Submitted to: Federation of European Microbiological Societies Microbiology Ecology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/24/2005
Publication Date: 9/20/2005
Citation: De La Fuente, L., Mavrodi, D.V., Landa, B.B., Thomashow, L.S., Weller, D.M. 2005. Genetic diversity and detection of genotypes of 2,4-diacetylphloroglucinol-producing pseudomonas spp.fluorescens based on phld sequences. Federation of European Microbiological Societies Microbiology Ecology. 56:64-78. 2006. Interpretive Summary: Some Pseudomonas bacteria 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 bacteria also are responsible for the natural suppressiveness of certain soils to diseases such as take-all of wheat and black root rot. From a worldwide collection of DAPG producers, 18 distinct genotypes (A-Q) are known. Genotypes differed significantly in ability to colonize the roots of wheat and pea. Root colonizing ability is directly related to biocontrol activity against root diseases such as take-all. Isolates of multiple genotypes occur in a single soil but no information has been available about the interactions among genotypes. We developed allele-specific PCR primers that allow detection of DAPG producers belonging to a specific genotype, thus allowing simultaneous monitoring of the population dynamics of multiple genotypes in a single soil. Studies with these new molecular tools are revealing why certain genotypes out compete other genotypes in the pea and wheat rhizosphere resulting in the dominance of one or two genotypes out of the multiple genotypes present.
Technical Abstract: Diversity within a worldwide collection of 2,4-diacetylphloroglucinol (DAPG)-producing fluorescent Pseudomonas fluorescens was assessed by sequencing the phlD gene. Phylogenetic analyses based on phlD sequences of 70 isolates supported the previous classification into eighteen BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, allele-specific PCR primers were designed and used with a PCR-based dilution endpoint assay to quantify the population sizes of the A-, B-, D-, K-, L- and P- genotype strains grown individually or in pairs in vitro and in the rhizosphere of wheat and bulk soil. Except for P. fluorescens Q8r1-96, which strongly inhibited the growth of P. fluorescens Q2-87, inhibition between pairs of strains grown in vitro did not affect the accuracy of the method. The allele-specific primer-based technique is a rapid method for studies of the interactions between genotypes of 2,4-DAPG producers in natural environments.