Improving biocontrol activity ofPseudomonas fluorescens through chromosomal integration of 2,4-diacetylphloroglucinol biosynthesis genes

Abstract

Antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) produced by Pseudo11Wnas jl.uorescens CPF-IO and 2P24 is a principal factor enabling bacteria to suppress plant diseases caused by soilborne pathogens. In this study, a 2,4-DAPG biosynthesis locus phlACBDE cloned from strain CPF-IO was assembled into a mini-Tn5 transposon and introduced into the chromosome of P. jl.uorescens P32 (2,4-DAPG), CPF-IO and 2P24 to construct the 2,4-DAPG overproducing derivatives P32-38, CPFlO-9 and 2P24-48, respectively. All the transgenic strains showed an enhanced antibiosis capacity against plant microbial pathogens in vitro and two strains, P32-38 and CPFlO-9, provided significantly better protection against wheat take-all disease caused by Gaeumannomyces graminis var. tritici and tomato bacterial wilt caused by Ralstonia solanacearum in greenhouse. Compared to their parental strains, the 2,4-DAPG overproducing derivatives colonized to the same extent on the wheat tips in the autoclaved soil, but developed larger populations in natural soil. These results indicated that production of antibiotics 2,4-DAPG by biological control pseudomonads can contribute not only to their disease suppression capacities but also to the ecological competence in the resident microflora. Our research also suggests that it is a realistic approach to improve biocontrol capacity of P. fluorescens through the genetic modification of its antibiotic 2,4-DAPG production.

Interaction between 2,4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing Pseudomonas brassicacearum LBUM300 and Clavibacter michiganensis subsp. michiganensis in the Tomato Rhizosphere

“…Production of DAPG in Pseudomonas fluorescens P32 increased colonization capacity on wheat roots in natural soils in comparison to the parental strain, which produced HCN and siderophores but not DAPG (30). Additionally, a DAPG overproducer (due to a phlF gene mutation) was able to colonize the rhizosphere of tomato plants at a higher rate than the wild type (31).…”

Enhancing plant disease suppression by Burkholderia vietnamiensis through chromosomal integration of Bacillus subtilis chitinase gene chi113

“…The transposon delivery vector pUTkm1 (Zhou et al 2005) was used for chromosomal integration of chi113 in P418. Primers ChiSDN-F (5 0 -CGGCGGCCGCAG-GAGGTTGATATGAAAAAAGTGTTTTCA-3 0 ) and ChiN-R (5 0 -CGGCGGCCGCTTATTTGCAATCACC AATT-3 0 ) were designed (this study) to include NotI sites (underlined nucleotides) to facilitate insertion of chi113 in pUTkm1 and Shine-Dalgarno sequences (bold nucleotides) to potentially enhance ribosome binding of transcripts in the recombinants (Driss et al 2005).…”

“…Conjugal transfer of pUTkm1/chi113 from E. coli S17-1 (k-pir) to P418 and chromosomal transposition were performed according to Zhou et al (2005). Putative transformants were selected for kanamycin resistance (Km R , 50 mg kanamycin l -1 ) on P418 ?…”

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

“…The standard PCR reaction involved 5 min at 94°C, the following 35 cycles of 40 s at 94°C, 40 s at 60°C and 1 min at 72°C, and finally 10 min at 72°C. After being digested with relevant restriction enzymes, the two fragments were inserted into pBSNot6 (Zhou et al 2005) to create pBSNDIA and pBSNDIB. The Pst1-BamH1 fragment from pBSNDIA was inserted into pBSNDIB resulting in pBSNDI, from which a 3.2-kb Not1 fragment, including a pcoI gene with 270 bp deletion, was lifted and ligated into pSR47S (Andrews et al 1998).…”