 0.0 0.0 0.0 8.8 0.0 0.0 11.3 14.3 12.3 35.1 17.6 Outer 0 7 5 0 1 4 1 1 1 0 0 1 0 4 0 0 0 7 4 2 0 0 0 3 6 0 0 N suspension (+Bb) or with sterile autoclaved water 15 10 20 % 0.0 0.0 3.0 2.3 0.0 0.0 1.0 5.8 0.0 9.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.8 0.0 0.0 0.0 1.8 0.0 2.9 2.9 0.0 16.5 20.6 Pod husk (%) Inner B. bassiana 0 0 4 3 0 0 1 6 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 7 1 0 N 17 10 Flowers sprayed with 0.1% Triton X-100 (control) ( -Bb) Flowers sprayed with 0.1% Triton 28 28 28 28 28 28 28 57 57 57 57 57 57 57 34 34 34 34 34 133 133 133 133 133 103 103 103 103 103 103 Total Total fungi % 0.0 7.8 5.9 2.2 1.1 6.5 6.0 0.0 7.5 3.0 1.5 0.0 0.0 0.0 6.5 1.6 0.0 0.0 11.1 49.0 37.3 19.6 52.2 18.5 62.7 19.4 91.9 37.0 33.3 18.5 (%) Pod+Ped 2 1 6 4 0 5 2 1 0 0 0 4 1 0 N 0 4 3 0 9 3 5 18 48 17 42 13 57 25 19 10 One month Two months Two Five months Four months Three months % 1.1 6.5 1.1 3.3 0.0 0.0 7.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.8 2.0 0.0 31.5 18.5 46.3 17.9 56.5 11.1 41.2 31.4 33.3 25.9 18.5 (+Bb) 0 4 1 1 6 1 3 0 0 5 0 0 0 0 0 0 0 0 0 9 7 3 5 N 21 16 29 17 31 12 35 Peduncle (%) B. bassiana % 0.0 0.0 3.9 2.0 2.0 1.1 4.3 0.0 0.0 0.0 6.0 0.0 0.0 1.5 3.0 1.5 0.0 0.0 0.0 1.6 0.0 0.0 0.0 3.7 7.4 0.0 0.0 13.0 16.4 14.5 Outer 0 0 2 1 1 1 4 0 0 0 4 0 0 1 2 1 0 0 9 0 1 0 0 0 1 2 0 0 N 11 12 % 0.0 0.0 3.9 3.9 2.0 0.0 0.0 0.0 0.0 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.8 1.6 0.0 0.0 0.0 0.0 0.0 0.0 0..0 16.3 21.0 Pod husk (%) Flowers sprayed with Inner 0 0 2 2 1 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 3 1 0 0 0 0 0 0 N 15 13 51 51 51 51 51 92 92 92 92 92 92 67 67 67 67 67 67 67 62 62 62 62 62 62 62 27 27 27 27 27 Total Total fungi sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. sp. Endophytic fungi B. bassiana Acremonium Penicilium Aspergillus Cladosporium B. bassiana Acremonium Penicilium Aspergillus Cladosporium Morphospecie 1 B. bassiana Acremonium Penicilium Aspergillus Cladosporium Morphospecie 1 Morphospecie 3 B. bassiana Acremonium Penicilium Aspergillus Cladosporium Morphospecie 1 Morphospecie 4 B. bassiana Acremonium Penicilium Aspergillus Cladosporium Table 1. Percentage of cocoa pod and peduncle pieces with endophytic fungi after the flowers were sprayed with a Table X-100 (control) (-Bb). Sampling was done monthly during a period of five months post spraying. Triton plus 0.1%

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post-spraying, respectively), Suryanarayanan & Thennarasa Beltsville, MD for outstanding laboratory assistance and MARS (2004) reported that in Plumeria rubra, a tropical ornamental and USDA-CRADA for financial support. tree, colonization frequencies of a particular naturally occurring endophytic fungus (i.e. Cladosporium sp. 1) can be as low as REFERENCES 0.5% at a particular time point. The colonization frequency of naturally occurring endophytes increases as the plant ages 1. AKELLO, J.; DUBOIS, T.; COYNE, D.; KYAMANYWA, S. because they seem to accumulate from the environment over 2009. The effects of Beauveria bassiana dose and time (Suryanarayanan & Thennarasan, 2004; Guo et al. 2008). exposure duration on colonization and growth of tis- Therefore, the establishment of B. bassiana on cocoa pods sue cultured banana (Musa sp.) plants. Biol. Control. of younger cocoa trees should be evaluated to avoid possible 49:6-10. antagonistic interactions with already established endophytic microorganisms. 2. ARNOLD, A.E.; MAYNARD, Z.; GILBERT, G.S. 2001. Fungal endophytes in dicotyledonous neotropical Plants produce chemical compounds for protecting the flowers trees: patterns of abundance and diversity. Mycol. Res. against infections caused by pests and diseases and also for 105:1502-1507. regulating pollen compatibility (Ngugi & Scherm, 2006). B. bassiana isolate Bb04005 was able to overcome this adverse 3. BARNETT, H.L.; HUNTER, B.B. 1998. Illustrated Genera of environment indicating that the flower inoculation method could Imperfect Fungi APS Press: Saint Paul. 218p. potentially be used to establish entomopathogenic biocontrol agents as endophytes in pods or fruits, sites that need constant 4. BOS, M.M.; STEFFAN-DEWENTER, I.; TSCHARNTKE, T. protection against economically important insect pests. 2007. Shade tree management affects fruit abortion, However, because the natural high abortion rate of flowers in insect pests and pathogens of cacao. Agriculture cocoa trees can be up to 95% (Young, 1994; Ramos et al. 2005), Ecosystems & Environment. 120:201-205. further experiments with higher number of sprayed flowers will be required to evaluate the colonization frequency of B. bassiana 5. BUTT, T.M.; CARRECK, N.L.; IBRAHIM, L.; WILLIAMS, I.H. on a greater number of cocoa pods. 1998. Honey-bee-mediated infection of pollen beetle (Meligethes aeneus Fab.) by the insect-pathogenic The diversity of fungi and bacteria found naturally as endophytes fungus, Metarhizium anisopliae. Biocontrol Science in cocoa pods and peduncles and the interactions among them and Technology. 8:533-538. and with B. bassiana is an additional component that needs to be studied in more detail. The presence of B. bassiana on cocoa 6. CHERRY, A.; BANITO, A.; DJEGUI, D.; LOMER, C. 2004. pods did not induce noticeable disease symptoms, therefore the Suppression of the stem-borer Sesamia calamistis fungus is apparently innocuous to the plant. Its capability to be (Lepidoptera; Noctuidae) in maize following seed established on the peduncle and move further into the pod as dressing, topical application and stem injection with it grows, demonstrated that it could spread systematically on African isolates of Beauveria bassiana. Internal J. Pest the pod. Further experiments are planned to select B. bassiana isolates with better endophytic capability and to improve Management. 50:67-73. formulations for the delivery of this entomopathogenic fungus. 7. EVANS, H.C.; HOLMES, K.A.; THOMAS, S.E. 2003. Endo- Using the flower inoculation method to establish B. bassiana phytes and mycoparasites associated with an indige- as an endophyte in cocoa pods brings several questions to the nous forest tree, Theobroma gileri, in Ecuador and a forefront: (1) Which mechanisms are used by B. bassiana to preliminary assessment of their potential as biocontrol colonize the flower? (2) Are some B. bassiana isolates better agents of cocoa diseases. Mycol. Progress. 2:149-160. suited than others to grow endophytically in cocoa pods? (3) Will B. bassiana also become established on the cocoa seeds? 8. FISHER, P.J.; PETRINI, O. 1987. Location of fungal endo- (4) Can B. bassiana move from the pod to other parts of the phytes in tissues of Suaeda fruticosa - a preliminary plant? Answers to these questions, along with improvements in study. Transactions of the British Mycological Soc. the successful establishment of B. bassiana in the cocoa pods 89:246-249. might result in a novel biocontrol method against C. cramerella in Southeast Asia and Monalonium spp. in America. 9. FLOOD, J.; GUEST, D.; HOLMES, K.A.; KEANE, P.; PADI, B.; SULISTYOWATI, E. 2004. Cocoa under attack. In: Acknowledgments. We thank Ann Simpkins from the US Flood, J.; Murphy R. eds. Cocoa Futures: A Source Department of Agriculture, Agriculture Research Service, in Book of Some Important Issues Confronting the Cocoa

76 Artículo Científico Posada, F.J.; Chaves, F.C.; Gianfagna, T.J.; Pava-Ripoll, M.; Hebbar, P.: Beauveria bassiana as endophyte in cocoa

Industry. Chinchiná, Colombia. CABI-FEDERACAFÉ- 21. QUESADA-MORAGA, E.; LANDA, B.B.; MUNOZ-LEDES- USDA, p.33-53. MA, J.; JIMENEZ-DIAZ, R.M.; SANTIAGO-ALVAREZ, C. 2006. Endophytic colonisation of opium poppy, Papa- 10. GÓMEZ-VIDAL, S.; LOPEZ-LLORCA, L.V.; JANSSON, ver somniferum, by an entomopathogenic Beauveria H.B.; SALINAS, J. 2006. Endophytic colonization of bassiana strain. Mycopathologia 161:323-329. date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron. 37:624-632. 22. RAMOS, A.R.; VENTURIERI, G.; CUCO, S.M.; CASTRO, N.M. 2005. The site of self-incompatibility action in 11. GUO, L.D.; HUANG, G.R.; WANG, Y. 2008. Seasonal and cupuassu (Theobroma grandiflorum). Rev. Brasil. Bot. tissue age influences on endophytic fungi of Pinus 28:69-578. tabulaeformis (Pinaceae) in the Dongling Mountains, Beijing. J. Integrative Plant Biol. 50:997-1003. 23. RUBINI, M.R.; SILVA-RIBEIRO, R.T.; POMELLA, A.W.V.; MAKI, C.S.; ARAÚJO, W.L.; DOS SANTOS, D.R.; 12. KOVACH, J.; PETZOLDT, R.; HARMAN, G.E. 2000. Use AZEVEDO, J.L. 2005. Diversity of endophytic fungal of honey bees and bumble bees to disseminate Tricho- community of cacao (Theobroma cacao L.) and bio- derma harzianum 1295-22 to strawberries for Botrytis logical control of Crinipellis perniciosa, causal agent control. Biol. Control. 18:235-242. of Witches’ Broom Disease. Internal J. Biological Sci- 13. LIM, T.K.; RITA, M.; OOI, P.A.C.; CHIN, C.L. 1988. Some ences. 1:24-33. entomopathogenic fungi isolated from Conopomorpha cramerella. Planter 64:548-554. 24. SHAFIR, S.; DAG, A.; BILU, A.; ABU-TOAMY, M.; ELAD, Y. 2006. Honey bee dispersal of the biocontrol agent 14. MAHESHWARI, R. 2006. What is an endophytic fungus? Trichoderma harzianum T39: effectiveness in sup- Current Science 90:1309. pressing Botrytis cinerea on strawberry under field conditions. European J. Plant Pathology. 116:119-128. 15. MONTEALEGRE, R.J.; RODRÍGUEZ, D.A. 1989. Pato- genicidad del hongo Beauveria bassiana (Bals.) Vuill. 25. ST. GERMAIN, G.; SUMMERBELL, R. 1996. Identifying sobre la chinche Monalion dissimulatum Distant plaga filamentous fungi: A clinical laboratory handbook Star del cacaotero Theobroma cacao L. Acta Agronomica. Publishing Company: Belmont, CA. 314p. 39:88-96. 26. STEPHENSON, A.G. 1981. Flower and Fruit Abortion - 16. NGUGI, H.K.; SCHERM, H. 2006. Biology of flower- Proximate Causes and Ultimate Functions. Ann. Rev. infecting fungi. Ann. Rev. Phytopathology. 44:261-282. Ecology and Systematics. 12:253-279.

17. PENG, G.; SUTTON, J.C.; KEVAN, P.G. 1992. Effective- 27. SURYANARAYANAN, T.S.; THENNARASAN, S. 2004. Tem- ness of honey-bees for applying the biocontrol agent poral variation in endophyte assemblages of Plumeria Gliocladium roseum to strawberry flowers to suppress rubra leaves. Fungal Diversity. 15:197-204. Botrytis cinerea. Can. J. Plant Pathology-Revue Cana- dienne de Phytopathologie. 14:117-129. 28. VEGA, F.E.; DOWD, P.F.; LACEY, L.A.; KLEIN, M.G. 2007. Dissemination of beneficial microbial agents by 18. POSADA, F.; AIME, M.C.; PETERSON, S.W.; REHNER, insects. . En: Lacey, L.A.; Kaya, H., eds. Field Manual S.A.; VEGA, F.E. 2007. Inoculation of coffee plants of Techniques in Invertebrate Pathology. Springer, The with the fungal entomopathogen Beauveria bassiana Netherlands. p.18. (Ascomycota : Hypocreales). Mycol. Res. 111:748-757.

19. POSADA, F.; VEGA, F.E. 2005. Establishment of the fungal 29. VEGA, F.E.; POSADA, F.; AIME, M.C.; PAVA-RIPOLL, M.; entomopathogen Beauveria bassiana (Ascomycota INFANTE, F.; REHNER, S.A. 2008. Entomopathogenic : Hypocreales) as an endophyte in cocoa seedlings fungal endophytes. Biol. Control. 46:72-82. (Theobroma cacao). Mycologia 97:1195-1200. 30. VEGA, F.E.; SIMPKINS, A.; AIME, M.C.; POSADA, F.; 20. POSADA, F.; VEGA, F.E. 2006. Inoculation and colonization PETERSON, S.W.; REHNER, S.A.; INFANTE, F.; CAS- of coffee seedlings (Coffea arabica L.) with the fungal TILLO, A.; ARNOLD, A.E. 2010. Fungal endophyte entomopathogen Beauveria bassiana (Ascomycota: diversity in coffee plants from Colombia, Hawai’i, Mexico Hypocreales). Mycoscience. 47:284-289. and Puerto Rico. Fungal Ecology. 3:122-138.

77 Revista U.D.C.A Actualidad & Divulgación Científica 12 (3): 71-78 2010

31. WAGNER, B.L.; LEWIS, L.C. 2000. Colonization of corn, 34. YOUNG, A.M. 1994. The Chocolate Tree: A Natural History Zea mays, by the entomopathogenic fungus Beauveria of Cacao Smithsonian Institution Press: Washington, bassiana. Applied and Environmental Microbiology. DC. 200p. 66:3468-3473. 35. YU, H.; SUTTON, J.C. 1997. Effectiveness of bumble bees 32. WATANABE, T. 2002. Pictorial atlas of soil and seed fungi. and honeybees for delivering inoculum of Gliocladium Morphologies of cultured fungi and key to species CRC roseum to raspberry flowers to control Botrytis cinerea. Press: Boca Raton, FL. 486p. Biol. Control 10:113-122.

33. WIRYADIPUTRA, S. 2001. The use of entomopathogenic fungus (Beauveria bassiana) to control cocoa pod borer (Conopomorpha cramerella) in the field. En: In- Received: August 25, 2010 coped 3rd International Seminar. Kota Kinabalu Sabah, Accepted: November 3, 2010 Malaysia, p.27-32.