Antibiotic gene specific characterization and ARDRA analysis of native isolates of Pseudomonas spp. from Jammu, India

Abstract

The genus Pseudomonas is well known biocontrol agent established for its diverse role and antagonistic properties against phytopathogens due its ability of producing antibiotics. The studies were conducted to characterize Pseudomonas spp. for antibiotic production on the basis of gene specific primer. 150 isolates of Pseudomonas spp. were isolated from different agricultural fields of Jammu on King’s B medium using plate dilution method. Out of the total, thirty isolates were selected. The antibiotic i.e. 2,4-Diacetylphloroglucinol (2,4-DAPG) extracted from the various isolates showed spots having Rf values in the range of 0.7–0.89 which is very close to the Rf value of synthetic phloroglucinol (0.89). 2,4-DAPG positive isolates were tested against Fusarium oxysporum using dual culture assay and they were found to inhibit the mycelial growth of F. oxysporum by 52.76–65.45%. PCR analysis with 2,4-DAPG gene (phlD) specific primer indicated that a DNA fragment of approximately 750 bp in size was obtained in all the strains except PS 3 and 5 which is closely corroborated with the already reported size of 2,4-DAPG gene in literature. This confirms that 28 isolates out of 30 isolates have the genetic ability for the production of 2,4-DAPG antibiotic and thus these isolates can be promoted as potential biocontrol agents. The genetic diversity of anti-fungal producing rhizobacteria Pseudomonas spp. was investigated using amplified 16S rDNA restriction analysis. 16S rDNA was amplified using PCR technique and digestion with restriction endonuclease HaeIII and dendrogram analysis was carried out by the SHAN clustering UPGMA method (NTSYS 2.1). ARDRA analysis revealed the variability of the Pseudomonas spp. based on the restriction sites and the amplified 16S rDNA distinguished into 18 clusters/types including twelve minor groups and six major groups.