First Report of Waitea circinata var. circinata causing Brown Ring Patch on Agrostis stolonifera in Maryland

Brown ring patch, caused by Waitea circinata var. circinata, was first identified in Japan on Agrostis stolonifera L. (creeping bentgrass) (Toda et al. 2005) and later on Poa annua (annual bluegrass) in the United States (de la Cerda et al. 2007). In mid-May 2024, symptoms consistent with a brown ring patch were observed on A. stolonifera L. cv. L-93 on a fairway at the Paint Branch Turfgrass Research Facility, University of Maryland, College Park, USA. Affected turf exhibited circular to irregular chlorotic to necrotic rings. Disease incidence was estimated at 30 to 50% of the approximately 1200 m² fairway, with individual symptomatic patches measuring 5 to 15 cm in diameter. Disease severity was moderate, with 60 to 80% tissue necrosis within affected rings. Symptomatic tissue samples were collected from 10 diseased patches across the fairway. Symptomatic leaf and sheath tissues were surface disinfected in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile water, and plated on potato dextrose agar (PDA). Petri dishes were incubated at 25 ± 2 °C under continuous light. The pathogen was successfully isolated from eight of 10 samples (80% isolation rate). One representative isolate (WC-BP1) was selected for morphological characterization, molecular identification, and pathogenicity testing. Colonies on PDA were white to orange after 7 days, gradually turning brown with age, and produced minute sclerotia (≤3 mm) within 14 days. Genomic DNA was extracted from the isolate using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The internal transcribed spacer (ITS) region and β-tubulin gene were amplified using primers ITS1/ITS4 and B36F/B12R, respectively. BLASTn analysis of the ITS sequence (GenBank accession no. PV261139) revealed 99.46% (585/589 bp; 99.44%) with W. circinata var. circinata isolate C297 (GenBank accession no. OL765274.1). The β-tubulin sequence (GenBank accession no. PX965784) revealed 98.72% identity (385/390 bp; 85%) with W. circinata var. circinata isolate BK1 (GenBank accession no. KT347131.1). Pathogenicity tests were performed on four-week-old A. stolonifera cv. L-93 plants grown in pots under controlled conditions. Plants (n = 5) were inoculated with five autoclaved wheat grains colonized by isolate WC-BP1, while control plants were inoculated with non-infested autoclaved wheat grains. All plants were maintained in a moist chamber at 28 to 32°C and 88 to 90% relative humidity. Chlorosis and necrosis, similar to field symptoms, developed on inoculated plants within 4 to 5 days, and orange sclerotia formed by 8 days after inoculation. Control plants remained asymptomatic. The pathogen was re-isolated from all inoculated plants but not from control plants. Reisolates matched the original isolate morphologically and via β-tubulin sequence identity. The experiment was repeated once with similar results. To the best of our knowledge, this is the first report of W. circinata var. circinata infecting A. stolonifera in Maryland. This finding expands the known geographic distribution and host association of the pathogen and highlights its potential significance for turfgrass management in the region.