MOLECULAR AND INSECTICIDAL CHARACTERIZATION OF NOVEL CRY TOXINS FROM Bacillus thuringiensis (BERLINER) AGAINST THE FALL ARMY WORM (Spodoptera frugiperda) AND BRINJAL ASH WEEVIL (Myllocerus subfasciatus)
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Date
2021-12-31
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University of Agricultural Sciences, Bangalore
Abstract
Twelve Bt strains were isolated from root nodules using six approaches, viz.,analysis of crystal protein production by microscopy; detection of cry gene content by PCR, Plasmid profiling, SDS-PAGE profiling; cloning and sequencing, phylogenetic analysis; and toxicity testing. Under a light microscope, all 12 Bt strains examined were gram-positive, endospore-forming, and had normal Bt crystal structures. A bipyramidal inclusion was predominant in 42.2 %. Five distinct plasmids were observed in the present
study. Universal primers and gene specific primers were used to detect Cry-type genes by the PCR. The nucleotide sequences of amplified new cry genes were BLASTed against NCBI database sequences, and they were labeled as native Bt strains with high homology (60–100%) to existing Bt strains. The nucleotide sequences of new Bt cry genes were deposited in the NCBI GenBank database. Three new cry1, cry3A and cry26 complete crygenes were isolated. Cry genes were cloned and expressed in the pRSET vector. The inferred 3-D structural model of the novel cry gene, which was predicted using Phyre2 homology modeling, demonstrates that the gene has three domains that participate in the development of a pore and define the receptor's binding selectivity. The new Bt strains were highly pathogenic, with pathogenicity ranging from 93 to 100 percent against Spodoptera frugiperda larvae and Myllocerus subfasciatus adults. In conclusion, native Bt strains from root nodules were shown to have bio-insecticidal activity on larvae of S. frugiperda and adults of M. subfasciatus