Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

A technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae

The entomopathogenic fungus Metarhizium anisopliae (strain ME1) failed to swell or form germ-tubes in distilled water. However, a period of soaking in distilled water (10–44 h) accelerated the process of germination when a suitable nutrient source was provided. The implications of this novel observation are discussed in terms of mechanisms of germination and the use of parasitic fungi for insect pest control.     

Laboratory studies on Australian isolates of Metarhizium anisopliae as a biopesticide for the cattle tick Boophilus microplus

Thirty-one isolates of Metarhizium anisopliae were bioassayed against the cattle tick (Boophilus microplus). More than half of the isolates showed a high degree of virulence to ticks. Radial growth curves for growth between 20 degrees C and 40 degrees C were obtained for all isolates. This information together with information on virulence will be important for the selection of isolates suitable to kill ticks on the surface of cattle. A biopesticide for cattle ticks must kill ticks rapidly at temperatures within the upper end of most isolates' growth curves. It was also found that the time taken to achieve 100% tick mortality in vitro using a virulent isolate could be halved by applying conidia in a 10% oil emulsion. Scanning electron microscopy and light microscopy were used to investigate and compare the germination and penetration of conidia formulated in aqueous and oil formulations. It was found that conidia in both formulations were able to germinate and produce appressoria on the surface of ticks in less than 11h. Marked weakness within 26h, followed by extensive hyphal growth on the cuticle characterised the invasion of ticks by M. anisopliae.     

Genetic diversity of Metarhizium anisopliae var. anisopliae in southwestern British Columbia

The abundance and genetic diversity of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, in southwestern British Columbia (BC) and southern Alberta was examined. The fungus was found to be widespread in soil throughout southwestern BC, and was recovered from 56% of 85 sample sites. In contrast to southwestern BC, no M. anisopliae isolates were recovered in southern Alberta. An automated fluorescent amplified fragment length polymorphism (AFLP) method was used to examine genetic diversity. In excess of 200 isolates were characterized. The method identified 211 polymorphic amplicons, ranging in size from ≈92 to 400 base pairs, and it was found to be reproducible with a resolution limit of 86.2% similarity. The AFLP method distinguished Metarhizium from other entomopathogenic fungal genera, and demonstrated considerable genetic diversity (25 genotypes) among the reference strains of M. anisopliae isolates examined (i.e. recovered from various substrates and geographical locations). Although 13 genotypes of M. anisopliae var. anisopliae were recovered from southwestern BC soils, the vast majority of isolates (91%) belonged to one of two closely-related genotypes. Furthermore, these two genotypes predominated in urban, agricultural and forest soils. The reasons for the limited diversity of M. anisopliae var. anisopliae in southwestern BC are uncertain. However, findings of this study are consistent with island biogeography theory, and have significant implications for the development of this fungus for microbial control of pest insects.     

Reduction in fitness of female Asian longhorned beetle (Anoplophora glabripennis) infected with Metarhizium anisopliae

Bioassays were conducted to document the effects of Metarhizium anisopliae infection on adult female Asian longhorned beetle (Anoplophora glabripennis) reproduction before death and subsequent survival of offspring. The effect of infection on fecundity was evaluated for females already laying eggs and for newly eclosed females using M. anisopliae isolates ARSEF 7234 and 7711, respectively. Decreased longevity and oviposition compared with controls were observed in females that were already laying eggs when exposed to M. anisopliae ARSEF 7234. Newly eclosed females exposed to M. anisopliae ARSEF 7711 displayed shortened longevity (10.0 ± 0.7 days vs 74.3 ± 6.8 days for controls) and decreased oviposition (1.3 ± 0.7 eggs per ARSEF 7711-exposed female vs 97.2 ± 13.7 eggs per female for controls) compared with controls. Percentages of eggs that did not hatch were greater for both groups of fungal-treated females compared with controls and 60.0% of unhatched eggs contained signs of fungal infection. The percentage of larvae dying within 9 weeks of oviposition was higher for sexually mature females exposed to ARSEF 7234 compared with controls and >40% of dead larvae displayed signs of fungal infection. Thus, for both stages of females and both fungal isolates, fewer surviving larvae were produced after female fungal infection compared with controls. M. anisopliae infection affects female fitness by decreasing female longevity, by decreasing female oviposition before death and through horizontal transmission of M. anisopliae to offspring.     

Growth of Metarhizium anisopliae on non-preferred carbon sources yields conidia with increased UV-B tolerance

Conidia of the insect-pathogenic fungus Metarhizium anisopliae var. anisopliae produced on different growth substrates (culture media or insect cadavers) demonstrate reproducibly altered tolerance to UV-B radiation [Rangel, D.E.N., Braga, G.U.L., Flint, S.D., Anderson, A.J., Roberts, D.W., 2004. Variations in UV-B tolerance and germination speed of M. anisopliae conidia produced on artificial and natural substrates. J. Invertebr. Pathol. 87, 77-83]. In the current study, the fungus was grown on potato dextrose agar with yeast extract (PDAY), on minimal medium [(MM)=Czapek medium without saccharose], or on MM with one of 16 different carbon sources. The conidia produced on these media were exposed to UV-B radiation. Great amplitude in phenotypic plasticity for UV-B tolerance was demonstrated, viz., conidia produced under nutritive stress [MM or MM supplemented with non-preferred carbon sources (e.g., fructose, galactose, lactose etc.)] had at least two times higher tolerance than conidia produced on the rich medium (PDAY). Endogenous trehalose and mannitol accumulated at least two times more in conidia produced on MM (or MM with lactose, a non-preferred carbon source), as compared to conidia from MM plus glucose. High accumulations of these two carbohydrates in fungal spores are known to protect them against a wide range of stresses. Sporulation, however, was most profuse on PDAY, second best on MM plus d-mannose and least on MM or MM containing non-preferred carbon sources. Taken together, the results illustrate that nutritive stress generated by MM or MM plus a non-preferred carbon source greatly improved UV-B tolerance, but reduced conidial yield; while, on the other hand, preferred carbon sources improved conidial yield, but reduced UV-B tolerance.     

Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis

Second and third instar Chilo partellus larvae were infected with Beauveria bassiana and Metarhizium anisopliae (both at 1x10(8)conidia/ml) and daily consumption of maize leaves was measured. Infection by the fungi was associated with reduced mean daily food consumption. Reduction in food consumption became evident 3-4 days after treatment with the fungi for second instar larvae and 4-5 days for third instar larvae. Four conidial concentrations, 1x10(5), 1x10(6), 1x10(7), and 1x10(8)conidia/ml, were tested against second instar larvae. Food consumption dropped by 70-85% when the second instar larvae were treated with the fungi at 1x10(8)conidia/ml. Reduction in food consumption by C. partellus larvae infected with B. bassiana and M. anisopliae may offset the slow speed of kill of the fungi. The effect of artificial versus natural diets on mortality and mycoses of second instar larvae treated with the fungi at 1x10(8)conidia/ml was determined. Larvae provided with artificial diet suffered little mortality and mycoses than larvae provided with maize leaves. The LT(50) was longer for larvae provided with artificial diet.     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

Circular dichroism analysis of destruxins from Metarhizium anisopliae

Destruxins are secondary metabolites secreted by Metarhizium anisopliae [Y. Kodaira, Toxic substances to insects, produced by Aspergillus ochraceus and Oopsra destructor, Agric. Biol. Chem., 25 (1961) 261-262. D.W. Roberts, Toxins from the entomogenous fungus Metarhizium anisoplaie: Isolation from submerged cultures, J. Invertebr. Pathol., 14 (1969) 82-88. D.W. Roberts, Toxins from the entomogenic fungi in microbial control of pest and plant disease, Academic press, New York, 1981, pp441-464.]. In recent research, other than being used as insecticides, destruxins exhibited great potential in therapeutical applications such as antitumor, antivirus, and animal cell immunization effectiveness, etc. In this study, the conformations purified destruxins were determined by circular dichroism (CD). The results indicated that these cyclic peptides have the type I beta-turn conformation. In addition, different types of destruxins exhibited different CD spectra in acetonitrile. Therefore, these characters can be used as fingerprints to identify each type of destruxin. To further investigate the interactions among destruxins, various combinations of destruxins in 10 mM phosphate-buffered saline (PBS) were also studied by CD. The results strongly suggested that destruxins might work independently in vivo. To our knowledge, this is the first report presenting the CD analysis of purified destruxins.     

Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae)

Virulence of Metarhizium anisopliae (Metschnikoff) Sorokin strain EAMa 01/121-Su against the German Cockroach, Blatella germanica (L.), was determined using four concentrations ranging from 4.2 x 10(6) to 4.2 x 10(9) spores per milliliter. The LD50 value was 1.4 x 10(7) spores per milliliter (56,000 spores per cockroach) and LT50 values were 14.8 days and 5.3 days for 4.2 x 10(8) and 4.2 x 10(9) spores per milliliter, respectively. An experiment was conducted to evaluate whether a fungal transmission could exist among infected and healthy cockroaches. Percentage mortality at a ratio of 1:10 of infected to unexposed cockroaches was 87.5% and LT50 was 12.2 days, which indicated the potential of this strain to be horizontally transmitted and to rapidly spread the infection in the insect population. The effect of a sublethal dose (ca. LD60) of M. anisopliae EAMa 01/121-Su strain, applied topically on German cockroaches, was studied by reciprocal crossing. Othecal production, oothecal hatchability, and nymphal production declined upon exposure to M. anisopliae EAMa 01/121-Su strain. The mean number of oothecae laid by female was progressively and significantly reduced by fungal treatment from second oviposition period onwards. Oothecal hatch of fungally challenged females was reduced by 46-49%, oothecal viability by 48-85%, and nymphal production by 22-35%. Only treated females showed an effect on oothecal production, oothecal hatch, and nymphal production, although oothecal hatch was also governed by treated males at a higher significance level. Our results on virulence and horizontal transmission of fungal conidia of M. anisopliae EAMa 01/121-Su strain and its sublethal reproductive effects on German cockroach females are discussed in terms of its potential to decrease the pest status of B. germanica in the short and long terms.     

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130±102 and 1200±97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35% of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels.     

Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Differentially-expressed glycoproteins in Locusta migratoria hemolymph infected with Metarhizium anisopliae

Glycoproteins play important roles in insect physiology. Infection with pathogen always results in the differential expression of some glycoproteins, which may be involved in host-pathogen interactions. In this report, differentially-expressed glycoproteins from the hemolymph of locusts infected with Metarhizium anisopliae were analyzed by two-dimensional electrophoresis (2-DE) and PDQuest software. The results showed that 13 spots were differentially expressed, of which nine spots were upregulated and four were downregulated. Using MS/MS with de novo sequencing and NCBI database searches, three upregulated proteins were identified as locust transferrin, apolipoprotein precursor, and hexameric storage protein 3. These proteins have been reported to be involved in the insect innate immune response to microbial challenge. Due to the limited available genome information and protein sequences of locusts, the possible functions of the other 10 differentially-expressed spots remain unknown.     

Persistence of Metarhizium anisopliae incorporated into soilless potting media for control of the black vine weevil, Otiorhynchus sulcatus in container-grown ornamentals

The objective of this study was to determine the persistence of Metarhizium anisopliae (F52), measured as infectivity against black vine weevil larvae, in a soilless potting medium at six wholesale nursery locations across the Willamette Valley, Oregon. A granule formulation (0.30 and 0.60 kg/m(3)) was incorporated into media at planting and fungal persistence determined over two growing seasons. The fungus persisted in the potting media over the duration of the experiment with 50-60% of the larvae exposed to treated media becoming infected at the end of the experiment. The percentage of infected larvae gradually declined from > or = 90% on week 3 to 40-60% by week 19. Larval infection rebounded over the fall and winter months of 2004 to 75-80% followed again by a slow decline over the course of the second growing season.     

Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae

The gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolated from the entomopathogenic fungus, Metarhizium anisopliae. The deduced amino acid sequence predicted a protein of 447 amino acids with a molecular weight of 49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1 cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assays showed that the purified recombinant protein had ornithine decarboxylase activity. The optimum pH of the purified Odc1 protein was 8.0-8.5, and the optimum reaction temperature was 37 °C. The apparent K_m for ornithine at a pyridoxal phosphate concentration of 20 mM was 22 μM. The competitive inhibitor of ODC activity, 1,4-diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNA showed an increase at the beginning of appressorium formation in vitro. During the M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNA showed a discrete increase within the germinating spore and during appressorium formation. The second expression peak was higher and prolonged during the invasion and death of the insect. The ODC1 gene complements the polyamine auxotrophy of Yarrowia lipolytica odc null mutant.     

Variability in conidial thermotolerance of Metarhizium anisopliae isolates from different geographic origins

Notable variability in thermotolerance was found among conidia of 16 isolates of the insect-pathogenic fungi Metarhizium anisopliae var. anisopliae and one M. anisopliae var. acridum isolated from latitudes 61 degrees N to 54 degrees S. Conidial suspensions were exposed to 40 or 45 degrees C for 2, 4, 8, and 12 h. Most of the isolates tolerated 40 degrees C very well, with relative germination (germination relative to unheated controls) above 90% after 12 h of exposure. Exceptions were three isolates originating from high latitude, viz., ARSEF 2038 (38 degrees N, South Korea), 4295 (54.4 degrees S, Australia), and 5626 (61.2 degrees N, Finland) that had approximately 80% germination. High variability, however, was observed among isolates at 45 degrees C; viz., after 2 h exposure, relative germination was above 80% for six isolates, between 50 and 70% for three isolates, and between 0 and 30% for eight isolates. After 8 and 12 h at 45 degrees C, only two M. anisopliae isolates pathogenic to grasshoppers, viz., ARSEF 324 (latitude 19 degrees S, Australia) and 3609 (15 degrees N, Thailand), had high relative germination (91.6 and 79.4%, respectively, for 8 h exposures; and 90 and 47.1%, respectively, for 12 h). These isolates also were the most tolerant to UV-B radiation [J. Invertebr. Pathol. 78 (2001) 98-108]. The median lethal dose (LD50) for isolate ARSEF 324 was 49.4 and 47.9 degrees C, for 2 and 4 h of exposures, respectively. Exposure of conidia to wet-heat greatly delayed germination of some isolates. In general, isolates from higher latitudes demonstrated greater heat susceptibility than isolates from nearer the equator. Dry conidia tolerated 50 degrees C better than 45 degrees C in aqueous suspension.     

Inhibition of fungal growth in thermoregulating locusts,Locusta migratoria,infected by the fungus Metarhizium anisopliae var acridum

The locust, Locusta migratoria, has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum. We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.     

Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor,a honey bee parasite

The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives. In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H. thompsonii and 5.85 (5.48-7.43) days for M. anisopliae at 1.1 x 10(3) conidia mm(-2). At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H. thompsonii [LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7] and M. anisopliae [LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7] both showed significant virulence against V. destructor. The applications of H. thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments. The treatments did not significantly affect the mite population in sealed brood. However, the fungus must have persisted because infected mites were still observed [82.97+/-(0.6)%] 42 days post-treatment. In addition, the fungus was found to sporulate on the host. A small percentage [2.86+/-(0.2)%] of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus. H. thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens. Microbial control with fungal pathogens provides promising new avenues for control of V. destructor and could be a useful component of an integrated pest management program for the honey bee industry.