Molecular typing of native <i>Bacillus thuringiensis</i> isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Abstract

IntroductionBacillus thuringiensis is a rod-shaped, gram-positive, endospore-forming bacterium. It is distinguished from three other closely related Bacillus spp., viz. B. cereus, B. anthracis and B. mycoides, because of its ability to synthesize delta endotoxins (Cry proteins) as protein inclusion crystals during sporulation (Hofte and Whiteley, 1989). The presence of a parasporal crystal, which is outside the exosporium of the endospore, is indicative of production of the toxin, and serves as a marker for this species. The bacterium was initially discovered as a pathogen of various insects and was fi rst used as an insecticidal agent. It is found in soil where it leads a saprophytic existence, but becomes an opportunistic pathogen of insects when ingested. The delta-endotoxin causes midgut paralysis and disruption when ingested by the insect host. The specifi c activities of the toxin towards insects and nontoxic nature toward animals have made this organism a useful biocontrol agent. The insecticidal crystal (Cry) J. Gen. Appl. Microbiol., 58, 83 94 (2012) Bacillus thuringiensis is a bacterium of great agronomic and scientifi c interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specifi city. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specifi c origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.

Molecular characterization of Bacillus thuringiensis strains to control Spodoptera eridania (Cramer) (Lepidoptera: Noctuidae) population

“…As a consequence, the correlation between the collection places and toxicity was influenced by this genetic divergence for the last group. Katara et al (2012) report that despite REP-PCR and ERIC-PCR did not generate similar fragment patterns among 113 strains found at the same location in India, such techniques may still be useful for distinguishing B. thuringiensis strains. A high genetic diversity was also observed among B. thuringiensis isolates present in different regions of Mexico, in which 39 fingerprints were identified in 40 B. thuringiensis isolates using ERIC-PCR (García et al, 2015).…”

“…Just a few papers have been published using REP-PCR to study the genetic diversity of B. thuringiensis isolates. Usually, REP and ERIC sequences are used for genetic diversity analysis of many organisms including bacteria (Ahmadi et al, 2018;Katara et al, 2012;Mishra et al, 2017), fish (Fernández-Álvarez et al, 2018), and plants (Rampadarath et al, 2015). REP and ERIC primers have been useful in distinguishing B. thuringiensis isolates found in different locations (Katara et al, 2012) and roots of various legumes (Mishra et al, 2017).…”

“…Usually, REP and ERIC sequences are used for genetic diversity analysis of many organisms including bacteria (Ahmadi et al, 2018;Katara et al, 2012;Mishra et al, 2017), fish (Fernández-Álvarez et al, 2018), and plants (Rampadarath et al, 2015). REP and ERIC primers have been useful in distinguishing B. thuringiensis isolates found in different locations (Katara et al, 2012) and roots of various legumes (Mishra et al, 2017). Based on the similarity for individuals distributed in the same group, it was possible to see a small correlation between B. thuringiensis strain-induced mortality and collection place, even regarding some strains originally isolated at a particular region being clustered together after the genetic diversity analysis (Figure 3).…”

Activity of vegetative insecticidal proteins Vip3Aa58 and Vip3Aa59 of Bacillus thuringiensis against lepidopteran pests

“…The ERIC-PCR method was used as described previously by Katara et al (2012). PCR products were separated by electrophoresis through 1.5% agarose gel (Sigma) in Tris-borate-EDTA buffer (0.089 M Tris, 0.089 M, H 3 BO 3 , 0.002 M EDTA).…”

Short Communication Operational taxonomic units (OTUs) of endophytic bacteria isolated from banana cultivars in the Amazon

“…Similarly, ERIC-PCR has shown great applicability in the diversity analysis of microorganisms. For example, Katara et al (2012) conducted a molecular typing analysis of Bacillus thuringiensis isolates obtained from diverse habitats in India using repetitive extragenic palindromic PCR (REP-PCR) and ERIC-PCR, and identified a sufficient number of patterns to discriminate among the various isolated B. thuringiensis strains.…”