Isolation and characterization of microsatellite markers from the entomopathogenic fungus Metarhizium anisopliae

Fourteen polymorphic microsatellite markers were isolated from the entomopathogenic fungus, Metarhizium anisopliae, based on enriched genomic libraries. In order to assess allelic variability, the microsatellite loci were analysed in a collection of 34 isolates sampled from across Switzerland. The number of detected alleles in 14 loci ranged from two to eight and expected heterozygosity from 0.265 to 0.808. Because of the high expected heterozygosity, the 14 microsatellite loci are very useful for ecological studies and analysis of population diversity, and to identifying, monitoring, and tracking M. anisopliae strains applied as biological control agents.     

Susceptibility of the Hymenopteran Parasitoids Apoanagyrus ( = Epidinocarsis ) lopezi (Encyrtidae) and Phanerotoma sp. (Braconidae) to the Entomopathogenic Fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes)

Laboratory and so-called extended laboratory bioassays were conducted in Benin, West Africa, to investigate the pathogenicity and virulence of the entomopathogenic fungus Metarhizium anisopliae var. acridum , a biocontrol agent against locusts and grasshoppers, to two hymenopteran parasitoids, Apoanagyrus ( = Epidinocarsis ) lopezi and Phanerotoma sp. Treatments were carried out under simulated field conditions at standard field dose rates of 2.5 and 5.0 ×10 12 conidia ha -1 . Test organisms were continuously (3 weeks) exposed to spray residues in artificial or simulated natural environments. The standard strain IMI 330 189 of the mycopesticide Green Muscle caused a significant reduction of 24% in the longevity ( = average survival time, AST) of A. lopezi , relative to the untreated control. Mycosis was confirmed in 16% of all cadavers. AST was shorter under low relative humidity (RH) conditions, and these conditions seemed to enhance susceptibility to fungal infection. However, this effect was only marginally significant. In contrast, average longevity of untreated A. lopezi was slightly yet significantly shorter at low RH (50-60%) than at high RH (80-90%). In the extended laboratory assay, the same isolate had no significant effect on mortality, parasitoid emergence ( = beneficial capacity) and sex ratio. In a further screening test with isolates IIBC I91 609, IIBC I93 833 and IMI 330 189 (reference), no infection of A. lopezi was confirmed. Similarly, Phanerotoma sp. was not susceptible to IMI 330 189. It is concluded that mycopesticides based on the three strains tested pose a low risk to parasitic hymenopterans under field conditions.     

Oils for Increased Efficacy of Metarhizium anisopliae to Control Whiteflies

The efficacy of Metarhizium anisopliae in combination with sublethal concentrations of oils and potassium-oleate for biological control of whiteflies was tested under controlled conditions. Three commercial products (Biola ® , Naturen ® , Neudosan ® ) and five experimental formulations of plant oils were tested. The efficacy of M. anisopliae against Trialeurodes vaporariorum and Bemisia tabaci without additives was about 50%. At 1/20 of their recommended dosages, all compounds tested significantly increased the efficacy of M. anisopliae for the control of T. vaporariorum , with the formulated sunflower oil Biola ® giving the highest synergistic effect, reaching nearly 100% control. Not only was the level of control increased but also the speed of action was improved, resulting in a higher reliability of control. Three of seven additives showed no effects on the viability of conidia on the leaf surface, whereas the formulations of the other oils and oleates reduced the longevity of spores. The synergistic effect of Biola resulted from the more even distribution of M. anisopliae conidia on leaves and insects. Other positive effects of oils on the efficacy of M. anisopliae are discussed in relation to an extended spectrum of environmental conditions and pests to be controlled.     

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.     

Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa

Studies were conducted tosearch for fungal strains with potentialpathogenicity against Diabrotica speciosa(Germar) (Coleoptera: Chrysomelidae).Among sixteen fungal isolates screenedthe most virulent was a Beauveria bassiana(Balsamo) Vuillemin isolate (FHD13) thatcaused 70% mortality of D. speciosathird instar larvae. The LC₅₀ value ofB. bassiana isolate FHD13 was3.48 × 10¹⁰ conidia/ml.Different temperatures (4, 17 and 26 °C)and vegetable oils (corn, sunflower and canola)used for storage did not significantly affectviability of conidia. A pathogenicity trialagainst D. speciosa larvae performed withthe corn oil formulation (1 × 10⁸ conidia/mlof oil) caused 65% of mortality.     

Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite

Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. This relationship becomes more complicated in social species, such as social insects, since the chance of disease transmission increases with the frequency of interactions among individuals. However, social insects may benefit from social immunity, whereby individual physiological defenses may be bolstered by collective‐level immune responses, such as grooming or sharing of antimicrobial substance through trophallaxis. We set out to determine whether differences in genetic diversity between colonies of the subterranean termite, Reticulitermes flavipes, accounts for colony survival against pathogens. We sampled colonies throughout the United States (Texas, North Carolina, Maryland, and Massachusetts) and determined the level of inbreeding of each colony. To assess whether genetically diverse colonies were better able to survive exposure to diverse pathogens, we challenged groups of termite workers with two strains of a pathogenic fungus, one local strain present in the soil surrounding sampled colonies and another naïve strain, collected outside the range of this species. We found natural variation in the level of inbreeding between colonies, but this variation did not explain differences in susceptibility to either pathogen. Although the naïve strain was found to be more hazardous than the local strain, colony resistance was correlated between two strains, meaning that colonies had either relatively high or low susceptibility to both strains regardless of their inbreeding coefficient. Overall, our findings may reflect differential virulence between the strains, immune priming of the colonies via prior exposure to the local strain, or a coevolved resistance toward this strain. They also suggest that colony survival may rely more upon additional factors, such as different behavioral response thresholds or the influence of a specific genetic background, rather than the overall genetic diversity of the colony.     

Transformation of the entomopathogenic fungus Metarhizium anisopliae to benomyl resistance

Abstract Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta . Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.     

House fly (Musca domestica) (Diptera: Muscidae) mortality after exposure to commercial fungal formulations in a sugar bait†

House flies are pests of medical and veterinary importance. They are known to be susceptible to fungal conidia presented in sugar baits. The purpose of this study was to evaluate the potential for commercial formulations of entomopathogenic fungi to be used as bait for fly control. Differences in sporulation by product were found. Current formulations may influence fly feeding on baits, but because formulations may protect conidia. Additional research into more attractive formulations is warranted.     

绿僵菌及其与dsRNA混合使用对褐飞虱的防治效果

褐飞虱Nilaparvata lugens Stl是我国重要的迁飞性水稻害虫,本文研究了金龟子绿僵菌Metarhizium anisopliae及其与dsRNA混合使用对褐飞虱的防治效果。绿僵菌悬浮液1.6×108孢子/m L至8×106孢子/m L对褐飞虱2龄、4龄和成虫进行喷药,发现1.6×107孢子/m L对各个虫态虫龄均有良好致死效果,并且成虫和4龄若虫均好于2龄若虫。在交配行为上来看,绿僵菌处理过的褐飞虱成虫活跃度非常低,从配对开始一直到交配结束的各个阶段都受到明显影响,处理组3 h的交配率只有3.70%,而对照组的交配率为24.44%。还把褐飞虱几丁质合成酶基因A的dsRNA与绿僵菌混合使用防治褐飞虱2龄和4龄若虫,结果表明0.5μg/μL ds CHSA与绿僵菌混合使用的防治效果最好,2龄若虫的死亡率为89.63%,4龄若虫的死亡率达到93.94%。而0.2μg/μL ds CHSA与绿僵菌的混合,对2龄和4龄若虫的致死率为65.56%-76.52%。研究结果为褐飞虱的生物防治提供了新的思路。