Identification And Antimicrobial Mechanism Of Plant Growth-Promoting Rhizobacteria Lyc2 And XW10

Pathogenic microorganisms affecting plant health are a major and chronic threat to food production and ecosystem stability worldwide. Biocontrol is a kind of green and safe disease control strategy which can effectively reduce the losses caused by pests and diseases of crops and promote plant growth, improve plant disease resistance and increase crops yields. In this study, plant growth promoting rhizobacteria(PGPR) were screened using the indicator pathogens. To understand the antimicrobial mechanism of the PGPR strains, a random mutation library was constructed, and putative antimicrobial genes were amplified for function analysis. The main results were as followed:Identification the taxonomic status of strain Lyc2. Based on the characteristics of physiological and biochemical, together with phylogenetic analysis, bacterial strain Lyc2 was identified to be Burkholderia pyrrocinia.A draft genome sequence of strain Lyc2 was generated for mining the gene clusters encoding the biosynthetic pathways for the secondary metabolism. Fifteen biosynthetic gene clusters were predicted from the genome of Lyc2 which grouped into eight categories, including two aryl polyene clusters, five terpene clusters, one Phosphonate cluster, one Homoserine lactone cluster, two Nonribosomal peptide synthetase clusters, two bacteriocin or other unspecified ribosomally synthesised and post-translationally modified peptide product(RiPP) clusters, one nonribosomal peptide synthetase cluster- Type I PKS cluster and one cluster containing a secondary metabolite-related protein that does not fit into any other category.To identify the genes associated with antifungal activity, strain Lyc2 was randomly mutated using an EZ-Tn5 Transposome Kit. Sequence analysis revealed the presence a 55.2-kb gene cluster ocfABCDEFGHIJKLMN was responsible for the production of the antifungal compounds occidiofungin which was a cyclic peptide containing eight amino acid residues. Gene function analysis and complementary assay of the disrupted orfI gene which was predicted to encode a flavin-dependent monooxygenase responsible for biosynthesis of a novel amino acid indicated that the uncharacterized orfI gene was essential for the antifungal activity of strain Lyc2. Antibacterial analysis indicated that mutant was defective in antibacterial activity. Sequence alignment of mutant suggested that the disrupted gene was homology(93.3%) to the glutathione synthase gene Bamb-2918 of strain Burkholderia ambifaria AMMD. Gene function analysis and complementary assay of the disrupted gene which was predicted to encode glutathione synthase indicated that the gene was essential for the antibacterial activity of strain Lyc2.Strain XW10 was isolated from soybean root rhizosphere and showed a broad-spectrum of antimicrobial activity especially against Ralstonia solanacearum. Phylogenetic analysis revealed that strain XW10 was apparently difference from the previously reported Pseudomonas strains, which indicated that XW10 could be a new species of Pseudomonas.To characterize the genes dedicated to antibacterial activities against R. solanacearum, a Tn5-mutation library of strain XW10 was constructed. Genetic and sequencing analyses revealed that a homology of TatA gene in twin-arginine translocase(Tat) secretion system was disrupted by transposon in mutant XW10-5206 which was responsible for the antibacterial activity against R. solanacearum. Gene complementary assay of TatA gene restored the antibacterial activity of the mutant against R. solanacearum to wild-type level. Thus the Tat pathway-mediated translocation of the sec pathway is essential for antibacterial activity of P. beanensis XW10 against R. solanacearum.