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Comparison of Three Methods for Monitoring Populations of Different Genotypes of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere

February 2002 , Volume 92 , Number  2
Pages  129 - 137

Blanca B. Landa , Henricus A. E. de Werd , Brian B. McSpadden Gardener , and David M. Weller

U.S. Department of Agriculture, Agricultural Research Service, Root Disease and Biological Control Research Unit, Washington State University, Pullman 99164

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Accepted for publication 11 October 2001.

Pseudomonas fluorescens strains producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) have biocontrol activity against a broad spectrum of root and seedling diseases. In this study, we determined the effect of genotype on the ability to isolate and quantify introduced 2,4-DAPG producers from the rhizosphere of wheat using three different methods: traditional dilution plating on selective media, colony hybridization followed by polymerase chain reaction (PCR), and phlD-specific PCR-based dilution endpoint assay. Regression analysis of the population densities of 10 2,4-DAPG-producing P. fluorescens, representing five genotypes, determined by the three different methods demonstrated that the relationship was linear (P < 0.001) and the techniques were very similar (i.e., slopes equal to 1.0). The phlD-specific PCR-based assay had a slightly lower limit of detection than the other two methods (log 3.3 versus log 4.0 CFU/g of fresh root weight). With the colony hybridization procedure, we observed that the phlD probe, derived from strain P. fluorescens Q8r1-96, hybridized more strongly to colonies of BOX-PCR genotypes D (strains W2-6, L5.1-96, Q8r1-96, and Q8r2-96) and K (strain F113) compared with strains of genotypes A (Pf-5 and CHA0), B (Q2-87), and L (1M1-96 and W4-4). Colony hybridization alone overestimated the actual densities of some strains, thus requiring an additional PCR step to obtain accurate estimates. In contrast, population densities estimated for three of the bacterial treatments (strains CHA0, W2-6, and Q8r2-96) with the PCR-based assay were significantly (P < 0.041) smaller by 7.6 to 9.2% and 6.4 to 9.4% than population densities detected by the dilution plating and colony hybridization techniques, respectively. In this paper, we discuss the relative advantages of the different methods for detecting 2,4-DAPG producers.

Additional keywords: antibiotics, plant growth-promoting rhizobacteria.

The American Phytopathological Society, 2002