Rhizosphere plant-microbe interactions under water stress.

Climate change, with its extreme temperature, weather and precipitation patterns, is a major global concern of dryland farmers, who currently meet the challenges of climate change agronomically and with growth of drought-tolerant crops. Plants themselves compensate for water stress by modifying aerial surfaces to control transpiration and altering root hydraulic conductance to increase water uptake. These responses are complemented by metabolic changes involving phytohormone network-mediated activation of stress response pathways, resulting in decreased photosynthetic activity and the accumulation of metabolites to maintain osmotic and redox homeostasis. Phylogenetically diverse microbial communities sustained by plants contribute to host drought tolerance by modulating phytohormone levels in the rhizosphere and producing water-sequestering biofilms. Drylands of the Inland Pacific Northwest, USA, illustrate the interdependence of dryland crops and their associated microbiota. Indigenous Pseudomonas spp. selected there by long-term wheat monoculture suppress root diseases via the production of antibiotics, with soil moisture a critical determinant of the bacterial distribution, dynamics and activity. Those pseudomonads producing phenazine antibiotics on wheat had more abundant rhizosphere biofilms and provided improved tolerance to drought, suggesting a role of the antibiotic in alleviation of drought stress. The transcriptome and metabolome studies suggest the importance of wheat root exudate-derived osmoprotectants for the adaptation of these pseudomonads to the rhizosphere lifestyle and support the idea that the exchange of metabolites between plant roots and microorganisms profoundly affects and shapes the belowground plant microbiome under water stress.

Antimicrobial Activity of, and Cellular Pathways Targeted by, p-Anisaldehyde and Epigallocatechin Gallate in the Opportunistic Human Pathogen Pseudomonas aeruginosa.

Plant-derived aldehydes are constituents of essential oils that possess broad-spectrum antimicrobial activity and kill microorganisms without promoting resistance. In our previous study, we incorporated p-anisaldehyde from star anise into a polymer network called proantimicrobial networks via degradable acetals (PANDAs) and used it as a novel drug delivery platform. PANDAs released p-anisaldehyde upon a change in pH and humidity and controlled the growth of the multidrug-resistant pathogen Pseudomonas aeruginosa PAO1. In this study, we identified the cellular pathways targeted by p-anisaldehyde by generating 10,000 transposon mutants of PAO1 and screened them for hypersensitivity to p-anisaldehyde. To improve the antimicrobial efficacy of p-anisaldehyde, we combined it with epigallocatechin gallate (EGCG), a polyphenol from green tea, and demonstrated that it acts synergistically with p-anisaldehyde in killing P. aeruginosa We then used transcriptome sequencing to profile the responses of P. aeruginosa to p-anisaldehyde, EGCG, and their combination. The exposure to p-anisaldehyde altered the expression of genes involved in modification of the cell envelope, membrane transport, drug efflux, energy metabolism, molybdenum cofactor biosynthesis, and the stress response. We also demonstrate that the addition of EGCG reversed many p-anisaldehyde-coping effects and induced oxidative stress. Our results provide insight into the antimicrobial activity of p-anisaldehyde and its interactions with EGCG and may aid in the rational identification of new synergistically acting combinations of plant metabolites. Our study also confirms the utility of the thiol-ene polymer platform for the sustained and effective delivery of hydrophobic and volatile antimicrobial compounds.IMPORTANCE Essential oils (EOs) are plant-derived products that have long been exploited for their antimicrobial activities in medicine, agriculture, and food preservation. EOs represent a promising alternative to conventional antibiotics due to their broad-range antimicrobial activity, low toxicity to human commensal bacteria, and capacity to kill microorganisms without promoting resistance. Despite the progress in the understanding of the biological activity of EOs, our understanding of many aspects of their mode of action remains inconclusive. The overarching aim of this work was to address these gaps by studying the molecular interactions between an antimicrobial plant aldehyde and the opportunistic human pathogen Pseudomonas aeruginosa The results of this study identify the microbial genes and associated pathways involved in the response to antimicrobial phytoaldehydes and provide insights into the molecular mechanisms governing the synergistic effects of individual constituents within essential oils.

Pseudomonas synxantha 2-79 Transformed with Pyrrolnitrin Biosynthesis Genes Has Improved Biocontrol Activity Against Soilborne Pathogens of Wheat and Canola.

A four-gene operon (prnABCD) from Pseudomonas protegens Pf-5 encoding the biosynthesis of the antibiotic pyrronitrin was introduced into P. synxantha (formerly P. fluorescens) 2-79, an aggressive root colonizer of both dryland and irrigated wheat roots that naturally produces the antibiotic phenazine-1-carboxylic acid and suppresses both take-all and Rhizoctonia root rot of wheat. Recombinant strains ZHW15 and ZHW25 produced both antibiotics and maintained population sizes in the rhizosphere of wheat that were comparable to those of strain 2-79. The recombinant strains inhibited in vitro the wheat pathogens Rhizoctonia solani anastomosis group 8 (AG-8) and AG-2-1, Gaeumannomyces graminis var. tritici, Sclerotinia sclerotiorum, Fusarium culmorum, and F. pseudograminearum significantly more than did strain 2-79. Both the wild-type and recombinant strains were equally inhibitory of Pythium ultimum. When applied as a seed treatment, the recombinant strains suppressed take-all, Rhizoctonia root rot of wheat, and Rhizoctonia root and stem rot of canola significantly better than did wild-type strain 2-79.

Exploring the Pathogenicity of Pseudomonas brassicacearum Q8r1-96 and Other Strains of the Pseudomonas fluorescens Complex on Tomato.

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.

Phenazine-1-carboxylic acid and soil moisture influence biofilm development and turnover of rhizobacterial biomass on wheat root surfaces.

Phenazine-1-carboxylic acid (PCA) is produced by rhizobacteria in dryland but not in irrigated wheat fields of the Pacific Northwest, USA. PCA promotes biofilm development in bacterial cultures and bacterial colonization of wheat rhizospheres. However, its impact upon biofilm development has not been demonstrated in the rhizosphere, where biofilms influence terrestrial carbon and nitrogen cycles with ramifications for crop and soil health. Furthermore, the relationships between soil moisture and the rates of PCA biosynthesis and degradation have not been established. In this study, expression of PCA biosynthesis genes was upregulated relative to background transcription, and persistence of PCA was slightly decreased in dryland relative to irrigated wheat rhizospheres. Biofilms in dryland rhizospheres inoculated with the PCA-producing (PCA+ ) strain Pseudomonas synxantha 2-79RN10 were more robust than those in rhizospheres inoculated with an isogenic PCA-deficient (PCA- ) mutant strain. This trend was reversed in irrigated rhizospheres. In dryland PCA+ rhizospheres, the turnover of 15 N-labelled rhizobacterial biomass was slower than in the PCA- and irrigated PCA+ treatments, and incorporation of bacterial 15 N into root cell walls was observed in multiple treatments. These results indicate that PCA promotes biofilm development in dryland rhizospheres, and likely influences crop nutrition and soil health in dryland wheat fields.

Differential Response of Wheat Cultivars to Pseudomonas brassicacearum and Take-All Decline Soil.

2,4-Diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. in the P. fluorescens complex are primarily responsible for a natural suppression of take-all of wheat known as take-all decline (TAD) in many fields in the United States. P. brassicacearum, the most common DAPG producer found in TAD soils in the Pacific Northwest (PNW) of the United States, has biological control, growth promoting and phytotoxic activities. In this study, we explored how the wheat cultivar affects the level of take-all suppression when grown in a TAD soil, and how cultivars respond to colonization by P. brassicacearum. Three cultivars (Tara, Finley, and Buchanan) supported similar rhizosphere population sizes of P. brassicacearum when grown in a TAD soil, however they developed significantly different amounts of take-all. Cultivars Tara and Buchanan developed the least and most take-all, respectively, and Finley showed an intermediate amount of disease. However, when grown in TAD soil that was pasteurized to eliminate both DAPG producers and take-all suppression, all three cultivars were equally susceptible to take-all. The three cultivars also responded differently to the colonization and phytotoxicity of P. brassicacearum strains Q8r1-96 and L5.1-96, which are characteristic of DAPG producers in PNW TAD soils. Compared with cultivar Tara, cultivar Buchanan showed significantly reduced seedling emergence and root growth when colonized by P. brassicacearum, and the response of Finley was intermediate. However, all cultivars emerged equally when treated with a DAPG-deficient mutant of Q8r1-96. Our results indicate that wheat cultivars grown in a TAD soil modulate both the robustness of take-all suppression and the potential phytotoxicity of the antibiotic DAPG.

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

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.

Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites.

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.

Biological control of wheat root diseases by the CLP-producing strain Pseudomonas fluorescens HC1-07.

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.

Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp.

Phenazine compounds represent a large class of bacterial metabolites that are produced by some fluorescent Pseudomonas spp. and a few other bacterial genera. Phenazines were first noted in the scientific literature over 100 years ago, but for a long time were considered to be pigments of uncertain function. Following evidence that phenazines act as virulence factors in the opportunistic human and animal pathogen Pseudomonas aeruginosa and are actively involved in the suppression of plant pathogens, interest in these compounds has broadened to include investigations of their genetics, biosynthesis, activity as electron shuttles, and contribution to the ecology and physiology of the cells that produce them. This minireview highlights some recent and exciting insights into the diversity, frequency and ecological roles of phenazines produced by fluorescent Pseudomonas spp.

Taxonomy and distribution of phenazine-producing Pseudomonas spp. in the dryland agroecosystem of the Inland Pacific Northwest, United States.

We investigated the taxonomic placement of phenazine-producing fluorescent Pseudomonas spp. in the Inland Pacific Northwest region of the United States. Five distinct species were identified, two of which were provisionally considered to be new. Agroclimatic zone and soil silt content affected the species diversity across the region.

pA506, a conjugative plasmid of the plant epiphyte Pseudomonas fluorescens A506.

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 MOBP6 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.

Ligand binding induces an ammonia channel in 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE.

PhzE utilizes chorismate and glutamine to synthesize 2-amino-2-desoxyisochorismate (ADIC) in the first step of phenazine biosynthesis. The PhzE monomer contains both a chorismate-converting menaquinone, siderophore, tryptophan biosynthesis (MST) and a type 1 glutamine amidotransferase (GATase1) domain connected by a 45-residue linker. We present here the crystal structure of PhzE from Burkholderia lata 383 in a ligand-free open and ligand-bound closed conformation at 2.9 and 2.1 Å resolution, respectively. PhzE arranges in an intertwined dimer such that the GATase1 domain of one chain provides NH(3) to the MST domain of the other. This quaternary structure was confirmed by small angle x-ray scattering. Binding of chorismic acid, which was found converted to benzoate and pyruvate in the MST active centers of the closed form, leads to structural rearrangements that establish an ammonia transport channel approximately 25 Å in length within each of the two MST/GATase1 functional units of the dimer. The assignment of PhzE as an ADIC synthase was confirmed by mass spectrometric analysis of the product, which was also visualized at 1.9 Å resolution by trapping in crystals of an inactive mutant of PhzD, an isochorismatase that catalyzes the subsequent step in phenazine biosynthesis. Unlike in some of the related anthranilate synthases, no allosteric inhibition was observed in PhzE. This can be attributed to a tryptophan residue of the protein blocking the potential regulatory site. Additional electron density in the GATase1 active center was identified as zinc, and it was demonstrated that Zn(2+), Mn(2+), and Ni(2+) reduce the activity of PhzE.

Irrigation differentially impacts populations of indigenous antibiotic-producing pseudomonas spp. in the rhizosphere of wheat.

This work determined the impact of irrigation on the seasonal dynamics of populations of Pseudomonas spp. producing the antibiotics phenazine-1-carboxylic acid (Phz(+)) and 2,4-diacetylphloroglucinol (Phl(+)) in the rhizosphere of wheat grown in the low-precipitation zone (150 to 300 mm annually) of the Columbia Plateau of the Inland Pacific Northwest. Population sizes and plant colonization frequencies of Phz(+) and Phl(+) Pseudomonas spp. were determined in winter and spring wheat collected during the growing seasons from 2008 to 2009 from selected commercial dryland and irrigated fields in central Washington State. Only Phz(+) bacteria were detected on dryland winter wheat, with populations ranging from 4.8 to 6.3 log CFU g(-1) of root and rhizosphere colonization frequencies of 67 to 100%. The ranges of population densities of Phl(+) and Phz(+) Pseudomonas spp. recovered from wheat grown under irrigation were similar, but 58 to 100% of root systems were colonized by Phl(+) bacteria whereas only 8 to 50% of plants harbored Phz(+) bacteria. In addition, Phz(+) Pseudomonas spp. were abundant in the rhizosphere of native plant species growing in nonirrigated areas adjacent to the sampled dryland wheat fields. This is the first report that documents the impact of irrigation on indigenous populations of two closely related groups of antibiotic-producing pseudomonads that coinhabit the rhizosphere of an economically important cereal crop. These results demonstrate how crop management practices can influence indigenous populations of antibiotic-producing pseudomonads with the capacity to suppress soilborne diseases of wheat.

Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland cereals.

Natural antibiotics are thought to function in the defense, fitness, competitiveness, biocontrol activity, communication, and gene regulation of microorganisms. However, the scale and quantitative aspects of antibiotic production in natural settings are poorly understood. We addressed these fundamental questions by assessing the geographic distribution of indigenous phenazine-producing (Phz(+)) Pseudomonas spp. and the accumulation of the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA) in the rhizosphere of wheat grown in the low-precipitation zone (<350 mm) of the Columbia Plateau and in adjacent, higher-precipitation areas. Plants were collected from 61 commercial wheat fields located within an area of about 22,000 km(2). Phz(+) Pseudomonas spp. were detected in all sampled fields, with mean population sizes ranging from log 3.2 to log 7.1 g(-1) (fresh weight) of roots. Linear regression analysis demonstrated a significant inverse relationship between annual precipitation and the proportion of plants colonized by Phz(+) Pseudomonas spp. (r(2) = 0.36, P = 0.0001). PCA was detected at up to nanomolar concentrations in the rhizosphere of plants from 26 of 29 fields that were selected for antibiotic quantitation. There was a direct relationship between the amount of PCA extracted from the rhizosphere and the population density of Phz(+) pseudomonads (r(2) = 0.46, P = 0.0006). This is the first demonstration of accumulation of significant quantities of a natural antibiotic across a terrestrial ecosystem. Our results strongly suggest that natural antibiotics can transiently accumulate in the plant rhizosphere in amounts sufficient not only for inter- and intraspecies signaling but also for the direct inhibition of sensitive organisms.

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.

Induced systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato by 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens.

Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 µM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.

Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96.

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.

Biological control of Rhizoctonia root rot on bean by phenazine- and cyclic lipopeptide-producing Pseudomonas CMR12a.

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.

Biological control of take-all by fluorescent Pseudomonas spp. from Chinese wheat fields.

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.