ARS | Publication request: APPLICATION OF REAL-TIME PCR QUANTIFICATION OF 2,4-DIACETYLPHLOROGLUCINAL-PRODUCING PSEUDOMONAS FLUORESCENS IN THE PLANT RHIZOSPHERE.

Submitted to: International Plant Growth Promoting Rhizobacteria Workshop
Publication Type: Abstract Only
Publication Acceptance Date: March 20, 2006
Publication Date: May 20, 2006
Citation: Mavrodi, O.V., Mavrodi, D.V., Thomashow, L.S., Weller, D.M. 2006. Application of real-time pcr quantification of 2,4-diacetylphloroglucinal-producing pseudomonas fluorescens in the plant rhizosphere.. International Plant Growth Promoting Rhizobacteria Workshop.

Technical Abstract: A quantitative real-time PCR SYBR Green assay was developed to quantify populations of DAPG-producing (phlD+) Pseudomonas spp. in the plant rhizosphere. Primers targeting the phlD gene were designed to specifically amplify four different BOX-PCR genotypes (A, B, D, and I) and PCR conditions were optimized for annealing temperature and MgCl2 concentration. Standard curves relating the threshold cycles (Ct) and copies of phlD gene were generated for P. fluorescens Pf-5 (A), Q2-87 (B), Q8r1-96 (D), FTAD1r34 (D) and FTAD1r36 (I) with dilution series of purified genomic DNA. Real-time PCR data were analyzed with LightCycler Software v.3, using the arithmetic baseline adjustment and second derivative maximum analysis. The detection limit for DNA purified from bacterial cultures was 60-600 fg (8-80 copies of phlD) depending on the strain. The optimized PCR conditions were then applied for quantification of phlD+ pseudomonads in bulk soil or the plant rhizosphere. Total DNA was extracted from soil and plant root washes with an UltraClean Soil DNA kit (MO BIO Labs) using minor modifications of the alternative protocol for wet soil samples. Target fragments amplified from different genotypes of DAPG-producing Pseudomonas spp. could be easily distinguished by melting curve analyses.