Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532

Abstract

Bacillus thuringiensis Berliner (Bt) being an eco-friendly bioinsecticide is effectively used in pest management strategies and, therefore, isolation and identification of new strains effective against a broad range of target pests is important. In the present study, four indigenous Bt isolates (T489, T491, T495 and T532) were taken and investigated for their properties such as crystal morphology, cry gene(s) content and insecticidal activity. Bipyramidal, cuboidal and spherical crystals were observed. Among the four isolates only one isolate was found positive for lepidopteran specific cry genes. The isolate T532 which showed the presence of cry1, cry2Aa and cry2Ab genes was selected for protein profiling and bioassay. The isolate showed the presence of two proteins of molecular weights at ~135 and ~65 kDa sizes. Artificial diet based bioassay resulted in 100% mortality of the neonate larvae of H. armigera and S. litura for the isolate T532 which was comparable to that of the positive control (HD1). The full length cry2A gene was amplified and cloned into E. coli DH5α strain. The recombinant plasmids were sequenced and the sequence was deposited in GenBank nucleotide database. The accession number of the gene is MH475905. The gene was named by the Bacillus thuringiensis delta endotoxin nomenclature committee as cry2Aa23 .