Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR
- 1 September 2007
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 73 (17), 5531-5538
- https://doi.org/10.1128/aem.00925-07
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 ( C T s) 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, r 2 = 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.Keywords
This publication has 40 references indexed in Scilit:
- Real time PCR quantification in groundwater of the dehalorespiring Desulfitobacterium dichloroeliminans strain DCA1Journal of Microbiological Methods, 2006
- Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCRApplied and Environmental Microbiology, 2006
- Distribution and Biocontrol Potential of phlD+ Pseudomonads in Corn and Soybean FieldsPhytopathology®, 2005
- Assessment of Genotypic Diversity of Antibiotic-Producing Pseudomonas Species in the Rhizosphere by Denaturing Gradient Gel ElectrophoresisApplied and Environmental Microbiology, 2005
- Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in SoilApplied and Environmental Microbiology, 2004
- Multiple sequence alignment with the Clustal series of programsNucleic Acids Research, 2003
- Comparison of Three Methods for Monitoring Populations of Different Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the RhizospherePhytopathology®, 2002
- Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCRJournal of Clinical Microbiology, 2001
- Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescens Strain Q8r1-96Applied and Environmental Microbiology, 2001
- Polymorphism of the Polyketide Synthase Gene phlD in Biocontrol Fluorescent Pseudomonads Producing 2,4-Diacetylphloroglucinol and Comparison of PhlD with Plant Polyketide SynthasesMolecular Plant-Microbe Interactions®, 2001