Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley

A L Reyaz,L Gunapriya,P Indra Arulselvi

doi:10.1007/s13205-017-0756-z

Abstract

Bacillus thuringiensis (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, new indigenous B. thuringiensis strains were isolated and investigated so that these could be used as an alternative and/or support the current commercial strains/cry proteins in use. For this, 159 samples including soil, leaf and spider webs were collected from ten districts of Kashmir valley (India). Of 1447 bacterial strains screened, 68 Bt strains were identified with 4 types of crystalline inclusions. Crystal morphology ranking among the Bt strains was spherical (69.11%) > spore attached (8.82%) > rod (5.88%) = bipyramidal (5.88%) > spherical plus rod (4.41%) > spherical plus bipyramidal (2.94%) = cuboidal (2.94%). SDS-PAGE investigation of the spore–crystal mixture demonstrated Bt strains contained proteins of various molecular weights ranging from 150 to 28 kDa. Insecticidal activity of the 68 indigenous Bt strains against Spodoptera litura neonates showed that Bt strain SWK1 strain had the highest mortality. Lepidopteron active genes (cry1, cry2Ab, cry2Ab) were present in six Bt strains. Further, analysis of a full-length cry2A gene (~1.9 kb) by PCR–RFLP in strain SWK1 revealed that it was a new cry2A gene in Bt strain SWK1 and was named as cry2Al1 (GenBank Accession No. KJ149819.1) using the Bt toxin nomenclature (http://www.btnomenclature.info). Insect bioassays with neonate larvae of S. litura and H. armigera showed that the purified Cry2Al1 is toxic to S. litura with LC50 2.448 µg/ml and H. armigera with LC50 3.374 µg/ml, respectively. However, it did not produce any mortality in third instar larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi larvae/pupae insects (100 µg/ml) at 28 ± 2 °C and 75 to 85% relative humidity under a photoperiod of 14L:10D.

Highlights

Bacillus thuringiensis is ubiquitous, Gram-positive, sporeforming bacterium that is characterized by the production of insecticidal crystal proteins known as d-endotoxin (Shishir et al 2014)
A modified diet overlay assay method was employed to test the susceptibility of Spodoptera litura neonates to indigenous Bacillus thuringiensis (Bt) strains
Bt strains were isolated from soil, leaf and spider web samples collected at Kashmir valley situated in the northwestern tip of the Himalayan biodiversity hotspot

Summary

Introduction

Gram-positive, sporeforming bacterium that is characterized by the production of insecticidal crystal proteins known as d-endotoxin (Shishir et al 2014). These have a great potential to control a number of insect pests belonging to Lepidoptera, Diptera and Coleoptera and are benign to the environment (Schnepf et al 1998; Zhong et al 2000; Vidyarthi et al 2002; Valicente et al 2010). Several reports of Bt and its toxicity against various insect pests have been documented (Ozturk et al 2008; de Escudero et al 2014; Neethu et al 2015). Commercial Bt-based bioinsecticides used worldwide are applied at 10–50 g/acre or about 1020 molecules/acre, while chemical pesticides such as organophosphates and pyrethroids are applied about 8 9 1024 and 3 9 1022 molecules/acre, 143 Page 2 of 11

Methods

Results

Conclusion