A rapid polymerase chain reaction-based assay characterizing rhizosphere populations of 2,4-diacetylphloroglucinol-producing bacteria

Pseudomonas species that produce 2,4-diacetylphloroglucinol (2,4-DAPG) play a significant role in the suppression of fungal root pathogens in the rhiz osphere of crop plants. To characterize the abundance and diversity of thes e functionally important bacterial populations, we developed a rapid polyme rase chain reaction (PCR)-based assay targeting phlD, an essential gene in the phloroglucinol biosynthetic pathway. The phlD gene is predicted to enco de a polyketide synthase that synthesizes monoacetylphloroglucinol, the imm ediate precursor to 2,4-DAPG. A major portion of the phlD open reading fram e was cloned and sequenced from five genotypically distinct strains, and th e sequences were screened for conserved regions that could be used as gene- specific priming sites for PCR amplification. Several new phlD-specific pri mers were designed and evaluated. Using the primers B2BF and BPR4, we devel oped a PCR-based assay that was robust enough to amplify the target gene fr om a diverse set of 2,4-DAPG producers and sensitive enough to detect as fe w as log 2.4 cells per sample when combined with enrichment from a selectiv e medium. Restriction fragment length polymorphism analysis of the amplifie d phlD sequence allows for the direct determination of the genotype of the most abundant 2,4-DAPG producers in a sample. The method described was usef ul for characterizing both inoculant and indigenous phlD(+) pseudomonads in habiting the rhizosphere of crop plants. The ability to rapidly characteriz e populations of 2,4-DAPG-producers will greatly enhance our understanding of their role in the suppression of root diseases.