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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 rhizosphere of crop plants. To characterize the abundance and diversity of these functionally important bacterial populations, we developed a rapid polymerase chain reaction (PCR)-based assay targeting phlD, an essential gene in the phloroglucinol biosynthetic pathway. The phlD gene is predicted to encode a polyketide synthase that synthesizes monoacetylphloroglucinol, the immediate precursor to 2,4-DAPG. A major portion of the phlD open reading frame was cloned and sequenced from five genotypically distinct strains, and the sequences were screened for conserved regions that could be used as gene-specific priming sites for PCR amplification. Several new phlD-specific primers were designed and evaluated. Using the primers B2BF and BPR4, we developed a PCR-based assay that was robust enough to amplify the target gene from a diverse set of 2,4-DAPG producers and sensitive enough to detect as few as log 2.4 cells per sample when combined with enrichment from a selective medium. Restriction fragment length polymorphism analysis of the amplified 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 useful for characterizing both inoculant and indigenous phlD(+) pseudomonads inhabiting the rhizosphere of crop plants. The ability to rapidly characterize populations of 2,4-DAPG-producers will greatly enhance our understanding of their role in the suppression of root diseases.
McSpadden Gardener, B.B. , Mavrodi, D.V. , Thomashow, L.S. , Weller, D.M.
crops , plant pathogenic fungi , rhizosphere fungi , biological control , Pseudomonas , diagnostic techniques , polymerase chain reaction , strain differences , pathotypes , genetic techniques and protocols , genes , rapid methods , methodology , nucleotide sequences , genetic variation , disease control , plant diseases and disorders , genotype
- Includes references
- Phytopathology Jan 2001. v. 91 (1)
Journal Articles, USDA Authors, Peer-Reviewed
- Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.