Molecular characterization of indigenous Bacillus thuringiensis strains isolated from Kashmir valley

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 LC 50 2.448 lg/ml and H. armigera with LC 50 3.374 lg/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 lg/ml) at 28 ± 2°C and 75 to 85% relative humidity under a photoperiod of 14L:10D.

Characterization ofBacillus thuringiensisBerl. indigenous from soil and its potency as biological agents ofSpodoptera litura(Lepidoptera: Noctuidae)

“…This number of protein was similar to the result reported by Konecka et al [5]. They found crystals contained 1-8 proteins with molecular weight of 36-155 kDa isolated during epizootics in Cydia pomonella L. Reyaz et al [6] found by SDS-PAGE investigation of the spore-crystal mixture demonstrated Bt strains contained proteins of various molecular weights ranging from 150 to 28 kDa. Two protein bands at 130 kDa and 65 kDa were detected with SDS-PAGE analysis from B. thuringiensis isolated from environments of boron [15].…”

“…Ninety percents of mortality in third instar larvae of diamond backmoth Plutella xylostella larvae treated for 72 hours was caused by a protein content of 11.1 g / l [16]. Research on insect bioassays with first larvae of S. litura showed that the purified Cry2Al1 was toxic to S. litura with LC 50 2.448 mg / ml [6]. Shah et al found that some isolates with cry protein range 30-150 kDa were toxic against Amsacta albistriga (Lepidoptera:Arctiidae) [17].Therefore, it was no doubt that B. thuringiensis isolates indigenous South Sumatera were toxic to diamond backmoth P. xylostella and armyworm S. litura.…”

“…Recent study showed new indigenous B. thuringiensis strains were isolated and examined so that these alternatives were used to support the current commercial strains, such as B. thurigiensis subsp. Aizawai / cry proteins in use [6]. Research on B. thuringiensis bacterial isolate from South Sumatera has been started since 15 years ago [7].…”

Molecular characterization of an indigenous lepidopteran toxic Bacillus thuringiensis strain T532

“…Bacillus thuringiensis is a ubiquitous bacterium found in various habitats such as soil, phylloplane, dead insects, dust, spider web etc. (Mizuki et al, 1999;Asokan and Puttaswamy, 2007;Ramalakshmi and Udayasuriyan, 2010;Unalmis et al, 2015 andReyaz et al, 2017). In the present study, the isolates preserved in glycerol stocks were revived and cultured in T 3 medium.…”

“…Only bipyramidal crystals were observed in T491 and T495 whereas cuboidal crystals were observed in T489. Jain et al (2017) and Reyaz et al (2017) reported the presence of various kinds of crystals such as spherical, bipyramidal, cuboidal and irregular shapes. Earlier the predominance of bipyramidal crystals was also reported by Bernhard et al (1997), Martin and Travers (1989) and Ramalakshmi and Udayasuriyan (2010).…”