Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp.

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.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

Mavrodi

Thomashow³

et al. 2007

“…BW11M1 (17). Bacteriocin production by biocontrol strains may contribute to their rhizosphere competence (64). If so, recombinant biocontrol strains equipped with (several) bacteriocin genes may perform better as plant growth-promoting inocula by outcompeting rhizosphere-inhabiting pseudomonads.…”

Section: Vol 71 2005mentioning

confidence: 99%

Novel Lectin-Like Bacteriocins of Biocontrol Strain Pseudomonas fluorescens Pf-5

Parret

Temmerman

Mot

2005

Bacteria living in a competitive environment are able to secrete proteinaceous toxins, known as bacteriocins, which can kill closely related bacterial competitors while causing little harm to the bacteriocinogenic cells. These bacterial inhibitors are produced by all major groups of Bacteria (53). Bacteriocin production has also been reported for Halobacteriaceae, a family of extremely halophilic Archaea (36). Bacteriocins constitute a structurally and functionally diverse group within the antimicrobials, ranging from small peptides, such as microcins of Enterobacteriaceae and antibiotics secreted by low-GC-content gram-positive bacteria (23, 31), to middle-sized polypeptides, such as colicins of Escherichia coli (34) and their counterparts in Pseudomonas aeruginosa (S pyocins) (39), to large phage tail-like multiprotein complexes, such as syringacin produced by Pseudomonas syringae (58) and R-and F-type pyocins of P. aeruginosa (39). Bacteriocin mode of killing can be either by membrane pore formation, nonspecific degradation of cellular DNA, cleavage of 16S rRNA or tRNA, or inhibition of peptidoglycan synthesis resulting in cell lysis (51).Bacteriocins from lactic acid bacteria, such as nisin, have received considerable attention, given their potential as food preservatives (41, 42). Among bacteriocins of gram-negative bacteria, colicins are the most intensively studied. Colicins are produced by, and attack, strains of E. coli. These proteins, as well as the S pyocins, are organized in functional domains, namely, regions for cell attachment, translocation, and bactericidal activity. Colicins and S pyocins parasitize specific membrane-bound receptors on target cells, resulting in a rather narrow spectrum of activity. The host strain is protected from its own bacteriocin through the action of a cognate immunity protein that is coproduced with the bacteriocin (39, 51).It has been proposed that bacteriocins may play a key role in bacterial population dynamics (52,53). In a recent study, Kirkup and Riley demonstrated that the production of colicins by E. coli colonizing the mouse colon gives the colicinogenic strains a competitive advantage by killing colicin-sensitive E. coli sharing the same ecological niche (33). Possible applications of bacteriocinogenic strains in agriculture include their use in biological control of soilborne or phyllosphere-inhabiting bacterial plant pathogens. In this way, heterologous production of the peptide bacteriocin trifolitoxin by an avirulent Agrobacterium strain effectively enhances biological control of Agrobacterium vitis crown gall (24). Expression of the trifolitoxin genes from Rhizobium leguminosarum bv. trifolii T24 in Rhizobium etli CE3 increases bean nodulation competitiveness of the recombinant strain in the presence of indigenous rhizobia under agricultural conditions (54). A phage tail-like bacteriocin (serracin P) produced by Serratia plymithicum may be used for biological control of fire blight caused by Erwinia amylovora (30), while Xanthomonas campestris pv. glycin...

“…Plants were grown in a growth chamber for two successive 2-week cycles in the same controlled environment, processed, and analyzed as described below. Population densities of the introduced strains were monitored by the modified dilution-endpoint method (43,71). Briefly, individual wheat root systems were placed in 10 ml of sterile distilled water, vortexed, and sonicated.…”

Section: Methodsmentioning

confidence: 99%

Role ofptsP,orfT, andsssRecombinase Genes in Root Colonization byPseudomonas fluorescensQ8r1-96

Mavrodi

Weller³

et al. 2006

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.Interest in biological control continues to increase in response to public concerns about the use of chemical pesticides and recognition of the need for environmentally benign plant disease control strategies (76). Plant growth-promoting rhizobacteria (PGPR) have potential for development as biopesticides, biofertilizers, or phytostimulants, but only a few such products have been marketed, in part because the performance of introduced strains varies among fields and years. Variable root colonization can contribute significantly to this inconsistency because the introduced bacteria must attain threshold population sizes in the rhizosphere in order to be effective. Considerable research during the past 25 years has been directed toward understanding the biotic and abiotic factors that influence root colonization.Our research has focused on colonization of the wheat rhizosphere by Pseudomonas fluorescens Q8r1-96, which produces the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG). 2,4-DAPG-producing strains of P. fluorescens suppress root and seedling diseases of a variety of crops and play a key role in the natural biological control of take-all disease of wheat known as take-all decline (4,19,28,49,50,65,75)....