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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.

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.

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.

Microbes can establish mutualistic interactions with plants and insects. Here we track the movement of an endophytic strain of Streptomyces bacteria throughout a managed strawberry ecosystem. We show that a Streptomyces isolate found in the rhizosphere and on flowers protects both the plant and pollinating honeybees from pathogens (phytopathogenic fungus Botrytis cinerea and pathogenic bacteria, respectively). The pollinators can transfer the Streptomyces bacteria among flowers and plants, and Streptomyces can move into the plant vascular bundle from the flowers and from the rhizosphere. Our results present a tripartite mutualism between Streptomyces, plant and pollinator partners., Microbes can establish mutualistic interactions with plants and insects. Here, Kim et al. show that Streptomyces bacteria can protect strawberry plants and honeybees from pathogens, can move into the plant vascular tissue from soil and from flowers, and are transferred among flowers by the pollinators.

Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.

Fusarium wilt disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense race 4, is a serious soil-borne fungal disease that currently threatens worldwide banana production. No single agricultural practice has yet been developed to effectively manage this disease. In the present study, greenhouse experiments were conducted to evaluate the effect of an integrated biofertilizer application after ammonia fumigation to enhance control of Fusarium wilt disease in severely infected banana mono-cropped soils. Quantitative PCR and high-throughput sequencing were used to characterise soil microbial community structure and the results from both two-season experimental studies showed that biofertilizer application after ammonia fumigation significantly reduced the incidence of banana Panama disease by approximately 55% and promoted the plant biomass, compared to the control application of cow manure to non-fumigated soil. Ammonia fumigation significantly reduced total fungal and F. oxysporum abundances and bacterial and fungal diversities. Biofertilizer application after fumigation further depleted the abundance of the pathogen. Biofertilizer application and fumigation altered the soil microbial community composition with the bacterial community responding first to fumigation, while the fungal community responded first to fertilization. Although Bacillus, including our inoculated strain, was not enriched after biofertilization, putative beneficial microbes such as Paenibacillus, Virgibacillus, Nitrosomonas, and Nitrobacter, were significantly enriched by ammonia fumigation and biofertilizer application, and were significantly correlated with disease suppression or increased plant biomass. Furthermore, fumigation and biofertilization significantly increased the soil pH and nutrient contents, with concomitant effects on the microbial community. Overall, the observed disease suppression and increased plant biomass resulting from both soil fumigation and biofertilization after banana cropping can be attributed to the reduced abundance of F. oxysporum and general suppression from altered soil properties that may enable the establishment of a beneficial soil microbiome.

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.

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.

Background: The physiology and growth of plants are strongly influenced by their associated microbiomes. Conversely, the composition of the phytobiome is flexible, responding to the state of the host and raising the possibility that it can be engineered to benefit the plant. However, technology for engineering the structure of the microbiome is not yet available.Results: Here we show that glutamic acid reshapes the plant microbial community and enriches populations of Streptomyces, a functional core microbe, both above and below ground, in strawberry and tomato. Upon application of glutamic acid, the population size of Streptomyces increased dramatically in the anthosphere and the rhizosphere. At the same time, diseases caused by species of Fusarium were significantly reduced in both habitats. Plant resistance-related genes were not activated, suggesting that glutamic acid modulates the microbiome community directly, rather than activating the host’s own protective mechanisms.Conclusions: Much is known about the structure of plant-associated microbial communities, but little has been learned about how the community composition and complexity are controlled. Our results demonstrate that the microbiome community can be engineered and unlock the mode of action of glutamic acid.

Phenazines are natural bacterial antibiotics that can protect crops from disease. However, for most crops it is unknown which producers and specific phenazines are ecologically relevant, and whether phenazine biodegradation can counter their effects. To better understand their ecology, we developed and environmentally-validated a quantitative metagenomic approach to mine for phenazine biosynthesis and biodegradation genes, applying it to >800 soil and plant-associated shotgun-metagenomes. We discover novel producer-crop associations and demonstrate that phenazine biosynthesis is prevalent across habitats and preferentially enriched in rhizospheres, whereas biodegrading bacteria are rare. We validate an association between maize and Dyella japonica, a putative producer abundant in crop microbiomes. D. japonica upregulates phenazine biosynthesis during phosphate limitation and robustly colonizes maize seedling roots. This work provides a global picture of phenazines in natural environments and highlights plant-microbe associations of agricultural potential. Our metagenomic approach may be extended to other metabolites and functional traits in diverse ecosystems.

Soil-borne plant diseases are increasingly causing devastating losses in agricultural production. The development of a more refined model for disease prediction can aid in reducing crop losses through the use of preventative control measures or soil fallowing for a planting season. The emergence of high-throughput DNA sequencing technology has provided unprecedented insight into the microbial composition of diseased versus healthy soils. However, a single independent case study rarely yields a general conclusion predictive of the disease in a particular soil. Here, we attempt to account for the differences among various studies and plant varieties using a machine-learning approach based on 24 independent bacterial data sets comprising 758 samples and 22 independent fungal data sets comprising 279 samples of healthy or Fusarium wilt-diseased soils from eight different countries. We found that soil bacterial and fungal communities were both clearly separated between diseased and healthy soil samples that originated from six crops across nine countries or regions. Alpha diversity was consistently greater in the fungal community of healthy soils. While diseased soil microbiomes harbored higher abundances of Xanthomonadaceae, Bacillaceae, Gibberella, and Fusarium oxysporum, the healthy soil microbiome contained more Streptomyces Mirabilis, Bradyrhizobiaceae, Comamonadaceae, Mortierella, and nonpathogenic fungi of Fusarium. Furthermore, a random forest method identified 45 bacterial OTUs and 40 fungal OTUs that categorized the health status of the soil with an accuracy >80%. We conclude that these models can be applied to predict the potential for occurrence of F. oxysporum wilt by revealing key biological indicators and features common to the wilt-diseased soil microbiome.

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.

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.

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.

Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs), and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24-48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography-mass spectrometry (SPME-GC/MS) included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy)-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.

Crops lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of the soil microbiome to defend their roots against pathogens and other pests. The best examples of this microbially based defense of roots are observed in disease-suppressive soils in which suppressiveness is induced by continuously growing crops that are susceptible to a pathogen, but the molecular basis of most is poorly understood. Here we report the microbial characterization of a Korean soil with specific suppressiveness to Fusarium wilt of strawberry. In this soil, an attack on strawberry roots by Fusarium oxysporum results in a response by microbial defenders, of which members of the Actinobacteria appear to have a key role. We also identify Streptomyces genes responsible for the ribosomal synthesis of a novel heat-stable antifungal thiopeptide antibiotic inhibitory to F. oxysporum and the antibiotic's mode of action against fungal cell wall biosynthesis. Both classical- and community-oriented approaches were required to dissect this suppressive soil from the field to the molecular level, and the results highlight the role of natural antibiotics as weapons in the microbial warfare in the rhizosphere that is integral to plant health, vigor and development.

Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs), and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24–48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography–mass spectrometry (SPME-GC/MS) included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy)-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.

Soils suppressive to soilborne pathogens have been identified worldwide for almost 60 years and attributed mainly to suppressive or antagonistic microorganisms. Rather than identifying, testing and applying potential biocontrol agents in an inundative fashion, research into suppressive soils has attempted to understand how indigenous microbiomes can reduce disease, even in the presence of the pathogen, susceptible host, and favorable environment. Recent advances in next-generation sequencing of microbiomes have provided new tools to reexamine and further characterize the nature of these soils. Two general types of suppression have been described: specific and general suppression, and theories have been developed around these two models. In this review, we will present three examples of currently-studied model systems with features representative of specific and general suppressiveness: suppression to take-all (Gaeumannomyces graminis var. tritici), Rhizoctonia bare patch of wheat (Rhizoctonia solani AG-8), and Streptomyces. To compare and contrast the two models of general versus specific suppression, we propose a number of hypotheses about the nature and ecology of microbial populations and communities of suppressive soils. We outline the potential and limitations of new molecular techniques that can provide novel ways of testing these hypotheses. Finally, we consider how this greater understanding of the phytobiome can facilitate sustainable disease management in agriculture by harnessing the potential of indigenous soil microbes.

The aim of this study was to inventory the types of plant growth-promoting rhizobacteria (PGPR) present in the rhizosphere of plants grown in soils contaminated with heavy metals, recalcitrant organics, petroleum sewage or salinity in China. We screened 1223 isolates for antifungal activity and about 24% inhibited Rhizoctonia solani or Sclerotinia sclerotiorum. Twenty-four strains inhibitory to R. solani, Gaeumannomyces graminis var. tritici and/or S. sclerotiorum and representing the dominant morphotypes were assayed for PGPR activity. Seven strains contained phlD, prnD, pltC or phzF genes and produced the antibiotics 2,4-diacetylphloroglucinol, pyrrolnitrin, pyoluteorin and phenazines respectively. Six strains contained acdS, which encodes 1-aminocyclopropane-1-carboxylic acid deaminase. Phylogenetic analysis of 16S rDNA and phlD, phzF and acdS genes demonstrated that some strains identified as Pseudomonas were similar to model PGPR strains Pseudomonas protegens Pf-5, Pseudomonas chlororaphis subsp. aureofaciens 30–84 and P. brassicacearum Q8r1-96. Pseudomonas protegens- and P. chlororaphis-like strains had the greatest biocontrol activity against Rhizoctonia root rot and take-all of wheat. Pseudomonas protegens and P. brassicacearum-like strains showed the greatest promotion of canola growth. Our results indicate that strains from contaminated soils are similar to well-described PGPR found in agricultural soils worldwide. Growth-promoting rhizobacteria in polluted soils

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.

Plant nutrient uptake is affected by environmental stress, but how plants respond to cation-nutrient stress is poorly understood. We assessed the impact of varying degrees of cation-nutrient stress on cation uptake in an experimental plant-mineral system. Column experiments, with red pine (Pinus resinosa Ait.) seedlings growing in sand/mineral mixtures, were conducted for up to 9 months. The Ca and K were supplied from both minerals and nutrient solutions with varying Ca and K concentrations. Cation nutrient stress had little impact on carbon allocation after 9 months of plant growth and K was the limiting nutrient for biomass production. Measurement of Ca/Sr and K/Rb ratios allowed independent estimation of dissolution incongruency and discrimination against Sr and Rb during cation uptake processes. The fraction of K in biomass from biotite increased with decreasing K supply from nutrient solutions. The mineral anorthite was consistently the major source of Ca, regardless of nutrient treatment. Red pine seedlings exploited more mineral K in response to more severe K deficiency. This did not occur for Ca since Ca was not limiting plant growth. Plant discrimination factors must be carefully considered to accurately identify nutrient sources using cation tracers.

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.

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 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.

This paper reviews strategies for manipulating plants and their root-associated microorganisms to improve plant health and productivity. Some strategies directly target plant processes that impact on growth, while others are based on our knowledge of interactions among the components of the rhizosphere (roots, microorganisms and soil). For instance, plants can be engineered to modify the rhizosphere pH or to release compounds that improve nutrient availability, protect against biotic and abiotic stresses, or encourage the proliferation of beneficial microorganisms. Rhizobacteria that promote plant growth have been engineered to interfere with the synthesis of stress-induced hormones such as ethylene, which retards root growth, and to produce antibiotics and lytic enzymes active against soilborne root pathogens. Rhizosphere engineering also can involve the selection by plants of beneficial microbial populations. For example, some crop species or cultivars select for and support populations of antibiotic-producing strains that play a major role in soils naturally suppressive to soil-borne fungal pathogens. The fitness of root-associated bacterial communities also can be enhanced by soil amendment, a process that has allowed the selection of bacterial consortia that can interfere with bacterial pathogens. Plants also can be engineered specifically to influence their associated bacteria, as exemplified by quorum quenching strategies that suppress the virulence of pathogens of the genus Pectobacterium. New molecular tools and powerful biotechnological advances will continue to provide a more complete knowledge of the complex chemical and biological interactions that occur in the rhizosphere, ensuring that strategies to engineer the rhizosphere are safe, beneficial to productivity, and substantially improve the sustainability of agricultural systems.

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.

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.

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.

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10 7 CFU per g of roots soon after sowing to less than 10 2 CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.

Rhizobial bacteria, endomycorrhizal fungi (also known as arbuscular my- corrhizas), and pseudomonad bacteria associated with plant roots can provide substantial benefits to the plants by fixing nitrogen, supplying phosphorus, or controlling root path- ogens, respectively. A significant fraction of plant photosynthetic carbon may be used by these associated microorganisms, both to support their beneficial activities and for microbial growth and reproduction. Because many microbial individuals are associated with each individual plant, the individual benefit to a microbe that allocates more resources to its own reproduction (thereby allocating less to fixing N 2, supplying P, or producing antifungal metabolites) would exceed its individual loss from any resulting reduction in collective benefits (mainly plant carbon substrates). An initially rare ''free rider'' mutant strain might therefore be expected to displace its more cooperative parental strain. Yet, the mycorrhizal and legume-rhizobium mutualisms have persisted (often coexisting with ''cheating'') for millions of years. This paper discusses the importance of microbial cooperation (with plants and with other microbes) and possible reasons for its evolutionary persistence in the rhi- zosphere. In undisturbed soils, spatial structure can favor kin selection, but this may be counterbalanced by the increased likelihood that future competitors will be among the beneficiaries of current cooperation. In loose associations, direct fitness benefits to micro- organisms may explain the evolutionary persistence of activities (e.g., production of an- tifungal compounds) that can benefit plants as a side effect. In closer, more symbiotic, relationships, host sanctions against individuals or clones that fail to perform their symbiotic function may be more important. New molecular methods and other research tools are facilitating research on this topic, and some of these conclusions soon may be revised.

▪ Abstract Agricultural soils suppressive to soilborne plant pathogens occur worldwide, and for several of these soils the biological basis of suppressiveness has been described. Two classical types of suppressiveness are known. General suppression owes its activity to the total microbial biomass in soil and is not transferable between soils. Specific suppression owes its activity to the effects of individual or select groups of microorganisms and is transferable. The microbial basis of specific suppression to four diseases, Fusarium wilts, potato scab, apple replant disease, and take-all, is discussed. One of the best-described examples occurs in take-all decline soils. In Washington State, take-all decline results from the buildup of fluorescent Pseudomonas spp. that produce the antifungal metabolite 2,4-diacetylphloroglucinol. Producers of this metabolite may have a broader role in disease-suppressive soils worldwide. By coupling molecular technologies with traditional approaches used in plant pathology and microbiology, it is possible to dissect the microbial composition and complex interactions in 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.

There is increasing interest in commercial application of genetically modified microorganisms (GMMs) with improved biocontrol properties toward soil-borne plant pathogens. Despite long-term experience with the introduction of nonmodified microorganisms, concern about the ecological impact of large-scale release of GMMs remains. To date, risk assessment studies under field conditions have focused mainly on microorganisms genetically modified with markers, such as antibiotic resistance, lacZY, or xylE, and attention has been given to the potential impact of GMMs on the indigenous soil microflora (8, 29). Biologically mediated processes are central to the ecological functioning of the soil system. Microorganisms play a crucial role in nutrient cycling, and they contribute to suppression of soil-borne plant pathogens (7, 28). The introduction of large numbers of GMMs into the soil could alter microbial populations and disturb microbially driven soil processes. Effects of introduced GMMs on soil ecosystems have been studied mainly in microcosm experiments. Transient perturbations have been observed in indigenous bacterial (26), fungal (30), and protozoal (3) populations, in carbon turnover (38), and in soil enzyme activities (24). However, these microcosms lack the full biotic and abiotic components of a field environment. Most studies of nontarget effects of GMMs examine only the culturable microflora. This limitation reduces the value of the results, because only a small proportion (0.5 to 2%) of the fungal and bacterial microflora can be cultured using currently available media (37). Techniques such as amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), temperature gradient gel electrophoresis (TGGE), or denaturing gradient gel electrophoresis (DGGE) (23) can be used to more accurately monitor microbial communities without cultivation. Shifts in microbial communities, at the 16S rDNA level, have been studied mainly in soil polluted with heavy metals or with pesticides (12, 13, 31). Reports on the effects of functional genetically modified bacteria on microbial communities, using molecular methods, are scarce. Robleto et al. (29) demonstrated a reduction in the diversity of trifolitoxin-sensitive bacteria after inoculation of field-grown Phaseolus vulgaris with Rhizobium strains differing in their trifolitoxin production. As far as we are aware, no reports have been published on the effects of genetically modified biocontrol bacteria on the composition of the fungal microflora at the 18S rDNA level. We modified Pseudomonas putida WCS358r to produce the antifungal agent phenazine-1-carboxylic acid (PCA) (35), resulting in improved biocontrol activity toward fungal pathogens, such as Gaeumannomyces graminis var. tritici (D. Glandorf et al., unpublished data). Our objective in this study was to determine if genetically improved biocontrol bacteria could exert effects on nontarget members of the fungal rhizosphere microflora by using both cultivation-dependent and cultivation-independent methods.

Fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) have biocontrol activity against damping-off, root rot, and wilt diseases caused by soilborne fungal pathogens, and play a key role in the natural suppression of Gaeumannomyces graminis var. tritici, known as take-all decline. Diversity within phlD, an essential gene in the biosynthesis of 2,4-DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 2,4-DAPG-producing isolates from six states in the United States and six other locations worldwide. Clusters defined by RFLP analysis of phlD correlated closely with clusters defined previously by BOX-polymerase chain reaction (PCR) genomic fingerprinting, indicating the usefulness of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Random amplified polymorphic DNA analyses of genomic DNA revealed a higher degree of polymorphism than RFLP and BOX-PCR analyses. Genotypic diversity in a subset of 30 strains representing all the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, 2,4-DAPG, or total phloroglucinol compounds. Twenty-seven of the 30 representative strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.

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.

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.

Fluorescent Pseudomonas isolated from the rhizosphere of diverse plants have been studied as biocontrol agents of soilborne pathogens worldwide. Certain strains of these bacteria are capable of exerting a variety of mechanisms of plant growth promotion and protection, including the production of the natural antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG) and derivatives of phenazine (Phz). These compounds are broadly active against plant pathogens and are produced by widely distributed taxonomically diverse Pseudomonas spp. that inhabit the rhizosphere of cereal crops and render certain soils naturally suppressive to soilborne plant diseases. There is mounting evidence that the role of 2,4-DAPG and phenazines in the rhizosphere is not limited to antibiosis but also involves regulatory and signaling functions, induction of systemic resistance, and reduction of minerals in soil.

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.

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.

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.

Rhizobacteria introduced to control soil-borne root diseases must establish metabolically active populations that mediate protection either by direct antagonism of pathogens or by stimulation of host plant defenses. Recent interest has focused on the genetic and biochemical basis of disease control and the influence of environmental factors on the expression and activity of biocontrol mechanisms. The cloning and sequencing of genes involved in the production of microbial metabolites playing key roles in plant defense opens new possibilities for improving the performance of biocontrol agents.

Introduction Why biological control? The past 15 years have witnessed a dramatic increase in research related to biological control. The current perception that biocontrol will have an important role in commercial agriculture in the future contrasts markedly with previously-held views that biocontrol agents perform too inconsistently, or are too narrow in their spectrum of activity, as compared with chemical pesticides, to be commercially feasible on a large scale. Renewed interest in biological control is in part a response to widespread concern about the potential negative impact of chemical pesticides on public health and the environment. Furthermore, the techniques of molecular biology have revolutionized the field by facilitating the identification of the molecular basis of pathogen suppression and by providing the means for construction of ‘superior’ biocontrol agents. New biocontrol agents resulting from recent intensive research are slowly becoming available to agriculture, and the trend should accelerate throughout this decade. One example is Gliocladium virens , which is being marketed in potting-mix to control Pythium and Rhizoctonia (see Lumsden and Walter, Chapter 25). This chapter deals with the potential benefits and risks from the introduction of biocontrol agents for the control of root diseases of wheat, as well as the impediments to the application of this technology in commercial agriculture. The focus of the chapter is on biological control of take-all of wheat by fluorescent Pseudomonas spp. because it is a model system for the study of the molecular basis of pathogen suppression, root colonization by introduced bacteria and field application of biocontrol agents.

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.

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