First report of ophiosphaerella agrostidis infecting creeping bentgrass in Wisconsin

Dead spot is a disease of golf course putting greens and is caused by the pathogen Ophiosphaerella agrostidis Dernoeden et al. (syn. O. agrostis) (Câmara et al. 2000). The disease is found on creeping bentgrass (Agrostis stolonifera L.) and hybrid bermudagrass (Cynodon dactylon [L.] Pers. × C. transvaalensis Burtt-Davy) in the eastern and southern United States as well as Canada (Kaminski and Dernoeden 2002; Kaminski and Hsiang 2006; Krausz et al. 2001). In June 2017, disease symptoms resembling dead spot were observed on a golf course in Wisconsin. Small (<3 cm) necrotic spots first appeared approximately 10 months after establishment of a 50/50 blend of ‘007’ and ‘SR1119’ creeping bentgrass on sand-based putting greens. The disease became more severe during the summer months with as many as 10 spots per square foot and spots up to 4 cm in diameter. The disease was less severe on greens infected in 2017, but new symptoms appeared in 2018 on greens that were seeded during the summer of 2017. Dark brown ectotrophic hyphae were observed on bentgrass stolons, and pseudothecia were observed embedded within necrotic tissue. A single fungal morphotype was consistently isolated from leaves, stem bases, and stolons, and the colonies were rose-quartz color when cultured on potato dextrose agar. To demonstrate pathogenicity, ‘Penn A-1’ creeping bentgrass seedlings were grown for 4 to 6 weeks in 2.5-cm Cone-tainers in an autoclaved sand-based growing medium with a mechanical analysis of 95% sand, 4% silt, and 1% clay. Inoculum was prepared by placing mycelia from a hyphal-tipped isolate on an autoclaved mix of seed of tall fescue (Festuca arundinacea Schreb.) and wheat (Triticum aestivum L.) germ (50% [vol/vol]) and grown at 20°C for 10 days. The inoculum (5 g) was set at the surface of the sand and surrounding the stem bases of the creeping bentgrass in the center of each Cone-tainer (n = 6). A noninfested inoculum and noninoculated grass each served as a nontreated control. The grass plants grown in individual Cone-tainers were enclosed in clear plastic bags to increase relative humidity and incubated at 20°C under 14 h of light per day from fluorescent lights. Pathogenicity tests were conducted twice. No symptoms developed in either of the nontreated control treatments. After 7 days, tissue along the periphery of each inoculation point became covered by pink mycelia, and newly infected leaves appeared tan or brownish-red. Reisolation of the pathogen from symptomatic tissue produced fungal colonies similar in color, morphology, and growth rate to the original isolate. The internal transcribed spacer (ITS) regions of DNA from the original and reisolated fungus were amplified using ITS primer pair ITS1/ITS4. Sequencing of the amplified region resulted in a 408-bp sequence (GenBank accession no. MK561423) matching 100% identity with GenBank accession numbers AF191548.1 and MF351996.1. Based on field symptoms, morphological characteristics, pathogenicity tests, and ITS sequences, the pathogen was identified as O. agrostidis. To our knowledge, this is the first report of dead spot on creeping bentgrass in Wisconsin.