First Report of Basal Leaf Blight of Kikuyugrass Caused by Waitea circinata var. prodigus in Southern California.

Kikuyugrass (Pennisetum clandestinum) is a C4 grass and invasive weed adopted for use as a primary turf species in some golf course fairways and roughs in southern California. In September 2008, a new Rhizoctonia-like fungus was isolated from a diseased kikuyugrass sample received from a golf course fairway in Oceanside, CA. The kikuyugrass was from a mature stand (>20 years old) that was maintained at a height of approximately 1.25 cm. Symptoms on kikyuygrass developed initially as irregular, blighted, chlorotic patches, several centimeters to one meter, which occurred during a period of warm, humid weather (27 to 29°C maximum daytime temperature, 75 to 85% average relative humidity) on a small part of one fairway. Affected areas became brown and necrotic as the disease progressed. Leaf chlorosis and stem rot were observed on affected plants. The organism was isolated by placing symptomatic leaves on acidified one-quarter-strength potato dextrose agar (PDA) (600 µl of 85% lactic acid per liter of medium) in a petri dish (1). A colony of a Rhizoctonia-like fungus with yellow aerial hyphae, multinucleate hyphal cells, and irregularly shaped, golden brown sclerotia (4 to 7 mm) developed within 30 days at 28°C. The rDNA internal transcribed spacer (ITS) sequence was obtained (GenBank Accession No. HQ850254) using PCR amplification with primers ITS1F and ITS4 (1,2), and a BLAST search showed 100% similarity with Waitea circinata var. prodigus (GenBank Accession No. HM597145), which had recently been described as the cause of basal leaf blight of seashore paspalum (Paspalum vaginatum), another C4 grass (3), in Florida. Colony morphology and other physical characteristics were similar but not completely identical to those from Florida, reflecting the reported morphological variation inherent in the pathogen (3). Koch's postulates were performed by growing this isolate on PDA in a petri dish for 7 days, homogenizing the culture with 100 ml of sterilized water, filtering the suspension through two layers of cheesecloth, and pipetting 10 ml of the mycelial suspension onto the foliage and stems of 4-week-old AZ-1 kikuyugrass plants grown in UC-soilless-mix in 7.5-cm-diameter pots (4). Control plants were treated with a homogenized and filtered dish of PDA only. There were three replicate pots for inoculated and noninoculated treatments and the experiment was repeated independently three times. All of the pots were incubated in a moist chamber with a 12-h light period at 28°C. Yellow lesions were observed on leaves and stems of inoculated plants 4 days postinoculation and necrosis developed 8 days later in all experiments. The same organism was isolated from symptomatic plants. The control plants did not exhibit any symptoms. To our knowledge, this is the first report of basal leaf blight caused by W. circinata var. prodigus on kikuyugrass in California and the first report of this pathogen affecting turfgrass in the western United States. References: (1) C. M. Chen et al. Plant Dis. 93:906, 2009 (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) S. J. Kammerer et al. Plant Dis. 95:515, 2011. (4) T. Toda et al. J. Gen. Plant Pathol. 73:379, 2007.

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. References: (1) C. M. Chen et al. Plant Dis. 91:1687, 2007. (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) T. Toda et al. Plant Dis. 89:536, 2005.

First Report of Rapid Blight Caused by Labyrinthula terrestris on Poa annua in Colorado.

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.

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). References: (1) C. M. Chen et al. Plant Dis. 93:906, 2009 (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Manage. 75(9):98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.

Leaf Scorch of Purple-Leafed Plum and Sweetgum Dieback: Two New Diseases in Southern California Caused by Xylella fastidiosa Strains with Different Host Ranges.

Sweetgum dieback and leaf scorch of purple-leafed plum are two new diseases of southern California landscape ornamentals. Samplings were conducted in 2003 and 2004 and 28 of 105 sweetgum (Liquidambar styraciflua) and 38 of 62 purple-leafed plum (Prunus cerasifera) plants tested positive for Xylella fastidiosa by enzyme linked immunosorbent assay. In all, 3 strains of X. fastidiosa were isolated from sweetgum and 13 from purple-leafed plum. All sweetgum strains and some purple-leafed plum strains grew on PW but not PD3 media. Strain PC045 from purple-leafed plum and strain LS022 from sweetgum were inoculated into their original hosts in addition to almond, oleander, and grapevine plants. Sweetgum plants also were inoculated with strains causing Pierce's disease, almond leaf scorch, and oleander leaf scorch. Strain PC045 caused symptoms in purple-leafed plum and almond plants within 6 months, and the pathogen was recovered from 93 and 100% of inoculated plants, respectively. Inoculation of grapevine and oleander plants with PC045 did not result in disease or recovery of the pathogen. In all, 5 of 25 sweetgum plants inoculated with LS022 showed symptoms after 9 months, and the pathogen was recovered from 3 of these plants. Inoculation of grapevine, oleander, and almond with LS022 resulted in no disease or recovery of the pathogen from the plants. A strain of Pierce's disease, a strain of oleander leaf scorch, and two strains from almond did not cause disease in sweetgum. These results confirm the role of X. fastidiosa strains as pathogens of purple-leafed plum and sweetgum, and that strains from sweetgum are unique in their host range.

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). References: (1) C. Chen et al. Plant Dis. 91:1687, 2007. (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) J. Kaminski and F. Wong. Golf Course Mgmt. 75(9):98, 2007. (4) T. Toda et al. Plant Dis. 89:536, 2005.

Concordance between side-stream end-tidal carbon dioxide and arterial carbon dioxide partial pressure in respiratory service setting.

OBJECTIVE: To explore the correlation and concordance between end-tidal carbon dioxide and arterial carbon dioxide partial pressure, and confirm the experience of the general consensus among service environments. DESIGN: A prospective cross-sectional analysis. SETTING: Two respiratory service units in Hong Kong. PARTICIPANTS: Two hundred respiratory patients were recruited, in whom 219 sets of observations were recorded. Patients deemed to require arterial blood gas determination also had their end-tidal carbon dioxide partial pressure measured at that time, using two LifeSense LS1-9R Capnometers. MAIN OUTCOME MEASURES: The agreement of end-tidal carbon dioxide partial pressure and arterial carbon dioxide partial pressure was studied by correlation coefficients, mean and standard deviation of their difference, and the Bland-Altman plot. RESULTS: Overall, the correlation was low and insignificant (r=0.1185, P=0.0801). The mean of the difference was 7.2 torr (95% confidence interval, 5.5-8.9) and significant (P<0.001). The limits of agreement by Bland-Altman analysis were -18.1 to 32.5 torr, which were too large to be acceptable. In the sub-group on room air, the mean difference was reduced to 2.26 torr, the correlation between end-tidal carbon dioxide partial pressure and arterial carbon dioxide partial pressure was 0.2194 (P=0.0068), though statistically significant, the extent of correlation was still low. CONCLUSION: End-tidal carbon dioxide partial pressure did not show significant correlation or concordance with arterial carbon dioxide partial pressure, especially when supplemental oxygen was used. End-tidal carbon dioxide partial pressure currently cannot replace arterial blood gas measurement as a tool for monitoring arterial carbon dioxide partial pressure. Possible reasons for the discrepancy with previous studies include small sample size in previous studies, lack of research facilities in service settings, and publication bias against negative studies.

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

First Report of Pyricularia grisea (Gray Leaf Spot) on Perennial Ryegrass (Lolium perenne) in Nevada.

In August of 2005, a golf course in Las Vegas, NV reported turf loss from an unknown disease on perennial ryegrass fairways. Samples from this course were examined, and diseased plants were found covered with lesions and sporulation typical of gray leaf spot as caused by Pyricularia grisea (Cooke) Sacc. With petroleum jelly, sporulating leaves were attached to the inside top surface of 100-mm petri dishes filled with 15 ml of 1.5% water agar. Conidia were allowed to drop onto the agar surface and 24 h later, individual germinating pyriform conidia were transferred to petri dishes containing one-quarter-strength potato dextrose agar (»-PDA) with the aid of a fine needle and stereomicroscope. Isolates of the fungus were maintained at 28°C with constant fluorescent light. Isolates were examined 7 to 10 days later, and morphology and conidia production were consistent with that described previously for P. grisea (1). Koch's postulates were performed using a single isolate (SSGC-1.1) grown for 14 days on »-PDA. The petri dish was flooded with 15 ml of sterile distilled water plus 0.05% Tween 20 and conidia dislodged into the solution with a rubber policeman to obtain a solution of approximately 5 × 103 conidia per ml. Using a modified thin-layer chromatography plate sprayer, the solution was misted onto six pots of 6-week-old perennial ryegrass (a mixture of approximately 33% each of varieties 'Kokomo', 'Cabo' and 'Secretaria'), seeded at a density of 2 kg per 93 m2 grown in 4- × 4-cm plastic pots filled with University of California soil mix. As a control treatment, six pots of perennial ryegrass (grown as previously described) were treated with water plus 0.05% Tween 20 only. Pots of plants were placed into closed, translucent, plastic containers lined with wet paper towels to provide a moist environment and held at 30°C for 48 h. Pots of plants were transferred to an incubator set at 30°C and 80% relative humidity with 12 h of alternating light and dark cycles. Four days after inoculation, plants misted with conidia developed symptoms typical of gray leaf spot. Plants were again placed into closed plastic containers lined with wet paper towels for 24 h, at which time, lesions on symptomatic plants developed abundant conidia characteristic of P. grisea. Water-only treated plants did not show any symptoms or signs of disease. P. grisea was reisolated from sporulating leaves as described above. The disease has been spreading in the midwestern and northeastern United States since first reported in 1991 on perennial ryegrass in Pennsylvania. It has only recently been found on turfgrass in California (2), and to our knowledge, this is the first report of this pathogen on perennial ryegrass in Nevada. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) W. Uddin et al. Plant Dis. 86:75, 2002.

Discovery and Characterization of Xylella fastidiosa Strains in Southern California Causing Mulberry Leaf Scorch.

Mulberry leaf scorch (MLS), caused by Xylella fastidiosa, is a disease of mulberry trees in the United States that has largely been documented from locations in the eastern and central areas of the country. MLS was recently detected for the first time in white mulberry (Morus alba) trees in southern California. Four MLS-strains were isolated from two locations and confirmed as X. fastidiosa by enzyme-linked immunosorbent assay (ELISA), direct isolation of the pathogen, and use of the X. fastidiosa-specific PCR primers RST31-33. Isolated strains were characterized by the sequencing of their 16S-23S rDNA intergenic spacer regions (ISR) and random amplified polymorphic DNA (RAPD) analysis and subsequent comparison with a previously characterized MLS-strain (Mulberry-VA) and representatives of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, and X. fastidiosa subsp. sandyi. MLS-strains isolated from California were distinct from strains causing almond leaf scorch, oleander leaf scorch, and Pierce's disease and similar to the Mulberry-VA-strain. The ISR sequences of two MLS-strains, MLS063 and MLS059, were 100% identical to that of the Mulberry-VA sequence, whereas MLS012 and MLS024 were 99.8 and 99.6% identical to the Mulberry-VA-strain and 99.4% identical among themselves. Genomic analysis using RAPD revealed no differences among the four strains. The pathogenicity of one strain, MLS063, was confirmed by inoculation of glasshouse-grown white mulberry plants. Three months after inoculation, the pathogen was recovered from 21 of 25 inoculated plants, and 5 of 25 plants were dead within a year of inoculation. Inoculation of grapevines and oleanders with MLS063 did not result in any disease or recovery of the pathogen up to 1 year later, showing that this strain was not cross-infective to these hosts.

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 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.