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. 2017 Dec;6(6):e00484.
doi: 10.1002/mbo3.484. Epub 2017 Oct 18.

Selection and characterization of Bacillus thuringiensis strains from northwestern Himalayas toxic against Helicoverpa armigera

Showkat A Lone  1   2 , Abdul Malik  1 , Jasdeep C Padaria  2
Affiliations

Selection and characterization of Bacillus thuringiensis strains from northwestern Himalayas toxic against Helicoverpa armigera

Showkat A Lone et al. Microbiologyopen. 2017 Dec.

Abstract

In this study, we present the selection and the characterization of Bacillus thuringiensis (Bt) strains with respect to their cry/cyt gene content and toxicity evaluation toward one of the most important polyphagous lepidopteran pest, Helicoverpa armigera. Fifty-six Bt isolates were obtained from 10 different regions of northwestern Himalayas, recording a total B. thuringiensis index of 0.62. Scanning electron microscopy revealed presence of bipyramidal, spherical, flat and irregular crystal shapes; SDS-PAGE analysis of spore-crystal mixtures showed the prominence of 130, 70, and 100 kDa protein bands in majority of the isolates; PCR analysis with primers for eight cry and cyt gene families and 13 cry gene subfamilies resulted in isolates showing different combinations of insecticidal genes. Strains containing cry1 were the most abundant (57.1%) followed by cyt2 (46.42%), cry11 (37.5%), cry2 (28.57%), cry4 (21.42%), cyt1 (19.64%), cry3 (8.9%), and cry7, 8 (7.14%). A total of 30.35% of the strains did not amplify with any of the primers used in this study. Median lethal concentration 50 (LC50 ) estimates of spore-crystal mixtures of Bt-JK12, 17, 22, 48, and 72 against second instar larvae of H. armigera was observed to be 184.62, 275.39, 256.29, 259.93 μg ml-1 , respectively. B. thuringiensis presents great diversity with respect to the presence of crystal protein encoding genes and insecticidal activity. Four putative toxic isolates identified in this study have potential application in insect pest control. B. thuringiensis isolate JK12 exhibited higher toxicity against H. armigera than that of B. thuringiensis HD1, hence can be commercially exploited to control insect pest for sustainable crop production. The results of this study confirm the significance of continuous exploration of new Bt stains from different ecological regions of the world.

Keywords: Bacillus thuringiensis; Helicoverpa armigera; LC 50; PCR; Bioassay; Cry proteins.

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Figures

Figure 1
Figure 1
Scanning electron micrographs of B. thuringiensis cells showing presence of crystals, spores, and vegetative cells. (a) bipyramidal crystal (bc), (b) spherical crystal (sc), (c) cuboidal crystal (cc), (d) irregular crystal (ic). sp and vc indicate spores and vegetative cells, respectively
Figure 2
Figure 2
Occurrence rate of cry and cyt‐type gene combinations in the B. thuringiensis strains of western Himalayas. (a) Distribution of strains harboring single cry/cyt gene, (b) distribution of isolates harboring combination of two or more cry/cyt genes, (c) distribution of cry1 and cry2 subtype gene combinations among cry1 and cry2 harboring isolates
Figure 3
Figure 3
SDS‐PAGE of spore‐crystal mixture of B. thuringiensis strains showing diverse protein profiles. Lanes: M, prestained molecular mass marker (Thermo Scientific, USA); 1, 5, 8, no major proteins; 2, 3, 4, major protein of 110 kDa; 6, 7, 9, major proteins of 130 kDa and 65 kDa; 10, major protein of 70 kDa; 11, Bt kurstaki (4D1) showing major protein of 130 kDa
Figure 4
Figure 4
Mortality of H armigera larvae fed with diets containing highly concentrated spore‐crystal mixtures (1,000 μg ml−1) of different B. thuringiensis isolates
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