Synthetic microbial consortia derived from rhizosphere soil protect wheat against a soilborne fungal pathogen.

Synthetic microbial communities (SynComs) could potentially enhance some functions of the plant microbiome and emerge as a promising inoculant for improving crop performance. Here, we characterized a collection of bacteria, previously isolated from the wheat rhizosphere, for their antifungal activity against soilborne fungal pathogens. Ten SynComs with different compositions from 14 bacterial strains were created. Seven SynComs protected wheat from Rhizoctonia solani AG8 infection, although SynComs were not more effective than single strains in reducing wheat root rot disease. Further, the mechanisms of interaction of the tested bacteria with each other and plants were explored. We found that nine bacteria and nine SynComs impacted the root growth of Arabidopsis. Nine bacteria and four SynComs significantly inhibited the growth of AG8 by producing volatiles. The cell-free supernatants from six bacteria inhibited the growth of AG8. Together, this study provided the potential for improving crop resilience by creating SynComs.

Fungal Pathogens Associated with Crown and Root Rot of Wheat in Central, Eastern, and Southeastern Kazakhstan.

Kazakhstan is the fourteenth largest wheat producer in the world. Despite this fact, there has not been a comprehensive survey of wheat root and crown rot. A quantitative survey was conducted for the purpose of establishing the distribution of fungi associated with root and crown rot on wheat (Triticum spp.). During the 2019 growing season, samples were taken from the affected plants' roots and stem bases. A total of 1221 fungal isolates were acquired from 65 sites across the central (Karagandy region), eastern (East Kazakhstan region), and southeastern (Almaty region) parts of the country and identified using morphological and molecular tools. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF1-α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences were successfully used to identify the species of fungal isolates. It was found that Bipolaris sorokiniana (44.80%) and Fusarium acuminatum (20.39%) were the most predominant fungal species isolated, which were present in 86.15 and 66.15% of the fields surveyed, respectively, followed by F. equiseti (10.16%), Curvularia spicifera (7.62%), F. culmorum (4.75%), F. oxysporum (4.10%), F. redolens (2.38%), Rhizoctonia solani AG2-1 (1.06%), Nigrospora oryzae (0.98%), C. inaequalis (0.90%), F. pseudograminearum (0.74%), F. flocciferum (0.74%), Macrophomina phaseolina (0.66%), F. cf. incarnatum (0.33%), Fusarium sp. (0.25%), and F. torulosum (0.16%). A total of 74 isolates representing 16 species were tested via inoculation tests on the susceptible Triticum aestivum cv. Seri 82 and the results revealed that F. culmorum and F. pseudograminearum, B. sorokiniana, Fusarium sp., R. solani, F. redolens, C. spicifera, C. inaequalis, and N. oryzae were virulent, whereas others were non-pathogenic. The findings of this investigation demonstrate the presence of a diverse spectrum of pathogenic fungal species relevant to wheat crown and root rot in Kazakhstan. To the best of our knowledge, this is the first report of F. pseudograminearum, Fusarium sp., C. spicifera, and C. inaequalis as pathogens on wheat in Kazakhstan.

Responses of Soil Fungal Communities to Lime Application in Wheat Fields in the Pacific Northwest.

Liming is an effective agricultural practice and is broadly used to ameliorate soil acidification in agricultural ecosystems. Our understanding of the impacts of lime application on the soil fungal community is scarce. In this study, we explored the responses of fungal communities to liming at two locations with decreasing soil pH in Oregon in the Pacific Northwest using high-throughput sequencing (Illumina MiSeq). Our results revealed that the location and liming did not significantly affect soil fungal diversity and richness, and the impact of soil depth on fungal diversity varied among locations. In contrast, location and soil depth had a strong effect on the structure and composition of soil fungal communities, whereas the impact of liming was much smaller, and location- and depth-dependent. Interestingly, families Lasiosphaeriaceae, Piskurozymaceae, and Sordariaceae predominated in the surface soil (0-7.5 cm) and were positively correlated with soil OM and aluminum, and negatively correlated with pH. The family Kickxellaceae which predominated in deeper soil (15-22.5 cm), had an opposite response to soil OM. Furthermore, some taxa in Ascomycota, such as Hypocreales, Peziza and Penicillium, were increased by liming at one of the locations (Moro). In conclusion, these findings suggest that fungal community structure and composition rather than fungal diversity responded to location, soil depth and liming. Compared to liming, location and depth had a stronger effect on the soil fungal community, but some specific fungal taxa shifted with lime application.

Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Rhizosphere community selection reveals bacteria associated with reduced root disease.

BACKGROUND: Microbes benefit plants by increasing nutrient availability, producing plant growth hormones, and protecting against pathogens. However, it is largely unknown how plants change root microbial communities. RESULTS: In this study, we used a multi-cycle selection system and infection by the soilborne fungal pathogen Rhizoctonia solani AG8 (hereafter AG8) to examine how plants impact the rhizosphere bacterial community and recruit beneficial microorganisms to suppress soilborne fungal pathogens and promote plant growth. Successive plantings dramatically enhanced disease suppression on susceptible wheat cultivars to AG8 in the greenhouse. Accordingly, analysis of the rhizosphere soil microbial community using deep sequencing of 16S rRNA genes revealed distinct bacterial community profiles assembled over successive wheat plantings. Moreover, the cluster of bacterial communities formed from the AG8-infected rhizosphere was distinct from those without AG8 infection. Interestingly, the bacterial communities from the rhizosphere with the lowest wheat root disease gradually separated from those with the worst wheat root disease over planting cycles. Successive monocultures and application of AG8 increased the abundance of some bacterial genera which have potential antagonistic activities, such as Chitinophaga, Pseudomonas, Chryseobacterium, and Flavobacterium, and a group of plant growth-promoting (PGP) and nitrogen-fixing microbes, including Pedobacter, Variovorax, and Rhizobium. Furthermore, 47 bacteria isolates belong to 35 species were isolated. Among them, eleven and five exhibited antagonistic activities to AG8 and Rhizoctonia oryzae in vitro, respectively. Notably, Janthinobacterium displayed broad antagonism against the soilborne pathogens Pythium ultimum, AG8, and R. oryzae in vitro, and disease suppressive activity to AG8 in soil. CONCLUSIONS: Our results demonstrated that successive wheat plantings and pathogen infection can shape the rhizosphere microbial communities and specifically accumulate a group of beneficial microbes. Our findings suggest that soil community selection may offer the potential for addressing agronomic concerns associated with plant diseases and crop productivity. Video Abstract.

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.

Decline in Soil Microbial Abundance When Camelina Introduced Into a Monoculture Wheat System.

Camelina [Camelina sativa (L.) Crantz] of the Brassicaceae family is a potential alternative and oilseed biofuel crop for wheat (Triticum aestivum L.)-based cropping systems of the Inland Pacific Northwest (PNW) of the United States. We investigated the effect of this relatively new rotational crop on soil microbial communities. An 8-year cropping systems experiment was initiated in 2007 at Lind, WA, to compare a 3-year rotation of winter wheat (WW)-camelina (C)-fallow (F) to the typical 2-year WW-F rotation. All phases of both rotations (total = 20 plots) were present every year to allow valid statistical analysis and data interpretations. Monoculture WW-F is the dominant system practiced by the vast majority of farmers on 1.56 million ha of cropland in the PNW drylands that receive <300 mm average annual precipitation. Microbial abundance and community composition were determined using phospholipid fatty acid analysis (PLFA) from soil samples collected during 3 consecutive years beginning in 2010. The abundance of fungi, mycorrhizae, Gram positive and negative bacteria, and total microbial abundance all declined over the 3-year period in the WW-C-F rotation compared to the WW-F rotation. All microbial lipid biomarkers were significantly less in fallow compared to WW of the WW-C-F rotation. The 2-year WW-F rotation demonstrated few differences in microbial lipid abundance and community structure between the rotation phases. Microbial abundance declined and community structure shifted in the 3-year WW-C-F rotation likely due to the combination of a Brassica crop followed by a 13-month-long fallow. The results of this study suggest that camelina in combination with a fallow period may disrupt microbial communities that have become stable under historical WW-F monocropping. Such disturbances have the potential to affect soil processes that have been provided by wheat-adapted microbial communities. However, the disruption appears to be short-lived with the microbial abundance of WW in the WW-C-F rotation, returning to similar levels observed in the WW-F rotation.

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.

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.

A mutualistic interaction between Streptomyces bacteria, strawberry plants and pollinating bees.

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.

Mining the Drilosphere: Bacterial Communities and Denitrifier Abundance in a No-Till Wheat Cropping System.

Earthworms play important roles in no-till cropping systems by redistributing crop residue to lower soil horizons, providing macropores for root growth, increasing water infiltration, enhancing soil quality and organic matter, and stimulating nitrogen cycling. The soil impacted by earthworm activity, including burrows, casts, and middens, is termed the drilosphere. The objective of this study was to determine the effect of earthworms on soil microbial community composition in the drilosphere at different landscape slope positions. Soil cores (50 cm depth) were extracted from three landscape locations (top, middle, and bottom slope positions) on a sloping aspect of a no-till wheat farm. Soil was sampled at the bottom of the soil core from inside multiple earthworm (Lumbricus terrestris) channels (drilosphere) and from adjacent bulk soil. Bacterial communities were characterized for 16S rRNA gene diversity using high-throughput sequencing and functional denitrifier gene abundance (nirK, nirS, and nosZ) by quantitative PCR. Bacterial communities were structured primarily by the landscape slope position of the soil core followed by source (bulk versus drilosphere soil), with a significant interaction between core position and source. The families AKIW874, Chitinophagaceae, and Comamonadaceae and the genera Amycolatopsis, Caulobacter, Nocardioides, and Variovorax were more abundant in the drilosphere compared to the bulk soil. Most of the individual bacterial taxa enriched in the drilosphere versus bulk soil were members of Actinobacteria, including Micrococcales, Gaiellaceae, Solirubrobacterales, and Mycobacterium. In general, the greatest differences in communities were observed in comparisons of the top and bottom slope positions in which the bottom slope communities had significantly greater richness, diversity, and denitrifier abundance than the top slope position. Populations of denitrifiers (i.e., ratio of nirK+nirS to 16S rRNA) were more abundant in earthworm-impacted soils and there was a significant impact of L. terrestris on soil community composition which was observed only in the top landscape position. There were significant correlations between the abundance of nirK and nirS and taxa within Proteobacteria, Acidobacteria, Actinobacteria, Verrucomicrobia, and Chloroflexi, suggesting a broad diversity of denitrifying bacteria. Earthworms influence the soil microbial communities, but the impact depends on the slope location in a variable landscape, which likely reflects different soil characteristics.

Soil Microbial Biomass and Fungi Reduced With Canola Introduced Into Long-Term Monoculture Wheat Rotations.

With increasing canola (Brassica napus L.) acreage in the Inland Pacific Northwest of the USA, we investigated the effect of this relatively new rotational crop on soil microbial communities and the performance of subsequent wheat (Triticum aestivum L.) crops. In a 6-year on-farm canola-wheat rotation study conducted near Davenport, WA, grain yields of spring wheat (SW) following winter canola (WC) were reduced an average of 17% compared to SW yields following winter wheat (WW). Using soil samples collected and analyzed every year from that study, the objective of this research was to determine the differences and similarities in the soil microbial communities associated with WC and WW, and if those differences were associated with SW yield response. Microbial biomass and community composition were determined using phospholipid fatty acid analysis (PLFA). The WC-associated microbial community contained significantly less fungi, mycorrhizae, and total microbial biomass than WW. Additionally, reduced fungal and mycorrhizal abundance in SW following WC suggests that the canola rotation effect can persist. A biocidal secondary metabolite of canola, isothiocyanate, may be a potential mechanism mediating the decline in soil microbial biomass. These results demonstrate the relationship between soil microbial community composition and crop productivity. Our data suggest that WC can have significant effects on soil microbial communities that ultimately drive microbially mediated soil processes.

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.

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.

An Improved Method for Establishing Accurate Water Potential Levels at Different Temperatures in Growth Media.

NaCl, KCl, or PEG (polyethylene glycol)-amended potato dextrose broth (PDB), and potato dextrose agar (PDA) are essential for pure culture studies of water stress on fungi. Direct information on the actual water potential (WP) of this salt-amended PDB and PDA is lacking. Much fungal research in the past calculated WP of these salt-amended growth media by adding the WP of their constituents taken from individual salt dilution studies. But the WP of any complex solution will be modified by the level of synergism between its solutes. This study presents evidence of change in NaCl concentration due to synergism for attaining the same level of WP in NaCl solution, and NaCl amended PDB and PDA. The relation between WP and temperature and WP and salt concentration is also modified depending on the number of solutes in a growth medium. The WP of PEG-amended PDB increases with rising temperature, while that of NaCl/KCl amended PDB and PDA decreases with the increase of temperature. These results can be useful for doing pure culture studies on the biology and modeling the growth of air, water, and soil-borne fungi important in the food and agriculture industry and in terrestrial and aquatic ecosystems.

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.

Resistance to Multiple Soil-Borne Pathogens of the Pacific Northwest, USA Is Colocated in a Wheat Recombinant Inbred Line Population.

Soil-borne pathogens of the Pacific Northwest decrease yields in both spring and winter wheat. Pathogens of economic importance include Fusarium culmorum, Pratylenchus neglectus, P. thornei, and Rhizoctonia solani AG8. Few options are available to growers to manage these pathogens and reduce yield loss, therefore the focus for breeding programs is on developing resistant wheat cultivars. A recombinant inbred line population, LouAu (MP-7, NSL 511036), was developed to identify quantitative trait loci (QTL) associated with resistance to P. neglectus and P. thornei This same population was later suspected to be resistant to F. culmorum and R. solani AG8. This study confirms partial resistance to F. culmorum and R. solani AG8 is present in this population. Six major and 16 speculative QTL were identified across seven measured traits. Four of the six major QTL were found within the same genomic region of the 5A wheat chromosome suggesting shared gene(s) contribute to the resistance. These QTL will be useful in breeding programs looking to incorporate resistance to soil-borne pathogens in wheat cultivars.

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.

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.