Identification Of Endophtic Bacterium Strain G5 And Role Of Regulation System In Its Biocontrol Activities

We have isolated a new strain of endophytic Pseudomonas G5 from the stems of Chinese parsley (Coriandrum sativum L.), and it is tentatively identified as Pseudomonas aurantiaca according to analysis of the entire substrate utilization profiles using BIOLOG MicrostationTM system (BIOLOG, Inc, Hayward CA.). The strain G5 produced several antifungal compounds, such as antibiotic phenazine-1-carboxylic acid (PCA), phenazine-1- carboxamide (PCN), hydrogen cyanide (HCN), siderophore(s) and extracelluar proteinase, and showed inhibitory to several pathogens in the dual culture. We have investigated of Indole-3-Acetic Acid (IAA) of strain G5 by HPLC. In greenhouse, strain G5 showed high efficiency to control plant diseases caused by Rhizoctomia solani and Botrytis cinerea.In this study, we have showed that Pseudomonas sp. strain G5 can produce several types of AHLs at a detectable level using Thin Layer Chromatography (TLC) analysis combined with bioreporter Chromobacterium violaceum CV026 bioassay, and N-hexanoyl-homoserine lactone (HHL, C6-HSL) with Rf value 0.4 is the major signal molecules. Furthermore, we have identified its quorum sensing system composed of PhzI and PhzR by cloning and sequencing of phzI-phzR. PhzI is responsible for synthesis of AHLs signal molecules, and PhzR is a transcriptional regulator. Finally, we heterologously expressed the recombinant plasmid pMD-phzIR in E. coli JM109 and verified it using C. violaceum CV026 bioassay. The phylogenetic analysis using MEGA4 reveals highly similarities exist among the phzIR homologs, suggesting it is evolutionary well conserved in the genus Pseudomonas.Using miniTn5 mutagenesis, AHL-deficient mutant (G5-6) have been screened on selective LA supplemented with rifampicin and kanamycin plates and tested for their ability to produce AHLs in an on-plate assay using Chromobacterium violaceum CV026 as the reporter. All derivative strains have been verified by PCR and gene mapping. The data suggests that gacA gene not only affected production of antifungal metabolites, such as HCN, siderophores and extracelluar proteinase, but significantly affected biofilm formation, colonization on bean rhizosphere and IAA. Furthermore, it influences biocontrol ability to phytopathogenic fungi in vitro and in vivo in greenhose. These date suggested gacA gene system was one important regulatory system pertinent to biocontrol ability in strain G5.