Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/3/2007
Publication Date: 9/20/2007
Citation: Mavrodi, O.V., Mavrodi, D.V., Thomashow, L.S., Weller, D.M. 2007. Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR.. Applied and Environmental Microbiology. 73:5531-5538. Interpretive Summary: Some strains of the bacterium Pseudomonas fluorescens produce the antifungal, biocontrol metabolite 2,4-diacetylphloroglucinol (DAPG). These bacteria suppress a wide spectrum of soilborne plant pathogens that cause wilts, damping-off and root diseases of food, fiber and ornamental crops. DAPG-producing Pseudomonas fluorescens also are responsible for the natural suppressiveness of certain soils to diseases such as take-all of wheat. From a worldwide collection of DAPG producers, 22 distinct genotypes (A-T, PfY and PfZ) are known. Genotypes differ significantly in ability to colonize the roots of wheat and pea: some are superior and some are average. Understanding root colonizing ability is important because it directly relates to biocontrol activity against root diseases such as take-all. The purpose of this study was to develop a Real Time-PCR assay to detect and quantify DAPG producers in the soil and rhizosphere by amplification of phlD, key gene in the DAPG biosynthesis locus. This research is important because it allows quantification of DAPG producers in the rhizosphere without culturing the bacteria and provides a rapid method to assess whether a wheat soil is suppressive to take-all, the most important root disease of wheat worldwide.
Technical Abstract: A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (CTs) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.