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Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates

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Abstract

The Western Ghats of Karnataka natural ecosystem are among the most diverse and is one of the eight hottest hotspots of biological diversity in the world, that runs along the western part of India through four states including Karnataka. Bacillus thuringiensis (Bt) strains were isolated from soils of Western Ghats of Karnataka and characterized by molecular and analytical methods as a result of which 28 new Bt-like isolates were identified. Bt strains were isolated from soil samples using sodium acetate selection method. The morphology of crystals was studied using light and phase contrast microscopy. Isolates were further characterized for insecticidal cry gene by PCR, composition of toxins in bacterial crystals by SDS-PAGE cloning, sequencing and evaluation of toxicity was done. As a result 28 new Bt-like isolates were identified. Majority of the isolates showed the presence of a 55 kDa protein bands on SDS-PAGE while the rest showed 130, 73, 34, and 25 kDa bands. PCR analysis revealed predominance of Coleopteran-active cry genes in these isolates. The variations in the nucleotide sequences, crystal morphology, and mass of crystal protein(s) purified from the Bt isolates revealed genetic and molecular diversity. Three strains containing Coleopteran-active cry genes showed higher activity against larvae Myllocerus undecimpustulatus undatus Marshall (Coleoptera: Curculionidae) than B. thuringiensis subsp. Morrisoni. Results indicated that Bt isolates could be utilized for bioinsecticide production, aiming to reduce the use of chemical insecticide which could be useful to use in integrated pest management to control agriculturally important pests for sustainable crop production.

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Acknowledgments

The authors are grateful to ICAR, New Delhi for funding this study under Network project on Application of microbes in agriculture and allied sectors (AMAAS). This project is funded under ICAR network project on Application of microbes for agriculture and allied sector through National Bureau of Agriculturally Important Micro organisms (NBAIM). This is a part of doctoral degree research work. The authors are also grateful to Geetha G. Thimmegowda, Division of Entomology & Nematology, Indian Institute of Horticultural Research (IIHR), Hessarghatta lake post, Bangalore 560089, Karnataka, India for providing the Mango ash weevil test insects for bio assay studies.

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Authors and Affiliations

  1. Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hessarghatta Lake Post, Bangalore, 560089, Karnataka, India

    H. M. Mahadeva Swamy, R. Asokan & S. N. Nagesha

  2. Post-Graduate Department of Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Jnanasahayadri, Shankaraghatta, Shimoga, 577451, Karnataka, India

    Riaz Mahmood

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  1. H. M. Mahadeva Swamy
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Correspondence to H. M. Mahadeva Swamy.

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Mahadeva Swamy, H.M., Asokan, R., Mahmood, R. et al. Molecular Characterization and Genetic Diversity of Insecticidal Crystal Protein Genes in Native Bacillus thuringiensis Isolates. Curr Microbiol 66, 323–330 (2013). https://doi.org/10.1007/s00284-012-0273-6

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