Wheat Rhizosphere-Derived Bacteria Protect Soybean from Soilborne Diseases.

Soybean (Glycine max [L.] Merr.) is an important oilseed crop with a high economic value. However, three damaging soybean diseases, soybean cyst nematode (SCN; Heterodera glycines Ichinohe), Sclerotinia stem rot caused by the fungus Sclerotinia sclerotiorum (Lid.) de Bary, and soybean root rot caused by Fusarium spp., are major constraints to soybean production in the Great Plains. Current disease management options, including resistant or tolerant varieties, fungicides, nematicides, and agricultural practices (crop rotation and tillage), have limited efficacy for these pathogens or have adverse effects on the ecosystem. Microbes with antagonistic activity are a promising option to control soybean diseases with the advantage of being environmentally friendly and sustainable. In this study, 61 bacterial strains isolated from wheat rhizospheres were used to examine their antagonistic abilities against three soybean pathogens. Six bacterial strains significantly inhibited the growth of Fusarium graminearum in the dual-culture assay. These bacterial strains were identified as Chryseobacterium ginsengisoli, C. indologenes, Pseudomonas poae, two Pseudomonas spp., and Delftia acidovorans by 16S rRNA gene sequencing. Moreover, C. ginsengisoli, C. indologenes, and P. poae significantly increased the mortality of SCN second-stage juveniles (J2), and two Pseudomonas spp. inhibited the growth of S. sclerotiorum in vitro. Further growth chamber tests found that C. ginsengisoli and C. indologenes reduced soybean Fusarium root rot disease. C. ginsengisoli and P. poae dramatically decreased SCN egg number on SCN-susceptible soybean 'Williams 82'. Two Pseudomonas spp. protected soybean plants from leaf damage and collapse after being infected by S. sclerotiorum. These bacteria exhibit versatile antagonistic potential. This work lays the foundation for further research on the field control of soybean pathogens.

Occurrence of Mummy Berry Associated with Huckleberry (Vaccinium membranaceum) Caused by Monilinia spp. in Oregon.

In this Short Communication we describe the occurrence of mummy berry associated with huckleberry (Vaccinium membranaceum) caused by Monilinia spp. in Oregon. To our knowledge, this is the first report of a Monilinia spp. associated with mummy berry of huckleberry in Oregon. Sequence data from our specimens reveal the closest identity was Monilinia vaccinii-corymbosi, a pathogen of commercial blueberry (Vaccinium corymbosum). This may be a new species of Monilinia, not previously reported on huckleberry, and further investigation is needed. Of specific importance, the huckleberry holds cultural importance as a sacred First Food of the Confederated Tribes of the Umatilla Indian Reservation and other Pacific Northwest tribes. Although plant pathogen management in natural landscapes presents unique challenges, we will work with tribal authorities to determine whether cultural management techniques may mitigate yield loss due to Monilinia spp.

Plant-Parasitic Nematodes Associated With Wheat in Central, Eastern, and Southeastern Kazakhstan.

Kazakhstan is one of the biggest wheat producers, however, its wheat production is far below the average international wheat production standard due to biotic and abiotic stressors. Plant-parasitic nematodes are devastating for cereal production systems worldwide. A comprehensive survey was conducted in 2019 to identify plant-parasitic nematodes associated with wheat in different locations of central, eastern, and southeastern Kazakhstan. The results revealed 33 root-lesion and 27 cyst nematode populations from the 77 localities sampled. These two genera occurred in separate or in mixed populations. The root-lesion populations were identified as Pratylenchus neglectus and P. thornei while all cyst nematodes were identified as Heterodera filipjevi. The identification of nematodes was firstly performed based on morphological and morphometric features and confirmed by BLAST and phylogenetic analyses based on the internal transcribed spacer and the D2-D3 expansion located in the 28S gene of ribosomal DNA for CCN and RLN populations, respectively. Pratylenchus neglectus and P. thornei populations from Kazakhstan showed a high similarity with the American, European, and Asian populations. Heterodera filipjevi populations formed a well-supported cluster with the corresponding populations from different countries and showed a slightly intraspecific polymorphism. Kazakhstan populations of H. filipjevi may have multiple introductions in Kazakhstan due to the divergence among them. The results of this study are of great importance for breeding programs and will enable awareness for extension advisors to develop measures to control these nematodes in cereal cropping areas in Kazakhstan.

First Report of Crown Rot Caused by Fusarium redolens on Wheat in Kazakhstan.

Fusarium crown rot, caused by several species within the genus, is a major constraint that results in significant losses in wheat production worldwide. In June 2019, diseased wheat plants with typical symptoms of crown rot, including discoloration on the first two or three internodes of the stem just above the soil line and stunted, dry rotted, and discolored roots were collected in several bread wheat fields during the maturity stage in Almaty, East Kazakhstan, and Karaganda Regions of Kazakhstan. For each field, approximately twenty tillers were randomly sampled. Symptomatic tissues were surface sterilized in 1% NaClO for 2 min, rinsed with sterile distilled water three times, air-dried in a laminar flow hood, and then transferred to Petri dishes containing one-fifth strength potato dextrose agar (PDA). After incubating in the dark at 23°C for 5 days, 79 single-spore isolates showing cultural and microscopic characteristics of Fusarium were obtained on PDA and Spezieller-Nährstoffarmer agar (SNA). Colonies were initially white but later produced a beige to pink diffusible pigment in PDA. Microconidia that formed on aerial monophialides were hyaline, 0 to 1 septum, oval- to kidney-shaped, and measured 4.3 to 10.3 × 1.9 to 3.4 µm (average 7.8 × 2.6 µm), whilst macroconidia were straight to slightly curved, 3 to 5 septate, and measured 18.7 to 38.8 × 2.9 to 6.6 µm (average 29.9 × 4.7 µm), with foot-shaped basal cells on SNA. Chlamydospores were present on PDA. Sequence analysis based on portions of translation elongation factor 1α (TEF1) and the nuclear ribosomal internal transcribed spacer region (ITS rDNA) loci with primers EF1/EF2 (O'Donnell et al. 1998) and ITS1/ITS4 (White et al. 1990) identified 29 of the 79 isolates as Fusarium redolens Wollenw. The sequences of the five representative isolates with 99.85% of similarity to those of F. redolens strains available in GenBank e.g., ITS (MT435063) and TEF1 (GU250584). The TEF1 (accession nos. MW403914-MW403918) and ITS rDNA (accession nos. MW397138-MW397142) sequences of the isolates were deposited in GenBank. The morphological features are consistent with the described features of F. redolens (Leslie and Summerell 2006). To confirm pathogenicity of the five isolates, five pre-germinated seeds of wheat cultivar Seri 82 were placed in a 9-cm-diameter pot filled with a sterile potting mix containing equal volumes of peat, vermiculite, and soil. An approximately 1-cm-diameter 7-day-old mycelial plug of each isolate was individually placed in contact with the seeds. Seeds were covered with the same potting mix, and then the pots were maintained for four weeks in a growth chamber at 23°C with a 12-h photoperiod. The experiment was conducted twice with three replicate 15-cm pots with 5 plants per pot. Controls were inoculated with sterile agar plugs using the same procedure. After four weeks, all the inoculated plants showed stunted growth with brown discoloration in most parts of the crown and roots, whereas no symptoms were observed in the control plants. The mean severity of the disease for each isolate was between 2.1 and 2.7 according to the scale of 1 to 5 described by Gebremariam et al. (2015). The pathogen was reisolated from crowns of diseased plants, but not from asymptomatic control tissues, and identified morphologically based on the methods described above, fulfilling Koch's postulates. Although several morphological features are shared by F. oxysporum and F. redolens, Baayen et al. (2001) showed that these species could be easily distinguished using molecular data. The pathogen was previously reported as F. redolens associated with crown rot of wheat in Turkey (Gebremariam et al. 2015) and Saskatchewan, Canada (Taheri et al. 2011). The presence of F. redolens causing crown rot is confirmed in the six wheat fields surveyed in Kazakhstan, for the first time. This pathogen may pose a risk for wheat production, and further studies needed to determine the impact on the crop in Kazakhstan.

Chickpea Seed Rot and Damping-Off Caused by Metalaxyl-Resistant Pythium ultimum and Its Management with Ethaboxam.

Metalaxyl and its isomer mefenoxam have been the primary fungicides used as seed treatments in managing Pythium seed rot and damping-off of chickpea (Cicer arietinum). However, outbreaks of seed rot and damping-off of metalaxyl-treated chickpea seeds were found in the dryland agriculture regions of southeastern Washington and northern Idaho. Pythium spp. isolated from rotten seeds and associated soils showed high levels of resistance to metalaxyl. Large proportions (31 to 91%) of Pythium isolates resistant to metalaxyl were detected in areas where severe chickpea damping-off occurred and were observed in commercial chickpea fields over several years. All metalaxyl-resistant (MR) isolates were identified as Pythium ultimum var. ultimum. The metalaxyl resistance trait measured by EC50 values was stable over 10 generations in the absence of metalaxyl, and no observable fitness costs were associated with metalaxyl resistance. Under controlled conditions, metalaxyl treatments failed to protect chickpea seeds from seed rot and damping-off after inoculation with MR Pythium isolates. In culture, ethaboxam inhibited mycelial growth of both MR and metalaxyl-sensitive isolates. Greenhouse and field tests showed that ethaboxam is effective in managing MR Pythium. Ethaboxam in combination with metalaxyl is commonly applied as seed treatments in commercial chickpea production.

Core Rhizosphere Microbiomes of Dryland Wheat Are Influenced by Location and Land Use History.

The Inland Pacific Northwest is one of the most productive dryland wheat production areas in the United States. We explored the bacterial and fungal communities associated with wheat in a controlled greenhouse experiment using soils from multiple locations to identify core taxa consistently associated with wheat roots and how land use history influences wheat-associated communities. Further, we examined microbial co-occurrence networks from wheat rhizospheres to identify candidate hub taxa. Location of origin and land use history (long-term no-till versus noncropped Conservation Reserve Program [CRP]) of soils were the strongest drivers of bacterial and fungal communities. Wheat rhizospheres were especially enriched in many bacterial families, while only a few fungal taxa were enriched in the rhizosphere. There was a core set of bacteria and fungi that was found in >95% of rhizosphere or bulk soil samples, including members of Bradyrhizobium, Sphingomonadaceae, Massilia, Variovorax, Oxalobacteraceae, and Caulobacteraceae Core fungal taxa in the rhizosphere included Nectriaceae, Ulocladium, Alternaria, Mortierella, and Microdochium Overall, there were fewer core fungal taxa, and the rhizosphere effect was not as pronounced as with bacteria. Cross-domain co-occurrence networks were used to identify hub taxa in the wheat rhizosphere, which included bacterial and fungal taxa (e.g., Sphingomonas, Massilia, Knufia, and Microdochium). Our results suggest that there is a relatively small group of core rhizosphere bacteria that were highly abundant on wheat roots regardless of soil origin and land use history. These core communities may play important roles in nutrient uptake, suppressing fungal pathogens, and other plant health functions.IMPORTANCE Plant-associated microbiomes are critical for plant health and other important agroecosystem processes. We assessed the bacterial and fungal microbiomes of wheat grown in soils from across a dryland wheat cropping systems in eastern Washington to identify the core microbiome on wheat roots that is consistent across soils from different locations and land use histories. Moreover, cross-domain co-occurrence network analysis identified core and hub taxa that may play important roles in microbial community assembly. Candidate core and hub taxa provide a starting point for targeting microbiome components likely to be critical to plant health and for constructing synthetic microbial communities for further experimentation. This work is one of the first examples of identifying a core microbiome on a major field crop grown across hundreds of square kilometers over a wide range of biogeographical zones.

Population Dynamics of Wheat Root Pathogens Under Different Tillage Systems in Northeast Oregon.

No-till or direct seeding can be described as seeding directly into the crop stubble from the previous season without use of tillage. A reduction in tillage can result in many benefits, including increased soil organic matter, increased water holding capacity, and reduced fuel costs. However, the effect of no-till and reduced tillage on crop root disease profiles is poorly understood. To study the effect of tillage on disease dynamics, soil samples were collected from commercial wheat fields representing a wide range of tillage strategies in fall 2016 and fall 2017. Because precipitation might affect soilborne diseases, wheat fields located across a diverse gradient of precipitation zones of the dryland Pacific Northwest were selected. Fusarium spp., Pythium spp., and Rhizoctonia spp. were quantified from soil samples using soil dilution plating and quantitative PCR (qPCR) assays. Results of dilution plating showed that the colony counts of Fusarium, Pythium, and Rhizoctonia at the genus level were negatively associated with tillage. However, the same patterns were not observed when specific causal agents of Fusarium, Pythium, and Rhizoctonia that are known to be pathogenic on wheat were quantified with qPCR. Furthermore, precipitation affected the population density of some fungal pathogens (F. culmorum, P. ultimum, and R. solani AG 8). Within the scope of inference of this study, results of this study indicate that the benefits of adopting reduced tillage likely outweigh potential risk for increased root disease.

Identity and Pathogenicity of Fungi Associated with Crown and Root Rot of Dryland Winter Wheat in Azerbaijan.

A comprehensive survey was performed to assess fungal populations associated with crown and root rot of wheat throughout the main wheat-growing areas of Azerbaijan. Samples were taken from 76 fields; 630 fungal strains were isolated, identified, and evaluated for pathogenicity. The identification was conducted with morphological and molecular tools such as species-specific PCR and DNA sequencing of the internal transcribed spacer (ITS) and translation elongation factor 1-α (EF1-α) loci. The fungus found in the greatest number of fields (44) was Fusarium culmorum with 192 isolates, followed by F. acuminatum. Other Fusarium spp. isolates were identified: F. equiseti, F. pseudograminearum, F. graminearum, F. incarnatum, F. avenaceum, F. hostae, F. oxysporum, F. proliferatum, F. algeriense, and F. brachygibbosum. Bipolaris sorokiniana, Curvularia spicifera, Exserohilum pedicellatum, Nigrospora oryzae, and Rhizoctonia spp. isolates were also identified, associated with underground parts of wheat. Phylogenetic analyses based on ITS and EF1-α sequences of the isolates showed that the isolates belonging to the same species were clearly separated in the dendrogram. Pathogenicity assays revealed that F. culmorum, F. pseudograminearum, and F. graminearum were most aggressive; F. avenaceum, F. hostae, F. algeriense, B. sorokiniana, C. spicifera, and R. solani isolates were moderately aggressive; C. inaequalis, E. pedicellatum, and N. oryzae were weakly aggressive; and others were nonpathogenic. The result of this study exhibited the existence of a wide range of species associated with crown and root rot of wheat in Azerbaijan. Additionally, this is the first report of F. hostae, F. algeriense, C. spicifera, C. inaequalis, and N. oryzae as pathogens on wheat in Azerbaijan. Azerbaijan is the second country after Algeria in which F. algeriense was detected.

Biosolids and Tillage Practices Influence Soil Bacterial Communities in Dryland Wheat.

Class B biosolids are used in dryland wheat (Triticum aestivum L.) production in eastern Washington as a source of nutrients and to increase soil organic matter, but little is known about their effects on bacterial communities and potential for harboring human pathogens. Moreover, conservation tillage is promoted to reduce erosion and soil degradation. We explored the impacts of biosolids or synthetic fertilizer in combination with traditional (conventional) or conservation tillage on soil bacterial communities. Bacterial communities were characterized from fresh biosolids, biosolid aggregates embedded in soil, and soil after a second application of biosolids using high-throughput amplicon sequencing. Biosolid application significantly affected bacterial communities, even 4 years after their application. Bacteria in the families Clostridiaceae, Norcardiaceae, Anaerolinaceae, Dietziaceae, and Planococcaceae were more abundant in fresh biosolids, biosolid aggregates, and soils treated with biosolids than in synthetically fertilized soils. Taxa identified as Turcibacter, Dietzia, Clostridiaceae, and Anaerolineaceae were highly abundant in biosolid aggregates in the soil and likely originated from the biosolids. In contrast, Oxalobacteriaceae, Streptomyceteaceae, Janthinobacterium, Pseudomonas, Kribbella, and Bacillus were rare in the fresh biosolids, but relatively abundant in biosolid aggregates in the soil, and probably originated from the soil to colonize the substrate. However, tillage had relatively minor effects on bacterial communities, with only a small number of taxa differing in relative abundance between traditional and conventional tillage. Although biosolid-associated bacteria persisted in soil, potentially pathogenic taxa were extremely rare and no toxin genes for key groups (Salmonella, Clostridium) were detectable, suggesting that although fecal contamination was apparent via indicator taxa, pathogen populations had declined to low levels. Thus, biosolid amendments had profound effects on soil bacterial communities both by introducing gut- or digester-derived bacteria and by enriching potentially beneficial indigenous soil populations.

Distribution of Cereal Cyst Nematodes (Heterodera avenae and H. filipjevi) in Eastern Washington State.

Cereal cyst nematodes (CCN; Heterodera avenae and H. filipjevi), cause substantial worldwide yield loss in small grain cereals such as wheat, barley, and oat. H. avenae was first detected in the United States in western Oregon in 1974 and had spread to northeast Oregon by the mid-1980s. Although H. avenae was detected in eastern Washington in 1984, extensive infestations were not recognized until 2010. H. filipjevi, first detected in Oregon in 2008, was found in eastern Washington in 2014. To gain more information about the distribution of these two species, an extensive survey was undertaken in eastern Washington, and methods were developed to distinguish species using DNA sequencing of single cysts. In this study, we surveyed 356 wheat and barley fields in eastern Washington from 2007 to 2017. CCN from the infested locations were identified to species level by sequencing the ribosomal internal transcribed spacers (ITS) and/or 28S ribosomal RNA (rRNA) genes. The sequences were compared in the GenBank database in the National Center for Biotechnology Information (NCBI) to identify species. The results show that H. filipjevi is primarily confined to southern Whitman County, WA; and H. avenae has a wider distribution across the higher precipitation annual cropping area of eastern Whitman County. Knowledge of species identification is critical for deployment of host resistance as an effective means of management, since resistance genes for one species of CCN may not be effective against the other.

Location, Root Proximity, and Glyphosate-Use History Modulate the Effects of Glyphosate on Fungal Community Networks of Wheat.

Glyphosate is the most-used herbicide worldwide and an essential tool for weed control in no-till cropping systems. However, concerns have been raised regarding the long-term effects of glyphosate on soil microbial communities. We examined the impact of repeated glyphosate application on bulk and rhizosphere soil fungal communities of wheat grown in four soils representative of the dryland wheat production region of Eastern Washington, USA. Further, using soils from paired fields, we contrasted the response of fungal communities that had a long history of glyphosate exposure and those that had no known exposure. Soil fungal communities were characterized after three cycles of wheat growth in the greenhouse followed by termination with glyphosate or manual clipping of plants. We found that cropping system, location, year, and root proximity were the primary drivers of fungal community compositions, and that glyphosate had only small impacts on fungal community composition or diversity. However, the taxa that responded to glyphosate applications differed between rhizosphere and bulk soil and between cropping systems. Further, a greater number of fungal OTUs responded to glyphosate in soils with a long history of glyphosate use. Finally, fungal co-occurrence patterns, but not co-occurrence network characteristics, differed substantially between glyphosate-treated and non-treated communities. Results suggest that most fungi influenced by glyphosate are saprophytes that likely feed on dying roots.

Succession of Fungal and Oomycete Communities in Glyphosate-Killed Wheat Roots.

The successional dynamics of root-colonizing microbes are hypothesized to be critical to displacing fungal pathogens that can proliferate after the use of some herbicides. Applications of glyphosate in particular, which compromises the plant defense system by interfering with the production of aromatic amino acids, are thought to promote a buildup of root pathogens and can result in a "greenbridge" between weeds or volunteers and crop hosts. By planting 2 to 3 weeks after spraying, growers can avoid most negative impacts of the greenbridge by allowing pathogen populations to decline, but with the added cost of delayed planting dates. However, the specific changes in microbial communities during this period of root death and the microbial taxa likely to be involved in displacing pathogens are poorly characterized. Using high-throughput sequencing, we characterized fungal and oomycete communities in roots after applications of herbicides with different modes of action (glyphosate or clethodim) and tracked their dynamics over 3 weeks in both naturally infested soil and soil inoculated with Rhizoctonia solani AG-8. We found that many unexpected taxa were present at high relative abundance (e.g., Pythium volutum and Myrmecridium species) in live and dying wheat roots and may play an under-recognized role in greenbridge dynamics. Moreover, communities were highly dynamic over time and had herbicide-specific successional patterns, but became relatively stable by 2 weeks after herbicide application. Network analysis of communities over time revealed patterns of interactions among taxa that were both common and unique to each herbicide treatment and identified two primary groups of taxa with many positive associations within-groups but negative associations between-groups, suggesting that these groups are antagonistic to one another in dying roots and may play a role in displacing pathogen populations during greenbridge dynamics.

Impacts of Repeated Glyphosate Use on Wheat-Associated Bacteria Are Small and Depend on Glyphosate Use History.

Glyphosate is the most widely used herbicide worldwide and a critical tool for weed control in no-till cropping systems. However, there are concerns about the nontarget impacts of long-term glyphosate use on soil microbial communities. We investigated the impacts of repeated glyphosate treatments on bacterial communities in the soil and rhizosphere of wheat in soils with and without long-term history of glyphosate use. We cycled wheat in the greenhouse using soils from 4 paired fields under no-till (20+-year history of glyphosate) or no history of use. At each cycle, we terminated plants with glyphosate (2× the field rate) or by removing the crowns, and soil and rhizosphere bacterial communities were characterized. Location, cropping history, year, and proximity to the roots had much stronger effects on bacterial communities than did glyphosate, which only explained 2 to 5% of the variation. Less than 1% of all taxa were impacted by glyphosate, more in soils with a long history of use, and more increased than decreased in relative abundance. Glyphosate had minimal impacts on soil and rhizosphere bacteria of wheat, although dying roots after glyphosate application may provide a "greenbridge" favoring some copiotrophic taxa.IMPORTANCE Glyphosate (Roundup) is the most widely used herbicide in the world and the foundation of Roundup Ready soybeans, corn, and the no-till cropping system. However, there have been recent concerns about nontarget impacts of glyphosate on soil microbes. Using next-generation sequencing methods and glyphosate treatments of wheat plants, we described the bacterial communities in the soil and rhizosphere of wheat grown in Pacific Northwest soils across multiple years, different locations, and soils with different histories of glyphosate use. The effects of glyphosate were subtle and much less than those of drivers such as location and cropping systems. Only a small percentage of the bacterial groups were influenced by glyphosate, and most of those were stimulated, probably because of the dying roots. This study provides important information for the future of this important tool for no-till systems and the environmental benefits of reducing soil erosion and fossil fuel inputs.

Disease Suppressive Soils: New Insights from the Soil Microbiome.

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.

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.

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.

Timing of Glyphosate Applications to Wheat Cover Crops to Reduce Onion Stunting Caused by Rhizoctonia solani.

Stunting caused by Rhizoctonia spp. is economically important in irrigated onion bulb crops in the semiarid Columbia Basin of Oregon and Washington, where cereal winter cover crops commonly are planted the previous fall to prevent wind erosion of soil. The cover crop is killed with herbicide application just before or shortly after onion seeding, so that the dead rows of cereal plants provide a physical barrier tall enough to protect onion seedlings against wind and sand blasting but not tall enough to shade onion seedlings. However, the cover crop also serves as a green bridge for Rhizoctonia spp. on cereal roots to colonize the onion roots, potentially resulting in severe stunting of onion seedlings. To determine the effect of timing of application of the herbicide glyphosate to reduce this green bridge effect and, subsequently, onion stunting, three herbicide application intervals preceding onion planting were evaluated in a grower's onion field in each of 2012 and 2014 in the Columbia Basin. The wheat cover crop was killed with a glyphosate application 27, 17, and 3 days before onion seeding in 2012 and 19, 10, and 3 days before seeding in 2014. As the interval between herbicide application and onion planting increased from 3 days to 19 and 27 days, the number of patches of stunted onion plants decreased by ≥55%, total area of stunted patches decreased by 54 to 63%, and patch severity index decreased by 59 to 65%. Similarly, the Rhizoctonia solani AG 8 DNA concentration in soil sampled from the dead cover crop rows declined as the interval between glyphosate application and onion seeding increased in the 2012 trial but not in the 2014 trial. R. solani AG 3 and AG 8 DNA concentrations in soil sampled from the cover crop rows were significantly positively correlated with the number of patches of stunted onion plants (r = 0.490 and 0.607 at P = 0.039 and 0.008, respectively), total area of stunted patches (r = 0.496 and 0.659 at P = 0.035 and 0.003, respectively), and patch severity index (r = 0.492 and 0.635 at P = 0.038 and 0.005, respectively) in the 2012 trial; however, these variables were only correlated significantly with R. solani AG 3 DNA concentration in the 2014 trial. Increasing the interval between herbicide application to the cover crop and onion planting provides a practical management tool for stunting in onion bulb crops.

Pythium Species Associated with Damping-off of Pea in Certified Organic Fields in the Columbia Basin of Central Washington.

Organic vegetable production accounted for 19% of the total organic acreage in Washington State in 2013, with 1,700 ha of certified organic vegetable pea. However, production is challenged constantly with the threat of poor emergence after planting due to damping-off caused by Pythium spp. A survey of Pythium spp. in organic vegetable production areas of the semiarid Columbia Basin of central Washington was carried out in fall 2009 to identify species associated with damping-off during early spring planting. Of 305 isolates baited from soil sampled from 37 certified organic fields, 264 were identified to 16 Pythium spp. by sequencing the internal transcribed spacer region of ribosomal DNA. A soil DNA-CFU regression curve was developed using real-time quantitative polymerase chain reaction assays for each of the three predominant pathogenic species (Pythium abappressorium, the P. irregulare complex, and P. ultimum var. ultimum) found in soil sampled from the 37 fields. The P. irregulare complex, P. abappressorium, and P. ultimum var. ultimum were detected in 57, 78, and 100% of the fields sampled, respectively. A regression analysis was used to determine that P. ultimum var. ultimum ranged from 14 to 332 CFU/g of soil in the 37 fields, the P. irregulare complex ranged from 25 to 228 CFU/g of soil, and P. abappressorium DNA was below the quantifiable limit. In summary, P. ultimum var. ultimum was the most prevalent pathogenic Pythium sp. detected in certified organic fields in the semiarid Columbia Basin of central Washington but multiple Pythium spp. may be associated with damping-off in cool and wet, early spring planting conditions.

Stunted Patches in Onion Bulb Crops in Oregon and Washington: Etiology and Yield Loss.

Onion stunting caused by Rhizoctonia spp. is an important soilborne disease on very sandy soils in the Columbia Basin of Oregon and Washington. From 2010 to 2013, 251 isolates of Rhizoctonia or Rhizoctonia-like spp. were obtained from soil and onion plant samples collected from inside and outside patches of stunted plants in 29 onion fields in the Columbia Basin. Sequence analysis of the internal transcribed spacer (ITS) region was used to identify the isolates, with 13 anastomosis groups (AGs) or subspecies detected. The most frequent was Waitea circinata var. circinata (25%), followed by Rhizoctonia solani AG 3 (17%), R. solani AG 4 (14%), Ceratobasidium sp. AG A (10%), R. solani AG 8 (7%), Ceratobasidium sp. AG K (6%), R. solani AG 2-1 (6%), W. circinata var. zeae (6%), R. solani AG 5 (4%), Ceratobasidium sp. AG G (2%), R. solani AG 11 (2%), and R. solani AG 1-1B and AG 10 (each <1%). However, the distribution of AGs and subspecies varied depending on whether soil or onion plants samples were collected within or adjacent to patches of stunted onion plants. In an attempt to predict the risk of onion stunting for a field prior to planting, DNA concentrations of AG 2-1, AG 3, AG 4, and AG 8 were quantified from bulk soil samples collected from each of nine growers' fields approximately 1 month before onion sowing in 2012. The preplant DNA concentrations did not show a significant association with the amount of stunting observed in the fields during the growing season. In contrast, the frequency of isolation and DNA concentration of R. solani AG 8 detected in soil samples collected during the growing season were greater from inside patches of stunted onion plants than from adjacent healthy areas of an onion crop sampled in 2012, but not for soil samples collected similarly from an onion crop in 2013. AG 2-1, AG 3, and AG 4 DNA concentrations did not differ significantly in soil sampled inside versus outside stunted patches in the fields sampled in 2012 and 2013. Relationships between the number of bulbs harvested or bulb weight versus severity of stunting were defined using correlation and regression analyses for six onion cultivars grown in seven fields surveyed in 2012 and 2013. Onion stunting reduced the average marketable bulb yield by 25 to 60% within stunted patches of the six cultivars. Stunting did not reduce onion plant stand but consistently reduced the size of bulbs, and yield reduction increased with increasing disease severity.

Characterization and Pathogenicity of Rhizoctonia and Rhizoctonia-Like spp. From Pea Crops in the Columbia Basin of Oregon and Washington.

Isolates of Rhizoctonia and Rhizoctonia-like spp. (n = 179) were baited selectively from soil and plant samples collected from irrigated pea crops in the semiarid Columbia Basin of Oregon and Washington from 2011 to 2013, and characterized to species, subspecies, and anastomosis groups (AG) based on sequences of the internal transcribed spacer region of ribosomal DNA. Rhizoctonia solani comprised 76% of all isolates, and included isolates of AG 4 (31% of all isolates), AG 2-1 (18%), AG 3 (10%), AG 8 (8%), AG 5 (5%), AG 10 (3%), and AG 9 (1%). The isolates of Ceratobasidium spp. (20%) comprised four AGs: AG K (11%), AG A (6%), AG I (2%), and AG I-like (1%). Waitea circinata isolates (4%) comprised two subspecies: W. circinata var. circinata (approximately 4%) and W. circinata var. zeae (<1%). Repeated pathogenicity tests of isolates of the 10 most frequently detected AGs and subspecies on 'Serge' pea at 15°C revealed that R. solani AG 2-1 caused the greatest reduction in pea emergence, followed by R. solani AG 4. R. solani AG 4 caused the most severe root rot, stunting, and reduction in pea seedling biomass, followed by isolates of AG 2-1. R. solani AG 8 did not affect emergence, plant height, and total biomass compared with noninoculated control plants; however, root rot caused by isolates of AG 8 was ranked the third most severe among isolates of the 10 Rhizoctonia subgroups, after that caused by isolates of AG 4 and AG 2-1. Isolates of other AGs and subspecies were either weakly virulent or nonpathogenic on pea. The most common AGs (AG 4 and AG 2-1) detected in pea fields in the Columbia Basin were also the most virulent. In a growers' pea crop grown for seed ('Prevail') planted 5 days after herbicide application and incorporation of a preceding winter wheat crop, severe stunting caused by Rhizoctonia spp. resulted in an average 75% yield loss within patches of stunted plants. In contrast, the yield of processing pea from a green pea crop of Serge did not differ significantly for plants sampled within versus outside patches of stunted plants; however, plants within patches were significantly more mature. In the Prevail seed crop, a greater frequency of R. solani AG 8 was detected than AG 2-1 or AG 4 from within patches of stunted plants, indicating that isolates of AG 8 may be associated with the root rot complex in some pea crops in the Columbia Basin.