Cumulative impact of a clover cover crop on the persistence and efficacy of Beauveria bassiana in suppressing the pecan weevil (Coleoptera: Curculionidae)

The pecan weevil, Curculio caryae (Horn), is a key pest of pecan. Endemic levels of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin can occur in pecan orchards and contribute to natural control of C. caryae. Commercial formulations of the fungus can also be applied for suppression of C. caryae. We hypothesized that a clover cover crop enhances B. bassiana efficacy and persistence (e.g., by protecting the fungus against abiotic environmental stresses). The hypothesis was tested by conducting field trials in a pecan orchard in Byron, GA, in 2009 and 2010. The study included four treatments arranged in a factorial with two levels of fungus (endemic fungus only, and application of a commercial B. bassiana product), and two levels of clover (white clover, Trifolium repens L., and no clover). Fungal persistence was measured by determining the number of CFUs per gram of soil over time (during 42 d postapplication of B. bassiana in 2009 and 29 d in 2010). Efficacy was measured by capturing naturally emerging C. caryae and subsequently determining mortality and mycosis (over 24 d in 2009 and 17 d in 2010). In 2009, greater prevalence of B. bassiana conidia was detected in plots receiving fungal applications compared with no fungus applications, and no clear effect of clover was observed in plots receiving B. bassiana applications in either year. In 2010, B. bassiana prevalence in the endemic fungus plus clover treatment was higher than fungus without clover, and was similar to plots receiving additional B. bassiana applications. Given that we observed enhanced persistence of endemic B. bassiana in 2010 but not 2009, the impact of clover appears to be a cumulative effect. Mortality of C. caryae (averaged over the sampling periods) ranged between 68-74% in plots receiving B. bassiana applications and 51-56% in plots with endemic fungus only. C. caryae mortality and mycosis data also provided evidence that endemic B. bassiana efficacy was enhanced by clover relative to plots without clover (with no clear clover effect on plots receiving fungus applications). Thus, we conclude that natural control of C. caryae can increase when clover is grown in pecan orchards with endemic populations of B. bassiana.     

Interaction effects between Beauveria bassiana and imidacloprid against Thrips tabaci (Thysanoptera: Thripidae)

The onion thrips, Thrips tabaci Lindeman is a major pest of many horticultural crops in many parts of the world. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin and imidacloprid are used for the control of the onion thrips. The potential interactions between the fungus and the chemical against the onion thrips adults were evaluated in a laboratory and two greenhouse bioassays. The laboratory bioassay was a leaf dip bioassay for a mixture of B. bassiana and imidacloprid. The first greenhouse bioassay was a topical application of a mixture of the fungus and the chemical while the second bioassay was a drenching application of imidacloprid and a topical application of B. bassiana. In all bioassays, the combined application resulted in higher percentage mortalities than either B. bassiana or imidacloprid alone. In the laboratory bioassay, the highest mortality of 97% was achieved by using B. bassiana at the field rate with imidacloprid at 1/10th the field rate relative to 88%, 94%, 21% for B. bassiana alone, imidacloprid alone and the control, respectively. In the first greenhouse bioassay, the highest mortality of 80% resulted from the combined application of B. bassiana and imidacloprid at field rates compared with 55%, 75%, and 22% for B. bassiana alone, imidacloprid alone and the control, respectively. In the second greenhouse bioassay, the combined application of both control agents at field rates resulted in 85% mortality compared with 52%, 83%, and 18% for B. bassiana alone, imidacloprid alone and the control, respectively. The interaction effect between the two control agents was additive except when B. bassiana at 1/2 field rate was mixed with imidacloprid at 1/10th field rate in the laboratory bioassay, where the interaction was antagonistic. The above results indicate that combining imidacloprid with B. bassiana might reduce the rate of the insecticide application while enhancing the efficacy of the biological control agent.     

Selection of Beauveria bassiana isolates to control Alphitobius diaperinus

This paper presents results on isolates of the entomopathogenic fungus Beauveria bassiana for Alphitobius diaperinus (Coleoptera: Tenebrionidae) control. Of the 30 isolates tested, four (CG 71, CG 152, UNIOESTE 4, and UNIOESTE 40) resulted in mortality rates >or= 40% within 10 days. These mortality rates could be considered high because of the resistance of these species to B. bassiana. Tests were conducted using these isolates to estimate LC(50), mortality rate over time, vegetative growth, and conidial production on artificial medium and on insects. Isolates CG 71, CG 152, and UNIOESTE 4 showed the best performance and great potential to be used in an integrated management program in poultry farms to control A. diaperinus. Also, the molecular profiles of 12 isolates were analyzed using the RAPD technique. The high-virulence isolates presented a more homogeneous RAPD pattern than the others. Genetic sequencing of the ITS region was performed for one of the virulent isolates (UNIOESTE 4) and compared with sequences deposited at the NCBI database, confirming its taxonomical position as belonging to B. bassiana Clade A.     

Strain-specific detection of introduced Beauveria bassiana in agricultural fields by use of sequence-characterized amplified region markers

Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on polymerase chain reaction amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate density of propagules of a commercial strain of B. bassiana (strain GHA) in field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-14(0.44), OPA-15(0.44), and OPB-9(0.67), generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R(445), OPA15 F/R(441), and OPB9 F/R(677), respectively. All three SCAR primers were highly sensitive, capable of detecting 100pg B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field.     

Compatibility of the insect pathogenic fungus Beauveria bassiana with neem against sweetpotato whitefly, Bemisia tabaci, on eggplant

A study on the compatibility of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) with neem was conducted against sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), on eggplant. Initially, three concentrations of B. bassiana (10⁶, 10⁷, and 10⁸ conidia ml⁻¹) and three concentrations of neem (0.25, 0.5, and 1.0%) were used as individual treatments against B. tabaci . The highest concentration of B. bassiana yielded the highest B. tabaci egg (25.2%) and nymph mortalities (73.0%), but this was not significantly different from the mortalities caused by the 10⁷ conidia ml⁻¹ suspension. Similarly, the highest concentration of neem yielded the highest egg (27.3%) and nymph mortalities (75.5%), which was also not significantly different from the 0.5% suspension. Therefore, 0.5% neem was used along with 10⁷ B. bassiana conidia ml⁻¹ suspension as an integrated pest management program against B. tabaci . The combination of B. bassiana and neem yielded the highest B. tabaci egg (29.5%) and nymph mortalities (97.2%), and the lowest LT₅₀ (2.08 day) value. Moreover, an integrated combination of B. bassiana with neem caused 27.6 and 20.5% more nymphal mortality than individual treatments of B. bassiana and neem, respectively, 7 days post-application. Thus, a combined application of an entomopathogenic fungus and a botanical insecticide may benefit from both, and it has proven effective for the control of B. tabaci on eggplant.     

Development of a novel bioassay for estimation of median lethal concentrations (LC50) and doses (LD50) of the entomopathogenic fungus Beauveria bassiana, against western flower thrips, Frankliniella occidentalis

To conduct laboratory experiments aimed at quantifying secondary acquisition of fungal conidia by western flower thrips (Frankliniella occidentalis), an efficient assay technique using Beauveria bassiana as the model fungus was developed. Various application protocols were tested and it was determined that the percent mortality did not vary among protocols. Peak mortality of second-instar nymphs, under constant exposure to conidia, occurred 5 days post-inoculation. Second-instar thrips that were exposed to conidia within 24 h of the molt to second instar were more susceptible to Beauveria bassiana than thrips exposed after times greater than 24 h post-molt. Conidia efficacy, which was monitored at 24 h intervals, did not differ significantly within 72 h. A test of the final bioassay system was conducted in a series of assays aimed at determining the LD50 of B. bassiana technical powder against second-instar western flower thrips. It was determined that B. bassiana (strain GHA) is highly effective at very low doses (LD50 of 33-66 conidia/insect).     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.     

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.     

Establishment of the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales) as an endophyte in cocoa seedlings (Theobroma cacao)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in in vitro-grown cocoa seedlings tested for up to 2 mo after inoculation to the radicle with B. bassiana suspensions. The fungus was recovered in culture from stems, leaves and roots. B. bassiana also was detected as an epiphyte 1 and 2 mo postinoculation. Penicillium oxalicum and five bacterial morphospecies also were detected, indicating that these were present as endophytes in the seed.     

Field Evaluation of Beauveria bassiana: Its Persistence and Effects on the Pea Aphid and a Non-target Coccinellid in Alfalfa

Several strains of the entomopathogenic fungus Beauveria bassiana have been considered for use as microbial insecticides. Experimental sprays were conducted in an alfalfa field with an aphid-derived strain of B. bassiana to determine its persistence and its effects on pea aphids, Acyrthosiphon pisum (Homoptera: Aphididae) and a non-target aphid predator, Hippodamia convergens (Coleoptera: Coccinellidae). B. bassiana conidia persisted in the field for at least 28 days, when approximately 10% of the original inoculum was still present. In the lower canopy, more conidia were present than on other plant parts and they persisted longer on the leaves in this location. However, conidia were still abundant in the upper canopy, where 97.9% of the aphids and 95.5% of H. convergens larvae were found. Thus, both insect species were exposed to the fungus for at least 1 month. However, pea aphid populations were not affected by the fungus. The predator's incidence was reduced by 75-93% (depending on application rate) early in the season, but was not affected later in the season. Insect life history patterns and weather conditions are likely causes for the differences seen in field effects.     

Enhanced efficacy of Beauveria bassiana for red flour beetle with reduced moisture

Red flour beetle, Tribolium castaneum (Herbst), is a major pest of stored and processed grains that is tolerant of Beauveria bassiana (Balsamo) Vuillemin under most conditions. Laboratory assays demonstrated that desiccating conditions improved the efficacy of the fungus. For T. castaneum larvae exposed to B. bassiana at different vapor pressure deficits (VPDs), the LC50 potency ratios were 2.3 for 2.42 kPa and 3.9 for 3.31 kPa compared with 1.06 kPa. There were significant effects of moisture on B. bassiana-associated mortality even with brief exposure to low VPD before or at the beginning of exposure to fungus. When T. castaneum larvae were held at 3.31 kPa 1 d before exposure to B. bassiana and/or in the first 1 or 2 d of exposure to the fungus, there was significantly greater mortality for all of the low moisture treatments than occurred with continuous incubation at 1.06 kPa. B. bassiana efficacy for adult T. castaneum was significantly better for VPDs of 3.56 or 3.78 than 1.06 kPa. Weight gain over 4 d of incubation with moisture held constant was significantly less at a VDP of 3.31 kPa than at 1.87 or 1.06 kPa. Treatment with fungus did not affect weight gain significantly. There was no significant difference in larval water content among treatments.     

Antagonism between Beauveria bassiana and imidacloprid when combined for Bemisia argentifolii (Homoptera: Aleyrodidae) control

Imidacloprid and the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin are both used to control the whitefly Bemisia argentifolii Bellows & Perring. We tested whether the two control strategies acted additively, synergistically, or antagonistically when combined for whitefly control. We found antagonism in that B. bassiana inhibited the effectiveness of imidacloprid. When B. bassiana was combined with imidacloprid, insect response was either less than or similar to (depending on B. bassiana rates) that when imidacloprid was used alone. Adding imidacloprid to B. bassiana treatments always increased mortality, but the increase was less than additive. Beauveria bassiana spore germination and colony formation were not inhibited by imidacloprid in vitro, and B. bassiana did not adsorb or degrade imidacloprid in a tank mix. We hypothesize that B. bassiana caused a behavioral response that reduced insect feeding and uptake of imidacloprid.     

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.     

Biocontrol of almond bark beetle (Scolytus amygdali Geurin-Meneville, Coleoptera: Scolytidae) using Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes)

AIMS: To formulate the entomopathogenic fungus Beauveria bassiana in invert emulsion, then apply it against adults of almond bark beetle (Scolytus amygdali) under laboratory and field conditions. METHODS AND RESULTS: The effect of formulated B. bassiana in invert emulsion against S. amygdali adults was shown by comparing the mortality percentage of adults exposed to the formulated fungus using a Petri dish treatment method and by field applications to infested peach trees with mortality of adults exposed to the unformulated fungus or the untreated control. Results obtained from both exposure methods have indicated that treatment of S. amygdali adults with the formulated fungus resulted in a significantly higher mean mortality percentage (P < 0.05) when compared with the treatment with the unformulated fungus or the untreated control. This mortality ranged from 81.2 to 100%, 10 days after treatment with the formulated fungus when compared with 6.7 to 49.6% mortality, 10 days after treatment with the control or the unformulated fungus, respectively. Viability of the fungus conidia in invert emulsion was assessed by calculating the germination percentage of the conidia over time. Results indicated a high storage stability shown by a small loss of germination percentage for the formulated conidia of both strains (5.8 to 8.4% over a 12-week period) vs a low storage stability shown by a high loss of germination percentage for the unformulated conidia of the same strains (58.9 to 61.0% over the same period). The presence of B. bassiana in the galleries of beetles following the treatment of infested trees was shown in the present research. CONCLUSIONS: The results obtained have demonstrated a significantly higher level of efficacy of formulated B. bassiana in invert emulsion against S. amygdali adults under laboratory and field conditions. The ingredients of invert emulsion used in the formulation of the fungus had a negligible effect on the viability of formulated conidia when compared with the unformulated. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained in the present research are promising and may be exploited commercially to control S. amygdali adults on various species of stone fruit trees, especially peach trees. This type of biocontrol of this insect may be used as an alternative means to chemical control for management of the insect. No adverse environmental impacts of the fungus or its formulation have been observed during application.     

Comparison of application methods for suppressing the pecan weevil (Coleoptera: Curculionidae) with Beauveria bassiana under field conditions

The pecan weevil, Curculio caryae (Horn), is a key pest of pecans. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin is pathogenic to C. caryae. One approach to managing C. caryae may be application of B. bassiana directed toward adult weevils as they emerge from the soil to attack nuts in the tree canopy. Our objective was to compare different application methods for suppression of C. caryae adults. Treatments included direct application of B. bassiana (GHA strain) to soil under the tree canopy, soil application followed by cultivation, soil application in conjunction with a cover crop (Sudan grass), direct application to the tree trunk, and application to the trunk with an UV radiation-protecting adjuvant. The study was conducted in a pecan orchard in Byron, GA, in 2005 and 2006. Naturally emerging C. caryae adults, caught after crawling to the trunk, were transported to the laboratory to determine percentage mortality and signs of mycosis. When averaged over the 15-d sampling period, weevil mortality and signs of mycosis were greater in all treatments than in the nontreated control in 2005 and 2006; >75% average mortality was observed with the trunk application both years and in the trunk application with UV protection in 2005. Results indicated trunk applications can produce superior efficacy relative to ground application, particularly if the ground application is followed by cultivation. Efficacy in the cover crop treatment, however, did not differ from other application approaches. Future research should focus on elucidating the causes for treatment differences we observed and the extent to which B. bassiana-induced C. caryae mortality reduces crop damage.     

Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

The pathogenicity of Beauveria bassiana: what happens after an endophytic phase in plants?

The banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is a serious constraint to banana (Musa spp.) production throughout the world. The entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) offers a potential weevil management option, but conventional delivery mechanisms have limited its success. As an endophyte, however, B. bassiana can be efficiently delivered to banana planting materials for the potential management of C. sordidus. However, entomopathogens can change morphology and efficacy against their target host when successively sub-cultured on artificial media or when exposed to certain physical and chemical environmental conditions. Whether such changes occur in B. bassiana after an endophytic phase inside a banana plant remains unknown. The primary aim of our study was to evaluate the viability, growth, sporulation and pathogenicity of endophytic B. bassiana. To attain this, two sets of experiments, namely morphological characterization and larval bioassays, were conducted under laboratory conditions. In these experiments, growth and pathogenicity of the wild-type B. bassiana strain G41, obtained originally from banana farms, was compared with the endophytic B. bassiana strain G41, re-isolated from the rhizome of B. bassiana-inoculated banana plants at one month post-inoculation. Morphological characterization, conidial germination, colony growth and sporulation rate was assessed on SDAY media while pathogenicity was determined 15 days after immersing the larvae of C. sordidus in different conidial doses. No differences were observed in colony appearance and growth rate between the endophytic and wild-type strain. Percentage conidial germination for the endophytic strain (91.4-94.0%) was higher than for the wild-type (86.6-89.7%). LD50 equated 1.76 x 10(5) and 0.71 x 10(5) conidia/ml for the wild-type and endophytic B. bassiana strains, respectively, but did not differ between strains. Our study demonstrated that, after an endophytic phase inside the banana plant, B. bassiana retains it morphology and pathogenicity against the banana weevil larvae; and thus can offer protection against the damaging larvae feeding inside the rhizome.     

Colonization of corn, Zea mays, by the entomopathogenic fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Effect of Beauveria bassiana and Metarhizium anisopliae (Deuteromycetes) upon the coffee berry borer (Coleoptera: Scolytidae) under field conditions

The effect of three strains of the fungus Beauveria bassiana (Balsamo) Vuillemin and two strains of Metarhizium anisopliae (Metschnikoff) Sorokin upon the coffee berry borer, Hypothenemus hampei (Ferrari), was studied in three coffee farms at different altitudes (450-1,100 m above sea level) in Soconusco, Chiapas, Mexico. The maximum average percentage mycosis varied according to altitude. At 450 m asl (El Rincon) mycosis was 14.3% for B. bassiana and 6.3% for M. anisopliae; at 880 m asl (Santa Anita) mycosis was 40.6% for B. bassiana and 12.6% for M. anisopliae, and at 1,100 m asl (Alpujarras) 33.9% for B. bassiana and 22. 1% for M. anisopliae. The effect of fungal mycosis through time was not significant (P > 0.01) in any of the farms, but there was a significant difference between the strains of the fungus (P < 0.01); the best strains being Bb25 and Ma4 at the lower altitude, Bb26 and Ma4 for the middle altitude and Bb26 and Ma4 at the higher altitude. Environmental factors such as temperature, relative humidity and rain were not correlated with the percentage mycosis caused by B. bassiana and M. anisopliae. However, in the case of B. bassiana there was a significant, positive correlation (P < 0.01) between the infestation levels of the pest and the mycosis response of the entomopathogen.