Screening of new isolates of Bacillus thuringiensis for cry1 genes and testing of toxicity against Dichocrocis punctiferalis (Family: Pyralidae, Order: Lepidoptera)

Resumo

In order to identify the more toxic novel cry gene, the cry1 gene was screened in six indigenous isolates of Bt by PCR with degenerate primers showed amplification in all the Bt isolates. Subsequent screening of cry1 subfamily gene(s) by gene specific primer showed amplification of cry1A gene in the five Bt isolates, three out of the six cry1 positive isolates showed the presence of cry1Aa gene. One of the six Bt isolates showed the presence of cry1Ab gene. Five Bt isolates showed amplification for cry1Ac gene and a variation in size of amplification was observed in one of the Bt isolates Bt, T27. Further, SDS-PAGE analysis of a spore crystal mixture isolated from new isolates of Bt, T27 showed a single band of ~135 kDa indicating presence of cry1Ac gene. The toxicity analysis of Bt strain T27 against Dichocrocis punctiferalis showed 100 per cent mortality on the fifth day after treatment. The varied ~925 bp amplicon of cry1Ac gene of Bt, T27 was amplified and cloned in a T/A vector. Comparison of nucleotide sequence data generated from the cry1Ac (~925 bp) gene showed 99 percent homology and two amino acid variation when comparison with its holotype sequence of Cry1Ac1.