Phenotypic, genotypic and colonization properties of 2,4-diacetylphloroglucinol-producing Pseudomonas spp. isolated from roots of wheat

Fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) inhibit a variety of soilborne plant pathogens and play an important role in the suppression of take-all disease of wheat. Diversity within phlD, an essential gene in the biosynthesis of DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 DAPG-producing isolates from throughout the USA and worldwide. Clusters (genotypes) defined by RFLP analysis of phlD correlated closely with clusters defined by rep-PCR of total genomic DNA, validating the utility of phlD as a marker of genetic diversity and population structure among DAPG producers. Additional population diversity was revealed by random amplified polymorphic DNA (RAPD) analysis of genomic DNA. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Genotypic diversity among 30 strains representing all of the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, DAPG or total phloroglucinol compounds. Twenty-seven of these 30 strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.; Members of the D genotype of DAPG-producing strains of P. fluorescens are exceptionally aggressive root colonizers of wheat and pea. Four genes broadly implicated in microbe-host interactions, sss, dsbA, ptsP, and orfT, were investigated for their contribution to this aggressive colonization phenotype. These genes influence global processes including phenotypic plasticity, secretion, organic nitrogen utilization, and transmembrane transport, respectively, and were identified in an ordered genomic library of Q8r1-96 by colony hybridization and PCR. All four genes were mapped, sequenced and the corresponding gene replacement mutants were constructed and characterized. Mutants in dsbA, sss, and orfT colonized the roots of wheat grown in natural soil as effectively as did the parental strain when introduced separately, but were less competitive root colonists when introduced together with the parental strain. However, the ptsP mutant, colonized wheat roots less effectively than Q8r1-96 whether introduced alone or in combination with the parental strain, indicating that this gene is critical for effective rhizosphere colonization by P. fluorescens Q8r1-96.