Establishment of the Fungal Entomopathogen Beauveria Bassiana (Ascomycota: Hypocreales) As an Endophyte in Cocoa Seedlings (Theobroma Cacao)
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Mycologia, 97(6), 2005, pp. 1195–1200. # 2005 by The Mycological Society of America Lawrence, KS 66044-8897 Establishment of the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) as an endophyte in cocoa seedlings (Theobroma cacao) Francisco Posada endophyte in woody plant hosts, can spread within Fernando E. Vega1 host tissues and can protect host plants against Insect Biocontrol Laboratory, U.S. Department of herbivores, such as the cocoa pod borer, has not Agriculture, Agricultural Research Service, Bldg. 011A, been evaluated. Our ultimate goal is to determine BARC-W, Beltsville, Maryland 20705 whether cocoa plants inoculated with B. bassiana in the seedling stage can sustain the fungus in the field, and more importantly, whether B. bassiana can be Abstract: The fungal entomopathogen Beauveria detected in the pod, where it ideally would help bassiana became established as an endophyte in in control the cocoa pod borer. vitro-grown cocoa seedlings tested for up to 2 mo The cocoa pod borer is believed to be endemic to after inoculation to the radicle with B. bassiana Southeast Asia and to have shifted from rambutan suspensions. The fungus was recovered in culture (Nephelium lappaceum L.), pulasan (N. mutabile from stems, leaves and roots. B. bassiana also was Blume) and nam-nam (Cynometra cauliflora L.) to detected as an epiphyte 1 and 2 mo postinoculation. cocoa, which was introduced from the American Penicillium oxalicum and five bacterial morphospecies continent in the 16th century (Malaysian Plant also were detected, indicating that these were present Protection Society 1987). as endophytes in the seed. Female cocoa pod borers can lay up to 100 eggs on Key words: biological control, cacao, cocoa pod the surface of the cocoa pod. Upon hatching, larvae borer, Conopomorpha, entomopathogens, insect bore into the pod where they feed on the pulp and pathogens placental tissue, which leads to deformed and clumped seeds and premature pod ripening, thus INTRODUCTION greatly reducing yield and quality (Malaysian Plant Protection Society 1987, Santoso et al 2004). Approx- One possible pest management mechanism against imately 2 wk after hatching, larvae emerge from the the cocoa pod borer Conopomorpha cramerella (Snel- pod to pupate on the leaf litter or directly on the pod, len) (Lepidoptera: Gracillariidae), the most impor- followed by adult emergence (Santoso et al 2004). tant insect pest attacking cocoa (Theobroma cacao L.) Because these insects are small and larval develop- in Malaysia, Indonesia and the Philippines (Bradley ment takes place inside the pod, they have proven to 1986), involves the inoculation of cocoa plants with be extremely difficult to control and have caused yield the fungal entomopathogen Beauveria bassiana (Bal- losses of up to 80% (Day 1989). Various techniques samo) Vuillemin (Ascomycota: Hypocreales) in an have been used in an attempt to reduce pest attempt to establish the fungus as an endophyte incidence, including harvesting of all available pods (Arnold and Lewis 2005). Bing and Lewis (1991) have to interrupt the life cycle (termed ‘‘rampassen’’, shown that B. bassiana can be introduced as an Wood et al 1992), chemical insecticides (Wardojo endophyte of Zea mays, where it causes mortality of 1980, Day and Mumford 1994), pheromone-baited the European corn borer, Ostrinia nubilalis (Hu¨bner) traps (Beevor et al 1993), biocontrol agents (Nagaraja (reviewed in Arnold and Lewis 2005). B. bassiana has et al 1986, Awang and Lee 1998) and bagging of been reported as an endophyte of maize (Arnold and cocoa pods (Vanialingam et al 1982). In this paper we Lewis 2005), potato ( Jones 1994), tomato (Leckie describe the in vitro introduction of B. bassiana as an 2002) and on Theobroma gileri, a relative of cocoa endophyte in cocoa seedlings, establishing an impor- (Evans et al 2003). Recently endophytic B. bassiana tant first step in the possible use of this fungus as has been isolated from coffee plants in Colombia; a biocontrol agent. preliminary laboratory results demonstrate the possi- bility of inoculating coffee plants with B. bassiana MATERIALS AND METHODS using various methods (Posada and Vega unpubl data). Whether B. bassiana can persist as an Cocoa seeds removed from ripe pods were surface sterilized by submerging them in 10% bleach for 2 min, followed by submersion in 95% ethanol for 2 min and rinsed in sterile Accepted for publication 16 Aug 2005. water three times. The seeds were placed on sterile filter 1 Corresponding author. E-mail: [email protected] paper moistened with sterile water and placed inside 100 3 1195 1196 MYCOLOGIA 15 mm Petri dishes. The dishes were sealed with parafilm At all sampling times, the control was processed first, and kept in an unlit growth chamber at 25 6 2.0 C. followed by CS16-1 and then IC-5486. To assess for the When the seeds had germinated and the roots were ca. presence of endophytes tissues (roots, stems, leaves) were 3 cm long, they were transferred to a laminar flow hood for surface-disinfected by submersion in 0.5% sodium hypo- inoculation with one of two isolates of B. bassiana: (i) Ivory chlorite for 2 min, 70% ethanol for 2 min and then rinsed Coast 5486 (IC-5486), isolated from an infected coffee berry with sterile water (Arnold et al 2001). The tissues were dried borer (Hypothenemus hampei (Ferrari); Coleoptera: Curculio- on sterile paper towels and, after cutting off the edges to nidae); or (ii) CS16-1, isolated as an endophyte from coffee remove the dead tissue originating from the disinfection berries in Colombia. Both isolates are highly pathogenic to process, cut into 4 to 9 mm2 sections, six of which were the coffee berry borer (Posada and Vega 2005) and have been placed in each of two Petri dishes containing YMA+3A. deposited in the Insect Biocontrol Laboratory fungal en- Tissues were examined four d later, and any fungal growth tomopathogens collection. Isolates were retrieved from was subcultured onto individual plates containing YMA+3A single-spore stocks stored in 10% glycerol and grown on for subsequent identification. To evaluate the quality of the yeast-maltose agar (YMA; Sigma-Aldrich Co., St Louis, surface sterilization method 10 mL of the water used to Missouri) to which a 0.1% stock antibiotic solution was added rinse the tissues after surface sterilization was taken and (YMA+3A). The antibiotic stock consisted of 0.02 g of each of 15 mL aliquots of a 1023 dilution were plated on YMA+3A three antibiotics (tetracycline, streptomycin and penicillin) and spread with a sterile glass rod. For each suspension two dissolved in 10 mL sterile distilled water, followed by filter dishes were plated and after incubation for 4 d at 25 6 sterilization through a 0.2 mm filter (Nalgene Disposable 2.0 C, colonies were counted and expressed as colony- Filterware, Nalge Nunc International, Rochester, New York); forming units (CFU) per mL. from this, 1 mL was added to each liter of medium. Spores The experiment was organized as a completely random- were less than 30 d old when used. Before inoculation, spore ized design with a factorial arrangement. The factors were germination rate was determined on 2.5% Noble agar (BD the isolates (CS16-1, IC-5486 and the control), two sampling DifcoTM Agar, Noble; Becton, Dickinson & Co., Franklin periods (1 and 2 mo after inoculation) and three types of Lakes, New Jersey) as follows: a 15 mL aliquot from a 1023 tissues sampled (leaves, stem and root). Thus, at each dilution was plated on the agar surface, spread with a sterile sampling period, 432 subsamples of tissues were plated, glass rod and incubated at 25 6 2.0 C. At 24 h three groups of based on the combination of two isolates and one control by 100 spores were counted and the data were expressed as four plants by three tissues (leaves, stems and roots) by two percentages. replicates for each tissue type and six subsamples plated on To inoculate each individual seedling, 100 mLofa13 each Petri dish. The data obtained from the evaluation were 107 mL21 B. bassiana spore suspension were inoculated on expressed as colonization frequency using Fisher and the main radicle of each seedling with a pipette. The seeds Petrini’s (1987) formula: colonization frequency 5 number then were placed inside individual 100 3 15 mm sterile of colonized segments divided by the total number of Petri dishes and allowed to dry in the laminar flow hood segments 1003. The data were square root-transformed and before transferring to sterile 25 3 250 mm test tubes analyzed with ANOVA (SAS Institute, Inc. 2000). (Kimble Glass Inc., New Jersey) containing 20 mL of sterile 10% water agar (Difco Bacto Agar, Becton, Dickinson & Co., Franklin Lakes, New Jersey). Using sterile tweezers the RESULTS germinated seeds were inserted individually in each tube Spore germination of B. bassiana isolates used to making sure the radicle was inserted in the agar. Tubes were inoculate the radicles was .85%. Neither B. bassiana capped with sterile sponges overlaid with sterile aluminum nor any other microorganism was ever observed in the foil and kept in the laboratory at ca. 12 : 12 photoperiod and ca. 25 6 2.0 C. water agar medium used to grow the seedlings. The Evaluations of B. bassiana colonization of cocoa tissues water used to rinse tissues after sterilization yielded were carried out 30 and 60 d after spore inoculation. Twelve no microorganisms in either sampling period; thus plants were used for each evaluation: four plants for each B. any ensuing fungal growth from surface-sterilized bassiana isolate and four for the control, which received tissues is inferred to have originated from internal 100 mL of sterile water at the moment of inoculation.