Circinata Var Research Articles

Specific detection of Waitea circinata var. zeae using conventional and real-time PCR

Specific detection of Waitea circinata var. zeae using conventional and real-time PCR

Anastomosis Grouping and Phylogenetic analysis of Rhizoctonia Isolates on Wheat in Türkiye

The aim of this study is to determine the species and to evaluate the genetic diversity of the pathogenic and nonpathogenic Rhizoctonia spp. and anastomosis groups (AG) from wheat plants and rhizosphere soils in Turkey. Rhizoctonia species were isolated from plants and rhizosphere soils in wheat fields in 5 provinces in the Central Anatolian Region of Türkiye. As a result of the isolations, a total of 88 multinucleate (MN) and binucleate (BN) Rhizoctonia isolates were obtained. Identifications of the isolates were determined by rDNA-ITS sequence analyzes. The identified isolates belonged to MN Waitea circinata var. zeae, W. circinata var. oryzae, W. circinata var. circinata, MN Rhizoctonia solani AG 2-1, 2-2, 3, 4 HGII, 4 HGIII, 5, 8, 11 and BN AG A, DI, E, G, H, I, I-like, K. The most isolated group was W. circinata var. circinata. In pathogenicity studies, the most virulent group was determined as R. solani AG 4. Among the binucleate isolates, groups other than R. cerealis AG DI were not found to be pathogenic. Neighbor-joining phylogenetic trees of isolates were constructed from rDNA-ITS sequences. As a result of this study, the regional distribution of MN and BN Rhizoctonia AG isolates in important wheat production areas in the Central Anatolia Region, Turkey was determined. In addition, this study is the first comprehensive study in which the genetic diversity of Rhizoctonia AGs isolates obtained from wheat and rhizosphere soils in the region was evaluated with a molecular approach.

Waitea circinata var. zeae - prouzrokovač truleži korena kupusa i uljane repice

Waitea circinata var. zeae (anamorf Rhizoctonia zeae) is an important pathogen of mainly monocotyledonous plants characterized by its soil-borne nature and capability for the long-term preservation. The host range of W. circinata var. zeae recently expanded to several dicotyledonous crops. The latest research conducted in Serbia revealed this pathogen as the causal agent of root and root neck rot, as well as plant decay of cabbage and oilseed at the localities of Futog and Rimski Šančevi. In this paper the available data on the distribution, preservation, dissemination pattern and control of W. circinata var. zeae, as well as research in Serbia are discussed.

Morphopathological and Molecular Morphometric Characterization of Waitea circinata var. prodigus Causing a Novel Sheath Spot Disease of Maize in India.

Maize brown sheath spot (MBSS), a new disease of maize, was discovered while surveying for maize leaf and sheath blight diseases in the Indian states of Assam, Jharkhand, Meghalaya, Manipur, and Odisha. Maize is the third most important cereal after rice and wheat in India. Unlike banded leaf and sheath blight disease caused by Rhizoctonia solani, MBSS symptoms on maize were discrete and limited to sheaths only. Symptoms of MBSS in the field were initially water-soaked necrotic lesions of 1 to 2 cm in diameter on the lowermost leaf sheaths, which then progressed to the upper sheaths. Lesions coalesced and covered approximately 2 to 5% of the sheath area. Infected dried lower leaves were shed, whereas infected upper leaves remained on the stem. The pathogen was isolated, characterized morphologically, pathologically, and molecularly, and identified as Waitea circinata var. prodigus, a basidiomycete known to cause basal leaf blight of seashore paspalum. The internal transcribed spacer (ITS) sequence 2 (ITS2) of rDNA from MBSS isolates formed a well supported clade with known W. circinata var. prodigus isolates. Molecular morphometric analysis of the ITS2 regions of the five known varieties of W. circinata detected distinguishing variations in GC content, compensatory base changes (CBCs), hemi- CBCs, indels, and altered base-pairing of helices. Variation in these characteristics may indicate that varieties are distinct biological species within W. circinata sensu lato. The geographical distribution and potential impacts of MBSS on the maize crop in India necessitate further investigations of pathogen identification and disease management.

Management of leaf and sheath spot (mini ring) on hybrid bermudagrass in Hawaii

AbstractLeaf and sheath spot (mini ring) is a turfgrass fungal disease that has been reported in some states in the USA. The causal pathogen is Waitea circinata var. zeae, also known as Rhizoctonia zeae. This disease typically causes thinned turf areas and/or rings of full, half, or quarter circles. Turfgrass under stress or irrigated with high salinity water is more prone to infestation. Seven fungicide programs were evaluated on ‘TifEagle’ hybrid bermudagrass [Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt Davy] at a golf course on Maui, Hawaii. Four replications (plots) per treatment were arranged in a randomized complete block design. Disease severity and turfgrass quality were measured prior to treatment and after each application. Azoxystrobin showed the best suppression of disease and improvement of turfgrass quality, followed by chlorothalonil + acibenzolar‐S‐methyl. These two fungicide programs can be rotated to minimize possible resistance, and should also be used in conjunction with beneficial cultural practices to manage mini ring disease.

Waitea circinata var. zeae Causing Root Rot of Cabbage and Oilseed Rape.

Cabbage, a widely used and popular vegetable, and oilseed rape, the second most valuable oilseed crop in the world, are two important species from the Brassicaceae family. Two geographically separated outbreaks of cabbage and oilseed rape root rot with estimated incidence of 15 and 20%, respectively, were recorded during 2017 in the Vojvodina region, Serbia. Twelve hyphal-tip isolates were obtained from symptomatic cabbage and oilseed rape plants and identified as Waitea circinata var. zeae based on morphological and molecular features. This indicates that W. circinata var. zeae has expanded its host range to the Brassicaceae family. Sequence analyses of internal transcribed spacer (ITS) and large subunit of the ribosomal DNA, RPB2, and β-tubulin genes revealed the highest similarity with multiple W. circinata var. zeae. Neighbor-joining analyses of ITS sequences resulted in a phylogenetic tree with one well-defined branch of W. circinata var. zeae, with two separate groups. All Serbian isolates and the majority of isolates originating from natural infection of dicotyledonous plants grouped together in group I. Following artificial inoculation, W. circinata var. zeae isolates caused mild to medium root necrosis of seedlings of 2 monocotyledonous and 12 dicotyledonous plant species, implying a wider host range than was known for W. circinata var. zeae. Additionally, this is the first occurrence of W. circinata var. zeae on dicotyledonous host plants in Europe. Because cabbage and oilseed rape are important crops grown worldwide, the occurrence of this new soilborne pathogen with a broad host range imposes the necessity for changes in routine disease control practices, particularly crop rotation.

First Report of Dollar Spot Caused by Clarireedia jacksonii and Brown Ring Patch Caused by Waitea circinata var. circinata on Agrostis stolonifera in Spain

First Report of Dollar Spot Caused by <i>Clarireedia jacksonii</i> and Brown Ring Patch Caused by <i>Waitea circinata</i> var. <i>circinata</i> on <i>Agrostis stolonifera</i> in Spain

Anastomosis group and pathogenicity of Rhizoctonia spp. associated with seedling damping-off of sugar beet in China

Between 2010 and 2015, 204 Rhizoctonia isolates were obtained from diseased sugar beet seedlings which collected from the sugar beet growing regions of China. On the basis of the morphological characteristics and sequence analysis of the internal transcribed spacers of the ribosomal DNA (rDNA ITS), 166 were identified as multinucleate Rhizoctonia (MNR), and 38 were binucleate Rhizoctonia (BNR), belonging to 15 species, anastomosis groups (AGs) or subgroups. The most prevalent was Rhizoctonia solani AG-4, with 49 isolates attributed to the subgroup HGIII, 47 to HGII, and 45 to HGI, followed by AG-K (31), AG-2-2IIIB (7), AG-A (6), AG-1-1C (4), AG-1-1B (3), AG-2-1 (3), AG-5 (3), AG-11 (2), AG-3 TB (1), Waitea circinata var. circinata (1), W. circinata var. zeae (1), and AG-C (1). Seventy-four representative isolates of all species, AGs or subgroups from different geographic origins were able to cause brown, dry lesions on roots of sugar beet seedlings or even seedling death with the disease incidence and the disease index ranging from 13.3 to 100% and from 1.9 to 97.1, respectively. The AG-4 isolates were the most aggressive on sugar beet seedlings. Moreover, the aggressiveness of the R. solani isolates was higher than that of the other isolates. To the best of our knowledge, this is the first detailed description of the AG composition of Rhizoctonia causing seedling damping-off on sugar beet in China.

Diversity and Pathogenicity of Rhizoctonia Species from the Brazilian Cerrado.

Eighty-one Rhizoctonia-like isolates were identified based on morphology and nuclei-staining methods from natural and agricultural soils of the Cerrado (Brazilian savanna). The nucleotide similarity analysis of ITS1-5.8S-ITS2 regions identified 14 different taxa, with 39.5% of isolates assigned to Waitea circinata (zeae, oryzae, and circinata varieties), while 37.0% belonged to Thanatephorus cucumeris anastomosis groups (AGs) AG1-IB, AG1-ID, AG1-IE, AG4-HGI, and AG4-HGIII. Ceratobasidium spp. AG-A, AG-F, AG-Fa, AG-P, and AG-R comprised 23.5%. Rhizoctonia zeae (19.8%), R. solani AG1-IE (18.6%), and binucleate Rhizoctonia AG-A (8.6%) were the most frequent anamorphic states found. Root rot severity caused by the different taxa varied from low to high on common beans, and tended to be low to average in maize. Twenty-two isolates were pathogenic to both hosts, suggesting difficulties in managing Rhizoctonia root rots with crop rotation. These results suggest that cropping history affects the geographical arrangement of AGs, with a prevalence of AG1 in the tropical zone from central to north Brazil while the AG4 group was most prevalent from central to subtropical south. W. circinata var. zeae was predominant in soils under maize production. To our knowledge, this is the first report on the occurrence of W. circinata var. circinata in Brazil.

Characterization, Pathogenicity, and In Vitro Sensitivity of Rhizoctonia spp. Associated with Leaf and Sheath Spot of Bermudagrass Putting Greens in North Carolina and Alabama

Twenty isolates of a fungus resembling Rhizoctonia zeae were obtained from three golf courses with bermudagrass [Cynodon dactylon (L.) Pers.] greens that were exhibiting leaf and sheath spot symptoms in 2013 and 2014. Morphological and molecular identification showed that 17 isolates were identified as R. zeae and three were identified as Waitea circinata var. zeae. Pathogenicity of the 20 isolates was determined by inoculating hybrid bermudagrass (‘Champion’) plants in a growth chamber. After inoculation, plants were incubated in a growth chamber at 34/30°C (day/night) and sealed in a closed container to ensure 100% humidity. After 7 d, disease was visually estimated using a modified Horsfall‐Barrett scale (0–10). All isolates were pathogenic to the hybrid bermudagrass cultivar Champion and aggressiveness among isolates varied. Sensitivity of seven isolates collected in 2013 to 16 fungicides (flutolanil, pyraclostrobin, fluoxastrobin, azoxystrobin, propiconazole, triticonazole, triadimefon, penthiopyrad, fluxapyroxad, azoxystrobin + difenoconazole, tebuconazole, difenoconazole, iprodione, polyoxin‐D zinc salt, fluxapyroxad + pyraclostrobin, and chlorothalonil) was tested. The sensitivity of 13 isolates collected in 2014 to five fungicides (chlorothalonil, azoxystrobin, pyraclostrobin, difenoconazole, and flutolanil) was tested. In general, all the isolates were extremely sensitive to demtheylation and succinate dehydrogenase inhibitor fungicides (50% effective concentration [EC50] &lt; 1 mg a.i. L−1) and all isolates showed insensitivity to quinone outside inhibitor fungicides (EC50 &gt; 10 mg a.i. L−1). These results demonstrated that R. zeae is a pathogen of ultradwarf bermudagrass under the imposed environmental conditions. Rhizoctonia zeae is sensitive to many fungicides, but based on in vitro sensitivity data presented here, some may not perform well under field conditions.

Occurrence of Brown Blight Caused by Waitea circinata var. zeae on Cool Season Turfgrass in Korea

In 2010, disease symptoms, including necrotic lesions on stems and leaves with circular yellow-brown or irregular brown color patches, were observed on cool-season turfgrass at golf courses (OHCC) and the Daegu University research farm in Gyeongbuk, Korea. We isolated the causal agent and identified it as Waitea circinata var. zeae by morphological characterization and molecular analysis. To the best of our knowledge, this is the first report of brown patch caused by W. circinata var. zeae on cool-season turfgrass in Korea.

Molecular identification and pathogenicity of Rhizoctonia spp. from tobacco growing areas in northwestern Argentina

In Argentina, more than 60 % of the tobacco crops are grown in the northwestern part of the country and where Rhizoctonia solani leads to a reduction in crop yield and quality. In this study, 35 isolates of Rhizoctonia were obtained from 32 tobacco fields in northwestern Argentina and characterized by both morphological and molecular approaches. Based on the variability in the ITS region, isolates were identified as R. solani (80 %), Waitea circinata var. zeae (Rhizoctonia zeae) (8 %) and binucleate Rhizoctonia (8 %). Most isolates of R. solani belonged to the anastomosis groups (AGs) AG 4 HG-I (44 %), AG 2-1 (41 %) and AG 4 HG-III (13 %). Isolates of binucleate Rhizoctonia belonged to AG-F and AG-P of Ceratobasidium sp. Morphological variability was higher within isolates of AG 2-1 and AG 4 HG-III than within those of AG 4 HG-I. Aggressiveness of the isolates towards tobacco seedlings was assessed in the greenhouse. Isolates of AG 2-1 were the most aggressive on leaves, causing target spot, whereas isolates of AG 4 HG-I were the most aggressive on stems and roots, causing damping-off.

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

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

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

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

First Report of Leaf and Sheath Spot Caused by Waitea circinata var. zeae on Paspalum vaginatum and Zoysia tenuifolia in China.

Waitea circinata var. zeae was previously reported as the causal agent of leaf and sheath spot on Festuca arundinacea (1) and Panicum tennesseense (2) in the United States. In late May to mid-September 2013, a disease resembling leaf and sheath spot was observed on Paspalum vaginatum in fairways from several golf courses in Hainan Province, China. Affected plants initially had large yellow and brown spots on leaves and sheathes, then the whole plant turned yellowish-brown and eventually died. The same symptoms were also observed on a lawn established with Zoysia tenuifolia in Hainan University. Symptomatic leaves were surface sterilized in 1% hypochlorite for 1 min, rinsed with sterile water three times, and plated onto potato dextrose agar (PDA) amended with 0.01% gentamicin sulfate. Two Rhizoctonia-like fungal isolates were obtained from the diseased P. vaginatum (Isolate no. ML-WC1) and Z. tenuifolia (Isolate no. HNU-1) samples. After incubation on PDA for 1 week at 25°C, white mycelial colonies developed and eventually turned a salmon color with age. Small, white, spherical sclerotia (0.5 to 1 mm in diameter) were observed submerged throughout the agar media after incubation for 1 week and turned a reddish-brown color within 4 weeks. The two isolates were tentatively identified as W. circinata var. zeae based on these characteristics. The internal transcribed spacer (ITS) region of the ribosomal DNA gene was amplified and sequenced using primer pair ITS1/ITS4. The sequences of the two isolates (GenBank Accession Nos. KJ717943 and KJ717944) were more than 99% similar to W. circinata var. zeae (JQ688056 and JQ688059) sequences deposited in GenBank. To confirm pathogenicity, inocula were prepared by incubating autoclaved rye grains with strains ML-WC1 and HNU-1 for 2 weeks at 25°C. Ten colonized rye grains were uniformly spread around the crowns of 6-week-old P. vaginatum and Z. tenuifolia seedlings grown in 10-cm-diameter pots. Each isolate was placed in four separate pots and four control plants were treated with non-inoculated grain. All pots were covered with translucent plastic bags and placed in a greenhouse at 24 ± 2°C with a 12-h light/dark cycle. By 1 week post-inoculation, significant blighting of leaves and sheaths was observed, while non-inoculated plants showed no symptoms. W. circinata var. zeae was successfully re-isolated from symptomatic plants and confirmed by its Rhizoctonia-like mycelium and small, reddish-brown, spherical sclerotia on the PDA. Recently a related species, W. circinata var. circinata, causing brown ring patch on two cool-season grasses was reported in China (3). However, the isolates of W. circinata var. zeae were distinguished from W. circinata var. circinata base on ITS sequence data and morphological characters. To our knowledge, this is the first report of W. circinata var. zeae infecting P. vaginatum and Z. tenuifolia in China.

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.

RDNA-ITS DIVERSITY OF WAITEA CIRCINATA VAR. ZEAE (ANAMORPH: RHIZOCTONIA ZEAE)

In this study, we analysed a collection of Waitea circinata comprising 20 isolates (17 var. zeae; one var. circinata and two var. oryzae) from different localities and hosts with macroscopic anastomosis tests, PCR-RFLP and phylogenetic analysis of the 18S-28S rDNAITS region. Macroscopic anastomosis was not useful for the identification of W. circinata isolates because all isolates, even those of the same variety, gave tuft reactions indicating unrelatedness. However, varieties of W. circinata showed unique RFLP patterns with HhaI, MseI and TaqI enzymes, suggesting that this molecular method may be suitable for quick identification. 18S-28S rDNA-ITS phylogeny revealed two main monophyletic groups within W. circinata var. zeae isolates. The first main monophyletic group appeared global as it contained isolates from Asia, the Americas and Europe, and the second main monophyletic group was only comprised of isolates from the Americas. Because the genetic diversity of W. circinata var. zeae isolates from the Americas was higher than that from other continents, it may have originated there and later it may have disseminated from Far East to Eastern Europe.

DNA fingerprinting and anastomosis grouping reveal similar genetic diversity in Rhizoctonia species infecting turfgrasses in the transition zone of USA

Rhizoctonia blight is a common and serious disease of many turfgrass species. The most widespread causal agent, Thanatephorus cucumeris (anamorph: R. solani), consists of several genetically different subpopulations. In addition, Waitea circinata varieties zeae, oryzae and circinata (anamorph: Rhizoctonia spp.) also can cause the disease. Accurate identification of the causal pathogen is important for effective management of the disease. It is challenging to distinguish the specific causal pathogen based on disease symptoms or macroscopic and microscopic morphology. Traditional methods such as anastomosis reactions with tester isolates are time consuming and sometimes difficult to interpret. In the present study universally primed PCR (UP-PCR) fingerprinting was used to assess genetic diversity of Rhizoctonia spp. infecting turfgrasses. Eighty-four Rhizoctonia isolates were sampled from diseased turfgrass leaves from seven distinct geographic areas in Virginia and Maryland. Rhizoctonia isolates were characterized by ribosomal DNA internal transcribed spacer (rDNA-ITS) region and UP-PCR. The isolates formed seven clusters based on ITS sequences analysis and unweighted pair group method with arithmetic mean (UPGMA) clustering of UP-PCR markers, which corresponded well with anastomosis groups (AGs) of the isolates. Isolates of R. solani AG 1-IB (n = 18), AG 2-2IIIB (n = 30) and AG 5 (n = 1) clustered separately. Waitea circinata var. zeae (n = 9) and var. circinata (n = 4) grouped separately. A cluster of six isolates of Waitea (UWC) did not fall into any known Waitea variety. The binucleate Rhizoctonia-like fungi (BNR) (n = 16) clustered into two groups. Rhizoctonia solani AG 2-2IIIB was the most dominant pathogen in this study, followed by AG 1-IB. There was no relationship between the geographic origin of the isolates and clustering of isolates based on the genetic associations. To our knowledge this is the first time UP-PCR was used to characterize Rhizoctonia, Waitea and Ceratobasidium isolates to their infra-species level.

Identification of Waitea circinata as a Pathogen of the Moss Bryum argenteum var. argenteum on a Golf Course Fairway.

Waitea circinata Warcup and Talbot (also referred to as W. circinata var. circinata) is an important fungal pathogen of amenity turfgrasses and is especially problematic on Poa annua in putting greens in the late spring or early summer. The pathogen was first identified in 2005 from Japan and has since been seen widely throughout the United States (1,2). On occasion, the pathogen has been observed attacking creeping bentgrass (Agrostis stolonifera) but is typically less virulent. Disease symptoms include prominent yellow rings appearing throughout established turf and moderate leaf necrosis. In the summer of 2012, moss from a section of fairway on a golf course in Edwards, CO was observed to be rapidly killed by a fungal pathogen producing copious amounts of aerial mycelium and appearing similar to Waitea microscopically. Aerial mycelium was transferred to acidified potato dextrose agar (PDA) (1 ml lactic/L). After 1 day at 25°C, mycelia were transferred to PDA. After 2 weeks, plates were covered with white aerial mycelium and separate, spherical, 0.5-mm diameter, salmon-colored sclerotia, which turned dark brown within a few days and were produced submerged throughout the media. Spores were never produced and right-angled branching of mycelia, characteristic of Waitea, was observed in mature cultures. Mycelial plugs were incubated in nutrient broth and DNA was extracted using a MoBio Power Plant DNA extraction kit. Amplification of ribosomal ITS sequences with ITS4 and ITS5 resulted in a 100% identity match with GenBank sequence HM807352, W. circinata var. circinta (3). To demonstrate pathogenicity on Bryum argenteum, unaffected moss from the submitted sample (identified as B. argenteum var. argenteum via 100% sequence identity with the published GenBank sequence GU907062) was removed from the originally submitted sample and placed in separate growth chambers at 95% humidity and 21, 26, and 31°C. An additional experiment employed local B. agenteum plants collected from the URI Kingston, RI campus. Agar plugs from the isolated W. circinata were placed on top of the moss and within 2 days the fungus had caused complete mortality at all three temperatures. The experiment was also undertaken using the same environmental conditions with 5-week-old annual bluegrass (P. annua) and creeping bentgrass cv. A4 grown from seed. Plants were inoculated with infected rye grains at 31, 26, and 21°C. After 1 week, the P. annua plants showed significant mortality at 26 and 31°C with little infection at 21°C and the A. stolonifera plants showed moderate mortality at 26°C and little infection at the other two temperatures. All experiments utilized an additional uninoculated control treatment that showed no moss/turf necrosis or mortality. Experiments were all repeated once and used three replicates per experiment. While moss is not intentionally cultivated on golf courses, it does occur with regularity and often presents itself as a difficult pest to manage. This particular isolate of W. circinata has identical ribosomal and physiological characteristics of the reported P. annua pathogen but can attack one moss species and may be a possible candidate for selective biological control of moss in golf course settings. It is unclear how widespread moss pathogenicity is within W. circinata.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Agrostis stolonifera and Poa pratensis in China.

Brown ring patch, caused by Waitea circinata var. circinata, is a recently described disease of turf grass (1,2). The disease was first reported in Japan in 2005 (2) and then in the United States (1). In late May to early September 2011, large yellow rings (20 to 30 cm in diameter) were observed on creeping bentgrass (Agrostis stolonifera) and Kentucky bluegrass (Poa pratensis) growing at the Qinghe Bay golf course, Beijing, China. Leaf blades turned from yellow to brown as the disease developed, and eventually died. The disease incidence was estimated at 20 to 30%. The rings became continuous on creeping bentgrass and Kentucky bluegrass in several putting greens. The same symptom was observed on the lawn of China Agricultural University. Symptomatic leaves were collected and incubated in high humidity at 25°C until mycelia developed. The leaves were then disinfested in 1% NaClO for 1 min, rinsed with sterile water three times, and placed on potato dextrose agar (PDA). Four isolates were obtained, including one isolate from the lawn of China Agricultural University (cau-1), and three from Qinghe Bay golf course (qhw-1, qhw-2, and qhw-3). The colonies that formed on PDA changed from white to orange over time, and minute orange to brown sclerotia (approx. 2 to 3 mm in diameter) formed after 2 weeks at 25°C. These characteristics were similar to W. circinata var. circinata (1,2). DNA was extracted from each isolate using a CTAB extraction method (3) and the internal transcribed spacer (ITS) regions were amplified with the ITS1/ITS4 universal primers. The ITS sequences of the isolated fungi (Accession Nos. JQ964235 and JQ964236) had 99 to 100% homology with the sequences of W. circinata in GenBank (Accession Nos. EU591763 and HQ207169). Pathogen inocula were prepared by inoculating autoclaved oat grains with strains qhw-1 and cau-1 respectively, followed by 4 days of incubation at 25°C. Each inoculum was placed in five spots in a uniform arrangement (5 g grain inoculum per spot) on soil in a 40 × 60 cm tray, followed by sowing bluegrass seed. In another experiment, 4-week-old bentgrass was transplanted into soil infested with 5 g grain inoculum in the middle of a 20-cm diameter pot (non-colonized grain was used as a control). There were five replicates for each isolate. Plants were then incubated in a growth chamber at 26°C and high relative humidity (>90%). After 5 to 6 days, the grass in the inoculated pots and trays began to turn yellow, and then became chlorotic and necrotic as the disease developed. Orange sclerotia were observed on the bluegrass leaves by the eighth day, and all the bentgrass turned chlorotic by the tenth day. After 2 weeks, brown ring patches formed in the trays with inoculated bluegrass. Waitea circinata var. circinata was reisolated from all inoculated plants and confirmed by morphological observation and the ITS sequences analysis as described above, while no symptoms were observed on the control plants and no isolate was obtained from them. To our knowledge, this is the first report of W. circinata var. circinata infecting turf grass in China.