Molecular And Biochemical Detection Of Antibiotics Produced By The Rhizobacteria Antagonistic Against Phytophthora Parasitica Var.nicotianae

Adopted rhizosphere soil samples of tobacco from Gui Zhou, screened22strainsantagonistic against Phytophthora parasitica var. nicotianae strongly and stably, carried outphysio-biochemical tests, extraction of total DNA, the amplification of16S rRNA gene andthe Blast alignment to completed determining the genera,14Bacillus sp. were obtained.The22antagonistic bacterial strains demonstrating effective inhibition againstPhytophthora parasitica var. nicotianae, were examined for the presence of antibioticbiosynthetic genes. Primers specific for iturin A, bacillomycin D, fengycin, surfactin wereused to amplify biosynthetic genes from these bacteria using PCR.9strains of the22antagonistic bacterial strains harbored iturin A biosynthetic genes,5strains harboredbacillomycin D biosynthetic genes,13strains harbored surfactin biosynthetic genes, and nonestrain of them harbored fengycin biosynthetic genes. Among14Bacillus sp. strains,6strains,3strains and11strains showed the presence of the iturin A, bacillomycin D and surfactinbiosynthetic gene and surfactin biosynthetic gene respectively. ZAH3was positive for iturin Aand surfactin, WCH1was positive for bacillomycin D, MTH23was positive for surfactin,RHH48was positive for bacillomycin D, WCH21showed the presence of the iturin Abiosynthetic gene, YQH34and WCH18showed the presence of the iturin A biosynthetic geneand surfactin biosynthetic gene respectively.Production of particular antibiotics in strains ZAH3, WCH1, MTH23, RHH48, WCH21,YQH34, WCH18was confirmed through matrix-assisted laser desorption ionization-time offlight-mass spectroscopy (MALDI-TOF-MS). Surfactin was detected from ZAH3bacterialcell-surface extracts, bacillomycin D was detected from WCH1, surfactin was detected fromMTH23, bacillomycin D and fengycin were detected from RHH48, iturin A and fengycinwere confirmedfrom WCH21, surfactin was confirmed from YQH34, surfactin and fengycin were confirmed from WCH18.MALDI-TOF-MS results differed form PCR amplification results to some extent. IturinA was not detected through MALDI-TOF-MS from ZAH3after confirmation of iturin Abiosynthetic genes. Fengycin was detected through MALDI-TOF-MS from RHH48、WCH21、WCH18although they did not harbore fengycin biosynthetic genes.Differences existed in the types of antibiotics dominating in the mass spectra of differentstrains. This indicates that, although these strains have multiple biosynthetic genes, only1or2antibiotics are produced at high concentrations at a certain time, and the major antibiotic inaction under a given set of conditions may vary from strain to strain. The results of this studysuggest that the major antibiotic produced by a particular strain might be induced by thegenetic makeup of the bacterium and by the environmental signals and affected.To the best of our knowledge, this is the first report of a Bacillus altitudinis,Agrobacterium tumefaciens, Delftia tsuruhatensis, Kocuria palustris, Bacillus flexusproducing antibiotics.The synergistic effect of several mechanisms along with antibiotics exists in biocontrol.However, at a certain time and under certain environmental conditions, antibiotic productionmay play the main role in its biocontrol of plant pathogens. Understanding the mechanisms ofbiocontrol at the molecular and biochemical level will contribute to developing rationalstrategies for the application of the antagonists and their metabolites within an agroecosystem.