Molecular characterization of mosquitocidal Bacillus sphaericus isolated from Tamil Nadu, India

• Published in: Acta tropica Vol. 127; no. 3; pp. 158 - 164
• Main Authors: Immanual Gilwax Prabhu, D., Gowri Sankar, S., Thirumalai Vasan, P., Sobana Piriya, P., Karpanai Selvan, B., John Vennison, S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2013
• Subjects: Animals; Anopheles stephensi; Bacillus - classification; Bacillus - isolation & purification; Bacillus - metabolism; Bacillus sphaericus; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Binary toxin; bioassays; Culex - microbiology; Culex quinquefasciatus; Dendrogram; Gene Expression Regulation, Bacterial; genes; genetic heterogeneity; India; insect larvae; Larva - microbiology; Mosquito Control - methods; Mosquitocidal toxin; Multiplex PCR; Pest Control, Biological - methods; Phylogeny; polymerase chain reaction; Polymerase Chain Reaction - methods; REP-PCR; screening; soil; toxicity; India; India, Tamil Nadu; Multiplex PCR; Binary toxin; Mosquitocidal toxin; Dendrogram; REP-PCR; Bacillus sphaericus
• ISSN: 0001-706X; 1873-6254
• DOI: 10.1016/j.actatropica.2013.04.013

Forty-two Bacillus sphaericus strains were isolated from soil around Tamil Nadu, India. Phylogenetic relationship and presence of mosquito larvicidal genes in the isolates were analysed by REP-PCR and multiplex PCR respectively. Three isolates showed high level of toxicity and they were grouped distinctly from other B. sphaericus isolates by dendrogram generated from REP-PCR. The mosquito larvicidal activity was mainly attributed by BinA and BinB were observed. Among Mtx toxins, Mtx1 plays a vital role in toxicity was elucidated. Both REP-PCR and multiplex PCR could be developed in screening of novel highly toxic B. sphaericus strains from environment without bioassay. •Forty-two B. sphaericus strains were isolated from Tamil Nadu, India.•Phylogenetic relationship of B. sphaericus isolates was analysed through REP-PCR.•Mosquito larvicidal genes of B. sphaericus isolates were detected by multiplex PCR.•Three isolates showed high level of toxicity and they were grouped distinctly from other B. sphaericus isolates by dendrogram generated from REP-PCR.•The mosquito larvicidal activity was mainly attributed by BinA, BinB and Mtx1 toxins. Forty-two Bacillus sphaericus strains were isolated from soil around Tamil Nadu, India. The phylogenetic relationship among the B. sphaericus isolates was analysed by REP-PCR and multiplex PCR was performed for the detection of mosquito larvicidal genes binA, binB, mtx1, mtx2 and mtx3 in B. sphaericus isolates. According to the REP-PCR band pattern, B. sphaericus isolates were divided into group A comprising I–XI clusters and group B comprising cluster XII. Three of the isolates BSTN01, 23 and 24 were gathered under cluster XII showed a high level of larvicidal activity against Culex quinquefasciatus and Anopheles stephensi, the other 39 isolates grouped under I–XI clusters were non-toxic or weak or moderately toxic to mosquito larvae. Even though BSTN23 and 24 were isolated from the same location and both contained all the five mosquito larvicidal genes, their intraspecies difference was clearly elucidated by REP-PCR analysis. Among high toxic isolates, BSTN23 and 24 were observed to contain all the five toxin genes and BSTN01 showed the presence of binary toxin and Mtx1 toxin genes. The isolates BSTN02, 03, 07, 14, 16, 19, 20, 21, 25, 31, 36 and 39 were found to contain mtx1 gene with combination of mtx2 and/or mtx3 showed moderate or low toxicity against mosquito larvae. binA, binB and mtx1 genes were not present in non-toxic isolates. The present study revealed the genetic heterogeneity between both toxic and non-toxic isolates and indicates that there is a good correlation between the presence of toxin genes and toxicity of the strains. These techniques could be developed in screening of novel highly toxic B. sphaericus strains from environment without bioassay on mosquito larvae.