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A Labyrinthula sp. was isolated from symptomatic rough bluegrass (Poa trivialis L.) and perennial ryegrass (Lolium perenne L.) from a golf course in Arizona. Initial symptoms were a water-soaked appearance and rapid collapse of small patches of turf foliage. The affected turf died, and patches coalesced to form large dead areas after several weeks. The symptoms were those of the disease recently termed “rapid blight” for which the causal agent has not been identified (1). Rapid blight was first observed in southern California in 1995 and has become increasingly problematic in 10 other states on several cool-season turfgrasses (1). In Arizona, it is associated with high salinity irrigation water. In microscopic examinations of symptomatic P. trivialis and L. perenne leaf tissue from November 2002 to February 2003, fusiform or spindle-shaped vegetative cells (4 to 5 × 15 to 20 μm) were observed in leaf cells. These cells are consistently associated with rapid blight (1) and are typical in size and shape of those described for Labyrinthula spp. (3,4). The fusiform cells were cultured in 1% horse serum water agar medium made with irrigation water (electrical conductivity [EC] = 3.5 to 4.0 dS/m) from a golf course in central Arizona with rapid blight. The cells readily formed colonies on this medium and exhibited gliding motility along a network of hyaline slime filaments as previously described for the genus Labyrinthula (3,4). Koch's postulates were fulfilled by inoculating P. trivialis and L. perenne seedlings with Labyrinthula sp. isolated from naturally infested P. trivialis in two experiments. The grasses were started from seed and grown as a lawn in containers in the laboratory. Both experiments were repeated once. In the first experiment, infested autoclaved leaf pieces of P. trivialis were used as inoculum. Inoculated leaf pieces were placed within each of several bundles of 4 to 6 leaves and held loosely in place with a 0.5-cm wide ring of tygon tubing. Seedlings were irrigated with sterilized irrigation water from the golf course (EC = 4.0 dS/m). In the second experiment, agar discs from Labyrinthula sp. colonies on 1% horse serum agar were used as inoculum by placing the agar discs in contact with leaves. Seedlings were irrigated with sterile tap water adjusted to 4.0 dS/m using synthetic sea salt (Instant Ocean, Aquarium Systems, Inc., Mentor, OH) Leaf tissue of all inoculated seedlings became water soaked within 3 to 7 days and collapsed within 10 days in both experiments. Fusiform cells were observed in inoculated leaf tissue cells, and the Labyrinthula sp. was reisolated from 100% of selected symptomatic seedlings. Control seedlings treated with noninfested leaf pieces or sterile agar pieces did not develop symptoms, and no fusiform cells were isolated from the leaf tissue. Labyrinthula spp. are usually associated with marine systems (3). Labyrinthula zosterae D. Porter & Muehlst. has been identified as the causal agent in a marine grass wasting disease (2), but to our knowledge, no Labyrinthula spp. have been described as pathogens of terrestrial plants. References: (1) S. B. Martin et al. Phytopathology (Abstr.) 92:(suppl)S52, 2002. (2) L. K. Muehlstein et al. Mycologia 83:180, 1991. (3) K. S. Porkorny. J. Protozool. 14:697, 1967. (4) D. Porter. Handbook of Protoctista. Jones and Bartlett, Boston, MA, 1990.

Pythium spp. were isolated from nonoverseeded bermudagrass and from bermudagrass overseeded with cool-season turfgrass species from October 2000 to July 2001 from two sites in Florida. Pythium spp. were isolated from nonoverseeded and overseeded, and fumigated and nonfumigated, bermudagrass plots from October 2001 to July 2002 from one of the two sites. The vast majority of isolates of Pythium spp. were obtained from the bermudagrass, rather than the cool-season turfgrass species (Poa trivialis or Lolium perenne) used as overseed. In the first year at the Ft. Lauderdale site, Pythium graminicola dominated (91% of isolates obtained). In the first year at the Gainesville site, P. graminicola (56% of isolates) and P. irregulare (36%) dominated; however, after fumigation and replanting, P. graminicola comprised only 11% of all isolates. P. irregulare comprised 30% of all isolates, but was found only in nonfumigated plots. A different species, P. ultimum var. ultimum, not isolated in the first year, was recovered (34% of isolates) from the replanted field in February and March. In preemergence pathogenicity tests, three of four isolates of P. irregulare were moderately to highly pathogenic on Poa trivialis, but not on L. perenne, and isolates of Pythium graminicola and P. ultimum var. ultimum were not pathogenic on either turfgrass species. Pathogenic Pythium spp. may survive from season to season on bermudagrass and, under favorable conditions, may cause damping-off or blight on the overseeded cool-season turfgrass.

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. References: (1) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (2) T. Toda et al. Plant Dis. 89:536, 2005.

A disease characteristic of rapid blight caused by the net slime mold, Labyrinthula terrestris (1,2), was observed on three annual bluegrass (Poa annua) putting greens from a golf course in Adams County, Colorado in April 2009. Symptoms included water-soaked lesions and browning and bronzing of leaves. With microscopic observation, the fusiform cells typically associated with Labyrinthula spp. (1) were detected inside symptomatic leaf tissue. The pathogen was isolated by placing symptomatic leaves on a selective medium modified from Bigelow et al. (1) (12 g of granulated agar [Fisher Scientific, Pittsburg, PA], 10 ml of horse serum [Hema Resource and Supply, Aurora, OR], and 250 μg of ampicillin, streptomycin sulfate, and penicillin G [Sigma, St. Louis, MO] in artificial seawater at 4.0 dS/m electrical conductivity [Instant Ocean, Atlanta, GA]). Irregular-shaped digitate colonies of fusiform cells developed within 1 to 2 days. The isolated organism was then used to fulfill Koch's postulates on 2-week-old Poa trivialis ‘Sabre III’ seedlings and 4-week-old Poa annua seedlings planted in a 90:10 peat moss/sand mixture in 6-cm-diameter pots. Plants were inoculated with a 9 × 106 cells/ml suspension of L. terrestris cells in artificial seawater (4.0 dS/m) as described by Peterson et al. (2) and irrigated daily with artificial seawater (4.0 dS/m). Negative controls consisted of either P. trivialis or P. annua plants irrigated with artificial seawater only. There were three replications for each treatment and the experiment was repeated for each grass species. Pots were maintained at 28 to 33°C in the greenhouse with ambient light. Within 8 to 10 days of inoculation, 95% of the plants showed symptoms of severe rapid blight, while noninoculated plants showed some minor salt stress symptoms but were otherwise healthy. The organism was successfully reisolated from several plants from each replication using the method described above. Results were the same for all experiments. Rapid blight is frequently associated with high soil salinity (>2.5 dS/m total dissolved salts) (1) and sodium levels above 110 mg/kg (Mehlich-3 extraction) in diagnostic samples. Soil salinity levels from the site affected by the disease were below this guideline. However, sodium levels measured an average of 184 mg/kg. The ability of this pathogen to cause disease on plants growing in soils not measuring high in salinity, and only with elevated sodium, should be considered when attempting to ascertain rapid blight as a cause of turf damage. References: (1) D. M. Bigelow et al. Mycologia 97:185, 2005. (2) P. D. Peterson et al. Online publication. doi:10.1094/ATS-2005-0328-01-RS. Applied Turfgrass Science, 2005.

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. References: (1) K. de la Cerda et al. Plant Dis. 91:791, 2007. (2) T. Toda et al. Plant Dis. 89:536, 2005.

Hybrid bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davy) is widely used on golf course putting greens in the southern United States. In March and April 1998, circular patches of dead grass 2 to 10 cm in diameter were observed on a bermudagrass putting green in College Station, TX, that had been overseeded with rough bluegrass (Poa trivialis L.) the previous October. Rapid death and deterioration of the rough bluegrass within the spot revealed extensive foliar and crown necrosis and root decay of the remaining bermudagrass. Diseased bermudagrass leaves in the patch were reddish brown to tan. Dark ectotrophic hyphae were not found on the roots or stolons, but dark hyphae were observed within the affected root tissues. Numerous pseudothecia were embedded in necrotic leaf and stolon tissues. The characteristics of the pseudothecia and ascospores coincide with the description of Ophiosphaerella agrostis Dernoeden, Camara, O'Neil, van Berkum, and Palm (1,2). This fungus was consistently isolated from stolons and roots, and single-ascospore isolates were obtained from pseudothecia. Inoculum was prepared by transferring fungal mycelium from a single-spore isolate grown in potato dextrose agar (PDA) to a moistened, autoclaved mixture of rice hulls (Oryza sativa L.) and milled rice (2:1, vol/vol) for 28 days at 24°C. ‘FloraDwarf’ bermudagrass was grown from stolons in 15-cm-diameter pots containing a mixture of sand, peat moss, and perlite (8:3:1, vol/vol). The bermudagrass was maintained at a height of 1 to 1.5 cm for ≈ 1 month. Plants were inoculated by forming a hole that was 0.8 cm in diameter and 7 cm deep in the center of the pot, using a rod and filling the hole with inoculum. Control pots received the same treatment, except uninoculated rice hull-milled rice mixture was used. The treatments were replicated three times, and the experiment was performed twice. The pots were maintained in a greenhouse for 6 weeks. In all inoculated pots, patches of dead bermudagrass 6 to 10 cm in diameter developed. Roots, stolons, and leaves were necrotic, and pseudothecia were abundant in stolon and leaf sheath tissues. O. agrostis was consistently reisolated from infected root and stolon tissues. All isolates produced colonies identical in appearance to the culture used for inoculation. To our knowledge, this is the first report that O. agrostis is pathogenic to hybrid bermudagrass. References: (1) M. P. S. Camara et al. Mycologia 92:317, 2000. (2) P. H. Dernoeden et al. Plant Dis. 83:397, 1999.