First Report of Xanthomonas translucens Causing Etiolation on Creeping Bentgrass Turf in Illinois, Kentucky, and North Carolina.

Symptoms of etiolation, which is an abnormal elongation and yellowing of tillers, have been observed on creeping bentgrass [Agrostis stolonifera L. (CBG)] putting greens for decades; however, symptoms are typically transient and non-problematic. Reports of etiolation have become more frequent recently and research supports the involvement of bacteria (1). During stressful summer periods in 2011 and 2012, 62 CBG putting green samples were submitted to the NCSU Turf Clinic exhibiting symptoms of etiolation, chlorosis, and/or general decline. Microscopic examination of stem and leaf tissue often showed bacterial streaming from the xylem tissue. Symptomatic tissue was surface disinfested in sodium hypochlorite (10% Clorox) for 5 min, blotted dry, and rinsed in sterile dH2O. Disinfested tissue was placed in a small drop of sterile dH2O on a glass microscope slide and cut to allow bacteria to stream into the water for 2 min. The resulting bacterial suspension was streaked onto three nutrient agar (NA) plates and incubated at 30°C overnight. Bacterial colonies varied in morphology and those present in the greatest number based on morphology were re-streaked to isolate individual colonies. Bacterial isolates were tentatively identified to species using rDNA sequencing of 16S and ITS regions (3). Sequencing results showed isolates obtained from 6 locations (in Illinois, Kentucky, and North Carolina) having a positive match (≥99% 16S and ≥93% ITS) to Xanthomonas translucens (GenBank accessions AY572961, HM181927, JX976312, AY253329, and AB680445). Additional research is needed to confirm pathovar designation as X. translucens isolates were similar to both poae and graminis pathovars. A representative isolate (LW10-12A) was also examined for carbon source utilization using the BIOLOG 3rd Gen Microplate (Biolog Inc., Hayward, CA) resulting in a positive identification of X. translucens. Isolate LW10-12A was used to inoculate 6-week-old seeded creeping bentgrass cv. A1 plants maintained at 1 cm height in 3.5 cm diameter containers. Scissors were dipped in a cell suspension (~109 CFU ml-1 in sterile dH2O) and used to cut healthy CBG plants at 1 cm height and the remaining suspension was applied to the foliage until runoff using an atomizer bottle. Non-inoculated plants were cut and misted using sterile dH2O. After inoculation, plants were placed in a sealed clear plastic Camwear container (Cambro Co., Huntington Beach, CA) for 48 h and then transferred to the growth chamber bench (30°C) receiving irrigation twice daily with dH2O. Etiolation was rated within each of the four replicates by counting the number of etiolated leaves that were easily observed as significantly higher than the rest of the turf canopy. Plants inoculated with X. translucens exhibited etiolation of the youngest leaf within 48 h, whereas the non-inoculated plants did not. Symptoms were similar to observations in the field, as etiolated leaves were chlorotic and easily extracted from the turf surface. Microscopic examination showed bacterial streaming and identification of bacteria, using the previously described methods, was positive for X. translucens. Etiolation symptoms persisted over multiple weeks, but a decline in turf quality was not observed. Etiolation has been previously suggested as a precursor to bacterial wilt, caused by X. translucens pv. poae, on annual bluegrass [Poa annua L. f. reptans (Hausskn) T. Koyama] (2) and Acidovorax avenae has also been shown to produce etiolation on CBG (1). To our knowledge, this is the first confirmation of X. translucens as a cause of etiolation in CBG. References: (1) P. R. Giordano et al. Plant Dis. 96:1736, 2012. (2) N. A. Mitkowski et al. Plant Dis. 89:469, 2005. (3) N. W. Schaad et al. Lab. Guide for Ident. of Plant Path Bac., 2001.

Evaluation of Preventive Fungicide Applications for Fairy Ring Control in Golf Putting Greens and In Vitro Sensitivity of Fairy Ring Species to Fungicides.

Fairy ring species induce symptoms on putting greens mostly indirectly, by modifying the soil physical or chemical properties. Therefore, preventive rather than curative fungicide applications may be more effective in managing fairy ring. Two field experiments were conducted on a creeping bentgrass research green to evaluate fairy ring control from preventive fungicide applications. A 3-year study investigated the optimal rate and soil temperature-based timing of a preventive application of triadimefon and tebuconazole. A 2-year study evaluated the impact of irrigation timing and fungicide + surfactant tank mixtures on the efficacy of preventive applications of triadimefon and triticonazole. Fungicide-treated plots in both studies exhibited less fairy ring severity than untreated plots. Data suggest that a 5-day average soil temperature range of 13 to 16°C may be suitable for initiating preventive applications. Symptoms occurred earlier in plots treated with a surfactant tank mix than in those treated with fungicide alone. Irrigation timing had no effect on fungicide performance. The sensitivity of 16 isolates representing major fairy ring species to flutolanil, propiconazole, tebuconazole, triadimefon, and triticonazole was determined with a mycelial growth assay. No significant differences in fungicide sensitivity were detected among species. Isolates had significantly higher 50% effective concentration values for triadimefon than for the other fungicides tested.

Identification and Distribution of Fungi Associated with Fairy Rings on Golf Putting Greens.

Traditional methods for identification of fairy ring fungi rely on the morphology of mature basidiocarps, which are ephemeral and often do not reach maturity on golf greens due to management practices. From 2007 to 2009, basidiocarps and soil samples were collected from 15 hybrid bermudagrass and 30 bentgrass greens exhibiting fairy ring symptoms in California, Florida, Hawaii, Illinois, Oklahoma, North Caroline, South Carolina, and Wisconsin. Genomic DNA was extracted from 122 unknown samples. Extractions were made from mycelium isolated from puffball or mushroom tissue, from mycelium isolated from a soil block, or through direct DNA extraction from infested soil. DNA also was extracted from 16 reference isolates. The internal transcribed spacer (ITS) region of ribosomal DNA was amplified and sequenced using the basidiomycete-specific primer sets ITS1f/ITS4b and Basid0001/2R. Phylogenetic trees were constructed with the neighborjoining algorithm, with nodes evaluated by bootstrap analysis. Most samples grouped into one of three clades corresponding to species within the family Lycoperdaceae: Arachnion album, Bovista dermoxantha, and Vascellum curtisii. Although over 60 different basidiomycetes have been associated with fairy rings in turfgrasses, relatively few species were found on golf putting greens in this study. Presently, DNA sequencing may be the most efficient method for attempting speciation of fairy ring fungi from infested soil.

First Report of Marasmiellus mesosporus Causing Marasmiellus Blight on Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum Sw.) is a newly cultivated C4 turfgrass that has exceptional salinity tolerance and is highly suited for use on golf courses in coastal areas. In October 2008 and June 2009, circular patches of blighted seashore paspalum ranging from 30 cm to >3 m in diameter were observed in fairways, tees, and roughs established with 'Supreme' seashore paspalum at Roco Ki Golf Club in Macao, Dominican Republic. Affected patches were initially chlorotic followed by reddish brown necrosis of leaves and leaf sheaths. Reddish brown-to-gray lesions were also observed on leaf sheaths during the early stages of necrosis. During periods of wet or humid weather from June through October, basidiocarps were produced on necrotic plant tissue and identified as Marasmiellus mesosporus Singer (2). Three isolates were obtained by plating symptomatic leaf sheaths that were surface sterilized with a 0.5% NaOCl solution on potato dextrose agar amended with 50 ppm each of streptomycin, chloramphenicol, and tetracycline (PDA+++). Sequences of the internal transcribed spacer (ITS) region of rDNA, obtained from these three isolates and three stipes of basidiocarps, were identical to each other and 99% similar to a M. mesosporus sequence deposited in the NCBI database (Accession No. AB517375). To confirm pathogenicity, a M. mesosporus isolate obtained from symptomatic plant tissue was inoculated onto 6-week-old P. vaginatum ('Seaspray') planted (0.5 mg seed/cm2) in 10-cm-diameter pots containing a mixture of 80% sand and 20% reed sedge peat. Two weeks prior to inoculation, the isolate was grown on a sterilized mixture of 100 cm3 of rye grain, 4.9 ml of CaCO3, and 100 ml of water. Infested grains were placed 0.5 cm below the soil surface for inoculation. Pots were inoculated with five infested grains or five sterilized, uninfested grains with three replications of each treatment. After inoculation, pots were placed in a growth chamber with a 12-h photoperiod set to 30°C during the day and 26°C at night. Approximately 20% of plants in inoculated pots were necrotic 7 days postinoculation and this increased to 75% by 21 days postinoculation. Diseased plants in inoculated pots exhibited symptoms similar to those observed in the field. Leaves were initially chlorotic with brown lesions on lower leaf sheaths and eventually turned necrotic, reddish brown, and collapsed. Pots receiving uninfested grains were healthy and showed no symptoms on all rating dates. At 21 days postinoculation, basidiocarps were observed emerging from three colonized plants at the base of the oldest leaf sheath near the crown. Three reisolations were made on PDA+++ from stem lesions surface sterilized with a 0.5% NaOCl solution. All reisolations were confirmed as M. mesosporus by culture morphology and ITS sequence data. M. mesosporus was previously reported causing disease on American beachgrass (Ammophila breviligulata Fernald) in North Carolina (1) and recently in Japan (3). The pathogen was initially placed in the genus Marasmius and reported as the cause of the disease Marasmius blight (1). Subsequent morphological observation found that the pathogen belonged in the genus Marasmiellus (2). To our knowledge, this is the first report of M. mesosporus causing Marasmiellus blight on seashore paspalum, a high-amenity turfgrass. References: (1) L. Lucas et al. Plant Dis. Rep. 55:582, 1971. (2) R. Singer et al. Mycologia 65:468, 1973. (3) S. Takehashi et al. Mycoscience 48:407, 2007.

Preventive Control of Pythium Root Dysfunction in Creeping Bentgrass Putting Greens and Sensitivity of Pythium volutum to Fungicides.

Pythium root dysfunction (PRD), caused by Pythium volutum, has been observed on golf course putting greens established with creeping bentgrass in the southeastern United States since 2002. To evaluate preventative strategies for management of this disease, a 3-year field experiment was conducted in Pinehurst, NC on a 'G-2' creeping bentgrass putting green. Fungicide treatments were applied twice in the fall (September and October) and three times in the spring (March, April, and May) in each of the 3 years. Applications of pyraclostrobin provided superior preventative control compared with the other fungicides tested. Azoxystrobin and cyazofamid provided moderate control of PRD in two of three seasons. Experiments were conducted to determine whether the disease suppression provided by pyraclostrobin was due to fungicidal activity or physiological effects on the host. In vitro sensitivity to pyraclostrobin, azoxystrobin, fluoxastrobin, cyazofamid, mefenoxam, propamocarb, and fluopicolide was determined for 11 P. volutum isolates and 1 P. aphanidermatum isolate. Isolates of P. volutum were most sensitive to pyraclostrobin (50% effective concentration [EC50] value = 0.005), cyazofamid (EC50 = 0.004), and fluoxastrobin (EC50= 0.010), followed by azoxystrobin (EC50 = 0.052), and mefenoxam (EC50 = 0.139). P. volutum isolates were not sensitive to fluopicolide or propamocarb. Applications of pyraclostrobin did not increase the foliar growth rate or visual quality of creeping bentgrass in growth-chamber experiments. This work demonstrates that fall and spring applications of pyraclostrobin, azoxystrobin, and cyazofamid suppress the expression of PRD symptoms during summer and that field efficacy is related to the sensitivity of P. volutum to these fungicides.

Influence of Temperature on Pathogenicity of Pythium volutum Toward Creeping Bentgrass.

Symptoms of Pythium root dysfunction (PRD) in creeping bentgrass (Agrostis stolonifera) are most common in the summer during periods of heat and drought stress. However, recent observations in North Carolina indicate that Pythium volutum, a causal agent of PRD, is most active during the fall and spring. Soil temperature thresholds for this pathogen are needed so that preventive fungicide applications can be timed accurately. A mycelial growth assay was performed by incubating 11 P. volutum isolates at 10 temperatures ranging from 10 to 31°C. To determine the optimal temperature range for infection by P. volutum, five isolates of P. volutum were used to inoculate 5-week-old 'Penn A-1' creeping bentgrass plants. Inoculated plants were transferred to growth chambers at constant 12, 16, 20, 24, 28, or 32°C (12-h day/night cycles) for 4 weeks to permit root infection, then the temperature in all chambers was increased to 32/26°C day/night to induce foliar symptoms. P. volutum grew most rapidly in vitro when temperatures were between 18 and 26°C. Typical PRD foliar symptoms developed in the 12, 16, 20, and 24°C treatments 2 weeks after the temperature was elevated to 32/26°C day/night. Disease severity was greatest when plants were incubated at 16°C after inoculation. Reductions in root depth and/or root mass were observed prior to raising the temperature to 32/26°C in the 12, 16, and 20°C temperature treatments. Once exposed to 4 weeks of heat treatment, extensive root dieback occurred in the 12, 16, 20, and 24°C treatments. These results demonstrate that P. volutum is most active at temperatures prevalent during the fall and spring in North Carolina, supporting the hypothesis that the majority of root infection occurs during this time and that fungicides should be applied when soil temperatures are between 12 and 24°C to achieve preventative control of PRD symptoms in the summer.

Pathogenicity of Pythium Species Associated with Pythium Root Dysfunction of Creeping Bentgrass and Their Impact on Root Growth and Survival.

Symptoms resembling Pythium root dysfunction have been observed on golf course putting greens established with creeping bentgrass across the southeastern United States since 2002. Root isolations from 14 golf courses yielded 59 isolates of Pythium volutum and 16 isolates of Pythium torulosum. Pathogenicity of five isolates of P. volutum, two isolates of P. torulosum, and a combination of the two species was determined by inoculating mature 'A-1' creeping bentgrass plants. Inoculated plants were incubated for 4 weeks at 24/16°C (day/night) to permit root infection, then temperatures were increased to 32/26°C to induce foliar symptoms. No isolates impacted root depth, root mass, or foliar disease severity after 4 weeks at 24/16°C. After increasing the temperature to 32/26°C, isolates of P. volutum induced foliar disease severity ranging from 60 to 84%, whereas isolates of P. torulosum induced only 14 to 35% disease. Isolates of P. volutum consistently reduced root mass and root depth after 4 weeks at 32/26°C, but P. torulosum exhibited no effect. These results demonstrate that P. volutum is a pathogen of mature creeping bentgrass plants. Infections that occur during cool weather reduce the growth and survival of creeping bentgrass roots during hot weather and give rise to foliar symptoms.

First Report of Curvularia Blight of Zoysiagrass Caused by Curvularia lunata in the United States.

Symptoms of an unknown foliar blight have been observed in zoysiagrass (Zoysia matrella, Z. japonica, and hybrids) landscapes in North Carolina since 2002. Disease activity is most common during spring and summer when temperatures are between 21 and 30°C. Affected leaves initially exhibit small, chocolate brown spots, followed by dieback of leaves from the tips, and eventually blighting of entire tillers. Symptoms appear in small, irregular patches as much as 15 cm in diameter, but numerous patches may coalesce to impact large sections of turf. Infected turf appears tan or brown from a distance, but often turns black during periods of wet or humid weather. Microscopic analysis revealed profuse sporulation of Curvularia spp. on the surface of symptomatic leaves. Leaf sections were surface disinfested in 10% Clorox for 1 to 2 min, blotted dry, then plated on potato dextrose agar (PDA) containing 50 mg/l of tetracycline, streptomycin, and chloramphenicol. Twenty-eight fungal isolates were obtained from six locations. Examination of conidia produced in culture revealed 21 isolates of Curvularia, two isolates of Drechslera, one isolate of Nigrospora, and four unidentified sterile fungi. Curvularia isolates were identified to species on the basis of morphological characteristics (1) and ITS-rDNA sequences. Known isolates of C. eragrostidis, C. geniculata, C. inequalis, C. lunata, C. pallescens, and C. trifolii were obtained from the American Type Culture Collection for comparison. All unknown isolates produced conidia that were characteristic of C. lunata (lacking a protuberant hilum, smooth walled, tri-septate, predominantly curved, and mid- or dark brown, average dimensions 17 to 25 × 8 to 12 µm). Colonies on PDA lacked stroma or the zonate appearance indicative of C. lunata var. aeria. The pathogenicity of C. lunata isolates was tested on zoysiagrass cvs. El Toro (Z. japonica) and Emerald (Z. japonica × matrella). Cores (11.4 cm in diameter) of established zoysiagrass were potted in calcined clay (Turface Allsport; Profile Products LLC, Buffalo Grove, IL), and transferred to a greenhouse where the average temperature was 26°C. Five isolates were selected to represent the geographic range of Curvularia blight in North Carolina, and conidia were produced on PDA under continuous fluorescent illumination. Each isolate was inoculated to one pot of each zoysiagrass variety by spraying with 25 ml of a suspension containing 2 × 105 conidia/ml with an airbrush. Inoculated pots were placed in a sealed, nontransparent plastic container for 48 h at 28°C to encourage infection and then transferred back to the greenhouse bench. Pathogenicity tests were repeated four times over time. Isolates ZFB3 and ZFB28 were most virulent with initial symptoms of foliar dieback appearing within 1 week after inoculation. Continued disease progress resulted in necrosis of the entire plant. Other isolates induced symptoms within 2 to 3 weeks after inoculation; however, disease severity was lower as compared with ZFB3 and ZFB28 throughout each experiment. Cvs. Emerald and El Toro were equally susceptible to infection by C. lunata. To our knowledge, this is the first report of Curvularia blight of zoysiagrass in the United States. This disease was previously described in Japan where it is commonly referred to as 'dog footprint' (3) and Brazil (2). References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) F. B. Rocha et al. Australas. Plant Pathol. 33:601, 2004. (3) T. Tani and J. B. Beard. Color Atlas of Turfgrass Diseases. Ann Arbor Press, Chelsea, MI, 1997.

First Report of Spring Dead Spot of Zoysiagrass Caused by Ophiosphaerella korrae in the United States.

Since 2002, symptoms of an unknown disease have been observed in 'El Toro' zoysiagrass (Zoysia japonica Steud.) in several locations across North Carolina. Symptoms become evident in the spring as the zoysiagrass comes out of winter dormancy. Circular or irregularly shaped patches, 10 to 30 cm in diameter, remain dormant as the surrounding turf resumes growth. These patches eventually collapse and die, leaving sunken depressions in the turf stand. After the initial appearance of symptoms, the zoysiagrass slowly recolonizes the patches by spreading inward from the perimeter. Microscopic observation revealed necrotic stolon and root tissue that was colonized by ectotrophic fungal hyphae, whereas leaf and sheath tissue was colonized by species of Curvularia, Colletotrichum, Alternaria, Ascochyta, Drechslera, or Fusarium. Sections of necrotic root and stolon tissue were washed under flowing tap water for 10 min, submersed in 0.6% NaOCl for 5 min, rinsed with sterile dH2O, blotted dry, and placed on » strength potato dextrose agar amended with 100 µg/ml each of streptomycin sulfate and chloramphenicol. A total of 50 isolates were obtained from four locations during 2002 and 2003. A fungus resembling Ophiosphaerella spp. was consistently isolated and was confirmed to be Ophiosphaerella korrae by species-specific PCR assays (3) and rDNA internal transcribed spacer (ITS) sequencing. Pathogenicity tests were conducted in the field on 'El Toro' zoysiagrass at the Lake Wheeler Turfgrass Field Laboratory in Raleigh, NC. Autoclaved rye grain (Secale cereale L.; 200 g of grain, 5.75 g of CaCO3, and 220 ml of H2O) was infested with one of eight O. korrae isolates. Plots (1 × 1 m) were inoculated on 13 October 2004 by removing an 11-cm-diameter core from the center of each plot to a 5-cm depth, placing 10 cm3 of infested rye grain in the bottom of the hole, and replacing the core. Noninoculated and uninfested rye grain treatments served as controls, and each treatment was replicated eight times in a randomized complete block. No symptoms were observed in the experimental area during 2005. In April 2006, five isolates (Zrr20, Zrr36, Zrr57, Zrr58, and Zrr59) incited spring dead spot symptoms in at least four of eight inoculated plots. The average diameter of patches induced by these isolates ranged from 7.9 to 11.4 cm. In April 2007, three isolates (Zrr20, Zrr36, and Zrr57) incited symptoms in at least four plots, with average patch diameters ranging from 14.5 to 16.0 cm. These inoculation success rates and patch diameters were similar to those resulting from O. korrae inoculation of bermudagrass conducted on the same date (L. P. Tredway, unpublished data). No symptoms were observed in noninoculated plots or those amended with uninfested rye grain. O. korrae was consistently reisolated from symptomatic stolons and roots in May 2007 to complete Koch's postulates. To the best of our knowledge, this is the first report of spring dead spot of zoysiagrass caused by O. korrae in the United States. Previously, O. herpotricha was shown to induce spring dead spot symptoms on zoysiagrass in Kansas (1), and O. korrae was reported as a zoysiagrass pathogen in Japan (2). To date, we have only observed spring dead spot on the Zoysia japonica 'El Toro'. References: (1) D. E. Green et al. Plant Dis. 77:1040, 1993. (2) T. Tani. Color Atlas of Turfgrass Diseases. Ann Arbor Press, Chelsea, MI, 1997. (3) N. A. Tisserat et al. Phytopathology 84:478, 1994.

First Report of Pythium Root Dysfunction of Creeping Bentgrass Caused by Pythium volutum in North Carolina.

In July and August of 2002 and 2003, a disease of unknown etiology was observed in Charlotte, NC on 'A-1' creeping bentgrass (CRB; Agrostis stolonifera L.) putting greens that were constructed in 2000. Symptoms appeared in irregular patches ranging from 15 to 30 cm in diameter. Grass in the affected areas was initially wilted and chlorotic, but later exhibited a yellow-to-orange foliar decline. Similar symptoms were observed in Durham, NC in July and August of 2003 on CRB greens established in 2001 with a 1:1 blend of 'A-1' and 'A-4'. The disease was initially diagnosed as take-all patch, but attempts to isolate Gaeumannomyces graminis var. avenae and other ectotrophic root pathogens were unsuccessful. Symptoms of the disease reappeared during periods of warm, dry weather in the fall of 2003 and spring of 2004. At that time, examination of affected root tissue revealed bulbous root tips, loose cortical structure, absence of root hairs, and abundant Pythium oospores and hyphae. These signs and symptoms are typical of Pythium root dysfunction (PRD) as described by Hodges and Coleman (2) in 1985 and Feng and Dernoeden (3) in 1999. Isolation of Pythium spp. was performed by plating directly on V8 agar (4) or baiting with 'A-4' CRB seedlings. Eleven Pythium isolates were obtained from Charlotte (seven via baiting) and 10 were obtained from Durham (all via baiting). All isolates were transferred to grass leaf-blade cultures (4) to induce development of sporangia, oospores, and antheridia for identification using the keys and descriptions of Dick (1). All isolates produced lobate sporangia, large oospores (27 to 33 ± 2.8 µm), and three to nine diclinous antheridia typical of Pythium volutum. Cone-Tainers (3.8 × 20 cm) containing sand meeting USGA specifications were seeded with 'A-1' CRB and grown for 6 weeks in the greenhouse. Each Cone-Tainer was inoculated by cutting the root system at a 5 cm depth, placing five to seven infested grass blades onto the surface of fresh sand, and then replacing the turf. Cone-Tainers inoculated with one of three P. volutum isolates and an uninoculated control (six reps each) were placed in a growth chamber with 12 h of light/dark periods at 24/16°C for 4 weeks to allow pathogen infection and disease development. After 4 weeks, the chamber temperature was raised to 32/26°C to induce symptom development. Two weeks after raising the temperature, all P. volutum isolates caused significant (P = <0.0001) foliar chlorosis and dieback (70 to 100% disease) and reduced root depth and mass by 25 to 65% compared with the uninoculated control. Roots of inoculated plants were colonized with Pythium hyphae, contained numerous oospores, and consistently yielded P. volutum in isolations. To our knowledge, this is the first reported occurrence of PRD in North Carolina and provides further support for the importance of P. volutum as a pathogen of creeping bentgrass. On the basis of our observations, the majority of pathogen activity and disease development occurs in the fall and spring, with foliar symptoms being induced by heat or other stresses. References: (1) M. W. Dick. Keys to Pythium. University of Reading Press, Reading, UK, 1990. (2) C. F. Hodges and L. W. Coleman. Plant Dis. 69:336, 1985. (3) Y. Feng and P. H. Dernoeden. Plant Dis. 83:516, 1999. (4) F. N. Martin. Pythium. Pages 39-49 in: Methods for Research on Soilborne Phytopathogenic Fungi. L. L. Singleton et al., eds. The American Phytopathological Society, St. Paul, MN, 1992.

Genetic Relationships Among Magnaporthe poae Isolates from Turfgrass Hosts and Relative Susceptibility of 'Penncross' and 'Penn A-4' Creeping Bentgrass.

Isolates of Magnaporthe poae from turfgrass hosts were analyzed for mating type, genetic relatedness according to ITS sequences, reaction to a previously developed species-specific poly-merase chain reaction (PCR) assay, and virulence on two creeping bentgrass cultivars in growth chamber experiments. Analysis of internal transcribed spacer (ITS) sequences revealed three clades, designated A, B, and C. Clade A contained isolates of both mating types from creeping bentgrass, annual bluegrass, and Kentucky bluegrass. Clade B contained only mating type 'A' isolates from annual bluegrass, whereas Clade C contained only mating type 'a' isolates from creeping bentgrass. The M. poae PCR assay failed to positively identify several North Carolina isolates from annual bluegrass and creeping bentgrass. M. poae isolates from Kentucky blue-grass were most virulent toward creeping bentgrass in growth chamber experiments. Although isolates of M. poae are not host specific, certain ITS clades may have a limited host or geographical range. The improved creeping bentgrass cv. Penn A-4 was more susceptible to summer patch than cv. Penncross. Additional research is needed to develop methods for accurate diagnosis of summer patch and other patch diseases in creeping bentgrass and to determine how creeping bentgrass cultivars vary in their susceptibility to these root pathogens.

First Report of Summer Patch of Creeping Bentgrass Caused by Magnaporthe poae in North Carolina.

An unknown disease was observed in June 2002 and 2003 on creeping bentgrass (CRB [Agrostis stolonifera L.]) putting greens at The Country Club of Landfall in Wilmington, NC that were established in 2001 with a 1:1 blend of cvs. A-1 and A-4. Soil pH ranged from 7 to 8 at this location because of poor quality irrigation water. Symptoms appeared in circular patches of 0.3 to 1 m in diameter that exhibited signs of wilt followed by chlorosis and orange foliar dieback. The disease was initially diagnosed as take-all patch caused by Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. avenae (E.M. Turner) Dennis, based on the observation of necrotic roots and crowns that were colonized with dark, ectotrophic hyphae. However, the historical lack of take-all patch occurrence in this region led to the suspicion that G. graminis var. avenae was not involved. Sections of root and crown tissue were surface disinfested in 0.6% NaOCl for 5 min or 1% AgNO3 for 1 min and 5% NaCl for 30 s. Tissue was plated on SMGGT3 (2) or on potato dextrose agar containing 50 mg L-1 of tetracycline, streptomycin, and chloramphenicol. A fungus resembling Magnaporthe poae Landschoot & Jackson was consistently obtained regardless of isolation method. Teleomorph production was conducted on Sachs agar (4) overlaid with autoclaved wheat (Triticum aestivum L.) stem sections. Seven isolates were plated alone or paired with M. poae tester isolates 73-1 or 73-15 (3) and incubated at room temperature under continuous fluorescent illumination. Six isolates produced perithecia and ascospores typical of M. poae (3) when paired with 73-15 but not when plated alone or paired with 73-1; these isolates are, therefore, M. poae mating type 'a'. Isolate TAP42 did not produce perithecia and remains unidentified. Cone-Tainers (3.8 × 20 cm) containing calcined clay were seeded with 'A-4' CRB (9.7 g cm-2) and inoculated 8 weeks later by placing four M. poae-infested rye (Secale cereale L.) grains below the soil surface. Inoculated Cone-Tainers were placed in growth chambers with 12-h day/night cycles at 30/25°C, 35/25°C, or 40/25°C. Field plots (1 m2) of 'A-4' CRB in Jackson Springs, NC were inoculated on 19 June 2003 by removing a soil core (1.9 × 10.3 cm) from the center of each plot, adding 25 cm3 of M. poae-infested rye grains, and then capping the hole with sand. Growth chamber and field inoculations were arranged in a randomized complete block with four replications. Eight weeks after inoculation in the growth chamber, isolates TAP35, TAP41, and SCR4 caused significant foliar chlorosis and dieback at 12-h day/night cycles of 30/25°C and 35/25°C, but only TAP41 induced symptoms at 40/25°C. Isolate TAP42 did not induce symptoms at any temperature regimen. Orange patches (10 to 15 cm in diameter) were observed in field plots inoculated with TAP41 on 27 August 2003. No other isolates induced aboveground symptoms. Roots and crowns of plants exhibiting foliar symptoms in the greenhouse and field were necrotic and colonized with ectotrophic hyphae, and M. poae was consistently isolated from this tissue. Although M. poae has been associated with CRB in Florida (1), to our knowledge, this is the first report of summer patch of CRB within the normal zone of adaptation for this turfgrass species. Observation of this disease highlights the need for accurate methods for diagnosis of diseases caused by ectotrophic root-infecting fungi. References: (1) M. L. Elliott. Plant Dis. 77:429, 1993. (2) M. E. Juhnke et al. Plant Dis. 68:233, 1984. (3) P. J. Landschoot and N. Jackson. Mycol. Res. 93:59, 1989. (4) E. S. Lutrell. Phytopathology 48:281, 1958.

Genetic Structure of Magnaporthe grisea Populations Associated with St. Augustinegrass and Tall Fescue in Georgia.

ABSTRACT Amplified fragment length polymorphisms (AFLPs) were used to estimate phylogenetic relationships within Magnaporthe grisea and determine the genetic structure of M. grisea populations associated with tall fescue and St. Augustinegrass in Georgia. Sixteen clonal lineages were identified in a sample population of 948 isolates. Five lineages were isolated from tall fescue (E, G1, G2, G4, and H), with lineage G4 comprising 90% of the population. Isolates from tall fescue were closely related to those from perennial ryegrass, weeping lovegrass, and wheat. Two M. grisea lineages were isolated from St. Augustinegrass (C and K), with lineage C comprising 99.8% of the population. Populations from crabgrass were dominated (98%) by lineage K, but also contained a single lineage C isolate. Haplotype diversity indices ranged from 0.00 to 0.29 in tall fescue populations and from 0.00 to 0.04 in St. Augustinegrass populations. Selection due to host species was the primary factor determining population structure according to analysis of molecular variance; host cultivar and geographical region had no significant effect. The host range of M. grisea lineages from turfgrasses was determined in growth chamber experiments and supports the prominent role of host species in determining the genetic structure of M. grisea populations from turfgrasses in Georgia.

Components of Resistance to Magnaporthe grisea in 'Coyote' and 'Coronado' Tall Fescue.

The components of resistance in tall fescue to Magnaporthe grisea, the causal agent of gray leaf spot, were measured in growth chamber experiments. Cultivars ranging in susceptibility to M. grisea were selected: 'Kentucky 31' (susceptible), 'Rebel III' (moderately susceptible), 'Coronado' (resistant), and 'Coyote' (resistant). Plants were inoculated with nine M. grisea isolates representing five clonal lineages associated with tall fescue in Georgia. Compared to Kentucky 31, Coronado and Coyote exhibited longer incubation and latent periods, reduced rates of disease progress and lesion expansion, and lower final disease incidence, final foliar blight incidence, final mean lesion length, area under the lesion expansion curve, and area under the disease progress curve. No evidence of hypersensitive response was observed, all M. grisea isolates completed the disease cycle by producing secondary inoculum, and no differential response to isolates from different clonal lineages was detected in Coronado and Coyote. These results indicate that Coronado and Coyote have partial resistance to M. grisea. Measurement of resistance components using primary parameters and derived parameters yielded similar results. Foliar blight incidence data exhibited increased variation relative to other parameters and was less powerful for detection of M. grisea resistance. Measurements of incubation period, latent period, final disease incidence, and final mean lesion length were the most effective and efficient methods for detecting M. grisea resistance in tall fescue.

Pathogenicity of a Novel Biotype of Limonomyces roseipellis in Tall Fescue.

An unusual and undescribed foliar blight of tall fescue was observed in a home lawn and in turf grass research plots near Griffin, GA in May and June, 2000 and 2001. Isolation from lesions yielded mycelium of a basidiomycete with hyphal characteristics (binucleate cells, absence of clamp connections) associated with Laetisaria and Limonomyces spp. Isolates from blighted tall fescue and an isolate of Limonomyces roseipellis formed a clade distinct from isolates of Laetisaria fuciformis based on ribosomal DNA sequences. These data, in conjunction with cultural morphology, indicate that the basidiomycete from tall fescue represents a biotype of Limonomyces roseipellis that lacks clamp connections. In a controlled environment, isolates of the biotype induced foliar blight in the fescue cvs. Kentucky 31 and Rebel III. Histological observations revealed that the fungus colonized leaf surfaces as branched hyphae and aggregated hyphal strands. Penetration occurred via stomatal pores on the abaxial leaf surface. Colonization of leaf tissues was inter- and intracellular, with no evidence of papilla formation in response to invading hyphae. The name "cream leaf blight" is proposed for this new disease of tall fescue.

Mating Type Distribution and Fertility Status in Magnaporthe grisea Populations from Turfgrasses in Georgia.

Populations of Magnaporthe grisea associated with tall fescue and St. Augustinegrass in Georgia were analyzed for mating type distribution and fertility status in 1999 and 2000. A polymerase chain reaction based assay for mating type was developed to facilitate population analysis. M. grisea populations from St. Augustinegrass in Georgia were dominated by the Mat1-1 mating type, whereas populations from tall fescue were dominated by Mat1-2. The opposite mating type was found in low frequency (0 to 5.7%) associated with each host. The fertility status of isolates from two populations was determined using controlled crosses in vitro. Seventy-eight Mat1-1 isolates from St. Augustinegrass were sterile in test crosses, but a single Mat1-2 isolate from St. Augustinegrass was male fertile. Of 87 Mat1-2 isolates from tall fescue, 47 were male fertile in test crosses, but 19 produced perithecia that were barren. All Mat1-1 isolates from tall fescue were sterile. Although both mating types exist in M. grisea populations from turfgrasses in Georgia, no female fertile isolates were identified in sample populations. The predominance of one mating type in eight sample populations and absence of female fertile isolates in two sample populations indicates that sexual reproduction may not occur with significant frequency in M. grisea populations associated with turfgrasses in Georgia.