Characterization of native Bacillus thuringiensis strains by PCR-RAPD based fingerprinting

Kumar, Dharmender; Chaudhary, K.; Boora, K. S.

doi:10.1007/s12088-009-0011-3

Cited by 7 publications

(7 citation statements)

References 15 publications

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“…Vilas-Boas and Lemos (2004) also observed remarkable genetic diversity among 218 Bt isolates from Brazil, suggesing that the diversity resulted from the infl uence of different ecological factors and spatial separation between strains generated by conquest of different habitats. Kumar et al (2008) have also carried out RAPD-PCR of 70 Bt isolates collected from soil samples of a water fi eld and found that RAPD provides a high degree of discrimination.…”

Section: Discussionmentioning

confidence: 99%

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

Katara¹,

Deshmukh²,

Singh³

et al. 2012

J. Gen. Appl. Microbiol.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Katara¹,

Deshmukh²,

Singh³

et al. 2012

J. Gen. Appl. Microbiol.

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“…The results are consistent with the findings of Wei et al (2019) [ 16 ], showing the high specificity of XRE primers for identifying B. thuringiensis . Moreover, it has been well-documented that B. thuringiensis selectively produces insecticidal crystal proteins [ 16 , 23 ], which can be utilized as an appropriate marker for proper identification. Herein, we examined the presence of crystal proteins using crystal protein staining and observed the presence of these crystal proteins in isolate D-1 ( Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

Study on the Identification Methods for Effective Microorganisms in Commercially Available Organic Agriculture Materials

et al. 2020

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The identification of microorganisms in closely related groups is challenging. The present work focused on the different molecular methodology for the accurate microbial identification in the five commercially available organic agriculture materials enriched with effective microorganisms. From the tested five organic agricultural materials, a total of seven distinct bacterial colonies (A-1, B-1, C-1, D-1, E-1, E-2, and E-3) were isolated and processed for sequential identification utilizing HiCrome™ Bacillus agar, biochemical tests with API CHB50, 16S rRNA gene analysis, random amplified polymorphic DNA (RAPD), and species-specific PCR analysis. All the isolated microorganisms were Gram-positive rods and spore former belonging to Bacillus group and appeared as a differential characteristic feature on HiCrome™ Bacillus agar. All isolates showed high-percentage similarities with the different members of Bacillus species in biochemical testing and 16S rRNA gene analysis. The collective identification results revealed isolates, A-1, B-1, and C-1, close to B. velezensis. Further RAPD-PCR and species-specific PCR discriminated and provided confirmatory evidence for D-1 as B. thuringiensis and E-1, E-2, and E-3 as B. licheniformis, respectively. In addition, presence of B. thuringiensis was also confirmed by toxin crystal protein staining. In conclusion, the species-specific primers could be used as a rapid and accurate identification tool to discriminate closely related Bacillus species such as B. subtilis, B. licheniformis, and B. thuringiensis.

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“…PFGE is generally considered as an accurate and reproducible method for typing clinically relevant bacteria, and has been used for typing Bt strains [ 26 , 30 ]. Another typing method, RAPD, which is fast and simple, also has been applied for the discrimination of Bt strains [ 13 ]. Although RAPD has been successfully used for the differentiation of Bt isolates based on their location of origin, further investigation is still needed to understand their insecticidal spectrum [ 13 ].…”

Section: Discussionmentioning

confidence: 99%

“…Another typing method, RAPD, which is fast and simple, also has been applied for the discrimination of Bt strains [ 13 ]. Although RAPD has been successfully used for the differentiation of Bt isolates based on their location of origin, further investigation is still needed to understand their insecticidal spectrum [ 13 ]. For PFGE, it has been reported that the clonal structure of Bt isolates determined by PFGE may not correlate with their cry gene content [ 10 ], however, different conclusions have been made by Gaviria Rivera and Priest [ 26 ].…”

Section: Discussionmentioning

confidence: 99%

“…In this study, 60 Bt strains isolated from different origins including soil and granaries in Taiwan were characterized for their toxicity against P. xylostella . The genotypes of these strains were investigated by pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD), which have been proven to provide a high degree of discrimination for Bt strains [ 10 , 13 ]. Since the information on insecticidal activities of individual crystal proteins against selected arthropod has been reviewed by Frankenhuyzen [ 14 ], a collection of cry genes, including those coding for proteins biologically active to P. xylostella , were assayed based on polymerase chain reaction (PCR) for these strains.…”

Section: Introductionmentioning

confidence: 99%

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The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

Chen

Pang

et al. 2014

Toxins

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The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

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Characterization of native Bacillus thuringiensis strains by PCR-RAPD based fingerprinting

Cited by 7 publications

References 15 publications

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

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

Study on the Identification Methods for Effective Microorganisms in Commercially Available Organic Agriculture Materials

The Correlation of the Presence and Expression Levels of cry Genes with the Insecticidal Activities against Plutella xylostella for Bacillus thuringiensis Strains

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