Kurtis L. Schroeder

Role of Bacterial Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.)

ABSTRACT 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.

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.

Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae

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 Komadas 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.

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.

Evaluation of Brassica species for resistance to Rhizoctonia solani and binucleate Rhizoctonia (Ceratobasidum spp.) under controlled environment conditions

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.

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.

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.

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.