ARS | Publication request: Quantification of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens in the plant rhizosphere by real-time PCR.

Submitted to: ASM Conference
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2007
Publication Date: August 26, 2007
Citation: Mavrodi, O.V., Mavrodi, D.V., Thomashow, L.S., Weller, D.M. 2007. Quantification of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens in the plant rhizosphere by real-time PCR. ASM Conference.2007.P.88.

Technical Abstract: A real-time PCR SYBR green assay was developed to quantify populations of 2, 4-DAPG-producing (phlD+) Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed to specifically amplify the phlD gene from four different genotypes (A, B, D, and I) of phlD+ P. fluorescens and PCR conditions were optimized. Standard curves relating the threshold cycles (Ct) and copies of phlD gene were generated for P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), FTAD1r34 (genotype D) and FTAD1r36 (genotype I) with dilution series of purified genomic DNA and with genomic DNA extracted from washes of wheat roots spiked with each bacteria. The detection limit of the optimized real-time PCR assay was 60-600 fg (8-80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens, and 600 fg - 6.0 pg (80-800 CFU) for bacterial DNA extracted from plant root washes, which corresponds to log 4-5 phlD+ CFU/rhizosphere. The real-time PCR assay was applied for quantification of phlD+ pseudomonads in the wheat rhizosphere, and regression analysis of population densities detected by both the phlD-specific PCR-based dilution endpoint assay and real-time PCR indicated a significant linear relationship (P = 0.0016, r2=0.2).