Isolation, Characterization Of Antagonistic Strains J12and J2Against Tomato Bacterial Wilt And Study Of Antibiotic Synthesis,Regulation2,4-DAPG

Pseudomonas spp. can produce some antagonistic compounds such as2,4-diacetylphloroglucinol (2,4-DAPG)、HCN、pyoluteorin (Plt)、pyrrolnitrin (Prn). siderophore and protease etc. Pseudomonas spp. can inhibit many plant pathogens and has strong colonization ability in rhizosphere. So Pseudomonas plays an important role in biocontrol of plant diseases caused by soil-borne pathogens. Screening of new Pseudomonas strains and development of their application in biocontrol are important in agriculture.9isolates, antagonistic against Ralstonia solanacearum were obtained from tomato rhizosphere soil from Henan and Jiangsu Province by diduting plating method. They belonged to9genus after identification by morphological, physiology, biochemical characteristics and16S rRNA gene analysis. Two of them, Pseudomonas brassicacearum J12and Pseudomonas fluorescens J2which had stronger antagonistic activities against R. solanacearum were chosen as test strains in this work.Strain J12could produce2,4-DAPG、HCN、siderophore and protease, and had abilities of phosphate solubilization and biofilm formation. The maximum growth and antagonistic activity were recorded at30℃and pH8, glucose and tryptone as the carbon and nitrogen sources respectively. Strain J12could suppress tomato bacterial wilt by45.5%in the greenhouse experiment. The main antimicrobial compound of J12was identified as2,4-diacetylphloroglucinol (2,4-DAPG) by HPLC-ESI-MS analysis. The gene cluster phlACBD, which is responsible for2,4-DAPG synthesis, was identified and it showed99%homology with that of P. brassicacearum NFM421(CP002585). phlACBD cluster was cloned into pMD19-T and expressed in E. coli DH5a. The results showed that recombinant Escherichia coli DH5a could strongly inhibit the growth of R. solanacearum. The expressed product was identified as2,4-DAPG by LC-MS. gfp gene was inserted into the chromosome of strain J12by Tn5transposon element. The J12cells with gfp gene showed green fluorence under blue excitation light. gfp-marked strain J2-gfp showed the same characterictics such as antagonistic activity、 growth and biofilm formation as wild strain J12. gfp gene in J2-gfp was very stable. J2-gfp could well colonized in rhizosphere of tomato.Strain J2could also produce2,4-DAPG The phlACBD gene cluster from J2showed high homology with some known phlACBD sequences. phlF gene located upstream of phlACBD and negatively regulated2,4-DAPG synthesis. A phlF gene (0.7kb) was obtained by PCR and showed99%similarity to that of P. fluorescens2P24. A phlF disruption mutant J2-phlF was constructed by double-crossover homologous recombination. The production of2,4-DAPG in J2-phlF increased3times more than that in widetype J2. J2-phlF showed an enhanced inhibitory effect on pathogens Ralstonia solanacearum and Fusarium oxysporum. Biocontrol experiments conducted in a greenhouse indicated that the J2-phlF showed higher biocontrol efficiency than the wild type, but there was no significant difference. Disruption of the phlF gene in J2did not affect the growth of cells and biofilm formation. An assay to assess colonization showed that J2-phlF formed a larger population size than wild-type J2in the rhizosphere and on root tips.phlE, responsible for2,4-DAPG transferring, located downstream of the phlACBD cluster and encoded423aa, which showed95%and99%homology with that in P. fluorescens F113and2P24. PhlE had no signal peptide with45.03kDa of molecular weight. PhlE belonged to major facilitator superfamily (MFS) and showed characteristics of some transmembrane proteins. phlE gene was coloned into vector pET28a(+) and recombinant plasmid was transformed into E. coli BL21(DE3), a PhlE overexpression strain J2-phlE was constructed. The extracellular2,4-DAPG production in J2-phlE increased4times compared with that of wild type J2. It also enhanced antagonistic activity against R. solanacearum.