Molecular and insecticidal characterization of Vip3A protein producingBacillus thuringiensisstrains toxic againstHelicoverpa armigera(Lepidoptera: Noctuidae)

Abstract

Vegetative insecticidal proteins (Vip) represent the second generation of insecticidal proteins produced by Bacillus thuringiensis (Bt) during the vegetative growth stage of growth. Bt-based biopesticides are recognized as viable alternatives to chemical insecticides; the latter cause environmental pollution and lead to the emergence of pest resistance. To perform a systematic study of vip genes encoding toxic proteins, a total of 30 soil samples were collected from diverse locations of Kashmir valley, India, and characterized by molecular and analytical methods. Eighty-six colonies showing Bacillus-like morphology were selected. Scanning electron microscopy observations confirmed the presence of different crystal shapes, and PCR analysis of insecticidal genes revealed a predominance of the lepidopteran-specific vip3 (43.18%) gene followed by coleopteran-specific vip1 (22.72%) and vip2 (15.90%) genes in the isolates tested. Multi-alignment of the deduced amino acid sequences revealed that vip3 sequences were highly conserved, whereas vip1 and vip2 showed adequate differences in amino acid sequences compared with already reported sequences. Screening for toxicity against Helicoverpa armigera larvae was performed using partially purified soluble fractions containing Vip3A protein. The mortality levels observed ranged between 70% and 96.6% in the isolates. The LC50 values of 2 of the native isolates, JK37 and JK88, against H. armigera were found to be on par with that of Bt subsp. kurstaki HD1, suggesting that these isolates could be developed as effective biopesticides against H. armigera.

Insecticidal Activity and Histopathological Effects of Vip3Aa Protein from Bacillus thuringiensis on Spodoptera litura

“…Since the first vip gene was reported [17], a considerable number of new vip genes had been screened [3,6,23,28,30,31,35,42]. So far, more than 100 Vips have been recognized and classified into four groups, Vip1, Vip2, Vip3, and Vip4, based on the amino acid sequence similarity (http://www.…”

Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

“…The estimation of the success of Bt isolation ( Bt index) varied among the soil samples and the average Bt index observed in the study was 0.51. The average Bt index varies between soil samples all over the world as reported previously [25,21,33,34]. The likely reason for difference in Bt index is hard to access, however, the difference in geomorphology and the interaction of the bacterium with their insect hosts could influence their numbers in different environments.…”

“…It was observed that the obtained sequences showed 95–100% identity with the reported sequences on BLAST analysis (Table 2). The partial 16S rRNA gene sequences were submitted to GenBank and accession numbers were obtained [21].

Fig. 1Scanning electron micrographs of B. thuringiensis cells showing different stages of growth.

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“…A total of 86 isolates were analysed in this study which were obtained from various soil samples, collected from forests, lake sediments and agricultural fields of Kashmir valley in our previous study [21]. Sites with no history of Bt application were selected (Table 1).…”

“…The different stages of bacterial growth as well as the crystal production was analysed by Scanning Electron Microscopy (SEM) as described previously [21]. Bt index was calculated as described by Baig et al [25].…”