First Report of Pyricularia grisea Causing Gray Leaf Spot on Kikuyugrass (Pennisetum clandestinum) in the United States.

Kikuyugrass (Pennisetum clandestinum) is a warm-season turfgrass that has been adopted for use in fairways and roughs in a number of subtropical areas including southern California, Mexico, Australia, and South Africa. During August 2003, a foliar disease of Kikuyugrass was reported from a number of golf courses in southern California. Examination of diseased plants showed the presence of dark, olive green-to-brown lesions on the foliage. Incubation of these plants in a moist chamber for 12 h led to the production of numerous pyriform conidia from these lesions that were characteristic of Pyricularia grisea. Single-spore isolates of the fungus were obtained from infected kikuyugrass samples by transferring conidia to acidified 1.5% water agar and then transferring single, germinated conidia to one-quarter-strength potato dextrose agar. Colony morphology and conidia production were consistent with that described for P. grisea (1). Koch's postulates were performed separately for two single-spore isolates (OSGC-1 and CCCC-1) obtained from infected kikuyugrass. For each isolate, 2-week-old, glasshouse-grown seedlings of kikuyugrass (cv. 'AZ-1') and perennial ryegrass (Lolium perenne) grown in 75-mm pots in soilless media were inoculated with conidia from either OSGC-1 or CCCC-1. For each test, six pots of both kikuyugrass and ryegrass were inoculated, and the tests were conducted three times for each isolate. Conidia were obtained from isolates grown on clarified V8 agar in 100-mm petri plates for 14 days at 25°C. Suspensions were made by adding 10 ml of sterile distilled H2O (sdH2O) to the plates, scraping the surface of the media to dislodge the conidia, filtering the suspension through cheesecloth, and then adjusting the final concentration to 1 × 106 conidia/ml with sdH2O. Seedlings were inoculated with the conidial suspensions with an aerosol applicator, placed in plastic boxes lined with wet paper towels, and sealed to provide adequate moisture for infection. Boxes were incubated at 28°C for 48 h after which time the covers were removed and the plants maintained in ambient glasshouse conditions at approximately 28°C. In all three replicated experiments, kikuyugrass seedlings inoculated with OSGC-1 or CCCC-1 developed symptoms of disease approximately 5 days after inoculation, while inoculated perennial ryegrass did not, even 14 days after inoculation. Symptomatic kikuyugrass leaves were taken randomly from plants from each of the three replicated tests, surface disinfested in 0.3% sodium hypochlorite for 30 s, rinsed with sdH2O, blotted dry, and placed onto acidified water agar in petri plates. Twenty-four hours later, abundant sporulation was observed from symptomatic tissue with conidiophores bearing conidia typical of P. grisea. To our knowledge, this is the first report of gray leaf spot being caused by P. grisea on Pennisetum clandestinum in North America. Reference: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK, 1971.

First Report of a Labyrinthula sp. Causing Rapid Blight Disease of Rough Bluegrass and Perennial Ryegrass.

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.

First Report of Gaeumannomyces graminis var. graminis on Kikuyugrass (Pennisetum clandestinum) in the United States.

Kikuyugrass (Pennisetum clandestinum) is a warm-season grass and invasive weed in the landscape, but can be used for golf course fairways in southern California. In 1999, a decline of kikuyugrass was observed on golf courses in southern California beginning in late summer or early autumn. Symptoms included sunken, bleached patches of turf with individual plants having chlorotic foliage and reduced vigor. Roots and stolons were often covered with dark, ectotrophic fungi, and lobed hyphopodia were visible on the stolons. On colonized roots, the cortex was rotted, and the stele showed evidence of colonization by the fungus. In March 2002, a sample of kikuyugrass exhibiting decline symptoms was obtained from a golf course fairway in Los Angeles, CA. Sections of roots and stolons were surface sterilized for 60 s in a 0.3% sodium hypochlorite solution and placed on acidified water agar. Emerging colonies were transferred to potato dextrose agar (PDA). Isolates were characteristic of Gaeumannomyces spp. (2) with dark hyphae and curled colony edges. The rDNA internal transcribed spacer (ITS) regions of two isolates (HCC-5 and -6) were amplified by polymerase chain reaction (PCR) using universal fungal rDNA primers ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS 5 (5'-GGAAGTAAAAGTCG TAACAAGG-3') (3). PCR products were sequenced and exhibited 99% sequence identity to G. graminis var. graminis (GenBank Accession No. 87685). These isolates were grown separately on autoclaved sand and cornmeal media (1) for 21 days at 25°C. Styrofoam cups were partially filled with autoclaved medium-coarse sand, and 10 g of inoculum was spread evenly in a layer on top. This layer was covered by an additional centimeter of autoclaved sand and 5 g of kikuyugrass seed (cv. 'AZ-1'). Both isolates were tested separately using six replicate cups per isolate. Controls were prepared using only a 10 g layer of autoclaved sand and cornmeal. Cups were misted at 1 h intervals on a greenhouse bench maintained at 25°C. Seeds germinated and emerged after ≈10 days. In cups inoculated with isolate HCC-5 or -6, dark mycelia were evident on the coleoptiles of the emerging plants. Plants were removed and washed 21 days after planting. Inoculated plants were chlorotic and had reduced root and foliar growth compared to the controls. Coleoptiles, hypocotyls, and roots were covered with dark, ectotrophic fungi with lobed hyphopodia present on the hypocotyls. In colonized roots, cortical tissue was rotted with extensive colonization of the epidermis and penetration of the fungus into the root cortex. Sections of infected root tissue were surface disinfested, placed on acidified water agar, and the resulting colonies transferred to PDA. Isolates exhibited the same colony morphology and characteristics as those previously identified as G. graminis var. graminis. To our knowledge, this is the first report of this fungus as a pathogen of kikuyugrass. References: (1) M. J. C. Asher. Ann. Appl. Biol. 70:215, 1972. (2) P. C. Cunningham. Isolation and culture. Pages 103-123 in: Biology and Control of Take All. M. J. C. Asher and P. J. Shipton, eds. Academic Press, London, 1981. (3) T. J. White et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al. eds. Academic Press, San Diego, CA, 1990.

Diagnosis of turfgrass diseases.

The role of the professional disease diagnostician has become increasingly important in turf management. Responsible turfgrass disease diagnosis must incorporate the possibility of biotic, as well as abiotic, disorders and should consist of three components: the interview, identification of the stress factor, and a management recommendation. The concept of management groups is introduced to facilitate delivery of the rapid and effective solution required by turf managers. Recent advances in diagnostics, including immunoassay, PCR kits, and distance diagnostics, have had minimal effect on turfgrass diagnostic practices to date. However, continued emphasis on the application of technology rather than knowledge-based diagnostic procedures is contributing to the demise of applied plant pathology. Nevertheless, the demand for turfgrass disease diagnostic services continues to increase, making the future for the applied plant pathologist somewhat uncertain, but full of opportunities.