Suppression of Tomato Bacterial Wilt Incited by Ralstonia pseudosolanacearum Using Polyketide Antibiotic-Producing Bacillus spp. Isolated from Rhizospheric Soil

Abstract

Bacillus spp. has the potential to control bacterial and fungal diseases of crops. In vitro study, Bacillus amyloliquefaciens DSBA-11 showed best to inhibit the growth of Ralstonia pseudosolanacearum as compared to Bacillus cereus JHTBS-7, B. pumilus MTCC-7092, B. subtilis DTBS-5 and B. licheniformis DTBL-6.Three primers sets from nucleotide sequences of polyketide antibiotic synthase genes viz., macrolactin, difficidin and bacillaene of B. amyloliquefaciens FZB42 were designed and standardized protocol for simultaneous detection of polyketide antibiotics-producing strains of Bacillus spp. by multiplex—PCR with products size of 792 bp, 705 bp and 616 bp respectively. All the strains of B. amyloliquefaciens contained three polyketide antibiotic synthase genes, and B. subtilis possessed difficidin and macrolactin, whereas B. cereus JHTBS-7, B. pumilus MTCC-7092 and B. licheniformis DTBL-6 did not contain any polyketide antibiotic genes. By using this technique, polyketide-producing strains of Bacillus spp. were screened within a short period with high accuracy. These polyketide synthase genes were cloned by using a T&A vector to study the role of these genes in producing antibiotics that suppressed the growth of R. pseudosolanacearum under both in vitro and in vivo conditions. Bio-efficacy of cloned products of these genes macrolactin, bacillaene, and difficidin along with parent strain B. amyloliquefaciens DSBA-11 inhibited the growth of R. pseudosolanacearum and formed 1.9 cm2, 1.9 cm2, 1.7 cm2 and 3.3 cm2 inhibition area under in vitro conditions respectively. Minimum bacterial wilt disease intensity (29.3%) with the highest biocontrol efficacy (57.72%) was found in tomato cv. Pusa Ruby (susceptible to wilt disease) was treated with B. amyloliquefaciens DSBA-11 followed by cloned products of difficidin and macrolactin under glasshouse conditions. Hence, the developed multiplex protocol might be helpful for screening polyketide antibiotics producing potential strains of Bacillus spp. from soil which can apply for managing the wilt disease of tomatoes. The polyketide antibiotics produced by bacteria might have a significant role suppression of R. pseudosolanacearum due to the disintegration of cells.