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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.

Evaluation of soil-phosphorus (P) tests is critical to ensure the accuracy of fertilizer recommendations to optimize crop yield while minimizing negative environmental consequences. Olsen-P is the most commonly used soil-P test for alkaline calcareous soils found in Idaho and the Western United States. The Bray-1 test is commonly used in the Pacific Northwest (PNW) on neutral to acidic soils but underestimates P in alkaline calcareous soils. Mehlich-3 has been evaluated throughout various regions in the United States. Little data evaluating the test exists on soils in the Western United States. Additionally, the comparatively newly developed H3A test, a component of the soil health tool, has not been widely evaluated on alkaline calcareous soils. Soil samples from the 0- to 30-cm depth were collected from agricultural fields throughout Idaho and analyzed using Bray-1, H3A, Mehlich-3, and Olsen P extractants. Results suggested that Olsen P was strongly correlated with Mehlich-3, while Bray-1 and H3A were not correlated with Olsen P. Both the Bray-1 and H3A test underestimated extractable P when compared with the Olsen P test, whereas the Mehlich-3 overestimated. A threshold point in calcium carbonate (i.e., inorganic carbon (IC)) of 6.7 and 5.1 mg kg-1 for the Bray-1 and H3A was obtained, respectively, that indicated inorganic carbon concentrations at or above these levels result in underestimation of extractable soil P. Thus, Mehlich-3 was very strongly correlated to Olsen P and could be evaluated for use in alkaline calcareous soils whereas Bray-1 and H3A have notable issues that would limit their applicability.

Seed endophytes, as primary symbionts, may influence formation of secondary symbioses in maturing hosts. We hypothesize that such priority effects are a function of the presence and the identity of the primary symbiont, and are simulated via inoculation with primary symbionts. We sampled primary symbionts in winter wheat using culture-based techniques and next generation sequencing. Ninety-one percent of 6300 seeds had either one or no isolates. NGS data supported this with over 99% fewer reads detected in seeds having no isolates. Alternaria and Cladosporium spp., accounted for almost 80% of the primary symbionts isolated from seeds but only 36% of OTUs. Bacteria accounted for 13% of isolates but 27% of OTUs. We grew seedlings with or without primary symbiont isolates aseptically in vitro with roots in contact with a suspension of the rhizosphere symbiont Phialocephala fortinii. Seedlings having Cladosporium spp. or bacteria as primary symbionts increased the P. fortinii root colonization in 3 weeks by 30% and 50%, respectively, compared to seedlings lacking symbionts. Alternaria primary symbionts had no effect on root colonization by P. fortinii. However, Cladosporium and Alternaria inocula reduced P. fortinii colonization by 75% and 42%, respectively, at 3 weeks, so natural priority effects were not simulated by inoculation with fungi. Bacillus inoculum doubled P. fortinii colonization, but colonization was delayed. Primary symbionts of wheat seed can have strong, natural priority effects dependent on their presence and identity, but inoculation does not always simulate natural effects.

Hard red spring wheat is an important crop in the Pacific Northwest and is a component of a 3-year crop rotation involving soft white winter wheat; cool season pulse crops such as pea, lentil and chickpea; and canola. Pythium root rot and damping-off have long been observed as an annual problem, particularly in spring planted crops. As such, a seed treatment that includes metalaxyl is very common. Recently, an increase in the incidence of Pythium damping-off highlighted that there is an emerging population of Pythium spp. that are resistant to traditional seed treatments containing metalaxyl. The use of alternative chemistries to manage Pythium root rot and damping-off has gained regional interest to increase crop health and improve yields. The effectiveness of ethaboxam was evaluated along with two other seed treatment cocktails in controlling metalaxyl-resistant Pythium in replicated field experiments of spring wheat over three growing seasons in northern Idaho. Emergence, seedling characteristics, plant height, yield and test weight were assessed. The addition of ethaboxam to metalaxyl-containing seed treatment blends consistently increased emergence and yield of spring wheat compared to seed treatment blends containing metalaxyl only. First leaf length, an indirect measure of early season Pythium damage, was longest in seed treatments containing ethaboxam and the average number of tillers was lower, likely due to increased emergence, than non-treated seed. These results indicate that ethaboxam can improve disease control in spring wheat production when applied as a seed treatment in combination with metalaxyl, specifically in the presence of metalaxyl-resistant Pythium spp.

The cropping systems of northern Idaho and eastern Washington are dominated by a cereal-based (winter wheat) rotation that is exclusively rainfed. Following the green revolution, the productivity of wheat increased dramatically with semi-dwarf varieties and inexpensive synthetic fertilizers. However, long-term and frequent use of ammonium-based nitrogen fertilizers has resulted in the gradual acidification of fields in the region, with reduced productivity and symptoms of aluminum toxicity in some locations. A micronized liquid formulation of lime (calcium carbonate) was evaluated at three locations to mitigate soil acidification in comparison to a no-lime control. To examine how liming affects bacterial community structure, soil samples were taken in the spring in wheat and pea rotations 7 and 19 months after lime application, DNA was extracted and the V1-V3 portion of the 16S rRNA gene was amplified and sequenced with Illumina MiSeq. pH was only increased ∼0.2–0.3 units in most locations, and liming only had a significant effect on bacterial community composition at one location (Pullman). Liming had no consistent or significant effect on community richness or diversity. Location had a stronger effect on communities than liming or crop rotation, but some specific taxa increased in relative abundance in response to liming, including the families Cytophagaceae and Flavobacteriaceae (Phylum Bacteroidetes), A4b (Phylum Chloroflexi), and Opitutaceae (Phylum Verrucomicrobia). A number of operational taxonomic units (OTU) in Chitinophagaceae and Xanthomonadaceae were increased by liming at the Pullman location, which had 3–4 X more OTUs affected by liming than the other locations. Streptomycetaceae and Oxalobacteraceae were more abundant in root zone soil of wheat than pea. In conclusion, there was a strong interaction between liming and location in determining soil bacterial composition, but specific components of the bacterial community responded to even minimal increases in soil pH.

Cultural practices, such as tillage, often have widespread impacts on phytobiomes. No-till has been increasingly adopted by wheat growers in the dryland cropping areas of the inland Pacific Northwest in the United States to reduce soil erosion and decrease fuel and labor inputs, yet there are limited data on how conversion to no-till impacts plant-associated bacteria in this highly productive system. To address this knowledge gap, we evaluated bacterial communities in bulk and rhizosphere soil of wheat in two locations (Idaho and Washington) for 2 years, comparing long-term no-till plots and adjacent plots under conventional tillage. In this study, members of phylum Proteobacteria were relatively more abundant in rhizosphere soil, while Acidobacteria and Gemmatimonadetes were more abundant in bulk soil than in the rhizosphere. Bacteroidetes were more frequent under conventional than conservation tillage. In general, bacterial families were more affected by the position of the sample (rhizosphere versus bulk soil) than by tillage practices. Families generally regarded as copiotrophic (Oxalobacteriaceae, Pseudomonadaceae, and Cytophagaceae) were more abundant in rhizosphere soil than bulk in both years. On the contrary, oligotrophic families such as Gaiellaceae and those within Gemmatimonadetes were more abundant in bulk soil than in the rhizosphere. Families affected by tillage varied between the 2 years. These results suggest that bacterial communities in soil were more influenced by plant proximity (rhizosphere versus bulk soil) than by tillage practices, but that specific differences were not consistent and may vary among locations and years.

Paulitz, T. C., and Schroeder, K. L. 2005. A new method for the quantification of Rhizoctonia solani and R. oryzae from soil. Plant Dis. 89:767-772. Rhizoctonia solani anastomosis group (AG) 8 and R. oryzae are important root pathogens on wheat and barley in the dryland production areas of the inland Pacific Northwest. R. solani AG-8 is difficult to isolate from root systems and quantify in soil because of slow growth and low population densities. However, both pathogens form extensive hyphal networks in the soil and can grow a considerable distance from a food base. A quantitative assay of active hyphae was developed, using wooden toothpicks as baits inserted into sample soils. After 2 days in soil, toothpicks were placed on a selective medium, and the numbers of colonies that grew after 24 h were counted under a dissecting microscope. R. solani and R. oryzae could be distinguished from other fungi based on hyphal morphology. This method was tested in natural soils amended with known inoculum densities of R. solani AG-8 and R. oryzae. Regressions were used to compare the inoculum density or toothpick colonization curves to a predicted curve based on the volume of the toothpicks. The slopes and y intercept of log-log transformed regressions did not differ from the predicted curves in most cases. This technique was used to assess the hyphal activity of R. solani AG-8 and R. oryzae from soil cores taken from various positions in and around Rhizoctonia bare patches at two locations. Activity of R. solani was highest in the center and inside edge of the patch, but there was no effect of patch position on R. oryzae. This simple and inexpensive technique can be used for detection and diagnosis in grower fields and to study the ecology and epidemiology of Rhizoctonia spp.

Okubara, P. A., Harrison, L. A., Gatch, E. W., Vandemark, G., Schroeder, K. L., and du Toit, L. J. 2013. Development and evaluation of a TaqMan real-time PCR assay for Fusarium oxysporum f. sp. spinaciae. Plant Dis. 97:927-937. Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3–14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada’s agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

The Hessian fly Mayetiola destructor (Diptera: Cecidmyiidae) is a major pest of wheat, globally. We conducted a series of laboratory choice and no-choice assays to quantify Hessian fly host preference for barley (cv. Champion), oat (cv. Cayuse), susceptible (cv. Alturas), and resistant (cv. Hollis) wheat. In addition, larval survivorship and adult emergence were compared among the evaluated host plants. We then examined whether insect preference for a host can be explained by differences in plant spectral reflectance. Further, larval survivorship and adult emergence were compared among host plants in relation to phytohormone concentrations. Hessian flies laid more eggs on wheat compared to either oat or barley. Spectral reflectance measurements of leaves were similar between susceptible and resistant wheat cultivars but different from those of barley and oat. Our results suggested that higher reflectance in the near-infrared range and lower reflectance in the visible range may be used by females for host selection. Hessian fly larvae were unable to develop into the pupal stage on resistant wheat and oat. No significant difference in larval survivorship was detected between the susceptible wheat and barley. However, adult emergence was significantly higher on barley than the susceptible wheat. Phytohormonal evaluations revealed that salicylic acid (SA) may be an important contributor to plant defense response to larval feeding as relatively higher concentrations of SA were present in oat and resistant wheat. While resistance in the resistant wheat is achieved only through antibiosis, both antibiosis and antixenosis were in effect rendering oat as a non-host for Hessian flies.

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.

Wheat varieties from Turkey, Australia and the Pacific Northwest of the US were screened for resistance to Fusarium crown rot (Fusarium pseudograminearum and Fusarium culmorum) in inoculated growth chamber studies. Fungal DNA was also quantified from the lower stems (crowns) of seedlings using quantitative PCR and crown rot scores were correlated with DNA results. There were significant differences among the varieties for crown rot severity index (p = 0.0265), crown rot score (p = 0.0003), disease index on leaf sheaths (leaf sheath score) (p = 0.0013), lesion length (p = 0.0200), and plant height (p

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.

The necrotrophic soilborne fungal pathogens Rhizoctonia solani AG8 and R. oryzae are principal causal agents of Rhizoctonia root rot and bare patch of wheat in dryland cropping systems of the Pacific Northwest. A 3-year survey of 33 parcels at 11 growers' sites and 60 trial plots at 12 Washington State University cereal variety test locations was undertaken to understand the distribution of these pathogens. Pathogen DNA concentrations in soils, quantified using real-time polymerase chain reaction, were correlated with precipitation, temperature maxima and minima, and soil texture factors in a pathogen-specific manner. Specifically, R. solani AG8 DNA concentration was negatively correlated with precipitation and not correlated with temperature minima, whereas R. oryzae concentration was correlated with temperature minima but not with precipitation. However, both pathogens were more abundant in soils with higher sand and lower clay content. Principal component analysis also indicated that unique groups of meteorological and soil factors were associated with each pathogen. Furthermore, tillage did not affect R. oryzae but affected R. solani AG8 at P = 0.06. Lower soil concentrations of R. solani AG8 but not R. oryzae occurred when the previously planted crop was a broadleaf (P < 0.05). Our findings showed that R. solani AG8 concentrations were consistent with the general distribution of bare patch symptoms, based on field observations and surveys of other pathogens, but was present at many sites in which bare patch symptoms were not evident. Management of Rhizoctonia root rot and bare patch should account for the likelihood that each pathogen is affected by a unique group of agroecological variables.

Isolates of R. solani AG 2–1, AG 8, AG 10 and binucleate Rhizoctonia (Ceratobasidium spp.) were tested for virulence on Brassica crops in growth chamber experiments. Isolate virulence and genotype resistance were determined based on percent of seedling survival, shoot length reduction, and shoot fresh weight. Isolates had significant effects on all tested measurements, compared to the non-inoculated controls. Rhizoctonia solani AG 2–1 appears to be the most aggressive pathogen on all tested genotypes followed by R. solani AG 8, binucleate Rhizoctonia and R. solani AG 10, respectively. Genotype by isolate interaction effects were found to be significant for percent of seedling survival and shoot length reduction. None of the tested genotypes exhibited any level of resistance to R. solani AG 2–1, but three promising genotypes with moderate levels of resistance to R. solani AG 10, R. solani AG 8 and binucleate Rhizoctonia were identified. Moderate heritability (0.57) was observed for the percent of seedling survival in the resistant genotype KS4022.

The genus Pythium is one of the most important groups of soilborne plant pathogens, present in almost every agricultural soil and attacking the roots of thousands of hosts, reducing crop yield and quality. Most species are generalists, necrotrophic pathogens that infect young juvenile tissue. In fact, Cook and Veseth have called Pythium the “common cold” of wheat, because of its chronic nature and ubiquitous distribution. Where Pythium spp. are the cause of seedling damping-off or emergence reduction, the causal agent can easily be identified based on symptoms and culturing. In more mature plants, however, infection by Pythium spp. is more difficult to diagnose, because of the nonspecific symptoms that could have abiotic causes such as nutrient deficiencies or be due to other root rotting pathogens. Molecular methods that can accurately identify and quantify this important group are needed for disease diagnosis and management recommendations and to better understand the epidemiology and ecology of this important group. The purpose of this article is to outline the current state-of-the-art in the detection and quantification of this important genus. In addition, we will introduce the reader to new changes in the taxonomy of this group.

Rhizoctonia root rot, caused by Rhizoctonia solani AG-8 and R. oryzae, is considered one of the main deterrents for farmers to adopt reduced-tillage systems in the Pacific Northwest. Because of the wide host range of Rhizoctonia spp., herbicide application before planting to control weeds and volunteer plants is the main management strategy for this disease. To determine the effect of timing of glyphosate applications on the severity of Rhizoctonia root rot of barley, field experiments were conducted in 2007, 2008, and 2009 in a field naturally infested with a high level of both R. solani and R. oryzae. Crop volunteer plants and weeds were allowed to grow over the winter and plots were sprayed with glyphosate at 42, 28, 14, 7, and 2 days prior to planting. As the herbicide application interval increased, there were significant increases in shoot length, length of the first true leaf, and number of healthy seminal roots and a decrease in disease severity. Yield and the number of seminal roots did not show a response to herbicide application interval in most years. The activity of R. solani, as measured by toothpick bioassay and real-time polymerase chain reaction, declined over time in all treatments after planting barley. The herbicide application interval required to meet 80 and 90% of the maximum response (asymptote) for all plant and disease measurements ranged from 11 to 27 days and 13 to 37 days, respectively. These times are the minimum herbicide application intervals required to reduce disease severity in the following crop.

Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative-polymerase chain reaction (Q-PCR) assays specific to internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of R. solani and R. oryzae. The assays were diagnostic for R. solani AG-2-1, AG-8, and AG-10, three genotypes of R. oryzae, and an AG-I-like binucleate Rhizoctonia species. Quantification was reproducible at or below a cycle threshold (Ct) of 33, or 2 to 10 fg of mycelial DNA from cultured fungi, 200 to 500 fg of pathogen DNA from root extracts, and 20 to 50 fg of pathogen DNA from soil extracts. However, pathogen DNA could be specifically detected in all types of extracts at about 100-fold below the quantification levels. Soils from Ritzville, WA, showing acute Rhizoctonia bare patch harbored 9.4 to 780 pg of R. solani AG-8 DNA per gram of soil.. Blastn, primer-template duplex stability, and phylogenetic analyses predicted that the Q-PCR assays will be diagnostic for isolates from Australia, Israel, Japan, and other countries.

Soilborne pathogens are important biotic factors in yield reduction in the dryland cereal production region of the Pacific Northwest. Rhizoctonia solani AG-8, Rhizoctonia oryzae, and Pythium spp. are causal agents of root rot, bare patch, and damping-off of wheat (Triticum aestivum) and barley (Hordeum vulgare). Although these pathogens can be rapidly and specifically quantified using quantitative real-time PCR, the extraction of Rhizoctonia DNA from agricultural samples is often inconsistent, especially at low pathogen population densities. Using a novel extraction system that uses pressure cycling technology (PCT), we improved the extraction of R. solani AG-8 DNA up to 16-fold and of P. abappressorium DNA up to 2-fold from three types of agricultural soils compared with a bead beating extraction method. PCT also yielded quantifiable amounts of R. solani AG-8 and R. oryzae DNA from lyophilized wheat roots that were otherwise recalcitrant to homogenization. Furthermore, the extractions were so consistent ...

The use of direct seeding (no-till) in place of tillage can reduce soil erosion and improve water infiltration. However, despite these improvements in soil quality, growers in the Pacific Northwest are reluctant to adopt direct seeding, partially because of fears of increased root diseases caused by Gaeumannomyces graminis var. tritici, Rhizoctonia spp., and Pythium spp. To examine the effect of the transition from conventional tillage to direct seeding, field plots were established at two locations. One site had been managed with direct seeding for 12 years, and the second had been conventionally tilled. Over 4 years, a portion of each plot was tilled or direct seeded, and planted to wheat or barley. Plants in the tilled plots had consistently more crown roots than plants in direct-seeded plots. Rhizoctonia root rot and yield did not differ between tillage types during the first 2 years of the study. However, in the third and fourth years of the transition to direct seeding, a higher incidence of Rhizoctonia root rot, increased hyphal activity of R. solani, and reduced yields were observed in direct-seeded plots. Populations of R. oryzae and Pythium spp., and incidence of take-all were the same for both management practices.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root−1 or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.

Real-time, or quantitative, polymerase chain reaction (Q-PCR), offers a rapid, sensitive, and specific method for the diagnosis of plant pathogens in soil, water, air, and plant samples. Fluorescence is used to monitor the accumulation of the PCR product after each PCR cycle. The fluorescence data are used to extrapolate the amount of target DNA present in the sample before amplification so that detection and quantification are achieved in a single assay. Detection limits are generally in the femtogram range for purified pathogen DNA, in the picogram range for pathogen DNA in plant samples, and one to several spores for soil samples. Quantification is reliable over a dynamic range of five to seven orders of magnitude of target DNA, in which differences of two fold or more in target DNA can be detected. Real-time PCR has already been recognized as a valuable tool for epidemiological studies, disease management, detection of emerging pathogens, evaluation of resistance and tolerance to disease, and phytosan...

Rhizoctonia bare patch and root rot disease of wheat, caused by Rhizoctonia solani AG-8, develops as distinct patches of stunted plants and limits the yield of direct-seeded (no-till) wheat in the Pacific Northwest of the United States. At the site of a long-term cropping systems study near Ritzville, WA, a decline in Rhizoctonia patch disease was observed over an 11-year period. Bacterial communities from bulk and rhizosphere soil of plants from inside the patches, outside the patches, and recovered patches were analyzed by using pyrosequencing with primers designed for 16S rRNA. Taxa in the class Acidobacteria and the genus Gemmatimonas were found at higher frequencies in the rhizosphere of healthy plants outside the patches than in that of diseased plants from inside the patches. Dyella and Acidobacteria subgroup Gp7 were found at higher frequencies in recovered patches. Chitinophaga , Pedobacter , Oxalobacteriaceae ( Duganella and Massilia ), and Chyseobacterium were found at higher frequencies in the rhizosphere of diseased plants from inside the patches. For selected taxa, trends were validated by quantitative PCR (qPCR), and observed shifts of frequencies in the rhizosphere over time were duplicated in cycling experiments in the greenhouse that involved successive plantings of wheat in Rhizoctonia -inoculated soil. Chryseobacterium soldanellicola was isolated from the rhizosphere inside the patches and exhibited significant antagonism against R. solani AG-8 in vitro and in greenhouse tests. In conclusion, we identified novel bacterial taxa that respond to conditions affecting bare patch disease symptoms and that may be involved in suppression of Rhizoctonia root rot and bare batch disease.

There are no resistant varieties or chemical controls for the major soilborne pathogens of wheat in the Pacific Northwest of the United States. Root and crown diseases of wheat include Rhizoctonia root rot and bare patch (caused by R. solani and R. oryzae), Fusarium crown rot (caused by F. pseudograminearum and F. culmorum), Pythium root rot (caused by numerous Pythium spp.) and take-all (caused by Gaeumannomyces graminis var. tritici). Growers rely almost completely on cultural control measures. Our research program has evaluated many of these techniques, some of which are effective against Rhizoctonia, including greenbridge (weed and crop volunteer) management, fallow (both chemical and mechanical), seed opener disturbance, precision seed row placement, crop rotation, residue and nitrogen management, and chemical seed treatment. Until recently, there was no way of accurately detecting and quantifying pathogens in soil. We have developed real-time PCR assays to quantify nine species of Pythium and seven groups of Rhizoctonia, based on ITS sequences of the rDNA. In the last 3 years, soils were extensively sampled in eastern Washington, including grower fields and breeder variety-testing sites. By developing a pathogen profile for each testing site, breeders can focus on locations with high pathogen densities to select for tolerance. This survey has shown that pathogen species composition is affected by cropping system and rotation. With accurate detection and quantification of soilborne pathogens, growers can determine risk before planting and make management decisions to mitigate the effects of soilborne fungal pathogens.

Rhizoctonia spp. cause substantial yield losses in direct-seeded cereal crops compared with conventional tillage. To investigate the mechanisms behind this increased disease, soils from tilled or direct-seeded fields were inoculated with Rhizoctonia spp. at population densities from 0.8 to 250 propagules per gram and planted with barley (Hordeum vulgare). The incidence and severity of disease did not differ between soils with different tillage histories. Both R. solani AG-8 and R. oryzae stunted plants at high inoculum densities, with the latter causing pre-emergence damping-off. High inoculum densities of both species stimulated early production of crown roots in barley seedlings. Intact soil cores from these same tilled and direct-seeded fields were used to evaluate the growth of Rhizoctonia spp. from colonized oat seeds. Growth of R. oryzae was not affected by previous tillage history. However, R. solani AG-8 grew more rapidly through soil from a long-term direct-seeded field compared to tilled soils. The differential response between these two experiments (mixed, homogenized soil versus intact soil) suggests that soil structure plays a major role in the proliferation of R. solani AG-8 through soils with different tillage histories.

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.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD , which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.

Canola (Brassica napus L) production has gained renewed interest in Washington State over the past few years, primarily for the purpose of producing biofuel. Plants were observed to be showing symptoms of Rhizoctonia root rot and postemergence damping-off. In many cases, this was due to Rhizoctonia solani AG-2-1, which was previously documented (4). However, additional plants were occasionally observed that were stunted and had reduced vigor, but lacked the distinctive severe stem damage and postemergence damping-off, which are both symptoms of infection with R. solani AG-2-1. Isolates of R. solani AG-10 were collected from symptomatic plants or baited from root zone soil at various dryland production locations in eastern Washington, including sites near Colfax, Pullman, and Walla Walla. Initial identification was determined by quantitative (Q)-PCR using R. solani AG-10 specific primers (3). The identity was verified by sequencing random isolates identified by Q-PCR (GenBank Accessions Nos. JQ068147, JQ068148 and JQ068149). All sequenced isolates had 99% identity to previously reported isolates of R. solani AG-10. Six isolates were chosen to test pathogenicity on canola plants in the greenhouse. Sterilized oats were inoculated with each of six isolates of R. solani AG-10 and grown for 4 weeks. The soil was infested with ground oat inoculum (1% wt/wt) and spring canola cv. Sunrise was seeded into 3.8 × 21-cm containers. After 3 weeks of incubation at 15°C, plants were harvested and assessed. Emergence was reduced in the infested soil with 73 to 93% (average 81%) emergence compared with 100% emergence in the noninfested soil. There was no evidence of postemergence damping-off. However, all six isolates of R. solani AG-10 significantly reduced the plant height and top dry weights compared with the noninfested controls. The plant height in infested soil was 28 to 42% (average 34%) shorter and top dry weights were 37 to 70% (average 54%) lower than in noninfested soil. Roots of infected plants had a light brown discoloration along with reduced length and fewer lateral roots. Additional host plants were tested, including wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), chickpea (Cicer arietinum L.), and lentil (Lens culinaris Medik.). There was no significant reduction in plant height or plant dry weight for any of these hosts. R. solani AG-10 was previously found to be weakly virulent on canola and other cruciferous hosts in Australia (1,2). To our knowledge, this is the first report of R. solani AG-10 causing disease on canola in Washington State. Reference: (1) R. K. Khangura et al. Plant Dis. 83:714, 1999. (2) G. C. MacNish et al. Australas. Plant Pathol. 24:252, 1995. (3) P. A. Okubara et al. Phytopathology 98:837, 2008. (4) T. C. Paulitz et al. Plant Dis. 90:829, 2006.

Rhizoctonia root rot occurs commonly on canola (Brassica napus L.) in Washington State. Recently, isolates of an additional pathogen were found to be involved in this disease complex. Isolates of an AG-I-like Ceratobasidium sp. were collected from roots and root zone soil in central Washington near Ritzville. Identity of selected isolates was verified by sequencing the internal transcribed spacer (ITS) region of the rDNA (GenBank Accession Nos. JQ247570, JQ247571, and JQ247572), with a 90 to 93% identity to AG-I. All isolates also amplified with AG-I-like specific primers (1). Six isolates were included in pathogenicity assays conducted in the greenhouse. There were five replicates of three plants for each treatment and the experiment was conducted twice. Pasteurized soil was infested with ground oat inoculum (1%) and placed into containers (3.8 × 21 cm). Infested soils were seeded with canola, chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.), pea (Pisum sativum L.), barley (Hordeum vulgare L.), or wheat (Triticum aestivum L.). After 3 weeks of incubation at 15°C, the plants were destructively harvested. The emergence of canola was consistently reduced in soil infested with a Ceratobasidium sp., with reductions of 0 to 23% (average 11%). There was no postemergence damping-off, a symptom commonly associated with AG-2-1 (2). Plant height and top dry weights were significantly reduced for canola seeded into infested soil. Heights of plants growing in infested soil was reduced by 25 to 53% (average 42%) and top dry weight was reduced by 37 to 81% (average 61%) compared with the noninfested control. The legume hosts tested in this study were also affected by this Ceratobasidium sp., but to a lesser extent. Compared with the noninfested controls, there was evidence of preemergence damping-off in chickpea (0 to 27%, average 13%) and pea plants were consistently stunted (5 to 23%, average 12%). Chickpea and pea plants grown in infested soil also had reduced top dry weights of 9 to 28% (average 17%) and 13 to 35% (average 21%), respectively. The roots of all infected hosts had a characteristic brown discoloration with tapered, rotted root tips (spear tips). There was no reduction in emergence or plant height of wheat and barley; there was inconsistent reduction in dry weight of these plants. To our knowledge, this is the first report of a Ceratobasidium sp. causing disease on canola in Washington State. References: (1) P. A. Okubara et al. Phytopathology 98:837, 2008. (2) T. C. Paulitz et al. Plant Dis. 90:829, 2006.