Multifunction Of Burkholderia Spp.from Tobacco Rhizosphere And Identification Analysis Of Genes Involved In Phosphate-Solubilizing By Strain 71-2

Burkholderia cepacia complex(BCC)is very common organism presented in the natural environment,and species isolated from plant rhizosphere and soil are related to the promotion of plant growth and considered to be plant growth-promoting rhizobacteria(PGPR).Phosphorus(P)is an essential plant nutrient and involved in a wide range of life processes.A large portion of inorganic P applied in soil fixed as phosphates iron and calcium become insoluble form and,therefore,are not efficiently taken up by the plants.Some species of PGPR have the ability to convert insoluble inorganic phosphorus into soluble forms and enhanced absorption and availability for plant root.In this study we firstly isolated phosphate-solubilizing bacteria strains from tobacco rhizosphere.Subsequently,some strains were analyzed for their genomovar,virulence and antimicrobial activity.To analyze the mechanisms of phosphate-solubilizing of BCC strain,strain 71-2 was selected and the mutant library of 71-2 was constructed using Tn5 transposon,then genes related to phosphate-solubilizing were cloned.The main results are as follows:1.Isolation of phosphate-solubilizing bacteria and their biological activity.A total of 43bacteria isolates showing phosphate-solubilizing ability were obtained on PVK media from tobacco rhizosphere soil.The phosphate solubilizing efficiencies were quantified using insoluble Ca₃(PO₄)₂ in NBRIP broth medium and the maximum P solubilization was up to349.06mg/L for one of some strains.The antagonistic activities of these strains to plant pathogens were tested and most of the isolates showed antagonistic to Ralstonia solanacearum,Phytophthora parasitica and Thielaviopsis basicola.Also,siderophores and IAA(Indole acetic acid)production were tested for strains of 35-6,68-1 and 71-2,all of these isolates revealed the ability to produce IAA and siderophores,indicating that these strains had variety of biological activity.2.Identification of bacteria and their genomovar.Bacteria strains showing high phosphate-dissolving activities were identified using 16S rRNA and/or RecA sequences,and28 strains were identified as Burkholderia cepacia complex.All of the 22 isolates that identified as B.cenocepacia belong to genomovar?B and 6 isolates were identified as B.cepacia.3.Testing of virulence factors and pathogenicity of BCC strains.Some BCC strains are opportunistic bacteria and could cause lung disease in immunocompromised individuals.It is important to distinguish virulent BCC species with the beneficial ones.To assessed if the twenty-eight BCC strains in our study were risk when posed to environment,specific PCR were conducted firstly to amplify genes encoding two virulence factors,cblA(cable pili&adhesion)and esmR,(BCESM,an epidemic strain marker).The results showed that esmR and cblA genes could be amplified in 16 and 5 strains respectively,and 4 strains could be tested for both of genes esmR and cblA.Alfalfa infection assay was performed subsequently on alfalfa seedlings and no obviously symptoms on alfalfa seedlings were observed after inoculation with bacterial cells of different strains.Onion pathogenesis and hypersensitive response(HR)on tobacco leaves were assayed to analyze plant pathogenicity of BCC strains.Each strain could only cause colorless water stain spots at the inoculation site in detached onion bulbs and only one strain named 12-1 showed HR on tobacco leaves.These results showed that most BCC strains obtained in our study showed extremely low risk of opportunistic infection and plant pathogenicity.4.Mechanisms analyzing of phosphate-solubilizing of strain 71-2.To identify genes associated with P-solubilizing,strain 71-2 was randomLy mutated using EZ-Tn5Transposome Kit.Thirty three mutants showing deficiency or/and decrease in P-solubilizing activity were obtained in PVK plates and eight mutants showing significantly decrease in soluble P were quantified in NBRIP broth medium.Genes and sites inserted by Tn5 in 8mutants were analyzed using plasmid rescue technology and sequence analysis.The results revealed that insertion sites of Tn5 in MT6480 and other two mutants were at the same gene that encoding twin-arginine translocation protein(Tat).Tn5 inserted in gene encoding enolase in MT51.Other inserted genes associated with the other four mutants were predicated to encode choline dehydrogenase(choline dehydrogenase),halogen acid dehalogenase(HAD family hydrolase),MG2 protein(MG2 protein)and xylose isomerase(xylose isomerase)respectively.Complementation analyses were performed for mutant MT51 and MT6480.The intact ORFs of ENO and Tat,which were amplified using PCR from gDNA of wild strain 71-2,were connected to the broad host vector pBBR1MCS-5 respectively to construct recombinant plasmid.The recombinant plasmids pBBR1-ENO and pBBR1-Tat were transferred into mutants MT51 and MT6480 respectively,and the complementary strains 71-2-MT51R and71-2-MT6480R were obtained after verification by means of molecular biology.Phosphate solubilizing abilities of complementary strains were analyzed.Firstly,the diameters of the phosphate-solubilizing circles were determined in PVK plate.The diameters of complementary strains 71-2-MT51R and 71-2-MT6480R were 8.4 mm and 9.3 mm,which had decreased about 1.9 mm and 1.0 mm than that of wide type,while had increased about 2.2mm and 2.0 mm than that of mutant MT51 and MT6480,indicating that the complementary strain partially restored the ability of phosphate solubilizing.The phosphate solubilizing efficiencies of 71-2-MT51R and 71-2-MT6480R were quantified after 48h's culturing and the result showed that the maximum P solubilization were 305.43mg/L and 295.43 mg/L respectively,which had decreased about 10.29 mg/L and 20.29 mg/L than that of wild strain.,while had increased than that of MT51 and MT6480,indicating that the complementary strains could partially recover the mutants'ability of phosphorous dissolving.The above results showed that enolase and Tat are essential for the phosphate solubilization ability of strain 71-2.This is the first report about the relationship of bacteria phosphate solubilization with enolase and Tat mediated sec pathway.