Brown Ring Patch Research Articles

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

First Report of Brown Ring Patch Caused by Waitea circinata on Perennial Ryegrass (Lolium perenne) in Oklahoma

First Report of Brown Ring Patch Caused by <i>Waitea circinata</i> on Perennial Ryegrass (<i>Lolium perenne</i>) in Oklahoma

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.

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.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Creeping Bentgrass in Arizona.

A disease resembling brown ring (Waitea) patch was observed on a 'Dominant Extreme' creeping bentgrass (Agrostis stolonifera) green on a golf course in Maricopa County, Arizona in February 2010. The green was 17 months old and built with 95% sand and 5% peat moss. The superintendent reported seeing yellow rings, 12 to 16 cm in diameter, on several greens as early as 3 months postinstallation; the yellow rings developed into brown, necrotic rings. Symptoms started in the cool, cloudy, and moist conditions of December (5.0 to 6.7°C) and became persistent into the spring. Symptoms on the samples appeared to be yellowing of leaves and stems with the development of a dark, water-soaked appearance of the whole plant on older affected portions. The samples were incubated in a moist chamber at 22 to 25°C for 24 h. Foliar mycelium developed on the symptomatic leaves, and upon microscopic examination, the mycelium appeared to have the characteristics of Rhizoctonia spp.; i.e., a right-angled branching pattern, constriction of the hyphal branch near its point of origin, and the presence of a septum near the point of origin. The pathogen was recovered from chlorotic tissue by plating the symptomatic tissue on one-quarter-strength acidified potato dextrose agar (9.90 g of PDA and 11.26 g of granulated agar [Fisher, Lenexa, KS] and 600 ml of lactic acid [Sigma, St. Louis, MO] per liter of water) and incubating at ~27°C in light. A Rhizoctonia-like pathogen emerged from the tissue within 48 h and was tentatively identified as Waitea circinata var. circinata based on colony and bulbil morphology after 10 days of incubation (3). The recovered isolate was used for DNA extraction and subsequent amplification and sequencing of the rDNA internally transcribed spacer (ITS) region using ITS1F and ITS4 primers (2). The recovered sequence (HM807352) was compared with the National Center for Biotechnology Information (NCBI) nucleic acid database and was found to show 100% similarity to W. circinata var. circinata (FJ755879). To confirm pathogenicity, the isolate was used to fulfill Koch's postulates. The isolate was grown on autoclaved sand and corn meal (250 g of sand and 50 g of corn meal) for 4 weeks to produce inoculum. Eight grams of colonized sand and corn meal was broadcast on 4-week-old creeping bentgrass seedlings ('Penncross') planted in a 90:10 peat moss/sand mixture in 10-cm-diameter pots. There were three replications and the experiment was repeated twice. Negative controls consisted of plants inoculated with sand and corn meal only. Pots were maintained at 28 to 33°C in the greenhouse with ambient light. Within 4 days of inoculation, the plants showed chlorosis and necrosis, while noninoculated plants showed no symptoms. The pathogen was successfully reisolated from several plants from each replication using the method described above. This pathogen has been known to cause disease on annual and rough bluegrass (1,2) in the United States, but not confirmed as a pathogen on creeping bentgrass here. To our knowledge, this is the first report of brown ring patch on creeping bentgrass in Arizona.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua and Agrostis stolonifera in New Jersey.

Waitea circinata var. circinata was first reported as the causal agent of brown ring patch on annual bluegrass (Poa annua L.) in the United States in 2007 (2). In early April to mid-June of 2009, circular to irregularly shaped yellow rings resembling symptoms of this disease were observed on an annual bluegrass putting green at Rutgers University in North Brunswick, NJ. Severely infected foliage eventually turned brown as the disease progressed. During the same time period, similar disease symptoms were observed on creeping bentgrass (Agrostis stolonifera L.) from a golf course in Bedminster Township, NJ. The disease reappeared in both locations in April of 2010. Five additional samples with similar symptoms on creeping bentgrass and annual bluegrass were received at Rutgers Diagnostic Laboratory from Paramus, Madison, Allamuchy, and Farmingdale, NJ between late April and early May of 2010. Portions of diseased leaf and sheath tissue that displayed symptoms of the disease were disinfested for 1 min in 0.5% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate. At the first sign of fungal growth, single hyphal tips were transferred to PDA. After 1 week at 25°C, white-to-orange mycelial colonies formed in culture and eventually turned brown with age. Minute sclerotia (≤3 mm), which followed the same color development pattern, formed within 10 days. These features are consistent with those described of W. circinata var. circinata (2,3). The internal transcribed spacer (ITS) region of the ribosomal RNA gene was amplified using primer pair ITS1/ITS4 and sequenced with ITS4 (GenBank Accession Nos. HQ166065 to HQ166071). BLASTn analysis of the ITS sequences showed a 99 to 100% similarity to W. circinata var. circinata sequences deposited in GenBank (1,2). Pathogenicity tests were conducted in 2010 using 6-week-old creeping bentgrass seedlings cv. Declaration inoculated with colonized oat grain that had been autoclaved and then infested with the Bedminster Township isolate. Eight colonized oat grains were uniformly spread around the crowns of seedlings grown in 10-cm-diameter pots. Control plants were treated with autoclaved grain. Plants were incubated at 25°C and high humidity maintained by misting the plants three times per day. Within 3 days postinoculation, foliage near infested grain turned chlorotic. All foliage in pots became completely blighted and spherical orange-brown sclerotia were observed on leaf sheaths by the eighth day. W. circinata var. circinata was consistently reisolated from inoculated plants (as confirmed by isolate morphology and ITS sequencing) but not from control plants. The ITS sequence data, morphological characters of the isolates, and pathogenicity tests demonstrate that W. circinata var. circinata is present in New Jersey. To our knowledge, this is the first report of W. circinata var. circinata infecting turfgrass in New Jersey.

First Report of Brown Ring Patch on Poa annua Caused by Waitea circinata var. circinata in West Virginia.

In mid-November 2009, thin, yellow, and irregular-shaped scalloped rings 10 to 25 cm in diameter were observed on 5 to 10% of a golf course putting green in Charles Town, WV. The 20-year-old USGA-specification sand-based green was mowed at 3.1-mm height and consisted of 60% annual bluegrass (Poa annua L.) and 40% creeping bentgrass (Agrostis stoloniferous L. 'Putter'). Minimum and maximum daily air temperature ranged from 2 to 22°C, respectively, with 38 mm of rainfall during the appearance of rings symptoms. Only affected annual bluegrass plants exhibited a peculiar yellow chlorosis of the upper and lower leaves. A single fungal isolate was obtained from active mycelium found within symptomatic annual bluegrass leaves and grown on potato dextrose agar (PDA) amended with chloramphenicol (0.1 g/liter). Fungal colony morphology (i.e., light yellow with irregular-shaped 2- to 4-mm-diameter sclerotia first appearing off-white but progressing to brown by 21 to 28 days in culture) and sequencing of the internal transcribed spacer (ITS) 5.8S rDNA region with primers ITS1 and ITS4 confirmed the isolate as Waitea circinata var. circinata (Warcup & Talbot) with ≥99% sequence identity with GenBank Accession No. FJ755889 (1,2,4). To confirm pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 4-day-old culture grown on PDA and placed in contact with the lower leaves of 12-week-old annual bluegrass (0.001 g of surface-sterilized seed per cm2) grown in 5- × 5-cm plastic pots of autoclaved 85% sand and 15% potting soil. Six pots were inoculated with the isolate and six pots were inoculated with an isolate-free agar plug and then placed in a moist chamber at 28°C. Leaf chlorosis and aerial mycelium was observed in all six inoculated pots 8 to 10 days after inoculation, and symptoms were similar to those expressed in the field. All noninoculated plants remained healthy and asymptomatic. W. circinata var. circinata was reisolated from symptomatic leaves and again confirmed by colony traits and sequencing of the ITS-5.8S rDNA region and submitted as GenBank Accession No. HM807582. To our knowledge, this is the first report of brown ring patch in West Virginia and could be economically important because of intensive fungicide practices used to maintain high-quality putting greens on golf courses (3).

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Wisconsin and Minnesota.

In summer of 2008, two turfgrass samples were submitted to the Turfgrass Diagnostic Lab at the University of Wisconsin-Madison. The samples were from golf courses in Beaver Dam, WI on 12 June and Minneapolis, MN on 14 July. Both samples were collected from 40-year-old native soil putting greens mowed at 3.2 mm that had received annual sand topdressing since 1992. The putting greens were a mixture of approximately 75% annual bluegrass (Poa annua L.) and 25% creeping bentgrass (Agrostis stolonifera L.) Stand symptoms observed in the field were bright yellow, sunken rings that were approximately 5 cm thick and 15 to 35 cm in diameter. Some rings were incomplete, giving a scalloped appearance. Affected plants were severely chlorotic and lacked any discrete lesions or spots. Symptoms were more prominent on annual bluegrass than creeping bentgrass. Upon incubation of samples at room temperature in a moist chamber for 24 h, fungal mycelia with septations and right-angle branching were observed in the foliage and thatch layer. Two isolates were obtained from affected annual bluegrass in each sample. Isolations were performed by washing affected leaves in 0.5% NaOCl solution for 2 min, blotting the tissue dry, and plating the tissue on potato dextrose agar (PDA) amended with chloramphenicol (0.05 g/liter), streptomycin (0.05 g/liter), and tetracycline (0.05 g/liter). After incubation for 2 days at 23°C, isolates were transferred and maintained on PDA. All four isolates had multinucleate hyphae and displayed sclerotial characteristics similar to those reported for Waitea circinata var. circinata (2). Sequencing the ITS1F/ITS4-amplified rDNA internal transcribed spacer (ITS) region confirmed the isolates as W. circinata var. circinata, with ≥99% sequence similarity to published W. circinata var. circinata ITS sequences (GenBank Accession No. FJ755849) (1,2,4). To confirm pathogenicity, isolates were inoculated onto 6-week-old annual bluegrass (True Putt/DW184) grown in 10-cm-diameter pots containing calcined clay (Turface; Profile Products LLC., Buffalo Grove, IL). Two 4-mm-diameter agar plugs for each isolate were removed from the margins of 3-day-old colonies grown on PDA and placed near the soil surface to ensure contact with the lower leaf blades. Each isolate was placed in four separate pots to have four replicated tests per isolate, and four noninfested pots were utilized as negative controls. All pots were placed in moist chambers at 28°C with a 12-h light/dark cycle. Within 4 to 6 days, inoculated plants exhibited severe chlorosis and a minor amount of aerial mycelium was observed. Inoculated plants became necrotic after 15 to 20 days, while the noninoculated plants remained healthy. W. circinata var. circinata was reisolated from inoculated plants and its identity was confirmed by morphological and molecular characteristics. This pathogen was previously reported as a causal agent of brown ring patch of creeping bentgrass in Japan and annual bluegrass in the western United States (2,4). To our knowledge, this is the first report of brown ring patch in Minnesota and Wisconsin. Intensive fungicide practices are needed to control brown ring patch; therefore, this disease could have significant economic impact throughout the Upper Midwest (3).

First Report of Waitea circinata from Turfgrass in British Columbia, Canada

In Canada, Waitea circinata was first identified from buckwheat (Fagopyrum sp.) in 1965 in Ontario (4). In 2004, the fungus was found on diseased putting greens of Poa annua and Agrostis stolonifera near Toronto, Ontario (2). In late July 2009, symptoms on A. stolonifera resembling those of brown ring patch were seen at a golf course in Kelowna, British Columbia. Brown rings with light-colored, cottony growth were observed on a putting green with mixed P. annua and A. stolonifera, originally seeded with A. stolonifera cv. Penncross. Following a short incubation of the diseased grass at 25°C, hyphae of a Rhizoctonia-like fungus, not matching the characteristics of R. cerealis or R. solani, were seen. Symptomatic leaves were surface sterilized in 1% hypochlorite and plated onto potato dextrose agar (PDA) amended with streptomycin. After 1 week at 23°C, the plates contained white colonies that were 5 cm across. DNA was extracted and amplified with primers ITS1/ITS4 and sequenced with ITS1. The 600-bp sequence (deposited in GenBank as GU176409) from the internal transcribed spacer (ITS) region of ribosomal DNA showed a 100% match in the overlapping range with sequence FJ755879 from GenBank, which is annotated as W. circinata var. circinata. Pathogenicity was tested at 23°C by inoculating 3-week-old A. stolonifera 'Penncross' plants grown in Magenta boxes and incubated for 15 days after inoculation with ground wheat seed inoculum of W. circinata. Within 1 week, significant blighting of leaves and sheaths was observed as well as spherical orange brown sclerotia that were 2 to 5 mm in diameter on sheaths. These sclerotial features match characteristics of W. circinata var. circinata (1). Symptomatic leaves were plated on PDA and fungal growth characteristic of W. circinata was recovered. W. circinata was previously reported as the causal agent of brown ring patch on A. stolonifera in Japan (3), as a pathogen of P. annua in the United States (1), and as a pathogen of both species in Ontario, Canada (2). To our knowledge, this is the first report of W. circinata from turfgrass in western Canada.

Geographic Distribution and rDNA-ITS Region Sequence Diversity of Waitea circinata var. circinata Isolated from Annual Bluegrass in the United States.

Waitea circinata var. circinata is the causal agent of brown ring patch, an emergent disease of turfgrass in the United States. Forty-two isolates from annual bluegrass were obtained from California, Connecticut, Idaho, Illinois, Massachusetts, New York, Ohio, Oregon, and Rhode Island. Almost all isolates produced white to orange sclerotia (bulbils), 2 to 5 mm in size, that turned dark brown after 21 days on ¼-strength potato dextrose agar. The ribosomal DNA internal transcribed spacer regions and 5.8S region (ITS) were analyzed by restriction fragment length polymorphism (RFLP) analysis using MspI and sequencing to attempt identification of the isolates. Some isolates were heterozygous at the MspI restriction site, results not found in previous reports using the RFLP technique for identification. Four additional nucleotide positions were found to be variable within ITS based on sequence analysis, including two indels and two additional heterozygous positions. A total of 17 ITS haplotypes were found, and there was no obvious relationship between ITS haplotype and the geographic distribution of the isolates. Results of this work indicate that W. circinata var. circinata is present in multiple states and provide an initial understanding of the diversity of the pathogen in the United States.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Pennsylvania.

In late May and early June of 2008, bright yellow, thin, irregular-shaped rings that were 10 to 15 cm in diameter were observed on 30% of an annual bluegrass (Poa annua L.) putting green in Coopersburg, PA. The 46-year-old silt-loam soil green was mowed at a 3.1-mm height and consisted of 80% annual bluegrass and 20% creeping bentgrass (Agrostis stolonifera L., unknown cultivar). During the appearance of ring symptoms, the overall minimum and maximum daily air temperature ranged from 19.9 to 31.1°C, respectively, along with 40.3 mm of total rain accumulation. In late May, only individual affected annual bluegrass plants exhibited a bright yellow chlorosis of upper and lower leaf blades and crown. By early June, affected annual bluegrass plants appeared dark brown and water soaked, turning reddish brown and then tan as they dessicated, wilted, and died. Fungal mycelium, similar in appearance to Rhizoctonia spp., was found among affected leaf blades and within the thatch layer. A single fungal isolate was obtained from affected annual bluegrass tissue and grown on potato dextrose agar (PDA) plus 0.1 g of chloramphenicol per liter. Fungal colony morphology and sequencing of the ITS1F/ITS4-amplified rDNA internal transcribed spacer (ITS) region confirmed the isolate as Waitea circinata var. circinata, with ≥90% similar homology match to published W. circinata var. circinata ITS sequences (GenBank Accession No. DQ900586) (2,4). To confirm pathogenicity, the isolate was inoculated onto 6-week-old annual bluegrass (0.001 g of surface-sterilized seed per cm2) grown in 5- × 5-cm2 plastic pots containing autoclaved 70% sand and 30% potting soil. Plants were maintained daily at a 4.0-mm height using a hand-held scissors. One 6-mm-diameter plug of the isolate was removed from the active edge of a 5-day-old culture grown on PDA and placed in contact with the lower leaf blades of the target plants. Four pots were inoculated with the isolate and four pots were inoculated with an isolate-free agar plug for each of two experimental runs. After inoculation, all pots were placed in a moist chamber at 28°C. In both experiments leaf blade chlorosis and a modest amount of aerial mycelium was observed in all four isolate-introduced pots at 5 to 7 days after inoculation. Symptoms were similar to those expressed in the field, and by 21 to 28 days, all isolate-infected plants died, whereas the noninoculated plants remained healthy and nonsymptomatic. W. circinata var. circinata was reisolated from symptomatic tissue of those inoculated plants and again confirmed by colony traits and rDNA ITS region sequences. This pathogen was reported previously as the causal agent of brown ring patch on annual bluegrass and rough bluegrass (Poa trivialis L.) in the western United States. (1,2). To our knowledge, this is the first report of brown ring patch in Pennsylvania. The economic impact of this disease could be significant since intensive fungicide practices are used to produce high-quality putting green surfaces in the region (3).

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Virginia.

Brown ring patch was first described as a disease of cool-season turfgrass on creeping bentgrass (Agrostis palustris) (4) in Japan and later reported in California on annual bluegrass (Poa annua) (2). Brown ring patch symptoms were observed beginning in December 2007 through spring 2008 on 6 of 18 putting greens on a golf course in Reston, VA. Symptoms included yellow rings and patches of blighted turfgrass on the mixed stands of creeping bentgrass (A. palustris) and primarily annual bluegrass (Poa annua). Chlorosis and blight occurred predominantly on P. annua. A turfgrass sample was received from a consultant in April 2008, and disease severity on affected greens was estimated to be 40%. After incubating for 2 days in a moist chamber, Rhizoctonia-like aerial mycelia were observed. The pathogen was isolated on water agar and potato dextrose agar amended with thiophanate-methyl (100 mg/L), rifampicin (100 mg/L), and ampicillin (500 mg/L) from P. annua plants that had been surface sterilized with 70% ethanol for 15 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (2,4). Hyphae were stained with aniline blue and multiple nuclei were observed per cell. The teleomorph was not observed on plant material or in culture. Amplified fragments of rDNA including internal transcribed spacers from the isolate were amplified in three bacterial clones and sequenced bidirectionally (GenBank Accession Nos. FJ154894, FJ154895, and FJ154896) using primers ITS1/ITS4 (2,4). The consensus sequences matched, with 99% homology and 99% sequence overlap, isolate TRGC1.1 of W. circinata var. circinata (GenBank Accession No. DQ900586) (2). Annual bluegrass was not available for use in performing Koch's postulates, but previous studies have shown that W. circinata var. circinata is pathogenic to roughstalk bluegrass (P. trivialis) (1,3). Pots of P. trivialis cv. Cypress that were 1 week postemergence were inoculated with seven wheat grains that had been autoclaved and then infested with the isolate. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel on the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days, and more than 80% of the turf in pots was dead after 1 week. Control pots were inoculated with autoclaved wheat seed and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. W. circinata var. circinata was reisolated from affected plants in all replications of the test. To our knowledge, this is the first report of brown ring patch in Virginia. Additional research is needed to assess the prevalence and importance of this disease on golf course putting greens in Virginia.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa trivialis in Florida.

Brown ring patch is a newly described disease of cool-season turfgrass first reported in Japan on creeping bentgrass (Agrostis palustris) (2) and later reported in California on annual bluegrass (Poa annua) (1). The disease is characterized by either patches or rings of discolored to blighted turfgrass that can range from a few centimeters to a meter in diameter. Affected turfgrass plants turn chlorotic and can be blighted from the crown to the leaf tips. Blight symptoms have been associated with fluffy white-to-cream aerial mycelium after extended incubation of the sample. Symptoms including patches of blighted turfgrass approximately 10 cm in diameter were observed on roughstalk bluegrass (Poa trivialis) that had been overseeded onto a dormant 'Tifdwarf' bermudagrass (Cynodon dactylon) putting green in Palatka, FL. A sample was submitted by the superintendent in June 2005 because symptoms were confused with dollar spot and a fungicide resistance issue was suspected. The sample produced abundant aerial mycelium after incubation. The pathogen was isolated on potato dextrose agar amended with rifampicin (100 ppm) and streptomycin (100 ppm) from Poa plants surface disinfested with 70% ethanol for 30 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (1,2). The teleomorph W. circinata var. circinata was not observed on plant material or culture plates. Amplified fragments of rDNA including internal transcribed spacers from the isolate were sequenced bidirectionally from four bacterial clones. The consensus sequences (GenBank Accession Nos. FJ029103, FJ029104, FJ029105, and FJ029106) matched with 99% homology (99% sequence overlap) isolate TRGC1.1 of W. circinata var. circinata described by Wong, NCBI Accession No. DQ900586 (1). Pots of 'Cypress' roughstalk bluegrass that were 1 week postemergence were inoculated with the pathogen using 10 infested wheat grains. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel in the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days and greater than 90% of the turf in pots was dead after 1 week. The fungus was reisolated from affected plants. Control pots were inoculated with uninfested wheat grains and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. To our knowledge, this is the first report of brown ring patch on P. trivialis in Florida.

First Report of Waitea circinata var. circinata Causing Brown Ring Patch on Poa trivialis in California.

Rough bluegrass (Poa trivialis L.) is a C3 (cool-season) turfgrass used on golf course putting greens. It is often used to overseed C4 (warm-season) turf for fall through early-summer use. In March 2007, at maximium daytime air temperatures of approximately 30 to 35°C, irregular, thin, yellow rings approximately 10 to 20 cm in diameter were reported on P. trivialis in putting greens from two golf courses in the Coachella Valley of southern California. Affected plants had a blight of the leaves and stems and a rot of the crown, with initial symptoms being a yellowing of the tissue followed by the development of a dark, water-soaked appearance of the whole plant. Plants turned reddish brown as the water-soaked tissue dessicated. A Rhizoctonia-like fungus was found to be colonizing the leaves, stems, and upper roots and thatch. Three isolates were obtained from the diseased turf samples. All were identified as Waitea circinata var. circinata based on colony morphology (2) and rDNA internal transcribed spacer (ITS) region sequences (1). The sequences of the three isolates were more than 99% similar to those of W. circinata var. circinata deposited in the NCBI database (1,2). To confirm pathogenicity, each isolate was inoculated onto 4-week-old P. trivialis (2 mg of seed per cm2) grown in 10-cm-diameter pots containing steam-sterilized UC-mix at 28°C. Five 8-mm-diameter plugs of each isolate were taken from 7-day-old cultures grown on potato dextrose agar (PDA) and placed in direct contact with the bases of the stems. The pots were then incubated in a moist chamber at 28°C. Five noncolonized PDA plugs were used as a negative control. Three pots were used for each of the three isolates and the check treatment. For all isolates, chlorosis of leaf and stems were observed 5 days after inoculation and the tissue appeared water soaked after 7 days with the production of abundant aerial mycelia being observed. All plants, except the negative controls, died after 10 days. W. circinata var. circinata was reisolated from symptomatic tissue from the inoculated plants. W. circinata var. circinata was previously reported as the causal agent of brown ring patch on creeping bentgrass (Agrostis palustris) in Japan (2) and as a pathogen of annual bluegrass (P. annua) in the United States (1). To our knowledge, this is the first report of W. circinata var. circinata infecting P. trivialis in California.

Discovery and Characterization of Waitea circinata var. circinata Affecting Annual Bluegrass from the Western United States.

Waitea circinata var. circinata was identified as the causal agent of a new disease of annual bluegrass (Poa annua) in the United States. This pathogen is also known to cause brown ring patch on creeping bentgrass (Agrostis stolonifera) in Japan, but it had not been reported on any turf species outside of Japan. Symptoms on annual bluegrass caused by this fungus included regular to irregular yellow rings several centimeters to 1 m in diameter, typically at maximum daytime temperatures of 15 to 35°C. A total of 26 isolates were collected from diseased annual bluegrass. Twenty-two of these isolates were multinucleate, grew optimally at 25 to 30°C, and in culture formed irregular sclerotia approximately 2 to 5 mm in size that were white to orange and remained orange or turned brown to dark brown over a 28-day period. The remaining four isolates were characterized as being W. circinata var. zeae (Rhizoctonia zeae), which is a known pathogen of annual bluegrass in the United States. Sequence analysis of the internal transcribed spacer (ITS) regions of rDNA on a subset of isolates confirmed the identifications of W. circinata var. circinata (n = 8) and W. circinata var. zeae (n = 1) based on deposited sequences in GenBank. The identity of the remaining 14 isolates suspected to be W. circinata var. circinata was confirmed by HapII digestion of the amplified rDNA ITS region. Pathogenicity of four W. circinata var. circinata isolates was confirmed on both annual bluegrass and creeping bentgrass. This study is the first morphological and molecular confirmation of the presence of W. circinata var. circinata as a pathogen of turfgrass in the United States.

First report of Rhizoctonia zeae on turfgrass in Ontario

In May 2004, a disease appeared on Poa annua and Agrostis stolonifera at a golf course near Toronto. Narrow yellow rings enclosing areas up to 30 cm across appeared after air temperatures reached 25°C. The disease resembled yellow patch caused by Rhizoctonia cerealis, but the weather was too warm for normal occurrences of that disease. The rings persisted until the end of July. In late May 2005, the disease appeared again after the weather became hot. A mixture of azoxystrobin and chlorothalonil was applied which seemed to suppress the disease within a week, until it reappeared in July. Samples were collected, and leaves with symptoms were surface sterilized in 1% hypochlorite, and transferred to potato dextrose agar (PDA) amended with streptomycin. After one week at 25°C, the plates contained white colonies 5 cm diameter. Sequencing of the ITS region of ribosomal DNA showed a 99·6% match with a R. zeae sequence in GenBank. Pathogenicity was tested by inoculating 2-wk-old plants with mycelial plugs of R. zeae. Within one week at 25°C, significant blighting on leaves and sheaths was observed, together with spherical orange sclerotia on sheaths. Affected leaves were incubated on PDA, and hyphae of R. zeae were recovered. In Canada, R. zeae has been reported from turfgrass samples in British Colombia (Joshi, 2004), but this is the first report from Ontario. In the USA, this fungus has been reported to cause diseases of several turfgrass species (Smiley et al., 2005), which have been called variously hot weather brown patch, leaf and sheath spot/rot/blight or brown ring patch. We propose that it should be referred to as sheath spot. The taxonomy of this organism is similarly confused since R. zeae is considered to be a subspecies of Waitea circinata which contains at least two other subspecies including R. oryzae (Oniki et al., 1985; Leiner & Carling, 1994). More work is required to clarify the taxonomic disposition of R. zeae. We are grateful for the financial support of the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Agriculture and Food, as well as technical support from Darcy Olds and Russ Gowan.

Brown Ring Patch: A New Disease on Bentgrass Caused by Waitea circinata var. circinata.

Isolates of an unidentified Rhizoctonia sp. (NP isolates), obtained from creeping bentgrass (Agrostis stolonifera var. palustris) in Japan that exhibited symptoms of a new disease, were compared with isolates of three varieties of Waitea circinata var. oryzae, var. zeae, and var. circinata. NP isolates also were compared with isolates of R. oryzae obtained from creeping bent-grass exhibiting white patch-like symptoms (RW isolates). The color and size of sclerotia, color of mycelia, and pigment deposition of NP isolates was similar to that of RW isolates and W. circinata var. circinata, but distinctly different from W. circinata var. oryzae and W. circinata var. zeae. The optimal temperature for hyphal growth of NP isolates, RW isolates, and W. circinata var. circinata was 28°C, and for W. circinata var. oryzae and W. circinata var. zeae was 30°C. Pathogenicity tests on creeping bentgrass showed that the severity of disease caused by NP isolates, RW isolates, and W. circinata var. circinata was greater than with W. circinata var. oryzae, but lower than with W. circinata var. zeae. No significant differences in symptom expression were apparent among NP isolates, RW isolates, and W. circinata var. circinata. A phylogenic tree, obtained using the results of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), showed that isolates of W. circinata var. oryzae and W. circinata var. zeae separated into individual clusters, while NP isolates, RW isolates, and W. circinata var. circinata clustered together. The lengths of the rDNA internal transcribed spacer (ITS) region of NP isolates, RW isolates, and W. circinata var. circinata were identical but smaller than those of W. circinata var. oryzae and W. circinata var. zeae. Restriction fragment length polymorphism (RFLP) analysis of the rDNA-ITS region, using three enzymes (HapII, HinfI, and MboI), also showed that NP isolates were the same as RW isolates and W. circinata var. circinata, but different from W. circinata var. oryzae and W. circinata var. zeae. Based on these results, the NP isolates causing a new disease on bentgrass are W. circinata var. circinata, and that RW isolates are also W. circinata var. circinata but not R. oryzae. We propose that the name of the disease on creeping bentgrass caused by W. circinata var. circinata is brown ring patch.