Molecular characterization and toxicity evaluation of indigenous Bacillus thuringiensis isolates against key lepidopteran insect pests

Background: The indiscriminate use of synthetic insecticides is not affordable and poses collateral damage to humans, non-target organisms, and environmental health. Bacillus thuringiensis (Bt) is a cosmopolitan, antagonistic soil bacterium employed as an alternative to chemical insecticides in pest management. In this study, genetic diversity and insecticidal activity of 50 indigenous Bt isolates were investigated in order to find the effective Bt isolates with a broad spectrum of insecticidal activity against lepidopteran insects. Results: Most of the Bt isolates investigated appeared as creamy white in colour and fried egg or mucoid-type colonies having a flat or raised elevation with entire or undulated margins. Out of 50 isolates investigated, crystal shapes varied from cuboidal (40.00%), spherical (36.00%), bipyramidal (30.00%), rectangular (10.00%), and minute crystal attached to the spores (28.00%). SDS-PAGE analysis revealed that the molecular weight of the crystal proteins of Bt isolates ranged from similar to 30 to similar to 200 kDa in size. Among the Bt isolates screened, similar to 135 kDa size representing Cry1 protein was observed in 12 isolates (24.00%) and 65 kDa representing Cry2 was observed in 14 isolates (28.00%). PCR analysis was performed for cry1, cry2, cry3, cry4, cry9, vip1, vip2, and vip3 genes, which revealed the presence of cry1 gene alone in 5 isolates, and cry2 alone in 7 isolates, whereas 7 isolates were positive for cry1, cry2, and vip3 genes together. None of the Bt isolates showed the presence of cry3, cry4, cry9, vip1, and vip2 genes. Toxicity of Bt isolates was tested against four species of lepidopteran larvae, viz. Plutella xylostella Linnaeus, Helicoverpa armigera Hubner, Spodoptera litura Fabricius, and S. frugiperda Smith. Among them, 6 isolates (T29, T30, T31, T357, T381, and T388) produced 100% larval mortality against all four species of insects. Conclusion: The present study showed the diversity of Bt isolates and confirmed the significance of relentless exploration of Bt isolates for novel genes. Further investigations need to be carried out to disclose the hidden potential of these toxic isolates.