Deciphering the mechanism of fungal pathogen-induced disease-suppressive soil.

One model of a disease-suppressive soil predicts that the confrontation of plant with a phytopathogen can lead to the recruitment and accumulation of beneficial microorganisms. However, more information needs to be deciphered regarding which beneficial microbes become enriched, and how the disease suppression is achieved. Here, we conditioned soil by continuously growing eight generations of cucumber inoculated with Fusarium oxysporum f.sp. cucumerinum in a split-root system. Disease incidence was found to decrease gradually upon pathogen infection accompanied with higher quantity of reactive oxygen species (ROS mainly OH• ) in roots and accumulation of Bacillus and Sphingomonas. These key microbes were proven to protect the cucumber from pathogen infection by inducing high ROS level in the roots through enrichment of pathways, including a two-component system, a bacterial secretion system, and flagellar assembly revealed by metagenomics sequencing. Untargeted metabolomics analysis combined with in vitro application assays suggested that threonic acid and lysine were pivotal to recruit Bacillus and Sphingomonas. Collectively, our study deciphered a 'cry for help' case, wherein cucumber releases particular compounds to enrich beneficial microbes that raise the ROS level of host to prevent pathogen attack. More importantly, this may be one of the fundamental mechanisms underpinning disease-suppressive soil formation.

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.

Evaluation of the Phytotoxicity of 2,4-Diacetylphloroglucinol and Pseudomonas brassicacearum Q8r1-96 on Different Wheat Cultivars.

Pseudomonas brassicacearum Q8r1-96 and other 2,4-diacetylphloroglucinol (DAPG)-producing pseudomonads of the P. fluorescens complex possess both biocontrol and growth-promoting properties and play an important role in suppression of take-all of wheat in the Pacific Northwest (PNW) of the United States. However, P. brassicacearum can also reduce seed germination and cause root necrosis on some wheat cultivars. We evaluated the effect of Q8r1-96 and DAPG on the germination of 69 wheat cultivars that have been or currently are grown in the PNW. Cultivars varied widely in their ability to tolerate P. brassicacearum or DAPG. The frequency of germination of the cultivars ranged from 0 to 0.87 and 0.47 to 0.90 when treated with Q8r1-96 and DAPG, respectively. There was a significant positive correlation between the frequency of germination of cultivars treated with Q8r1-96 in assays conducted in vitro and in the greenhouse. The correlation was greater for spring than for winter cultivars. In contrast, the effect of Q8r1-96 on seed germination was not correlated with that of DAPG alone, suggesting that DAPG is not the only factor responsible for the phytotoxicity of Q8r1-96. Three wheat cultivars with the greatest tolerance and three cultivars with the least tolerance to Q8r1-96 were tested for their ability to support root colonization by strain Q8r1-96. Cultivars with the greatest tolerance supported significantly greater populations of strain Q8r1-96 than those with the least tolerance to the bacteria. Our results show that wheat cultivars differ widely in their interaction with P. brassicacearum and the biocontrol antibiotic DAPG.

Proteomics Reveals the Changes that Contribute to Fusarium Head Blight Resistance in Wheat.

Fusarium head blight (FHB) is a devastating disease of wheat, causing yield losses and quality reduction as a result of mycotoxin production. In this study, iTRAQ (isobaric tags for relative and absolute quantification)-labeling-based mass spectrometry was employed to characterize the proteome in wheat cultivars Xinong 538 and Zhoumai 18 with contrasting levels of FHB resistance as a means to elucidate the molecular mechanisms contributing to FHB resistance. A total of 13,669 proteins were identified in the two cultivars 48 h after Fusarium graminearum inoculation. Among these, 2,505 unique proteins exclusively accumulated in Xinong 538 (resistant) and 887 proteins in Zhoumai 18 (susceptible). Gene Ontology enrichment analysis showed that most differentially accumulated proteins (DAPs) from both cultivars were assigned to the following categories: metabolic process, single-organism process, cellular process, and response to stimulus. Kyoto Encyclopedia of Genes and Genomes analysis showed that a greater number of proteins belonging to different metabolic pathways were identified in Xinong 538 compared with Zhoumai 18. Specifically, DAPs from the FHB-resistant cultivar Xinong 538 populated categories of metabolic pathways related to plant-pathogen interaction. These DAPs might play a critical role in defense responses exhibited by Xinong 538. DAPs from both genotypes were assigned to all wheat chromosomes except chromosome 6B, with approximately 30% mapping to wheat chromosomes 2B, 3B, 5B, and 5D. Twenty single nucleotide polymorphism markers, flanking DAPs on chromosomes 1B, 3B, 5B, and 6A, overlapped with the location of earlier mapped FHB-resistance quantitative trait loci. The data provide evidence for the involvement of several DAPs in the early stages of the FHB-resistance response in wheat; however, further functional characterization of candidate proteins is warranted.

Control of Meloidogyne incognita in Three-Dimensional Model Systems and Pot Experiments by the Attract-and-Kill Effect of Furfural Acetone.

Meloidogyne incognita causes large-scale losses of agricultural crops worldwide. The natural metabolite furfural acetone has been reported to attract and kill M. incognita, but whether the attractant and nematicidal activities of furfural acetone on M. incognita function simultaneously in the same system, especially in three-dimensional spaces or in soil, is still unknown. Here, we used 23% Pluronic F-127 gel and a soil simulation device to demonstrate that furfural acetone has a significant attract-and-kill effect on M. incognita in both three-dimensional model systems. At 24 h, the chemotaxis index and the corrected mortality of nematodes exposed to 60 mg/ml of furfural acetone in 23% Pluronic F-127 gel were as high as 0.82 and 74.44%, respectively. Soil simulation experiments in moist sand showed that at 48 h, the chemotaxis index and the corrected mortality of the nematode toward furfural acetone reached 0.63 and 82.12%, respectively, and the effect persisted in the presence of tomato plants. In choice experiments, nematodes selected furfural acetone over plant roots and were subsequently killed. In pot studies, furfural acetone had a control rate of 82.80% against M. incognita. Collectively, these results provide compelling evidence for further investigation of furfural acetone as a novel nematode control agent.

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.

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

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.

Phenazine-1-Carboxylic Acid-Producing Bacteria Enhance the Reactivity of Iron Minerals in Dryland and Irrigated Wheat Rhizospheres.

Phenazine-1-carboxylic acid (PCA) is a broad-spectrum antibiotic produced by rhizobacteria in the dryland wheat fields of the Columbia Plateau. PCA and other phenazines reductively dissolve Fe and Mn oxyhydroxides in bacterial culture systems, but the impact of PCA upon Fe and Mn cycling in the rhizosphere is unknown. Here, concentrations of dithionite-extractable and poorly crystalline Fe were approximately 10% and 30-40% higher, respectively, in dryland and irrigated rhizospheres inoculated with the PCA-producing (PCA+) strain Pseudomonas synxantha 2-79 than in rhizospheres inoculated with a PCA-deficient mutant. However, rhizosphere concentrations of Fe(II) and Mn did not differ significantly, indicating that PCA-mediated redox transformations of Fe and Mn were transient or were masked by competing processes. Total Fe and Mn uptake into wheat biomass also did not differ significantly, but the PCA+ strain significantly altered Fe translocation into shoots. X-ray absorption near edge spectroscopy revealed an abundance of Fe-bearing oxyhydroxides and phyllosilicates in all rhizospheres. These results indicate that the PCA+ strain enhanced the reactivity and mobility of Fe derived from soil minerals without producing parallel changes in plant Fe uptake. This is the first report that directly links significant alterations of Fe-bearing minerals in the rhizosphere to a single bacterial trait.

Rapidly mitigating antibiotic resistant risks in chicken manure by Hermetia illucens bioconversion with intestinal microflora.

Antibiotic resistance genes (ARGs) in animal manure are an environmental concern due to naturally occurring bacteria being exposed to these wastes and developing multidrug resistance. The bioconversion of manure with fly larvae is a promising alternative for recycling these wastes while attenuating ARGs. We investigated the impact of black soldier fly (BSF, Hermetia illucens) larval bioconversion of chicken manure on the persistence of associated ARGs. Compared with traditional composting or sterile larval treatments (by 48.4% or 88.7%), non-sterile BSF larval treatments effectively reduced ARGs and integrin genes by 95.0% during 12 days, due to rapid decreases in concentrations of the genes and associated bacteria as they passed through the larval gut and were affected by intestinal microbes. After larval treatments, bacterial community composition differed significantly, with the percentage of Firmicutes possibly carrying ARGs reduced by 65.5% or more. On average, human pathogenic bacteria populations declined by 70.7%-92.9%, effectively mitigating risks of these bacteria carrying ARGs. Environmental pH, nitrogen content and antibiotic concentrations were closely related to both bacterial community composition and targeted gene attenuation in larval systems. Selective pressures of larval gut environments with intestinal microbes, larval bacteriostasis and reformulation of manure due to larval digestion contributed to ARG attenuation.

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.

Sensitivity of Rhizoctonia Isolates to Phenazine-1-Carboxylic Acid and Biological Control by Phenazine-Producing Pseudomonas spp.

Rhizoctonia solani anastomosis groups (AG)-8 and AG-2-1 and R. oryzae are ubiquitous in cereal-based cropping systems of the Columbia Plateau of the Inland Pacific Northwest and commonly infect wheat. AG-8 and R. oryzae, causal agents of Rhizoctonia root rot and bare patch, are most commonly found in fields in the low-precipitation zone, whereas R. solani AG-2-1 is much less virulent on wheat and is distributed in fields throughout the low-, intermediate-, and high-precipitation zones. Fluorescent Pseudomonas spp. that produce the antibiotic phenazine-1-carboxylic acid (PCA) also are abundant in the rhizosphere of crops grown in the low-precipitation zone but their broader geographic distribution and effect on populations of Rhizoctonia is unknown. To address these questions, we surveyed the distribution of PCA producers (Phz+) in 59 fields in cereal-based cropping systems throughout the Columbia Plateau. Phz+ Pseudomonas spp. were detected in 37 of 59 samples and comprised from 0 to 12.5% of the total culturable heterotrophic aerobic rhizosphere bacteria. The frequency with which individual plants were colonized by Phz+ pseudomonads ranged from 0 to 100%. High and moderate colonization frequencies of Phz+ pseudomonads were associated with roots from fields located in the driest areas whereas only moderate and low colonization frequencies were associated with crops where higher annual precipitation occurs. Thus, the geographic distribution of Phz+ pseudomonads overlaps closely with the distribution of R. solani AG-8 but not with that of R. oryzae or R. solani AG-2-1. Moreover, linear regression analysis demonstrated a highly significant inverse relationship between annual precipitation and the frequency of rhizospheres colonized by Phz+ pseudomonads. Phz+ pseudomonads representative of the four major indigenous species (P. aridus, P. cerealis, P. orientalis, and P. synxantha) suppressed Rhizoctonia root rot of wheat when applied as seed treatments. In vitro, mean 50% effective dose values for isolates of AG-8 and AG-2-1 from fields with high and low frequencies of phenazine producers did not differ significantly, nor was there a correlation between virulence of an isolate and sensitivity to PCA, resulting in rejection of the hypothesis that tolerance in Rhizoctonia spp. to PCA develops in nature upon exposure to Phz+ pseudomonads.

Rhizosphere Competence of Wild-Type and Genetically Engineered Pseudomonas brassicacearum Is Affected by the Crop Species.

2,4-Diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas brassicacearum Q8r1-96 is a highly effective biocontrol agent of take-all disease of wheat. Strain Z30-97, a recombinant derivative of Q8r1-96 containing the phzABCDEFG operon from P. synxantha (formerly P. fluorescens) 2-79 inserted into its chromosome, also produces phenazine-1-carboxylic acid. Rhizosphere population sizes of Q8r1-96, Z30-97, and 2-79, introduced into the soil, were assayed during successive growth cycles of barley, navy bean, or pea under controlled conditions as a measure of the impact of crop species on rhizosphere colonization of each strain. In the barley rhizosphere, Z30-96 colonized less that Q8r1-96 when they were introduced separately, and Q8r1-96 out-competed Z30-96 when the strains were introduced together. In the navy bean rhizosphere, Q8r1-96 colonized better than Z30-97 when the strains were introduced separately. However, both strains had similar population densities when introduced together. Strain Q8r1-96 and Z30-97 colonized the pea rhizosphere equally well when each strain was introduced separately, but Z30-97 out-competed Q8r1-96 when they were introduced together. To our knowledge, this is the first report of a recombinant biocontrol strain of Pseudomonas spp. gaining rhizosphere competitiveness on a crop species. When assessing the potential fate of and risk posed by a recombinant Pseudomonas sp. in soil, both the identity of the introduced genes and the crop species colonized by the recombinant strain need to be considered.

Molecular Characterization, Morphological Characteristics, Virulence, and Geographic Distribution of Rhizoctonia spp. in Washington State.

Rhizoctonia root rot and bare patch, caused by Rhizoctonia solani anastomosis group (AG)-8 and R. oryzae, are chronic and important yield-limiting diseases of wheat and barley in the Inland Pacific Northwest (PNW) of the United States. Major gaps remain in our understanding of the epidemiology of these diseases, in part because multiple Rhizoctonia AGs and species can be isolated from the same cereal roots from the field, contributing to the challenge of identifying the causal agents correctly. In this study, a collection totaling 498 isolates of Rhizoctonia was assembled from surveys conducted from 2000 to 2009, 2010, and 2011 over a wide range of cereal production fields throughout Washington State in the PNW. To determine the identity of the isolates, PCR with AG- or species-specific primers and/or DNA sequence analysis of the internal transcribed spacers was performed. R. solani AG-2-1, AG-8, AG-10, AG-3, AG-4, and AG-11 comprised 157 (32%), 70 (14%), 21 (4%), 20 (4%), 1 (0.2%), and 1 (0.2%), respectively, of the total isolates. AG-I-like binucleate Rhizoctonia sp. comprised 44 (9%) of the total; and 53 (11%), 80 (16%), and 51 (10%) were identified as R. oryzae genotypes I, II, and III, respectively. Isolates of AG-2-1, the dominant Rhizoctonia, occurred in all six agronomic zones defined by annual precipitation and temperature within the region sampled. Isolates of AG-8 also were cosmopolitan in their distribution but the frequency of isolation varied among years, and they were most abundant in zones of low and moderate precipitation. R. oryzae was cosmopolitan, and collectively the three genotypes comprised 37% of the isolates. Only isolates of R. solani AG-8 and R. oryzae genotypes II and III (but not genotype I) caused symptoms typically associated with Rhizoctonia root rot and bare patch of wheat. Isolates of AG-2-1 caused only mild root rot and AG-I-like binucleate isolates and members of groups AG-3, AG-4, and AG-11 showed only slight or no discoloration of the roots. However, all isolates of AG-2-1 caused severe damping-off of canola, resulting in 100% mortality. Isolates of Rhizoctonia AG-8, AG-2-1, AG-10, AG-I-like binucleate Rhizoctonia, and R. oryzae genotypes I, II, and III could be distinguished by colony morphology on potato dextrose agar, by PCR with specific primers, or by the type and severity of disease on wheat and canola seedlings, and results of these approaches correlated completely. Based on cultured isolates, we also identified the geographic distribution of all of these Rhizoctonia isolates in cereal-based production systems throughout Washington State.

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.

Plant-Driven Assembly of Disease-Suppressive Soil Microbiomes.

Plants have coevolved together with the microbes that surround them and this assemblage of host and microbes functions as a discrete ecological unit called a holobiont. This review outlines plant-driven assembly of disease-suppressive microbiomes. Plants are colonized by microbes from seed, soil, and air but selectively shape the microbiome with root exudates, creating microenvironment hot spots where microbes thrive. Using plant immunity for gatekeeping and surveillance, host-plant genetic properties govern microbiome assembly and can confer adaptive advantages to the holobiont. These advantages manifest in disease-suppressive soils, where buildup of specific microbes inhibits the causal agent of disease, that typically develop after an initial disease outbreak. Based on disease-suppressive soils such as take-all decline, we developed a conceptual model of how plants in response to pathogen attack cry for help and recruit plant-protective microbes that confer increased resistance. Thereby, plants create a soilborne legacy that protects subsequent generations and forms disease-suppressive soils.

Trapped intermediates in crystals of the FMN-dependent oxidase PhzG provide insight into the final steps of phenazine biosynthesis.

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.