Relative importance of fluorescent siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79 and M4-80R.

Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.     

Variation in Sensitivity of Gaeumannomyces graminis to Antibiotics Produced by Fluorescent Pseudomonas spp. and Effect on Biological Control of Take-All of Wheat

Isolates of Gaeumannomyces graminis var. tritici, the causal agent of take-all of wheat, varied in sensitivity in vitro to the antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) produced by fluorescent Pseudomonas spp. shown previously to have potential for biological control of this pathogen. None of the four isolates of G. graminis var. avenae examined were sensitive to either of the antibiotics in vitro at the concentrations tested. The single isolate of G. graminis var. graminis tested was insensitive to PCA at 1.0 (mu)g/ml. Pseudomonas fluorescens 2-79 and Pseudomonas chlororaphis 30-84, both of which produce PCA, effectively suppressed take-all caused by each of two PCA-sensitive isolates of G. graminis var. tritici. PCA-producing strains exhibited a reduced ability or complete inability to suppress take-all caused by two of three isolates of G. graminis var. tritici that were insensitive to PCA at 1.0 (mu)g/ml. P. fluorescens Q2-87, which produces Phl, suppressed take-all caused by three Phl-sensitive isolates but failed to provide significant suppression of take-all caused by two isolates of G. graminis var. tritici that were insensitive to Phl at 3.0 (mu)g/ml. These findings affirm the role of the antibiotics PCA and Phl in the biocontrol activity of these fluorescent Pseudomonas spp. and support earlier evidence that mechanisms in addition to PCA are responsible for suppression of take-all by strain 2-79. The results show further that isolates of G. graminis var. tritici insensitive to PCA and Phl are present in the pathogen population and provide additional justification for the use of mixtures of Pseudomonas spp. that employ different mechanisms of pathogen suppression to manage this disease.     

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.     

Role of antibiotics and siderophores in biocontrol of take-all disease of wheat

Both antibiotics and siderophores have been implicated in the control of soilborne plant pathogens by fluorescent pseudomonads. In Pseudomonas fluorescens 2–79, which suppresses take-all of wheat, the importance of the antibiotic phenazine-1-carboxylic acid was established with mutants deficient or complemented for antiobiotic production and by isolation of the antibiotic from the roots of wheat colonized by the bacteria. Genetic and biochemical studies of phenazine synthesis have focused on two loci; the first is involved in production of both anthranilic acid and phenazine-1-carboxylic acid, and the second encodes genes involved directly in phenazine synthesis. Because the antibiotic does not account fully for the suppressiveness of strain 2-79, additional mutants were analyzed to evaluate the role of the fluorescent siderophore and of an antifungal factor (Aff, identified as anthranilic acid) that accumulates when iron is limiting. Whereas strains producing only the siderophore conferred little protection against take-all, Aff⁺ strains were suppressive, but much less so than phenazine-producing strains. Iron-regulated nonsiderophore antibiotics may be produced by fluorescent pseudomonads more frequently than previously recognized, and could be partly responsible for beneficial effects that were attributed in the past to fluorescent siderophores.     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.     

Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens

Pseudomonas fluorescens 2-79RN(10) protects wheat against take-all disease caused by Gaeumannomyces graminis var. tritici; however, the level of protection in the field varies from site to site. Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol. In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN(10) (which produces phenazine-1-carboxylate [PCA(+)]) or a series of mutants with PCA(+) and PCA(-) phenotypes. Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest. Sixteen soil properties were correlated with disease suppression. Biocontrol activity of PCA(+) strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc. In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen. Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol). A model was identified with step-wise regression analysis (R(2) = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc. As predicted by our regression model, the biocontrol activity of 2-79RN(10) was improved by amending a soil low in Zn with 50 micro g of zinc-EDTA/g of soil. We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN(10).     

Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments

The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soilborne plant pathogens by various strains of fluorescent Pseudomonas spp. In this study, we described primers and probes that enable specific and efficient detection of a wide variety of fluorescent Pseudomonas strains that produce various phenazine antibiotics or Phl. PCR analysis and Southern hybridization demonstrated that specific genes within the biosynthetic loci for Phl and PCA are conserved among various Pseudomonas strains of worldwide origin. The frequency of Phl- and PCA-producing fluorescent pseudomonads was determined on roots of wheat grown in three soils suppressive to take-all disease of wheat and four soils conducive to take-all by colony hybridization followed by PCR. Phenazine-producing strains were not detected on roots from any of the soils. However, Phl-producing fluorescent pseudomonads were isolated from all three take-all-suppressive soils at densities ranging from approximately 5 x 10(sup5) to 2 x 10(sup6) CFU per g of root. In the complementary conducive soils, Phl-producing pseudomonads were not detected or were detected at densities at least 40-fold lower than those in the suppressive soils. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of these soils to take-all disease of wheat.     

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.     

Production of the Antibiotic Phenazine-1-Carboxylic Acid by Fluorescent Pseudomonas Species in the Rhizosphere of Wheat

Pseudomonas fluorescens 2-79 and P. aureofaciens 30-84 produce the antibiotic phenazine-1-carboxylic acid and suppress take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. To determine whether the antibiotic is produced in situ, wheat seeds were treated with strain 2-79 or 30-84 or with phenazine-nonproducing mutants or were left untreated and then were sown in natural or steamed soil in the field or growth chamber. The antibiotic was isolated only from roots of wheat colonized by strain 2-79 or 30-84 in both growth chamber and field studies. No antibiotic was recovered from the roots of seedlings grown from seeds treated with phenazine-nonproducing mutants or left untreated. In natural soils, comparable amounts of antibiotic (27 to 43 ng/g of root with adhering soil) were recovered from roots colonized by strain 2-79 whether or not the pathogen was present. Roots of plants grown in steamed soil yielded larger bacterial populations and more antibiotic than roots from natural soils. In steamed and natural soils, roots from which the antibiotic was recovered had significantly less disease than roots with no antibiotic, indicating that suppression of take-all is related directly to the presence of the antibiotic in the rhizosphere.     

Isolation, identification, and accumulation of 2-acetamidophenol in liquid cultures of the wheat take-all biocontrol agent Pseudomonas fluorescens 2-79

Pseudomonas fluorescens strain 2-79 (NRRL B-15132) is a classic biological control agent known to produce phenazine-1-carboxylic acid (PCA) as its primary means of suppressing take-all disease of wheat. In addition to PCA, an unknown metabolite was discovered in a liquid culture used to produce the biocontrol agent. The objective of the current study was to isolate, identify, and evaluate the accumulation of this compound in production cultures. Upon centrifugal fractionation of a production culture, thin-layer chromatography (TLC) analyses of extracts of the cells and cell-free supernatant indicated the compound to be primarily in the supernatant. Purified compound was obtained by extraction of culture supernatant, followed by flash chromatography of the extract and preparative TLC. The ¹H and ¹³C nuclear magnetic resonance and electron impact mass spectra indicated the compound to be 2-acetamidophenol (AAP). Measured by reversed-phase HPLC, the accumulations of AAP and PCA in cultures of strain 2-79 reached 0.05 g/l and 1 g/l, respectively. The accumulations of AAP and PCA in liquid cultures were linearly correlated (P < 0.001), as shown by studies of cultures stimulated to yield varying levels of PCA by controlling levels of oxygen transfer, pH, and growth medium composition. In this study, oxygen limitation, a defined amino-acid-free medium, and neutral pH stimulated maximal production of both AAP and PCA. Furthermore, a transposon mutant of 2-79 [2A40 2-79 (phz–)] unable to produce PCA did not accumulate AAP. These findings indicate that AAP and PCA are likely to share a common segment of biosynthetic pathway. This is the first report of AAP production by a strain of P. fluorescens. Possible routes of AAP production are discussed relative to current knowledge of the phenazine biosynthetic pathway of strain 2-79. The pertinence of AAP to the design of commercial seed inoculants of phenazine-producing bacteria for controlling wheat take-all is also considered. Received: 2 November 1999 / Received revision: 3 April 2000 / Accepted: 14 April 2000     

Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.     

Role of ptsP, orfT, and sss recombinase genes in root colonization by Pseudomonas fluorescens Q8r1-96

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.     

Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84.

Pseudomonas aureofaciens strain 30-84 suppresses take-all disease of wheat caused by Gaeumannomyces graminis var. tritici. Three antibiotics, phenazine-1-carboxylic acid, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine, were responsible for disease suppression. Tn5-induced mutants deficient in production of one or more of the antibiotics (Phz-) were significantly less suppressive than the parental strain. Cosmids pLSP259 and pLSP282 from a genomic library of strain 30-84 restored phenazine production and fungal inhibition to 10 different Phz- mutants. Sequences required for production of the phenazines were localized to a segment of approximately 2.8 kilobases that was present in both cosmids. Expression of this locus in Escherichia coli required the introduction of a functional promoter, was orientation-specific, and resulted in the production of all three phenazine antibiotics. These results strongly suggest that the cloned sequences encode a major portion of the phenazine biosynthetic pathway.     

Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. fluorescens strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed(-1) or soil(-1)) to establish high rhizosphere population densities (10(7) CFU g of root(-1)) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10(5) CFU g of root(-1) after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Population Structure and Diversity of Phenazine-1-Carboxylic Acid Producing Fluorescent Pseudomonas spp. from Dryland Cereal Fields of Central Washington State (USA)

Certain strains of the rhizosphere bacterium Pseudomonas fluorescens contain the phenazine biosynthesis operon (phzABCDEFG) and produce redox-active phenazine antibiotics that suppress a wide variety of soilborne plant pathogens. In 2007 and 2008, we isolated 412 phenazine-producing (Phz(+)) fluorescent Pseudomonas strains from roots of dryland wheat and barley grown in the low-precipitation region (<350 mm annual precipitation) of central Washington State. Based on results of BOX-PCR genomic fingerprinting analysis, these isolates, as well as the model biocontrol Phz(+) strain P. fluorescens 2-79, were assigned to 31 distinct genotypes separated into four clusters. All of the isolates exhibited high 16S rDNA sequence similarity to members of the P. fluorescens species complex including Pseudomonas orientalis, Pseudomonas gessardii, Pseudomonas libanensis, and Pseudomonas synxantha. Further recA-based sequence analyses revealed that the majority of new Phz(+) isolates (386 of 413) form a clade distinctly separated from P. fluorescens 2-79. Analysis of phzF alleles, however, revealed that the majority of those isolates (280 of 386) carried phenazine biosynthesis genes similar to those of P. fluorescens 2-79. phzF-based analyses also revealed that phenazine genes were under purifying selection and showed evidence of intracluster recombination. Phenotypic analyses using Biolog substrate utilization and observations of phenazine-1-carboxylic acid production showed considerable variability amongst members of all four clusters. Biodiversity indices indicated significant differences in diversity and evenness between the sampled sites. In summary, this study revealed a genotypically and phenotypically diverse group of phenazine producers with a population structure not seen before in indigenous rhizosphere-inhabiting Phz(+) Pseudomonas spp.     

The siderophores of Pseudomonas fluorescens 18.1 and the importance of cyclopeptidic substructures for the recognition at the cell surface

The structure of the pyoverdin siderophore of Pseudomonas fluorescens 18.1 was elucidated by spectroscopic methods and chemical degradation. By cross feeding studies structurally closely related pyoverdins containing a C-terminal cyclopeptidic substructure were tested regarding the mutual recognition by the producing strains. Partial recognition of foreign pyoverdins was observed.     

Mechanisms of natural soil suppressiveness to soilborne diseases

Suppressive soils are characterized by a very low level of disease development even though a virulent pathogen and susceptible host are present. Biotic and abiotic elements of the soil environment contribute to suppressiveness, however most defined systems have identified biological elements as primary factors in disease suppression. Many soils possess similarities with regard to microorganisms involved in disease suppression, while other attributes are unique to specific pathogen-suppressive soil systems. The organisms operative in pathogen suppression do so via diverse mechanisms including competition for nutrients, antibiosis and induction of host resistance. Non-pathogenic Fusarium spp. and fluorescent Pseudomonas spp. play a critical role in naturally occurring soils that are suppressive to Fusarium wilt. Suppression of take-all of wheat, caused by Gaeumannomyces graminis var. tritici, is induced in soil after continuous wheat monoculture and is attributed, in part, to selection of fluorescent pseudomonads with capacity to produce the antibiotic 2,4-diacetylphloroglucinol. Cultivation of orchard soils with specific wheat varieties induces suppressiveness to Rhizoctonia root rot of apple caused by Rhizoctonia solani AG 5. Wheat cultivars that stimulate disease suppression enhance populations of specific fluorescent pseudomonad genotypes with antagonistic activity toward this pathogen. Methods that transform resident microbial communities in a manner which induces natural soil suppressiveness have potential as components of environmentally sustainable systems for management of soilborne plant pathogens. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of fluorescent and nonfluorescent peptide siderophores produced by Pseudomonas syringae strains and their potential use in strain identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain.

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.