Microbiome Networks: A Systems Framework for Identifying Candidate Microbial Assemblages for Disease Management.

Network models of soil and plant microbiomes provide new opportunities for enhancing disease management, but also challenges for interpretation. We present a framework for interpreting microbiome networks, illustrating how observed network structures can be used to generate testable hypotheses about candidate microbes affecting plant health. The framework includes four types of network analyses. "General network analysis" identifies candidate taxa for maintaining an existing microbial community. "Host-focused analysis" includes a node representing a plant response such as yield, identifying taxa with direct or indirect associations with that node. "Pathogen-focused analysis" identifies taxa with direct or indirect associations with taxa known a priori as pathogens. "Disease-focused analysis" identifies taxa associated with disease. Positive direct or indirect associations with desirable outcomes, or negative associations with undesirable outcomes, indicate candidate taxa. Network analysis provides characterization not only of taxa with direct associations with important outcomes such as disease suppression, biofertilization, or expression of plant host resistance, but also taxa with indirect associations via their association with other key taxa. We illustrate the interpretation of network structure with analyses of microbiomes in the oak phyllosphere, and in wheat rhizosphere and bulk soil associated with the presence or absence of infection by Rhizoctonia solani.

Characterizing and Mapping Resistance in Synthetic-Derived Wheat to Rhizoctonia Root Rot in a Green Bridge Environment.

Root rot caused by Rhizoctonia spp. is an economically important soilborne disease of spring-planted wheat in growing regions of the Pacific Northwest (PNW). The main method of controlling the disease currently is through tillage, which deters farmers from adopting the benefits of minimal tillage. Genetic resistance to this disease would provide an economic and environmentally sustainable resource for farmers. In this study, a collection of synthetic-derived genotypes was screened in high-inoculum and low-inoculum field environments. Six genotypes were found to have varying levels of resistance and tolerance to Rhizoctonia root rot. One of the lines, SPBC-3104 ('Vorobey'), exhibited good tolerance in the field and was crossed to susceptible PNW-adapted 'Louise' to examine the inheritance of the trait. A population of 190 BC1-derived recombinant inbred lines was assessed in two field green bridge environments and in soils artificially infested with Rhizoctonia solani AG8. Genotyping by sequencing and composite interval mapping identified three quantitative trait loci (QTL) controlling tolerance. Beneficial alleles of all three QTL were contributed by the synthetic-derived genotype SPCB-3104.

Natural Suppression of Rhizoctonia Bare Patch in a Long-Term No-Till Cropping Systems Experiment.

The soilborne fungus Rhizoctonia solani AG-8 is a major concern for farmers who practice no-till in the inland Pacific Northwest of the United States. Bare patches caused by Rhizoctonia spp. first appeared in 1999 during year 3 of a 15-year no-till cropping systems experiment near Ritzville, WA (269 mm of annual precipitation). The extent and pattern of patches were mapped each year from 1999 to 2012 at the 8-ha study site with a backpack-mounted global positioning system equipped with mapping software. Bare patches appeared in winter and spring wheat (SW; Triticum aestivum), spring barley (SB; Hordeum vulgare), yellow mustard (Brassica hirta), and safflower (Carthamus tinctorius). At its peak in years 5 to 7, bare patches occupied as much as 18% of total plot area in continuous annual monoculture SW. The area of bare patches began to decline in year 8 and reached near zero levels by year 11. No measurable patches were present in years 12 to 15. Patch area was significantly greater in continuous SW compared with SW grown in a 2-year rotation with SB. Additionally, the 15-year average grain yield for SW in rotation with SB was significantly greater than for continuous SW. Russian thistle (Salsola tragus), a troublesome broadleaf weed with a fast-growing tap root, was the only plant that grew within patches. This article reports the first direct evidence of natural suppression of Rhizoctonia bare patch with long-term no-till in North America. This suppression also developed in a rotation that contained broadleaf crops (yellow mustard and safflower) in all but 5 years of the study, and the suppression was maintained when safflower was added back to the rotation.

Stunting of Onion in the Columbia Basin of Oregon and Washington Caused by Rhizoctonia spp.

During 2009 and 2010, 45 isolates of Rhizoctonia spp. were recovered from onion bulb crops in the semiarid Columbia Basin of Oregon and Washington, in which patches of severely stunted onion plants developed following rotation with winter cereal cover crops. Characterization of isolates recovered from naturally infested soil and roots was performed by sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region, with the majority of isolates (64%) identified as Rhizoctonia solani. In steam-pasteurized field soil, stunting of onion was caused by isolates of R. solani anastamosis groups (AGs) 2-1, 3, 4, and 8, as well as Waitea circinata var. circinata and binucleate Rhizoctonia AG E evaluated at 13 and 8 or 15 and 15°C day and night temperatures, respectively, typical of spring planting conditions in the Columbia Basin. Isolates of R. solani AG 5 as well as binucleate AG A and I were nonpathogenic. The most virulent isolates belonged to AG 8, although an AG 3 and an AG E isolate were also highly virulent. Isolates of AG 2-1 and 3 caused moderate levels of disease, while isolates of AG 4 and W. circinata var. circinata caused low levels of disease. Emergence was reduced by isolates of AG 2-1, 3, and E. When the various AGs were grown at temperatures of 5 to 30°C, the relative growth rate of the Rhizoctonia isolates was not positively correlated with virulence on onion within an AG.

First Report of Blackleg Caused by Leptosphaeria maculans on Canola in Idaho.

Canola (Brassica napus L.) is produced in the dryland agriculture areas of eastern Washington State and northern Idaho, often in rotation with cereal cropping systems. Canola is also used as a rotation crop in irrigated circles in the Columbia Basin of Washington and southern Idaho, where potato is the main cash crop. In 2011, 7,700 ha of canola were harvested in Idaho and 4,200 ha in Washington. One of the major diseases of canola around the world is blackleg, caused by Leptosphaeria maculans (aggressive) and L. biglobosa (non-aggressive). Both Washington and Idaho have been considered blackleg-free, and production of canola in Idaho is subject to government regulations. Canola seed originating from outside of Washington and Idaho should have a phytosanitary certificate. This disease is widespread in Canada and the U.S. Northern Plains, Midwest, and South, and is the major disease of canola in these areas. In August 2011, a sample from a canola field in Bonners Ferry, Idaho, was brought for diagnosis to Washington State University. The canola stems showed the typical gray to dark grey lesions with black pycnidia. The pycnidia and conidia were examined microscopically, and found to be similar to descriptions of Phoma lingam, the anamorph of L. maculans (2). Samples were sent to the University of Manitoba for confirmation with PCR. The pathogen was cultured out of stems on V8 juice agar amended with streptomycin and 22 single pynidiospore isolates were made from the cultures. DNA was extracted from the cultures using methods described in Fernando et al. (1) and a multiplex PCR was performed with species-specific primers for L. maculans and L. biglobosa. The reaction should produce a 330-bp amplicon for L. maculans and a 440-bp amplicon for L. biglobosa. Based on this, all 22 isolates were identified as L. maculans. The susceptible cultivar Westar was inoculated with the isolates, by wound inoculating 7-day-old cotyledons with a concentration of 107 spores/ml. Plants were kept in a moist chamber at 23°C. After 14 days, plants were rated for disease with a 0 to 9 scale, where 0 = no infection and 9 = tissue collapse and appearance of pycnidiospores. Isolates with rating ≥5 are considered virulent. All isolates produced a rating of 7 to 9, indicating a high level of virulence. The source of the seed used in the infested fields is not known at this time. This disease is seedborne, and may pose a threat to the two major vegetable and oilseed brassica seed production areas of Washington: the Skagit River valley of western Washington and the Columbia Basin area of central Washington. In addition, the susceptibility of Pacific Northwest varieties of canola and other brassica oilseeds is largely unknown. References: (1) W. G. D. Fernando et al. Plant Dis. 90:1337, 2006. (2) S. Roger Rimmer et al. Compendium of Brassica Diseases, APS Press, 2007.

Identification of quantitative trait loci (QTL) for resistance to Fusarium crown rot (Fusarium pseudograminearum) in multiple assay environments in the Pacific Northwestern US.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum and F. culmorum, reduces wheat (Triticum aestivum L.) yields in the Pacific Northwest (PNW) of the US by as much as 35%. Resistance to FCR has not yet been discovered in currently grown PNW wheat cultivars. Several significant quantitative trait loci (QTL) for FCR resistance have been documented on chromosomes 1A, 1D, 2B, 3B, and 4B in resistant Australian cultivars. Our objective was to identify QTL and tightly linked SSR markers for FCR resistance in the partially resistant Australian spring wheat cultivar Sunco using PNW isolates of F. pseudograminerarum in greenhouse and field based screening nurseries. A second objective was to compare heritabilities of FCR resistance in multiple types of disease assaying environments (seedling, terrace, and field) using multiple disease rating methods. Two recombinant inbred line (RIL) mapping populations were derived from crosses between Sunco and PNW spring wheat cultivars Macon and Otis. The Sunco/Macon population comprised 219 F(6):F(7) lines and the Sunco/Otis population comprised 151 F(5):F(6) lines. Plants were inoculated with a single PNW F. pseudograminearum isolate (006-13) in growth room (seedling), outdoor terrace (adult) and field (adult) assays conducted from 2008 through 2010. Crown and lower stem tissues of seedling and adult plants were rated for disease severity on several different scales, but mainly on a numeric scale from 0 to 10 where 0 = no discoloration and 10 = severe disease. Significant QTL were identified on chromosomes 2B, 3B, 4B, 4D, and 7A with LOD scores ranging from 3 to 22. The most significant and consistent QTL across screening environments was located on chromosome 3BL, inherited from the PNW cultivars Macon and Otis, with maximum LOD scores of 22 and 9 explaining 36 and 23% of the variation, respectively for the Sunco/Macon and Sunco/Otis populations. The SSR markers Xgwm247 and Xgwm299 flank these QTL and are being validated for use in marker-assisted selection for FCR resistance. This is the first report of QTL associated with FCR resistance in the US.

First Report of Root Rot Caused by Rhizoctonia solani AG-10 on Canola in Washington State.

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.

First Report of a Ceratobasidium sp. Causing Root Rot on Canola in Washington State.

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.

Hyaloperonospora camelinae on Camelina sativa in Washington State: Detection, Seed Transmission, and Chemical Control.

Camelina (Camelina sativa) plants with symptoms of downy mildew were obtained from three different locations in Washington State. Based on polymerase chain reaction (PCR) and sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, the causal pathogen was identified as Hyaloperonospora camelinae. The PCR primers consistently amplified 699-bp bands from the infected plants but not from the asymptomatic plants. A comparison of the sequences with those in GenBank revealed 100% sequence similarity to H. camelinae. Growth and development of the H. camelinae was observed in different tissues using light microscopy and scanning electron microscopy (SEM). Light microscopic observation revealed the presence of oospores in the infected leaves and SEM revealed the presence of conidia and conidiophores on the seed surface. To determine whether H. camelinae is a seed-transmitted pathogen, seed collected from infected plants were planted in Sunshine professional growing mix maintained in a growth chamber. Disease symptoms were observed in 96% of the seedlings compared with 3% of the seedlings grown from seed from asymptomatic plants, which indicates that H. camelinae is a seed-transmitted pathogen. Seed treated with mefenoxam, a fungicide specific for Oomycetes, significantly reduced the incidence of the disease.

Optimum Timing of Preplant Applications of Glyphosate to Manage Rhizoctonia Root Rot in Barley.

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.

Soilborne Pathogens of Cereals in an Irrigated Cropping System: Effects of Tillage, Residue Management, and Crop Rotation.

An irrigated cropping systems experiment was conducted for 6 years in east-central Washington State to examine agronomic and economic alternatives to continuous annual winter wheat (Triticum aestivum) with burning and plowing, and to determine how root diseases of cereals are influenced by management practices. The continuous winter wheat treatment with burning and plowing was compared with a 3-year no-till rotation of winter wheat-spring barley (Hordeum vulgare)-winter canola (Brassica napus) and three straw management treatments: burning, straw removal, and leaving the straw stubble standing after harvest. Take-all disease and inoculum increased from years 1 to 4 in the continuous winter wheat treatment with burning and plowing, reducing plant growth compared to the no-till treatments with crop rotations. Inoculum of Rhizoctonia solani AG-8 was significantly lower in the tilled treatment compared to the no-till treatments. Inoculum concentration of Fusarium pseudograminearum was higher than that of F. culmorum, and in one of three years, the former was higher in treatments with standing stubble and mechanical straw removal compared to burned treatments. Residue management method had no effect on Rhizoctonia inoculum, but spring barley had more crown roots and tillers and greater height with stubble burning. This 6-year study showed that irrigated winter wheat can be produced in a no-till rotation without major disease losses and demonstrated how cropping practices influence the dynamics of soilborne cereal diseases and inoculum over time.

Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction.

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.

Effect of inoculum density and soil tillage on the development and severity of rhizoctonia root rot.

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.

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction.

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.

Root Diseases of Wheat and Barley During the Transition from Conventional Tillage to Direct Seeding.

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.

First Report of Damping-Off of Canola Caused by Rhizoctonia solani AG 2-1 in Washington State.

In early September 2003, winter canola (Brassica napus L) cv. Inca was direct seeded into plots previously cropped with spring barley at the Washington State University Dryland Research Station at Lind, WA. Before planting, the plots received 80 mm of water by sprinkler irrigation, and 2 weeks later, volunteer barley was killed with Paraquat contact herbicide. In late September, 3 weeks after planting, canola seedlings exhibited postemergence damping-off and lesions on the hypocotyls, resulting in significant stand reductions. Rhizoctonia solani was isolated from infected hypocotyls using water agar amended with chloramphenicol (100 µg/ml). Cultures on potato dextrose agar produced dark brown colonies with dark brown microsclerotia. Three isolates were grown on autoclaved oat seed for 3 weeks in 1-liter Erlenmeyer flasks at 22°C, and colonized seed was air dried in a laminar flow hood, ground in a coffee grinder, and added to a Thatuna silt loam at 1% (w/w). The infested soil was placed into 4- × 20.5-cm plastic tubes and planted with five canola seeds per tube, five tubes per isolate. In the control treatment, soil was not infested. Plants were grown in a temperature-controlled room in a greenhouse at 16°C, 12-h light/dark. Isolates caused pre- and postemergence damping-off after 1 week, and the surviving seedlings had significantly less plant height and dry weight. Isolates were identified as AG 2-1 by pairing cultures with AG 8, 2-1, and 10 on agar-coated slides (1). Selected isolates were also identified as AG 2-1 by sequencing of the ITS 1 and 2 regions of the rDNA and matching them to sequences in GenBank. On a farm north of Pullman, WA in 2004, R. solani was isolated from soil in spring and winter wheat fields using a toothpick baiting method (2). R. solani was found primarily from sites previously cropped with winter and spring canola. These isolates were identified as AG 2-1 and five isolates were tested in the greenhouse, as described above, on canola (cv. Inca), lentil (Lens culinaris Medik. cv. Merrit), wheat (Triticum aestivum L. cv. Madsen), barley (Hordeum vulgare L. cv. Baronesse), pea (Pisum sativum L. cv. Stirling), and chickpea (Cicer arietinum L. cv. Sierra). Three of five isolates significantly reduced emergence of canola, and all isolates significantly reduced dry weight of canola seedlings and caused lesions on hypocotyls. None of the isolates reduced emergence of the other crops. All isolates reduced the dry weight of pea and three isolates reduced plant height. None of the isolates reduced the dry weight of lentil, chickpea, wheat, or barley. One of the isolates was also tested on Arabidopsis thaliana and found to be pathogenic. R. solani AG 2-1 has been reported as an important pathogen on canola in Canada and Australia, but has not been reported from the Pacific Northwest of the United States. R. solani AG 2-1 is also pathogenic on rapeseed, mustard, and subterranean clover and has been isolated from wheat, sugar beets, and potato (3). Canola is a minor rotation crop in cereal-based cropping systems in eastern Washington (1,600 ha in 2005), but there is increasing interest in this oilseed crop for biodiesel production. However, R. solani AG 2-1 may reduce stands and yield of canola. References: (1) W. C. Kronland and M. E. Stanghellini. Phytopathology 78:820, 1988. (2) T. C. Paulitz and K. L. Schroeder. Plant Dis.89:767, 2005. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society. St. Paul, MN, 1991.

Reduction of Rhizoctonia Bare Patch in Wheat with Barley Rotations.

Rhizoctonia bare patch caused by Rhizoctonia solani AG-8 is a major fungal root disease in no-till cropping systems. In an 8-year experiment comparing various dryland no-till cropping systems near Ritzville, WA, Rhizoctonia bare patch first appeared in year 3 and continued unabated through year 8. Crop rotation had no effect on bare patch during the first 5 years. However, from years 6 to 8, both soft white and hard white classes of spring wheat (Triticum aestivum L.) grown in a 2-year rotation with spring barley (Hordeum vulgare L.) had an average of only 7% of total land area with bare patches compared with 15% in continuous annual soft white wheat or hard white wheat (i.e., monoculture wheat). In years 6 to 8, average grain yield of both soft white wheat and hard white wheat were greater (P < 0.001) when grown in rotation with barley than in monoculture. Although both classes of wheat had less bare patch area and greater grain yield when grown in rotation with barley, monoculture hard white wheat was more severely affected by Rhizoctonia than soft white wheat. Soil water levels were higher in bare patches, indicating that roots of healthy cereals did not grow into or underneath bare patch areas. This is the first documentation of suppression of Rhizoctonia bare patch disease in low-disturbance no-till systems with rotation of cereal crops.

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction.

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.

A New Method for the Quantification of Rhizoctonia solani and R. oryzae from Soil.

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.

Virulence of Pythium Species Isolated from Wheat Fields in Eastern Washington.

Although Pythium root rot in wheat (Triticum aestivum) is well documented, limited information is available concerning which species of Pythium are most responsible for disease damage. The objective of this study was to examine the variation in virulence on wheat among isolates of Pythium collected from cereal grain fields in eastern Washington. Isolates of nine Pythium species were tested for virulence on spring wheat cultivars Chinese Spring and Spillman. Cultivars were planted in pasteurized soil infested with Pythium isolates and placed in a growth chamber maintained at a constant 16°C and ambient humidity. Plant height, length of the first true leaf, and number of seminal roots were recorded, and roots were digitally scanned to create computer files that were analyzed using WinRhizo software. Pythium isolates caused a significant reduction (P < 0.05) in the number of root tips, root length, and length of the first leaf. Differences in virulence were detected among species and among isolates within species. Isolate Pythium debaryanum 90136 and P. ultimum 90038 were the most virulent and may prove useful in future disease screening assays of Triticum germ plasm.