V. Yu. Kryukov

Synergistic action of entomopathogenic hyphomycetes and the bacteria Bacillus thuringiensis ssp. morrisoni in the infection of Colorado potato beetle Leptinotarsa decemlineata

A synchronous coinfection of the Colorado potato beetle Leptinotarsa decemlineata (Say) with the entomopathogenic bacteria Bacillus thuringiensis ssp. morrisoni Bonnifoi & de Barjak var. tenebrionis Krieg et al. and hyphomycete Metarhizium anisopliae (Metsch.) Sorokin or Beauveria bassiana (Bals.) Vuill leads to the rapid death of 95–100% of larvae. The bacteria arrest the nutrition of insects, while the fungal spores kill the weakened larvae. The synergistic effect of two pathogens is recorded at a relatively low hyphomycete titer (1–5 × 106 conidia/ml) and is evident in the mortality dynamics at all larval ages. These bacterial and fungal pathogens display no antagonism on artificial nutrient media. This microbial complex is highly efficient under natural conditions (80–90% larval mortality rate and no plant defoliation).

The activity of nonspecific esterases and glutathione-S-transferase in Locusta migratoria larvae infected with the fungus Metarhizium anisopliae (Ascomycota, Hypocreales)

The activity of nonspecific esterases and glutathione-S-transferase in whole body homogenates, hemolymph plasma, and fat body of the larvae of the locust Locusta migratoria was analyzed during development of infection with the fungus Metarhizium anisopliae. The lethal dose of the fungus (LC80) was found to enhance the activity of detoxifying enzymes in the whole body homogenate of the larvae on the 3rd day after infection. The activity of nonspecific esterases and glutathione-S-transferase in the plasma and fat body of the infected larvae increased on the 3rd day but dropped to the control levels by the 6th day, during the acute period of infection. The detoxifying enzymes may participate in defense reactions at the early stage of the acute fungal infection.

An increase in the immune system activity of the wax moth Galleria mellonella and of the Colorado potato beetle Leptinotarsa decemlineata under effect of organophosphorus insecticide

Several aspects of immune response in insects treated with an organophosphate (pirimiphosmethyl) have been assessed. Both humoral (phenoloxidase activity) and cellular (hemocyte count and encapsulation rate) immunity changes have been studied in larvae of the representatives of two insect orders, the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) and the wax moth Galleria mellonella (Lepidoptera, Pyralidae). A direct contact with sublethal and half-lethal doses of the insecticide results in stimulation of immune reactions: phenoloxidase activity and encapsulation rate are heightened, and hemocyte count increases.

Change in the temperature preferences of Beauveria bassiana sensu lato isolates in the latitude gradient of Siberia and Kazakhstan

The radial growth of twenty isolates of the entomopathogenic fungus Beauveria bassiana sensu lato from different natural zones of Western Siberia and Kazakhstan (from 65 to 43°N) was tested under different temperatures (5–35°C). It was shown that the thermotolerance of the fungal isolates increased significantly from the north to south. The cold activity of the cultures did not significantly correlate with the latitude of origin and the sum positive temperatures of the regions. A distinct group of the steppe thermotolerance isolates was shown by the analysis of genomic polymorphism using seven intermicrosatellite DNA markers (ISSR). The steppe isolates had high levels of virulence to the wax moth Galleria mellonella and the Colorado potato beetle Leptinotarsa decemlineata at high temperatures (>30°C) compared to that of the forest-steppe isolates. The obtained data indicate that the use of isolates from the steppe zone will be most promising for the insect pest control under the conditions of continental and arid climate.

Insecticidal and immunosuppressive effect of ascomycete Cordyceps militaris on the larvae of the Colorado potato beetle Leptinotarsa decemlineata.

The immunosuppressive and insecticidal activity of culture of the entomopathogenic fungus Cordyceps militaris on the larvae of the Colorado potato beetle Leptinotarsa decemlineata has been established for the first time. It was found that the peroral effect of the fungal culture resulted in dose-dependent decrease in survival, delayed in development time and molting, decreases in the total hemocyt counts, increased activity of phenoloxidases in the hemolymph, and reduced activity of the enzyme in the cuticle, as well as increased sensitivity of larvae to the fungus Beauveria bassiana at the level of the synergistic effect.

Dominating species of entomophilous ascomycetes anamorphs in West Siberia, Primorsky krai, and Kyrgyzstan

The paper studies mycobiota of the dead insects in West Siberia, Primorsky krai, and Kyrgyzstan. Ascomycetes anamorphs of 13 genera are revealed. In all regions Beauveria bassiana (Bals.-Criv.) Vuill. dominated comprising on average 68% of the total number of isolates. The fungus hosts list the insects of 7 orders and 32 families with Coleoptera, Lepidoptera, and Hemiptera dominating. The rarely found entomopathogens include Tolypocladium inflatum Gams (primarily on Lepidoptera), Metarhizium anisopliae (Metschn.) Sorokin (on Coleoptera). The mortality rate of the insects due to micromycetes is observed mainly on enzootic level. The study of the pathogenic properties of the dominating species (B. bassiana) show the absence of specificity of its environmental isolates for a number of representatives of Orthoptera, Lepidoptera, Coleoptera, and Diptera.

Local epizootics caused by teleomorphic cordycipitoid fungi ( Ascomycota: Hypocreales ) in populations of forest lepidopterans and sawflies of the summer-autumn complex in Siberia

Unique epizootic loci were revealed in the summer-autumn complex of the multispecies communities of forest lepidopterans and sawflies in two regions of Novosibirsk oblast and in the Baikal region at the border of the Buryat Republic and Irkutsk oblast. Mass insect mortality was caused by two species of cordycipitoid fungi: Cordyceps militaris and Cordyceps sp. At least 30 species from 7 families of millers (Macroheterocera) and sawflies from the family Cimbicidae were found to be C. militaris hosts. Lepidopterans from the family Thyatiridae are the hosts of the second species, Cordyceps sp. Total mortality was noted for the condition close to optimal for pupation, i.e., in places of mass pupa accumulation. It may be assumed on the basis of the results of the laboratory experiments that, under natural conditions, host insects may be infected by ascospores and conidia at the anamorphic stage.

Comparative analysis of immune responses in Colorado potato beetle larvae during development of mycoses caused by Metarhizium robertsii, M. brunneum, and M. pemphigi

Development of mycoses and progress of humoral and cellular immune responses were compared in larvae of the Colorado potato beetle Leptinotarsa decemlineata infected with entomopathogenic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found to be highly susceptible to the strains of M. robertsii and M. brunneum but weakly responsive to M. pemphigi. The extent of susceptibility to the pathogens was not related to the stimulating effect of epicuticular extracts on fungal growth. Metarhizium pemphigi, which is non-specific to the Colorado potato beetle, did not cause any significant changes in the immune response and did not colonize the hemocoel. When infected with M. robertsii and M. brunneum, the larvae exhibited an increase in hemocyte count during the early stage of mycosis (day 2) followed by a drastic decrease on day 3. The immunocompetent cells, plasmatocytes and granulocytes, exhibited the greatest decrease. Elevated phenoloxidase activity was recorded in the hemolymph and cuticle on days 2 and 3 post-infection. These changes in the immune responses correlated with strain-specific virulence. Thus, the immune response in Colorado potato beetle larvae is an important factor, which determines differences in the development of mycoses caused by different Metarhizium species.

Phenotypic variability of environmental isolates of the entomopathogenic fungus Beauveria bassiana

A total of 35 isolates of Beauveria bassiana (Bals.) Vuill. isolates obtained from various insects of Novosibirsk oblast were investigated. The fungal morphotypes were found to include cultures of high, medium, and low virulence. Low correlation (r < 0.48) was observed between virulence and the morphophysiological characteristics of the isolates (lipase and protease activity, biomass, radial growth rate, conidia productivity, and relief). Isolates exhibiting high virulence to insects of a certain order proved to be virulent to the insects of other orders. A high correlation (r > 0. 74) was revealed between the virulence of the isolates to the potato beetle Leptinotarsa decemlineata Say and the locusts Calliptamus barbarus Costa and Locusta migratoria L. Isolates obtained from insects of the same species in the same site may differ significantly in virulence.

Dynamics of mortality of the migratory locust under synchronous infection with entomopathogenic fungi (Beauveria bassiana, Metarhizium anisopliae) and bacteria Pseudomonas sp.

As a result of search for species and strains of entomopathogenic fungi and bacteria virulent to migratory locust (Locusta migratoria migratoria L.), combinations were found which cause high mortality of insect in a short time interval. Four or five days after the L. migratoria had been infected with Beauveria bassiana (Bals.) Vuill and Metarhizium anisopliae (Metsch.) Sorokin a sharp increase in nymphas’ mortality was observed, reaching 95–100% on the 13th to 17th day after inoculation. The mortality of L. migratoria after infection with Pseudomonas sp. bacteria was approximately 30–50% on the 3rd to 7th day of the experiment. Later deaths of the locusts were not observed. When we made synchronous inoculation with fungi and bacteria, the rate of nymphas’ mortality was higher in comparison with monoinfections, and LT50 was about three days. Microbiological analysis of the dead insects showed that both pathogens could coexist in the locust. To determine the antagonism between Pseudomonas and fungi on a synthetic nutrient medium, the blocking method was used. We showed that the fungi do not affect the development of the bacterium, and the Pseudomonas has an insignificant effect on the fungi growth.