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

ABSTRACT 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.

[1]  D. Weller,et al.  Exploiting Genotypic Diversity of 2,4-Diacetylphloroglucinol-Producing Pseudomonas spp.: Characterization of Superior Root-Colonizing P. fluorescensStrain Q8r1-96 , 2001, Applied and Environmental Microbiology.

[2]  G. Défago,et al.  Polymorphism of the polyketide synthase gene phID in biocontrol fluorescent pseudomonads producing 2,4-diacetylphloroglucinol and comparison of PhID with plant polyketide synthases. , 2001, Molecular plant-microbe interactions : MPMI.

[3]  L. Thomashow,et al.  Genetic Diversity of phlD from 2,4-Diacetylphloroglucinol-Producing Fluorescent Pseudomonas spp. , 2001, Phytopathology.

[4]  K. Qing.  Systemic resistance induced by rhizosphere bacteria , 2001 .

[5]  B. M. Gardener,et al.  A rapid polymerase chain reaction-based assay characterizing rhizosphere populations of 2,4-diacetylphloroglucinol-producing bacteria. , 2001, Phytopathology.

[6]  S. Kalloger,et al.  Genotypic and Phenotypic Diversity of phlD-ContainingPseudomonas Strains Isolated from the Rhizosphere of Wheat , 2000, Applied and Environmental Microbiology.

[7]  R. Fani,et al.  Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth , 2000, Applied and Environmental Microbiology.

[8]  F. O'Gara,et al.  Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. , 2000, Microbiology.

[9]  D. Weller,et al.  Effect of Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat. , 1999, Phytopathology.

[10]  L. Thomashow,et al.  Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87 , 1999, Journal of bacteriology.

[11]  C. Lacroix,et al.  Digoxigenin-labeled probe for rapid identification of nisinogenic Lactococcus lactis strains. , 1999, FEMS microbiology letters.

[12]  S. Kalloger,et al.  Genotypic and phenotypic diversity of PhlD-containing Pseudomonas , 1999 .

[13]  Kevin P. Smith,et al.  HOST VARIATION FOR INTERACTIONS WITH BENEFICIAL PLANT-ASSOCIATED MICROBES. , 1999, Annual review of phytopathology.

[14]  L. van der Fits,et al.  A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Weller,et al.  Natural plant protection by 2,4-diacetylphloroglucinol-producing Pseudomonas spp. in take-all decline soils , 1998 .

[16]  S. Kalloger,et al.  Distribution of 2,4-diacetylphloroglucinol-producing Pseudomonas spp. with extended monoculture. , 1998 .

[17]  B. Duffy,et al.  Zinc Improves Biocontrol of Fusarium Crown and Root Rot of Tomato by Pseudomonas fluorescens and Represses the Production of Pathogen Metabolites Inhibitory to Bacterial Antibiotic Biosynthesis. , 1997, Phytopathology.

[18]  M. Griffiths,et al.  Detection of pathogenic Yersinia enterocolitica in milk and pork using a DIG-labelled probe targeted against the yst gene. , 1997, International journal of food microbiology.

[19]  F. O'Gara,et al.  Ecological interaction of a biocontrol Pseudomonas fluorescens strain producing 2,4-diacetylphloroglucinol with the soft rot potato pathogen Erwinia carotovora subsp. atroseptica , 1997 .

[20]  W. Mahaffee,et al.  Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic marker (bioluminescence) for monitoring the environmental fate of rhizobacterium , 1997, Applied and environmental microbiology.

[21]  F. O'Gara,et al.  Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis , 1997, Applied and environmental microbiology.

[22]  L. Thomashow,et al.  Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments , 1997, Applied and environmental microbiology.

[23]  L. Thomashow,et al.  Quantification of 2,4-Diacetylphloroglucinol Produced by Fluorescent Pseudomonas spp. In Vitro and in the Rhizosphere of Wheat , 1997, Applied and environmental microbiology.

[24]  C. Keel,et al.  Conservation of the 2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse geographic locations , 1996, Applied and environmental microbiology.

[25]  L. Thomashow,et al.  Current Concepts in the Use of Introduced Bacteria for Biological Disease Control: Mechanisms and Antifungal Metabolites , 1996 .

[26]  C. Keel,et al.  Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities , 1995, Journal of bacteriology.

[27]  P. Bakker,et al.  Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments , 1994, Applied and environmental microbiology.

[28]  D. Weller,et al.  Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat , 1994 .

[29]  P. Gerhardt,et al.  Methods for general and molecular bacteriology , 1994 .

[30]  D. Weller,et al.  Purification of an antibiotic effective against Gaeumannomyces graminis var. tritici produced by a biocontrol agent, Pseudomonas aureofaciens , 1993 .

[31]  D. Kluepfel.  The Behavior and Tracking of Bacteria in the Rhizosphere , 1993 .

[32]  S. Lindow,et al.  Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae , 1992, Applied and environmental microbiology.

[33]  F. O'Gara,et al.  Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. , 1992, Microbiological reviews.

[34]  F. O'Gara,et al.  Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain , 1992, Applied and Environmental Microbiology.

[35]  M. Mazzola,et al.  Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats , 1992, Applied and environmental microbiology.

[36]  F. O'Gara,et al.  Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production , 1992, Applied and environmental microbiology.

[37]  C. Keel,et al.  Suppression of root diseases by Pseudomonas fluorescens CHA0 - importance of the bacterial seconday metabolite 2,4-diacetylphloroglucinol , 1992 .

[38]  T. Leser,et al.  Survival of Enterobacter Cloacae on Leaves and in Soil Detected by Immunofluorescence Microscopy in Comparison with Selective Plating , 1992 .

[39]  D. Weller,et al.  Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain , 1991, Applied and environmental microbiology.

[40]  J. Kloepper,et al.  PSEUDOMONAS INOCULATION TO BENEFIT PLANT PRODUCTION , 1988 .

[41]  G. Défago,et al.  Naturally occurring fluorescent pseudomonads involved in suppression of black root rot of tobacco , 1986 .

[42]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[43]  D. Weller.  Colonization of wheat roots by a fluorescent pseudomonad suppressive to take-all. , 1983 .

[44]  R. Cook,et al.  Suppression of take-all of wheat by seed treatments with fluorescent pseudomonads. , 1983 .

[45]  C. R. Howell.  Control of rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium. , 1979 .

[46]  J. K. Martin.  Comparison of agar media for counts of viable soil bacteria , 1975 .