Potential of a Dried Mycelium Formulation of an Indigenous Strain of Metarhizium anisopliae Against Subterranean Pests of Cranberry

We demonstrated that a dried mycelium formulation of an indigenous strain of Metarhizium anisopliae has good potential as an effective biopesticide against the black vine weevil (BVW), Otiorhynchus sulcatus (F.), and the cranberry girdler (CG), Chrysoteuchia topiaria (Zeller). The formulation was produced at moderate sized batches scales (~ 100g product per batch) using inexpensive and easily obtainable equipment (< US$1000). Levels of conidiation were consistent among production batches and storage intervals with the product remaining viable for long time periods at 4&#176;C, in one case for more than a year. Levels of contamination by yeasts and other benign fungi were also consistent. Fewer BVW and CG were found in small plots treated with the dried mycelium formulation than in untreated plots, but differences were not always significant. A waxmoth ( Galleria mellonella ) bait bioassay demonstrated the formulation remained viable in the soil over long time periods (> 7 months). Percentage of M. anisopliae infected waxmoth larvae was significantly affected by rate.     

Development of a Spray-drying Technique for Submerged Spores of Entomopathogenic Fungi

The stability of blastospores or submerged conidia of entomopathogenic fungi is one of the key problems associated with the practical use of fungal biopreparations. A spray-drying technique was developed which allows the drying of blastospores and/or submerged conidia, here called submerged spores, without a significant loss of viability and with only a slight delay in eY cacy. The method was tested successfully using the fungal species Metarhizium anisopliae , M. flavoviride, Beauveria bassiana and Paecilomyces fumosoroseus . For all experiments, a laboratory spray dryer was used. The optimum inlet and outlet temperatures were found to be 64 2oC and 48 2oC respectively. The best protective agent for submerged spores was skimmed milk powder at concentrations of 10 or 20%. The addition of sugars, especially 2.5% sugar-beet syrup, slightly improved the viability after spray-drying. Submerged spores of eight isolates of M. anisopliae, M. flavoviride, B. bassiana and P. fumosoroseus were suspended in 20% skimmed milk powder and 2.5% sugar-beet syrup, and spray-dried. Germination rates of about 90% were achieved, which were comparable to those of freshly produced submerged spores. Finally, the virulences of spray-dried submerged spores of M. anisopliae (Ma 97) and M. flavoviride (Mfl 5) were tested in bioassays using third- and fourth-instar nymphs of the African locust, Locusta migratoria . In contrast to M. flavoviride , there was no significant diVerence in the median lethal time between spray-dried and fresh submerged spores of M. anisopliae . The investigations demonstrated that spray-drying is possible to preserve sensitive submerged spores of entomopathogenic fungi.     

Toxicity of Xanthene Dyes to Entomopathogenic Fungi

Effects of xanthene dyes on mycelial growth and conidial germination in three species of entomopathogenic fungi, Beauveria bassiana, Metarhizium anispoliae and Paecilomyces fumosoroseus, were evaluated in a variety of assay systems. In a disk-diffusion assay, erythrosin B and phloxine B (but not eosin B) produced zones of inhibition in colonies of all three species under continuous exposure to light at disk-loadings of 100mug. None of the dyes produced zones of inhibition in the absence of light at disk loadings of 100mug. Both erythrosin B and phloxine B inhibited mycelial growth of all three species in the light in a dose-dependent manner. Weaker dose-responses for inhibition of growth in the dark were observed for some fungus/dye combinations. Erythrosin B, tested singly, completely inhibited conidial germination in the light in all eight fungal strains tested at 100mug ml-1 medium, but failed to inhibit conidial germination in any of these strains in the dark at the same concentration of dye. For single strains of each of the three fungi, erythrosin B and phloxine B inhibited conidial germination in a dose-dependent manner in the light with IC50s < 6.2mug dye ml-1 medium for all fungus/dye combinations. Phloxine B was a more potent inhibitor of germination than erythrosin B for all three fungal species. At fixed dosages of erythrosin B and phloxine B, inhibition of conidial germination in all three species increased with time of exposure to light. These results constitute the first quantitative demonstration of photodynamic inhibition of conidial germination in fungi by xanthene dyes.     

Influence of Pesticide Use on the Natural Occurrence of Entomopathogenic Fungi in Arable Soils in the UK: Field and Laboratory Comparisons

The spectrum and abundance of entomopathogenic fungi in agricultural soil receiving different pesticide applications were evaluated . Seven small field plots within a barley crop were selected . Each plot had received a different pesticide treatment at slightly higher than the field rate each year for the previous 12-19 years . The field plots received either benomyl (fungicide) , triadimefon (fungicide) , aldicarb (insecticide) , chlorfenvinphos (insecticide) , glyphosate (herbicide) , all five of these pesticides or no pesticides at all (control) . Soil sampled from each plot was baited with Galleria mellonella larvae at either 18 or 26 C . Five species of entomopathogenic fungi infected these larvae . Beauveria bassiana was the dominant species , and the only species for which infection levels were high enough to be analyzed statistically . Significantly fewer G. mellonella larvae became infected with B. bassiana in soil treated with benomyl than in other treatments . This deleterious effect was confirmed in in vitro experiments where benomyl inhibited fungal growth significantly . Chlorfenvinphos also reduced the proportion of G. mellonella becoming infected with B. bassiana slightly (although not statistically significantly) . This effect was much greater in in vitro experiments in which chlorfenvinphos inhibited fungal growth significantly . B. bassiana- induced mortality was significantly greater in G. mellonella larvae placed in soil treated with triadimefon than in other treatments . In vitro studies , however , demonstrated that triadimefon inhibited rather than stimulated growth . A similar anomaly between field and laboratory observations was seen in the case of aldicarb . There was no significant difference in the number of G. mellonella larvae found infected with B. bassiana in soil treated with aldicarb than in the control soil . However , in the in vitro studies , aldicarb stimulated fungal growth at all concentrations except 10 times the field rate . In general , significantly fewer larvae became infected with B. bassiana at 26 C than at 18 C in all treatments . Pesticides may have a direct impact on the natural occurrence , infectivity and population dynamics of entomopathogenic fungi . They also affect other macro - and microorganisms in the soil which may interact with the ento mopathogenic fungi . In combination with climate and soil variables , these effects are difficult to separate . Although any suppression of these fungi may be detrimental to their capacity as natural enemies , this study has demonstrated that pesticides used under field conditions are unlikely either to kill all the entomopathogenic fungi present in the treated area or to limit their recolonization . It seems likely , therefore , that pesticides have the potential to be used in conjunction with mycoinsecticides in integrated pest management systems .     

Entomopathogenic Fungi Associated with Natural Populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera: Gomphocerinae) and Other Acridoidea in Spain

The entomopathogenic fungi Beauveria bassiana Vuill . and Metarhizium anisopliae (Metschnikoff) have been found in natural populations of the Moroccan locust Dociostaurus maroccanus (Thunberg) and other species of acridoids that cohabit the same locust breeding areas in southern Spain . Infection levels of B. bassiana on insects collected in the field and maintained under laboratory conditions were relatively high (1 . 6 - 20 . 5%) . The prevalence of the disease extended from spring to summer in the three consecutive years monitored . A local isolate of this fungus demonstrated high virulence (LD = 440 conidia / insect) against the 50 locust D. maroccanus in the laboratory bioassay . The relatively wide host range of B. bassiana and its adaptation to the dry and hot conditions dominating the locust breeding area suggest that this isolate could be considered in the development of a biological control programme against D. maroccanus.     

Susceptibility of Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) to Entomopathogenic Fungi from Ethiopia

The efficacy of 13 isolates of entomopathogenic fungi belonging to Beauveria , Metarhizium or Paecilomyces spp. was assessed against Sitophilus zeamais (Coleoptera: Curculionidae) and Prostephanus truncatus (Coleoptera: Bostrichidae) using a total immersion bioassay technique in the laboratory. Fungi were applied at concentrations of 1 ×10 7 and 1 ×10 8 conidia mL -1 for P. truncatus and S. zeamais , respectively. All isolates tested were virulent to P. truncatus (98-100% mortality, and median survival time (MST) ranged from 2.85-4.05 days). Metarhizium anisopliae and B. bassiana were also virulent to S. zeamais (92-100% mortality, MST ranged from 3.58-6.28 days). The isolate of Paecilomyces sp. was found to be the least virulent against S. zeamais , causing only 26.32 ±4.29% mortality with MST of 10.38 ±0.29 days. P. truncatus proved more susceptible to the entomopathogenic fungi tested than S. zeamais . One M. anisopliae (PPRC-EE) and three B. bassiana isolates (PPRC-HH, PPRC-9609 and PPRC-9614) were selected for further study and dose-mortality relationships were assessed on S. zeamais . The tested concentrations ranged from 1 ×10 4 -1 ×10 7 conidia mL -1 . M. anisopliae (PPRC-EE) showed the lowest LC 50 (3.39 ×10 5 conidia mL -1 ) followed by B. bassiana PPRC-HH (2.04 ×10 6 conidia mL -1 ). PPRC-9609 and PPRC-9614 showed slight differences in LC 50 but not at LC 90 . The results revealed the higher potency of M. anisopliae as compared with the B. bassiana isolates tested. The study suggests that the use of entomopathogenic fungi may hold promise as an alternative method to control pests of stored-products in Ethiopia.     

Proteases Released by Entomopathogenic Fungi Impair Phagocytic Activity, Attachment and Spreading of Plasmatocytes Isolated from Haemolymph of the Greater Wax Moth Galleria mellonella

Reliable assays for the in vitro quantification of the attachment and spreading of isolated Galleria mellonella plasmatocytes have been established. The effects of extracellular proteases released by the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana on the phagocytic activity, spreading, attachment and cytoskeleton formation of isolated plasmatocytes of G. mellonella were examined to elucidate their putative role in the suppression of cellular immune responses in infected insects. In addition, the influence of different commercially available proteases on isolated plasmatocytes was studied. Among the proteases tested, the metalloprotease thermolysin produced the strongest inhibitory activity on plasmatocytes. The results obtained support the conclusion that invading fungal cells could interfere with the insect immune system via the release of proteases which affect cellular defence reactions. Isolated G. mellonella plasmatocytes incubated with fungal proteases had an impaired ability to ingest yeast cells and exhibited alterations in morphology and cytoskeleton formation. The effects were similar to those observed in plasmatocytes from infected larvae. The role of extracellular fungal proteases in the interactions of entomopathogens with the insect immune system is discussed.     

Mortality in Three African Tephritid Fruit Fly Puparia and Adults Caused by the Entomopathogenic Fungi, Metarhizium anisopliae and Beauveria bassiana

The pathogenicity of 13 isolates of Metarhizium anisopliae and two isolates of Beauveria bassiana to Ceratitis capitata and Ceratitis var. rosa fasciventris exposed as late third instar larvae in sand was evaluated in the laboratory. All isolates caused a significant reduction in adult emergence and a corresponding large mortality on puparia of both species. All isolates also induced large deferred mortality in emerging adults following treatment as late third instar larvae. On C. capitata , seven isolates ( M. anisopliae ICIPE 18, 20, 32, 60 and 69 and B. bassiana ICIPE 44 and 82) caused significantly higher mortality on puparia than other isolates. With the exception of ICIPE 32, the other four isolates of M. anisopliae above were the most pathogenic against C. r. fasciventris . Dose-response study carried out with these isolates of M. anisopliae on the two species of flies above plus another species, Ceratitis cosyra showed that the dose-mortality regression lines of ICIPE 18 and 20 were steeper with lower LC 50 values when compared with ICIPE 60 and 69 on the three species. When these two isolates were evaluated with regard to their pathogenicity to different pupal age, adult emergence was found to increase with increasing pupal age with a corresponding decrease in mortality in puparia and emerging adults in the three species of fruit flies. M. anisopliae ICIPE 18 and 20 were equally pathogenic to all pupal ages tested in C. capitata and C. cosyra but ICIPE 18 was more pathogenic to older puparia of C. r. fasciventris than ICIPE 20. Our results suggest that soil inoculation with M. anisopliae under mango trees might form an important component of integrated pest management strategies in areas where these three species of fruit fly coexist.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Control of Leaf-footed Bug Leptoglossus zonatus and Shield-backed Bug Pachycoris klugii with Entomopathogenic Fungi

The pathogenicity of three isolates of Beauveria bassiana (Bals.) Vuil. and one isolate of Metarhizium anisopliae (Metsch.) Sorok. (Deuteromycotina: Hyphomycetes) was assessed in the laboratory against adults of the leaf-footed bug Leptoglossus zonatus (Dallas) (Heteroptera: Coreidae) and the shield-backed bug Pachycoris klugii Burmeister (Heteroptera: Scutelleridae), the two most frequent pest species in physic nut ( Jatropha curcas L., Euphorbiaceae) plantations in Nicaragua. In a dipping bioassay, the median lethal concentration (LC) of the most efficient strain, M. anisopliae NB, was determined as 4.34 106 50 conidia/ml for adult P. klugii . In a field trial, a scheduled high-volume spray regime using B. bassiana increased fruit yield by 28%, and was more effective than malathion or an aqueous extract of ground neem seeds. The effectiveness of M. anisopliae was further tested in field cages covering entire trees and containing a predetermined number of insects. Mineral oilbased ultra-low volume controlled droplet applications of M. anisopliae at a rate of 1 1010 conidia/tree were made using hand-held Micron ULVA + sprayers. The corrected mortalities ranged from 65% in P. klugii to 94% in L. zonatus.     

The Effect of Cucurbitacin E Glycoside, a Feeding Stimulant for Corn Rootworm, on Biocontrol Fungi: Beauveria bassiana and Metarhizium anisopliae

Corn rootworms compulsively feed on cucurbitacins (bitter compounds found in many cucurbits), while most other pest insects are repelled by them. Several hypotheses have been proposed for this activity, but the results have been equivocal. One recent hypothesis suggested that cucurbitacin may provide protection against soil borne fungal entomopathogens, both in the adult corn rootworm and in the eggs laid in the soil. Any antifungal activity would preclude the use of this feeding stimulant to enhance the activity of fungal pathogens used in biocontrol. To test this hypothesis, we exposed two fungal pathogens of corn rootworm, Beauveria bassiana and Metarhizium anisopliae , to an extract from a bitter mutant of Hawkesbury watermelon, which contained about 0.05% cucurbitacin E glycoside. The extract inhibited the growth of both fungi. However, when the extract was sterilized by passing through a 0.45 micron filter, this inhibitory activity disappeared. Purified cucurbitacin E glycoside did not inhibit the growth of either fungus. Four Bacillus isolates (identified as Bacillus subtilis var. globigii and B. amyloliquefaciens ) were isolated from this extract which were able to inhibit the growth of both fungi. All of these bacteria excreted the inhibitory activity into the medium. Thus, the inhibition of fungal growth attributed to cucurbitacin may actually be due to bacteria incidentally associated with cucurbits.     

Susceptibility to Fungi of Cotton Boll Worms Before and After a Natural Epizootic of the Entomopathogenic Fungus Nomuraea rileyi (Hyphomycetes)

Epizootics caused by Beauveria bassiana and Nomuraea rileyi have been observed on boll worms and Spodoptera litura in south Indian fields during winter since the last 15 years. During the N. rileyi-induced natural epizootics, some boll worms were found surviving without infection. Whether they represent pathogen-resistant genotypes was investigated. Two insect populations, collected 3 months prior to and during the epizootic were established. Their sensitivity to both the fungi was compared in laboratory bioassays. No significant difference in sensitivity was observed between the two populations. It was concluded that the boll worm population surviving the epizootic was not genotypically resistant.     

Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to Entomopathogenic Fungi and Their Extracts

The effectiveness of seven strains of entomopathogenic fungi against Ceratitis capitata adults was evaluated in the laboratory. Adults were susceptible to five of seven aqueous suspensions of conidia. Metarhizium anisopliae and strain CG-260 of Paecilomyces fumosoroseus were the most pathogenic fungi, with 10-day LD₅₀ values of 5.1 and 6.1 × 10³ conidia/fly, respectively, when applied topically. Sublethal effects on fecundity and fertility of the fungal-exposed females were also studied. The most effective fungus in reducing fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at 1 × 10⁶ conidia/fly. M. anisopliae and Aspergillus ochraceus also showed significant reductions of fecundity (40–50% for most of the assayed concentrations). Fertility was moderately affected by the fungi. M. anisopliae at 1 × 10⁶ conidia/fly was the most effective fungus, showing egg eclosion reduction of over 50% compared with the control. In addition, culture broth dichloromethane extracts from the entomopathogenic fungi were tested for insecticide activity against C. capitata, including effects on fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in about 90% mortality at a concentration of 25 mg/g of diet; under these conditions, fecundity and fertility of treated females were reduced by 94 and 53%, respectively, compared with untreated controls.     

Evaluation of the Entomopathogenic Fungi Metarhizium anisopliae and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for Control of the Green Leafhopper Empoasca decipiens (Homoptera: Cicadellidae) and Potential Side Effects on the Egg Parasitoid Anagrus atomus (Hymenoptera: Mymaridae)

The efficacy of Metarhizium anisopliae strain Ma43 and Paecilomyces fumosoroseus strain Pfr12 (both Deuteromycotina: Hyphomycetes) against adults of Empoasca decipiens (Homoptera: Cicadellidae) and potential side effects on the egg parasitoid Anagrus atomus (Hymenoptera: Mymaridae), were investigated in greenhouse cage and laboratory experiments. Treating leafhopper-infested faba bean plants at a dose rate of 1×10⁷ conidia mL^-1 resulted in up to 97% mortality 7 days after application and a 100% infection rate. Experiments on the residual effects revealed a significant decrease in adult E. decipiens mortality with increasing time from application to insect release. The decrease in mortality over time corresponded well with data from conidia germination tests. The germination of conidia on agar medium after washing them from the surafce of sprayed plants declined significantly from 95 and 96% immediately after application for M. anisopliae Ma43 and P. fumosoroseus Pfr12, respectively, to 29 and 27% 5 days later. Experiments on potential side effects of the entomopathogenic fungi on A. atomus showed that the tested isolates had no influence on adult emergence and longevity; however, the rates of parasitism were significantly reduced by the fungal treatments. The latter might be due to either density effects and/or could indicate that A. atomus avoids fungal-treated plants. However, the parasitoid is substantially less susceptible to the fungal strains tested than the host itself.     

Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes

The influence of simulated sunlight on survival of conidia of 4 species of entomopathogenic Hyphomycetes was investigated. Conidia from 65 isolates ofBeauveria bassiana, 23 ofMetarhizium anisopliae, 14 ofMetarhizium flavoviride and 33 isolates ofPaecilomyces fumosoroseus were irradiated by artificial sunlight (295 to 1,100 nm at an ultraviolet-B irradiance of 0.3 W m^–2) for 0, 1, 2, 4 and 8 h. Survival was estimated by comparing the number of colony forming units (CFU) produced by conidia exposed to irradiation to the number of CFUs produced by an unexposed control. Survival decreased with increased exposure to simulated sunlight; exposure for 2 h or more was detrimental to all isolates tested. Overall, isolates ofM. flavoviride were the most resistant to irradiation followed byB. bassiana andM. anisopliae. Conidia ofP. fumosoroseus were most susceptible. In addition to the large interspecies differences in susceptibility to irradiation, there was also an intraspecies variation indicating that strain selection to irradiation tolerance may be important in the development of microbial control agents where increased persistence in an insolated environment is desirable.Abbreviations CFU Colony forming units - UV-B ultraviolet radiation-B     

Entomopathogenic fungi of Eurygaster integriceps Puton (Hemiptera: Scutelleridae): collection and characterization for development

Exploratory activities were done in Syria, Turkey, Iran, Uzbekistan, Kazakhstan, The Kyrghyz Republic, and Russia to locate entomopathogenic fungi of Eurygaster integriceps. Isolates from the entomopathogenic genera Beauveria, Paecilomyces, and Verticillium were collected. Beauveria bassiana was the most commonly recovered species. Thirty-one isolates of the 221 recovered were examined at 20, 25, 30, and 35 °C for 20 days for growth and sporulation. Growth and sporulation were generally highest at 25 °C. None of the isolates grew at 35 °C, and at 30 °C growth was retarded with no conidia being produced. Single- and multiple-concentration bioassays were conducted on greenhouse-grown wheat plants and in pine litter to evaluate virulence of fungi from several sources to E. integriceps. When tested at a single concentration, mortality after 15 days ranged from 66 to >95% in the litter assays and 50 to 91% in the plant assays. There was a distinct concentration response for most of the isolates tested in the multiple-concentration assay, particularly in the in-litter environment. In litter, mortality tended to develop earlier than in on-plant assays. Several isolates of B. bassiana and one Metarhizium anisopliae displayed consistently high virulence against E. integriceps and were more virulent than two commercial strains. Our results demonstrate the potential of entomopathogenic fungi for management of E. integriceps in overwintering sites and in wheat fields.     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。