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Pseudomonas brassicacearum and related species of the P. fluorescens complex have long been studied as biocontrol and growth-promoting rhizobacteria involved in suppression of soilborne pathogens. We report here that P. brassicacearum Q8r1-96 and other 2,4-diacetylphloroglucinol (DAPG)-producing fluorescent pseudomonads involved in take-all decline of wheat in the Pacific Northwest of the United States can also be pathogenic to other plant hosts. Strain Q8r1-96 caused necrosis when injected into tomato stems and immature tomato fruits, either attached or removed from the plant, but lesion development was dose dependent, with a minimum of 106 CFU ml−1 required to cause visible tissue damage. We explored the relative contribution of several known plant-microbe interaction traits to the pathogenicity of strain Q8r1-96. Type III secretion system (T3SS) mutants of Q8r1-96, injected at a concentration of 108 CFU ml−1, were significantly less virulent, but not consistently, as compared with the wild-type strain. However, a DAPG-deficient phlD mutant of Q8r1-96 was significantly and consistently less virulent as compared with the wild type. Strain Q8r1-96acc, engineered to over express ACC deaminase, caused a similar amount of necrosis as the wild type. Cell-free culture filtrates of strain Q8r1-96 and pure DAPG also cause necrosis in tomato fruits. Our results suggest that DAPG plays a significant role in the ability of Q8r1-96 to cause necrosis of tomato tissue, but other factors also contribute to the pathogenic properties of this organism.

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular-weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialogue between eight well-characterized beneficial strains of thePseudomonas fluorescensgroup andBrachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates ofB. distachyonand demonstrated the presence of multiple carbohydrates, amino acids, organic acids and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for thePseudomonasstrains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains’ pangenome, reflecting the importance of the variable gene content in the adaptation ofP. fluorescensto the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.

Plants live in association with microorganisms that positively influence plant development, vigor, and fitness in response to pathogens and abiotic stressors. The bulk of the plant microbiome is concentrated belowground at the plant root-soil interface. Plant roots secrete carbon-rich rhizodeposits containing primary and secondary low molecular weight metabolites, lysates, and mucilages. These exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. We addressed this gap by focusing on the molecular dialog between eight well-characterized beneficial strains of thePseudomonas fluorescensgroup andBrachypodium distachyon, a model for economically important food, feed, forage, and biomass crops of the grass family. We collected and analyzed root exudates ofB. distachyonand demonstrated the presence of multiple carbohydrates, amino acids, organic acids, and phenolic compounds. The subsequent screening of bacteria by Biolog Phenotype MicroArrays revealed that many of these metabolites provide carbon and energy for thePseudomonasstrains. RNA-seq profiling of bacterial cultures amended with root exudates revealed changes in the expression of genes encoding numerous catabolic and anabolic enzymes, transporters, transcriptional regulators, stress response, and conserved hypothetical proteins. Almost half of the differentially expressed genes mapped to the variable part of the strains’ pangenome, reflecting the importance of the variable gene content in the adaptation ofP. fluorescensto the rhizosphere lifestyle. Our results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant hosts.

The primary mechanism of biocontrol by Pseudomonas fluorescens strains HC1–07 and HC9–07 is production of a cyclic lipopeptide (CLP) and phenazine-1-carboxylic acid, respectively. We introduced the seven-gene operon for the synthesis of phenazine-1-carboxylic acid (PCA) from P. synxantha 2–79 into Pseudomonas fluorescens HC1–07rif to determine if the biocontrol activity of the recombinant strain HC1–07PHZ increased against Gaeumannomyces graminis var. tritici, causal agent of take-all disease of wheat. In vitro inhibition assays showed that strain HC1–07PHZ consistently inhibited the hyphal growth of three isolates of the take-all pathogen on plates of both PDA and KMB, and the recombinant consistently inhibited G. graminis. var. tritici to a greater extent than the wild type. Strain HC1–07PHZ applied at a dose of 102 CFU seed−1 suppressed take-all better than strains HC1–07rif and HC9–07rif applied either individually or in combination. When the dose of the bacteria was increased to 104 CFU seed−1, the strain combination HC1–07rif + HC9–07rif showed significantly better disease suppression than did HC1–07rif, HC9–07rif or HC1–07PHZ applied individually. However, when the bacterial dose was increased to 107 CFU seed−1, strains HC1–07rif and HC1–07PHZ showed significantly better disease suppression than HC9–07rif and the combination HC1–07rif + HC9–07rif.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence.

Take-all disease, caused by Gaeumannomyces graminis var. tritici (Ggt), is one of the most serious root diseases in wheat production. In this study, a proteomic platform based on 2-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Tandem Mass Spectrometry (MALDI-TOF/TOF MS) was used to construct the first proteome database reference map of G. graminis var. tritici and to identify the response of the pathogen to 2,4-diacetylphloroglucinol (DAPG), which is a natural antibiotic produced by antagonistic Pseudomonas spp. in take-all suppressive soils. For mapping, a total of 240 spots was identified that represented 209 different proteins. The most abundant biological function categories in the Ggt proteome were related to carbohydrate metabolism (21%), amino acid metabolism (15%), protein folding and degradation (12%), translation (11%), and stress response (10%). In total, 51 Ggt proteins were affected by DAPG treatment. Based on gene ontology, carbohydrate metabolism, amino acid metabolism, stress response, and protein folding and degradation proteins were the ones most modulated by DAPG treatment. This study provides the first extensive proteomic reference map constructed for Ggt and represents the first time that the response of Ggt to DAPG has been characterized at the proteomic level.

Bacteria in the diverse Pseudomonas fluorescens group include rhizosphere inhabitants known for their antifungal metabolite production and biological control of plant disease, such as Pseudomonas protegens Pf-5, and mushroom pathogens, such as Pseudomonas tolaasii. Here, we report that strain Pf-5 causes brown, sunken lesions on peeled caps of the button mushroom (Agaricus bisporus) that resemble brown blotch symptoms caused by P. tolaasii. Strain Pf-5 produces six known antifungal metabolites under the control of the GacS/GacA signal transduction system. A gacA mutant produces none of these metabolites and did not cause lesions on mushroom caps. Mutants deficient in the biosynthesis of the antifungal metabolites 2,4-diacetylphloroglucinol and pyoluteorin caused less-severe symptoms than wild-type Pf-5 on peeled mushroom caps, whereas mutants deficient in the production of lipopeptide orfamide A caused similar symptoms to wild-type Pf-5. Purified pyoluteorin and 2,4-diacetylphloroglucinol mimicked the symptoms caused by Pf-5. Both compounds were isolated from mushroom tissue inoculated with Pf-5, providing direct evidence for their in situ production by the bacterium. Although the lipopeptide tolaasin is responsible for brown blotch of mushroom caused by P. tolaasii, P. protegens Pf-5 caused brown blotch–like symptoms on peeled mushroom caps through a lipopeptide-independent mechanism involving the production of 2,4-diacetylphloroglucinol and pyoluteorin.

Take-all, caused by Gaeumannomyces graminis var. tritici, is an important soilborne disease of wheat worldwide. Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents of take-all and provide natural suppression of the disease during wheat monoculture known as take-all decline. To identify factors that could contribute to the effectiveness of 2,4-DAPG producers in take-all suppression, P. fluorescens strains Q8r1-96 (genotype D) and Q2-87V1 (genotype B; reduced antibiotic production) were tested against three pathogen isolates differing in sensitivity to 2,4-DAPG (LD5, ARS-A1 and R3-111a-1) and two wheat cultivars (Tara and Buchanan). The antibiotic sensitivity of the pathogen and cultivar significantly affected the level of take-all suppression by Q8r1-96 and Q2-87V1; suppression was greatest with LD5 and Tara. Q8r1-96 suppressed ARS-A1 and R3-111a-1 on Tara but not Buchanan, and Q2-87V1 failed to suppress either pathogen isolate on either cultivar. Q8r1-96 colonized the rhizosphere of Tara and Buchanan grown in soil similarly, but 2,4-DAPG accumulation was higher on the roots of Buchanan than Tara. 2,4-DAPG at 7.5 μg mL−1 reduced the growth of roots of both cultivars, and 10 μg mL−1 caused brown necrosis and tissue collapse of seedling roots and reduced root hair development. The half-life of 2,4-DAPG in the rhizosphere was estimated to be 0.25 days. These results suggest that several interconnected factors including sensitivity of G. graminis var. tritici to 2,4-DAPG, wheat cultivar, fluctuations in populations of 2,4-DAPG producers, and antibiotics accumulation in the rhizosphere will impact the robustness of take-all suppression by P. fluorescens in the field.

Pseudomonas fluorescens 2112, isolated in Korea as an indigenous antagonistic bacteria, can produce 2,4- diacetylphloroglucinol (2,4-DAPG) and the siderophore pyoveridin2112 for the control of phytophthora blight of red-pepper. P. fluorescens 2112 was classified into a new genotype C among the 17 genotypes of 2,4-DAPG producers, by phlD restriction fragment length polymorphism (RFLP). The colonizing ability of P. fluorescens 2112 in pea rhizosphere was equal to the well-known pea colonizers, P. fluorescens Q8r1 (genotype D) and MVP1-4 (genotype P), after 6 cycling cultivations for 18 weeks. Four tested 2,4- DAPG-producing Pseudomonas spp. could colonize with about a 96% dominance ratio against total bacteria in pea rhizosphere. The strain P. fluorescens 2112 was as good a colonizer as other Pseudomonas spp. genotypes in pea plant growth-promoting rhizobacteria.

Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto King's medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.

Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae . We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1 - 1 and hopM1 , as well as a novel type III effector gene designated ropB . These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses.

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation −1 and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.

Pseudomonas fluorescens strain 2-79, a natural isolate of the rhizosphere of wheat ( Triticum aestivum L.), possesses antagonistic potential toward several fungal pathogens. We report the draft genome sequence of strain 2-79, which comprises 5,674 protein-coding sequences.

Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A–Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in 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-diacetylphloroglucinol producers in natural environments.

Certain well-conserved genes in fluorescent Pseudomonas spp. are involved in pathogenic interactions between the bacteria and evolutionarily diverse hosts including plants, insects and vertebrate animals. One such gene, dsbA, encodes a periplasmic disulfide-bond-forming enzyme implicated in the biogenesis of exported proteins and cell surface structures. This study focused on the role of dsbA in Pseudomonas fluorescens Q8r1-96, a biological control strain that produces the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) and is known for its exceptional ability to colonize the roots of wheat and pea. The deduced DsbA protein from Q8r1-96 is similar to other predicted thiol : disulfide interchange proteins and contains a conserved DsbA catalytic site, a pattern associated with the thioredoxin family active site, and a signal peptide and cleavage site. A dsbA mutant of Q8r1-96 exhibited decreased motility and fluorescence, and altered colony morphology; however, it produced more 2,4-DAPG and total phloroglucinol-related compounds and was more inhibitory in vitro to the fungal root pathogen Gaeumannomyces graminis var. tritici than was the parental strain. When introduced separately into a natural soil, Q8r1-96 and the dsbA mutant did not differ in their ability to colonize the rhizosphere of wheat in greenhouse experiments lasting 12 weeks. However, when the two strains were co-inoculated, the parental strain consistently out-competed the dsbA mutant. It was concluded that dsbA does not contribute to the exceptional rhizosphere competence of Q8r1-96, although the dsbA mutation reduces competitiveness when the mutant competes with the parental strain in the same niche in the rhizosphere. The results also suggest that exoenzymes and multimeric cell surface structures are unlikely to have a critical role in root colonization by this strain.

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.Key words: biocontrol, 2,4-diacetylphloroglucinol, Pseudomonas fluorescens, rice, Xanthomonas oryzae pv. oryzae.

Many pseudomonads, including the generalist Pseudomonas aeruginosa and fluorescent soil forms, such as Pseudomonas fluorescens, Pseudomonas aureofaciens, and Pseudomonas chlororaphis, produce phenazine-1-carboxylic acid (PCA) or its derivatives (7, 11, 49). These secondary metabolites exhibit antimicrobial activity against a wide range of prokaryotic and eukaryotic microbes, and the synthesis and secretion of these compounds is thought to confer an advantage on the producer bacteria when in competition with other microflora in the soil habitat (29). Production of substituted phenazines is also required by these bacteria to fully protect plants against diseases caused by fungal and bacterial pathogens that are susceptible to the antibiotics (2, 7, 9, 33, 49). Such biocontrol properties are of considerable interest, since their application in the field could reduce reliance on the use of environmentally toxic antimicrobial chemicals. However, for such strategies to be successful, the antibiotics must be synthesized reliably in the appropriate environmental sites. Hence, it is important to identify the genetic determinants required for synthesis of the phenazines, as well as the mechanisms by which expression of these genes is regulated. The phz cluster is organized as an operon of seven genes, the products of which catalyze the synthesis of chorismate and its conversion to PCA (9, 27, 28, 35). This parent compound can be substituted at several positions, depending upon the producer strain, and the genes coding for the enzymes involved in such substitutions are restricted to those strains producing the modified compounds. While gene nomenclature differs with the isolate (for an example, see reference 27), the order of the seven genes and the amino acid sequences of their products are conserved in all phz operons examined to date (9, 27, 28, 35). Although regulation is complex and multifactorial, the phz operons of P. aureofaciens 30-84 and P. chlororaphis PCL1391 are controlled at the transcriptional level by a ligand-dependent transcription factor called PhzR (8, 34). This activator is a member of the LuxR family and, as such, requires a low-molecular-weight signal, an acyl-homoserine lactone (acyl-HSL), for activity (8, 56). Signal production in both pseudomonads is catalyzed by PhzI, an acyl-HSL synthase of the LuxI family (8, 56). The acyl-HSL most probably transits out of and back into the cell by diffusion and thus accumulates within the environment as the population size of the dividing bacteria increases. When the concentration of the signal reaches a critical level, which corresponds to a particular population size of the bacteria, it interacts with and activates PhzR, thus initiating expression of the phz operon. In P. aureofaciens 30-84 and P. chlororaphis PCL1391, phzR and phzI are adjacent and convergently oriented, with phzR being located just upstream of and in opposite orientation to the phz operon (8, 56). Similarly, the phz operon of P. fluorescens 2-79 is preceded by phzR and phzI homologs organized in identical manners (Fig. ​(Fig.1).1). Moreover, the three phzR genes and the three phzI genes, as well as their encoded protein products, are all strongly orthologous (8). FIG. 1. Gene structure of the phz locus from P. fluorescens 2-79 and its mutants. (A) The organizations of the phz operon and the two regulatory genes phzR and phzI are shown by arrows. Alterations in the three mutants—strain 2-79IR, in which phzR and ... Strain 2-79 has served as a model for the genetics, biochemistry, and enzymology of phenazine biosynthesis. We previously reported that this isolate produces at least six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-l-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-l-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL); and an active signal compound of unknown structure (45). However, P. aureofaciens 30-84 and P. chlororaphis PCL1391 both have been reported to produce and utilize the alkanoyl signal, C6-HSL (8, 57). Moreover, the last two strains have not been reported to produce 3-hydroxy-acyl-HSLs. Given the very close amino acid sequence relatedness of the PhzI and PhzR proteins from these three strains, it became important to determine what acyl-HSL signals are produced by PhzI of P. fluorescens 2-79 and which of these signals activates PhzR of this strain. In this report, we show that PhzI catalyzes synthesis of all of the 3-OH- and alkanoyl-acyl-HSLs produced by P. fluorescens 2-79. We also show that 3-OH-C6 accumulates to the highest concentration in culture supernatants and that PhzR from strain 2-79 responds with greatest sensitivity to this signal. In addition, we show that activation of the phz operon by PhzR is dependent upon an intact inverted repeat, the phz box, located in the divergent phzA-phzR promoter region and that PhzR weakly autoactivates its own transcription, also in a phz box-dependent manner.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5. Supplementary information The online version of this article (doi:10.1038/nbt1110) contains supplementary material, which is available to authorized users.

A seven-gene operon for the synthesis of phenazine-1-carboxylic acid was introduced into Pseudomonas fluorescens Q8r1-96, an aggressive root colonizer that produces 2,4-diacetylphloroglucinol and consistently suppresses take-all of wheat. The recombinant strains produced both antifungal metabolites and maintained population sizes comparable to those of Q8r1-96 over a seven-week period in the rhizosphere of wheat. The strains were no more suppressive of take-all or Pythium root rot than was Q8r1-96, but suppressed Rhizoctonia root rot at a dose of only 10(2) CFU per seed, one to two orders of magnitude lower than the dose of Q8r1-96 required for comparable disease control.

The efficient use of rhizospheric microorganisms to control plant pathogens has been reported worldwide in different plants. Pseudomonas fluorescens UP61 is a biocontrol strain isolated from the rhizosphere of Lotus corniculatus(birdsfoot trefoil) from Uruguayan soils. This strain exhibited in vitro antagonistic activity against a broad spectrum of fungal and bacterial phytopathogens. It was an effective biocontrol agent in different hosts, reducing the disease incidence caused by Sclerotium rolfsii in beans and Rhizoctonia solani in tomato. P. fluorescens UP61 produced three antibiotics possibly involved in its biocontrol activity: 2,4-diacetylphloroglucinol, pyrrolnitrin and pyoluteorin. Molecular techniques such as 16S rDNA RFLP, RAPD and rep-PCR, and partial sequence of the phlD gene, revealed the similarity of UP61 with other biocontrol strains isolated worldwide that are able to produce these antibiotics.

Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.

Pseudomonas fluorescens HC1-07, previously isolated from the phyllosphere of wheat grown in Hebei province, China, suppresses the soilborne disease of wheat take-all, caused by Gaeumannomyces graminis var. tritici. We report here that strain HC1-07 also suppresses Rhizoctonia root rot of wheat caused by Rhizoctonia solani AG-8. Strain HC1-07 produced a cyclic lipopeptide (CLP) with a molecular weight of 1,126.42 based on analysis by electrospray ionization mass spectrometry. Extracted CLP inhibited the growth of G. graminis var. tritici and R. solani in vitro. To determine the role of this CLP in biological control, plasposon mutagenesis was used to generate two nonproducing mutants, HC1-07viscB and HC1-07prtR2. Analysis of regions flanking plasposon insertions in HC1-07prtR2 and HC1-07viscB revealed that the inactivated genes were similar to prtR and viscB, respectively, of the well-described biocontrol strain P. fluorescens SBW25 that produces the CLP viscosin. Both genes in HC1-07 were required for the production of the viscosin-like CLP. The two mutants were less inhibitory to G. graminis var. tritici and R. solani in vitro and reduced in ability to suppress take-all. HC1-07viscB but not HC-07prtR2 was reduced in ability to suppress Rhizoctonia root rot. In addition to CLP production, prtR also played a role in protease production.

Phenazine compounds produced by fluorescent Pseudomonas species are biologically active metabolites that function in microbial competitiveness (37), the suppression of soilborne plant pathogens (1, 11, 55, 56), and virulence in human and animal hosts (35). The most widely studied phenazine-producing fluorescent pseudomonad is P. aeruginosa, a gram-negative opportunistic pathogen of animals, insects, nematodes, and plants (30, 33, 35, 46). In humans, P. aeruginosa infects immunocompromised, burned, or injured patients and can cause both acute and chronic lung disease. Strains of P. aeruginosa produce a variety of redox-active phenazine compounds, including pyocyanin, phenazine-1-carboxylic acid (PCA), 1-hydroxyphenazine (1-OH-PHZ), and phenazine-1-carboxamide (PCN) (7, 52, 57). From 90 to 95% of P. aeruginosa isolates produce pyocyanin (52), and the presence of high concentrations of pyocyanin in the sputum of cystic fibrosis patients has suggested that this compound plays a role in pulmonary tissue damage observed with chronic lung infections (64). This idea is supported by several recent studies which demonstrated that pyocyanin contributes in a variety of ways to the pathophysiological effects observed in airways infected by P. aeruginosa. Pyocyanin interferes with the regulation of ion transport, ciliary beat frequency, and mucus secretion in airway epithelial cells by altering the cytosolic concentration of calcium (15). It may interact with endothelium-derived relaxing factor or with nitric oxide (which plays a central role in the control of blood pressure, blood flow, and immune function) through the formation of a complex, or it may act by inhibition of nitric oxide synthase (29, 58, 59). Phenazines that are produced by P. aeruginosa also can stimulate alveolar macrophages to produce two neutrophil chemotaxins, IL-8 and leukotriene B4, that attract neutrophils into airways, causing an inflammatory response and neutrophil-mediated tissue damage (14, 33). The unusually broad range of biological activity associated with phenazines is thought to be due to their ability to undergo redox cycling in the presence of various reducing agents and molecular oxygen, which leads to the accumulation of toxic superoxide (O2−) and hydrogen peroxide (H2O2) and eventually to oxidative cell injury or death (6, 25). It also has been shown that pyocyanin can interact synergistically with the siderophore pyochelin and with transferrin cleaved by proteases secreted by both P. aeruginosa and neutrophils in infected lungs to catalyze the formation of the highly cytotoxic hydroxyl radical (·OH), which damages pulmonary endothelial cells (6, 38). In model pathogenesis systems, phenazine synthesis by P. aeruginosa is required for the generation of disease symptoms in plants and for effective killing of the nematode Caenorhabditis elegans and the greater wax moth, Galleria mellonella (30, 35, 46). Phenazine compounds produced in the rhizosphere of plants contribute to the biological control activity of P. aeruginosa against Fusarium wilt of chickpea and Pythium damping-off of bean (1). Although the pathophysiological effects of phenazines produced by P. aeruginosa in host organisms are well studied (6, 14, 15, 33, 34, 38, 64) and pyocyanin-deficient phenotypes frequently have been described (18, 19, 26, 32, 35, 46, 54), the biochemistry and genetics of phenazine synthesis in P. aeruginosa have remained unclear. We describe here cloning and functional analysis of two seven-gene phenazine operons and three phenazine-modifying genes from P. aeruginosa PAO1. Our results show that P. aeruginosa contains a complex phenazine biosynthetic pathway consisting of two homologous core loci (phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2) responsible for synthesis of PCA and three additional genes (phzM, phzS, and phzH) encoding unique enzymes involved in the conversion of PCA to pyocyanin and PCN. We detected the core biosynthetic operon by Southern hybridization in 21 phenazine-producing pseudomonads, including strains of P. aeruginosa, Pseudomonas fluorescens, Pseudomonas chlororaphis, and Pseudomonas aureofaciens, but not in seven phenazine-producing isolates of Burkholderia cepacia, Burkholderia phenazinium, and Brevibacterium iodinum. Thus, the core biosynthetic pathway is highly conserved in fluorescent Pseudomonas spp. but differs significantly from that in other phenazine-producing bacterial genera.

A disarmed Tn5 vector (pUT∷Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P. fluorescens∷phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which ( phlACBD ) comprise an operon that includes a set of three genes ( phlACB ) conserved between eubacteria and archaebacteria and a gene ( phlD ) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF , which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA , phlC , phlB , and phlD , individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.

Pseudomonas fluorescens 2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated phzABCDEFG that are sufficient for synthesis of PCA were localized within a 6.8-kb BglII-XbaI fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all phz genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the phzC, phzD, and phzE genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5*-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the phzA and phzB genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes, phzX and phzY, that are homologous to phzA and phzB, respectively, were cloned and sequenced from P. aureofaciens 30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the phz genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA. Certain members of the genus Pseudomonas produce diverse low-molecular-weight (“secondary”) metabolites including nitrogen-containing heterocyclic pigments known as phenazine compounds (5, 19). Phenazines are synthesized by a limited number of bacterial genera including Pseudomonas, Burkholderia, Brevibacterium, and Streptomyces (38). Almost all phenazines exhibit broad-spectrum activity against various species of bacteria and fungi (32). This activity is connected with the ability of phenazine compounds to undergo oxidation-reduction transformations and thus cause the accumulation of toxic superoxide radicals in the target cells (15). Some phenazine compounds can act as bacterial virulence factors. For example, pyocyanin, produced by the opportunistic pathogen Pseudomonas aeruginosa during cystic fibrosis, has been shown to inhibit the ciliary function of respiratory epithelial cells (40). Phenazine antibiotics produced by the biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 are major factors in the ability of these strains to inhibit the growth of fungal root pathogens. Moreover, studies involving phenazine-deficient mutants have clearly demonstrated that antibiotic production in natural habitats plays an important role in the ecological competence and long-term survival of these strains in the environment (21). Over 50 naturally occurring phenazine compounds have been described, and certain bacterial producers are able to synthesize mixtures of as many as 10 different phenazine derivatives at one time (32, 38). Growth conditions

Conjugative plasmids are known to facilitate the acquisition and dispersal of genes contributing to the fitness of Pseudomonas spp. Here, we report the characterization of pA506, the 57-kb conjugative plasmid of Pseudomonas fluorescens A506, a plant epiphyte used in the United States for the biological control of fire blight disease of pear and apple. Twenty-nine of the 67 open reading frames (ORFs) of pA506 have putative functions in conjugation, including a type IV secretion system related to that of MOB P6 family plasmids and a gene cluster for type IV pili. We demonstrate that pA506 is self-transmissible via conjugation between A506 and strains of Pseudomonas spp. or the Enterobacteriaceae . The origin of vegetative replication ( oriV ) of pA506 is typical of those in pPT23A family plasmids, which are present in many pathovars of Pseudomonas syringae , but pA506 lacks repA , a defining locus for pPT23A plasmids, and has a novel partitioning region. We selected a plasmid-cured derivative of A506 and compared it to the wild type to identify plasmid-encoded phenotypes. pA506 conferred UV resistance, presumably due to the plasmid-borne rulAB genes, but did not influence epiphytic fitness of A506 on pear or apple blossoms in the field. pA506 does not appear to confer resistance to antibiotics or other toxic elements. Based on the conjugative nature of pA506 and the large number of its genes that are shared with plasmids from diverse groups of environmental bacteria, the plasmid is likely to serve as a vehicle for genetic exchange between A506 and its coinhabitants on plant surfaces.

Strain 2–79 is a biocontrol agent of take-all, an important root disease of wheat caused by Gaeumannomyces graminis var. tritici. In the rhizosphere, strain 2–79 produces the antibiotic phenazine-1-carboxylic acid as the primary means of disease suppression. Barriers to the commercial use of phenazine-producing pseudomonads, such as strain 2–79, include the lack of liquid-culture and formulation technologies needed to optimize cost-effective mass production and application. For instance, there is little published research concerning the impact of growth culture physiological state and associated metabolites on the biocontrol qualities of the cells harvested and formulated in seed coatings, i.e., efficacy, phytotoxicity, and storage survival. To enable exploration of these issues, cells of strain 2–79 in various physiological states were obtained by harvesting fermentors at 24-h intervals after inoculation. Cells formulated in 0.5% methylcellulose suspended in either water (MW) or metabolite-bearing, spent culture broth (MSB) were applied as wheat-seed coatings, air dried, and stored at 4°C. Younger cells (24–48 h) had twice the drying survival rate but only half of the storage life demonstrated by older cells (72–96 h) (P?0.05). Cell populations surviving drying were 3.5 times higher in MW than in MSB formulations and they remained viable up to 3 times longer (P?0.05). This effect of formulation on viability was attributable to the culture nutrients but not the metabolites present in the spent broth. Disease suppression in bacterized seed treatments was significant (P?0.05) relative to unbacterized controls and averaged 9.1%, but did not vary significantly (P?0.24) with culture age, encapsulation medium, or storage time. Relative seedling height improvement increased with relative disease suppression (P=0.003) and significantly decreased with lengthening storage time (P=0.004). This latter decline in plant growth promotion coincided with the deterioration of biocontrol agent viability during storage. Seed batches inoculated with cells in both MW and MSB encapsulations suffered significant germination losses due to phytotoxic metabolites. The extent of loss was an interactive result of encapsulation medium and storage time (P?0.01), and the rate of loss was much higher for seeds with MSB than with MW coatings, i.e. 54% compared to 11% loss after 6 months storage.

Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases. "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host. Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var. tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat. The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria. Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline. There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes. Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol. The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens.

Phenazines produced by members of several bacterial genera are biologically active metabolites that function in microbial competitiveness, the suppression of soil-borne plant diseases and virulence in infectious disease. Despite recent progress towards understanding the biochemistry of phenazine synthesis, the key reactions leading to the formation of the heterocyclic scaffold common to all phenazine compounds remain obscure. Pseudomonas fluorescens 2-79 contains seven phenazine (phz) genes that encode components of the pathway for biosynthesis of phenazine-1-carboxylic acid. A central step in this pathway involves the condensation of two identical precursor molecules derived from chorismic acid and is catalysed by the product of the phzF gene. In this study, recombinant PhzF was purified and crystallized from PEG 4000/ammonium sulfate/sodium citrate pH 5.6. The crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 56.3, c = 156.4 A. They contain one monomer in the asymmetric unit and diffract to better than 1.7 A on synchrotron beamlines. Crystals of seleno-L-methionine-labelled PhzF have been obtained and SAD data are reported.

Phenazines are redox-active secondary metabolites that many bacteria produce and secrete into the environment. They are broad-specificity antibiotics, but also act as virulence and survival factors in infectious diseases. Phenazines are derived from chorismic acid, but important details of their biosynthesis are still unclear. For example, three two-electron oxidations seem to be necessary in the final steps of the pathway, while only one oxidase, the FMN-dependent PhzG, is conserved in the phenazine-biosynthesis phz operon. Here, crystal structures of PhzG from Pseudomonas fluorescens 2-79 and from Burkholderia lata 383 in complex with excess FMN and with the phenazine-biosynthesis intermediates hexahydrophen­azine-1,6-dicarboxylate and tetrahydrophenazine-1-carboxyl­ate generated in situ are reported. Corroborated with biochemical data, these complexes demonstrate that PhzG is the terminal enzyme in phenazine biosynthesis and that its relaxed substrate specificity lets it participate in the generation of both phenazine-1,6-dicarboxylic acid (PDC) and phen­azine-1-carboxylic acid (PCA). This suggests that competition between flavin-dependent oxidations through PhzG and spontaneous oxidative decarboxylations determines the ratio of PDC, PCA and unsubstituted phenazine as the products of phenazine biosynthesis. Further, the results indicate that PhzG synthesizes phenazines in their reduced form. These reduced molecules, and not the fully aromatized derivatives, are the likely end products in vivo, explaining why only one oxidase is required in the phenazine-biosynthesis pathway.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire., Author Summary We sequenced the genomes of seven strains of the Pseudomonas fluorescens group that colonize plant surfaces and function as biological control agents, protecting plants from disease. In this study, we demonstrated the genomic diversity of the group by comparing these strains to each other and to three other strains that were sequenced previously. Only about half of the genes in each strain are present in all of the other strains, and each strain has hundreds of unique genes that are not present in the other genomes. We mapped the genes that contribute to biological control in each genome and found that most of the biological control genes are in the variable regions of the genome, which are not shared by all of the other strains. This finding is consistent with our knowledge of the distinctive biology of each strain. Finally, we looked for new genes that are likely to confer antimicrobial traits needed to suppress plant pathogens, but have not been identified previously. In each genome, we discovered many of these new genes, which provide avenues for future discovery of new traits with the potential to manage plant diseases in agriculture or natural ecosystems.

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 (

2,4-Diacetylphloroglucinol (2,4-DAPG), an antibiotic produced by Pseudomonas fluorescens , has broad-spectrum antibiotic activity, inhibiting organisms ranging from viruses, bacteria, and fungi to higher plants and mammalian cells. The biosynthesis and regulation of 2,4-DAPG in P. fluorescens are well described, but the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a deletion library of Saccharomyces cerevisiae in broth and agar medium supplemented with 2,4-DAPG. We identified 231 mutants that showed increased sensitivity to 2,4-DAPG under both conditions, including 22 multidrug resistance-related mutants. Three major physiological functions correlated with an increase in sensitivity to 2,4-DAPG: membrane function, reactive oxygen regulation, and cell homeostasis. Physiological studies with wild-type yeast validated the results of the mutant screens. The chemical-genetic fitness profile of 2,4-DAPG resembled those of menthol, sodium azide, and hydrogen peroxide determined in previous high-throughput screening studies. Collectively, these findings indicate that 2,4-DAPG acts on multiple basic cellular processes.

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.

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.

Strains of Pseudomonas fluorescens that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are responsible for the natural suppression of take-all diseases of wheat (Gaeumannomyces graminis var. tritici), known as take-all decline (TAD) (34, 42, 43). These bacteria also provide biological control of many root and seedling diseases on a variety of crops (6, 13, 32, 39). Because 2,4-DAPG producers occur in soils worldwide (5, 14, 25) and have such an important role in plant defense, the genotypic and phenotypic diversity of these strains has been studied extensively. Phylogenetic comparisons based on amplified rRNA gene restriction analysis of 16S rRNA genes revealed three distinct lineages among 2,4-DAPG-producing Pseudomonas fluorescens strains (14, 25). A finer degree of variation was defined by genomic fingerprinting utilizing randomly amplified polymorphic DNA analysis (14, 21, 31, 33) or repetitive sequence-based PCR (18, 25) and by restriction fragment length polymorphism (RFLP) (18, 19, 21, 25), DNA sequence (5, 36), and denaturing gradient gel electrophoresis (2) analyses of phlD, a key gene in the biosynthesis of 2,4-DAPG. The terms “phlD+ fluorescent Pseudomonas spp.” and “2,4-DAPG producer” have been used synonymously because the detection of phlD correlates with the production of 2,4-DAPG by Pseudomonas spp. (42). Genotypes defined by RFLP and sequence analysis of phlD correlated closely with clusters defined by BOX-PCR of total genomic DNA, validating the utility of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers (5, 21). To date, these techniques have distinguished at least 22 different genotypes, designated as A to T, PfY, and PfZ (19, 22, 23, 42). Although most strains from different genotypes are phenotypically similar (25, 33), they differ considerably in their rhizosphere competence (4, 17-19, 33), and it appears that certain genotypes and crop species have a mutual preference or “affinity” for each other (19, 42). For example, genotype D strains have a preference for wheat (17, 33) and pea (18), which accounts for their dominance in Washington State soils that contain multiple genotypes but have undergone continuous wheat or pea monoculture. By understanding the population size and genotype composition of 2,4-DAPG-producing strains of P. fluorescens in a soil, it is possible to predict the soil's suppressiveness to certain soilborne plant pathogens (42). Two approaches have been used extensively for the quantification of 2,4-DAPG producers in situ: colony hybridization followed by confirmatory PCR with phlD-specific probes and primers (2, 35) and the phlD-specific PCR-based dilution endpoint assay (dilution endpoint assay) (19, 24). Landa et al. (16) employed these techniques and traditional dilution plating to quantify population densities of 10 strains from five genotypes introduced into the wheat rhizosphere and demonstrated significant linear relationships among the population sizes detected by the three methods. Each technique has strengths and weaknesses. For example, colony hybridization followed by phlD-specific PCR has a detection limit of log 4 phlD+ strain CFU/g of fresh root weight but is labor-intensive and time-consuming and does not discriminate among genotypes (16). In contrast, the phlD-specific PCR-based dilution endpoint technique with the enrichment step developed by McSpadden Gardener et al. (24) has a detection limit of log 3.1 phlD+ strain CFU/rhizosphere and allows the identification of the dominant genotype by RFLP analysis, but detection of subdominant genotypes is limited (5). To overcome this limitation, De La Fuente et al. (5) developed allele-specific PCR primers for genotypes A, B, D, K, and L for use with the dilution endpoint assay, enabling detection of multiple genotypes in a single sample. However, all of these approaches are culture dependent and require incubation of serially diluted root washes in medium selective for fluorescent pseudomonads (24), potentially altering the proportion of each genotype present in a sample (5). In addition, these approaches are not ideal for large-scale high-throughput studies of the biogeography of 2,4-DAPG producers in managed and unmanaged ecosystems. Culture-independent methods based on extraction and analysis of DNA from environmental samples are becoming more popular for assessing the population structure of indigenous or introduced bacterial communities. One such technique, real-time PCR, is based on the quantification of the amplified PCR product, which in turn is proportional to the concentration of the template DNA. Various real-time systems demonstrating specificity, sensitivity, and speed have been developed to detect and enumerate bacteria, fungi, viruses, and yeasts (8, 10-12, 20, 26, 28, 41). The objectives of this study were (i) to design real-time PCR primers for specific amplification of phlD from genotypes A, B, D, and I of P. fluorescens; (ii) to develop and optimize real-time PCR assays for the enumeration of 2,4-DAPG producers in bacterial cultures and rhizosphere samples; and (iii) to compare quantification of 2,4-DAPG-producing P. fluorescens strains by real-time PCR with that by phlD-specific dilution endpoint assay and traditional dilution plating.

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.

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.

Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in 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-diacetylphloroglucinol producers in natural environments.

Phenazines are broad-spectrum antibiotic metabolites produced by organisms such as Pseudomonas and Streptomyces. Phenazines have been shown to enhance microbial competitiveness and the pathogenic potential of the organisms that synthesize them. PhzA (163 residues, approximate molecular weight 18.7 kDa) is the product of the first of seven genes of the operon responsible for phenazine biosynthesis in P. fluorescens 2-79. This enzyme is thought to catalyse one of the final steps in the formation of phenazine-1-carboxylic acid, the end product of phenazine biosynthesis in P. fluorescens 2-79. Here, the purification and crystallization of recombinant PhzA are reported. Crystals diffracting to 2.1 angstroms were obtained using 1.6 M magnesium sulfate and 2-morpholinoethanesulfonic acid monohydrate (MES) buffer pH 5.2-5.6. Crystals of both native and seleno-L-methionine-labelled protein belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 66.8, b = 75.3, c = 84.5 angstroms. The asymmetric unit contains one dimer of PhzA.

Certain 2,4-diacetylphloroglucinol-producing strains of Pseudomonas fluorescens colonize roots and suppress soilborne diseases more effectively than others from which they are otherwise phenotypically almost indistinguishable. We recovered DNA fragments present in the superior colonizer P. fluorescens Q8r1-96 but not in the less rhizosphere-competent strain Q2-87. Of the open reading frames in 32 independent Q8r1-96-specific clones, 1 was similar to colicin M from Escherichia coli , 3 resembled known regulatory proteins, and 28 had no significant match with sequences of known function. Seven clones hybridized preferentially to DNA from strains with superior rhizosphere competence, and sequences in two others were highly expressed in vitro and in the rhizosphere.

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root) −1 over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a major determinant in the biological control of a wide range of plant diseases by fluorescent Pseudomonas spp. A protocol was developed to readily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants. Extraction with ethyl acetate at an acidic pH was suitable for both in vitro and in situ sources of Phl. For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar organic soil components, such as humic and fulvic acids, which can interfere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 54.6%, and a second extraction added another 6.1%. These yields were substantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil. For the first time Phl was isolated from the rhizosphere environment in raw soil. Following application of Pseudomonas fluorescens Q2-87 and the Phl-overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2.4 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Ritzville silt loam; 0.47 and 1.3 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Shano silt loam. However, when the amount of Phl was calculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.

The antibiotic 2,4-diacetylphloroglucinol (Phl) is an important factor in the biological control by fluorescent Pseudomonas spp. of many soilborne diseases including take-all disease of wheat. A 6.5-kb genomic DNA fragment from Pseudomonas fluorescens Q2-87 conferred production of Phl and of a red pigment distinct from Phl, but which typically is present when Phl is produced, upon all of 13 Phl-nonproducing recipient Pseudomonas strains into which it was introduced. Larger fragments that included flanking DNA sequences did not transfer this capability, suggesting that they contain negative regulatory element(s). Analysis of the 6.5-kb fragment by Tn3HoHo1 mutagenesis further localized the sequences required for Phl production to a segment of approximately 5 kb and revealed the presence of at least two divergently oriented transcriptional units. Insertions within the smaller unit or within about 3 kb of the 5' end of the larger unit caused loss of production of both Phl and the red pigment. Other insertions within the distal 1.5 kb of the larger transcriptional unit abolished production of only the red pigment. Pleiotropic changes in secondary metabolism or colony morphology were not observed in Pseudomonas strains containing the 6.5-kb fragment, although some Phl-producing derivatives grew more slowly and gave rise to smaller colonies than did the wild-type parental strains. The size of the genomic region involved in Phl production, and the consistency and specificity with which these sequences transferred Phl biosynthetic capability, support the conclusion that the 6.5-kb fragment contains the Phl biosynthetic locus.

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.

The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by certain strains of fluorescent pseudomonads that control soilborne plant pathogens. Phl production is required for the suppression of take-all disease of wheat by Pseudomonas fluorescens (formerly P. aureofaciens) Q2-87, and a locus involved in synthesis of the antibiotic was described previously ( Vincent et al., 1991, Appl. Environ. Microbiol. 57:2928-2934). The objective of this study was to characterize the cloned locus with respect to structure and function. A 7-kb subclone was sufficient to transfer Phl biosynthetic capability to each of 17 heterologous Phl-nonproducing strains of Pseudomonas from the rhizosphere of wheat. Tn3HoHol mutagenesis identified a region of 5 kb with at least one promoter and up to three transcriptional and/or translational units necessary for Phl biosynthesis. Three open reading frames located within a 2-kb segment predicted proteins designated PS 1, PS2 and PS3 respectively. PS 1 and PS2 show similarity to the chalcone synthase/stilbene synthase family of enzymes from plants and PS3 shows similarity to sterol-carrier protein from animals. The structure of these predicted proteins suggests that they function directly in the biosynthesis of the antibiotic.