Involvment Of GacS/GacA Two-Component Regulatory System And Quorum-Sensing System In Biocontrol Activity Of Pseudomonas Fluorescens 2P24

Bacterial strains 2P24 and CPF-10, two biocontrol agents isolated from wheat take-all decline soil, were classified into Pseudomonas fluorescens biovar I and biovarV, respectively, by analyzing the 16S rDNA sequences and detection of physiological and biochemical characteristics. Both 2P24 and CPF-10 produced several antifungal compounds, such as antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide (HCN), siderophore(s) and extracellular proteinase, and showed inhibitory to Ralstonia solanacearum, Rhizoctonia solani and other pathogens in the dual-culture test on plates. In greenhouse, strain 2P24 and CPF-10 showed high efficiency to control soil-borne disease such as tomato bacterial wilt and wheat take-all. For further understanding the biocontrol mechanism, the roles of 2,4-DAPG, quorum-sensing(QS) system and GacS/GacA two-component regulatory system in biocontrol activity were studied in P. fluorescens 2P24.The 2,4-DAPG in strain 2P24 was investigated as a functional agent in antibiosis and biocontrol activity using genetic methods. A 2,4-DAPG negative mutant, constructed through site-directed mutangensis, lost the ability to inhibit the growth of plant pathogens and control tomato bacterial wilt. A plasmid-bearing 2,4-DAPG biosynthetic locus could restore 2,4-DAPG production and antibiosis activity against the phytopathogens. Moreover, the efficacy of the complemented mutant for control of tomato bacterial wilt was also recovered to the level of the wild type. Our results indicate that the 2,4-DAPG was one of the key biocontrol factors in P. fluorescens 2P24 and played an important role in controlling tomato bacterial wilt.The pool and pcoR, luxl and luxR homologs which were two components of QS system, were cloned from genomic library of P. fluorescens 2P24 by heterologous expression in E.coli DH5 a . In this experiment, A. tumefaciens NTL4(pZLR4) was used as AHLs reporter strain. The pooI deleted mutant did not evidently affect production of antifungal metabolites, such as 2,4-DAPG, HCN and siderophores, but significantly affected biofilm formation, colonization on wheat rhizosphere and adaption of environment. Furthermore, it affected biocontrol ability of P. fluorescens 2P24 in greenhouse. These date suggested QS system was one important regulatory system pertinent to biocontrol ability in P. fluorescens 2P24.The-gacS gene, one component of GacS/GacA two-component system, was cloned from P. fluorescens 2P24 by PCR mediate genomic library screening. The further research revealed that the knock-out mutant of gacS gene was unable to produce antifungal metabolites such as 2,4-DAPG, HCN and proteinase. It also lost the ability to inhibit the growth of phytopathogens in Petri dish and to control the development of wheat take-all in greenhouse. But the complemented strain containing a plasmid-bearing gacS gene could restore all of the lost phenotypes. In addition, GacS positively monitored the production of QS signal and regulated pcol gene at transcriptional level. All of the results indicated that GacS/GacA was a globe regulatory system that directly controlled biocontrol ability in P. fluorescens 2P24.