First report of melting out on creeping bentgrass caused by Bipolaris sorokiniana in China.

Abstract

Creeping bentgrass (Agrostis stolonifera L.), is one of the major cool-season turfgrass species, which is widely planted in putting greens at golf courses in China (Zhou et al. 2022). In June 2022, an unknown disease with reddish-brown spots (2-5 cm in diameter) were observed on 'A4' creeping bentgrass putting greens at Longxi golf course in Bejing. As the disease progressed, the spots coalesced and formed irregular patches (15-30 cm in diameter). When looking closely, the leaves were wilting, yellowing and melting out from the foliar tips to the crowns. The disease incidence was estimated up to 10-20 % of each putting green and a total of five putting greens had similar symptoms as described above. Three to five symptomatic samples were collected from each green. Diseased leaves were cut into pieces, surface sterilized in 0.6% sodium hypochlorite (NaClO) for 1 min, washed three times with sterilized water, air dried and placed on potato dextrose agar (PDA) containing 50 mg L-1 streptomycin sulfate and tetracycline. Plates were incubated in the dark at 25 °C for 3 days, and fungal isolates with similar morphology (irregular cultures with dark-brown back and light-brown to white surface) was consistently recovered. Pure cultures were obtained by repeat hyphal-tip transfer. The fungus did not grow well on PDA medium, radial growth was estimated 1.5 mm/d, and dark-brown colony was surrounded by light-white margin. However, it grew fast on creeping bentgrass leaf extract (CBLE) medium, CBLE medium was produced by adding 0.75 g potato powder, 5 g agar and 20 mL creeping bentgrass leaf juice (with 1 g fresh creeping bentgrass leaf) into 250 mL sterile water. The colony was sparse and light-white, and radial growth was roughly 9 mm/d on CBLE medium. Conidia were spindle-shaped, olive to brown in color, pointy or obtuse at both ends, 4 to 8 septa, and a size range of 9.85 to 20.20 × 26.26 to 45.64 μm (14.85 μm × 40.62 μm average, n = 30). Genomic DNA of two representative isolates (HH2 and HH3) was extracted, the nuclear ribosomal internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) region were amplified with primers ITS1/ITS4 (White et al. 1990) and gpd1/gpd2 (Berbee et al. 1999), respectively. The ITS (OQ363182 and OQ363183) and GAPDH (OQ378336 and OQ378337) sequences were deposited in GenBank. BLAST analyses revealed that the sequences shared 100% and 99% similarity with the published ITS (CP102792) and GAPDH (CP102794) of B. sorokiniana strain LK93, respectively. To complete Koch's postulates, 3 plastic pots (15 cm height × 10 cm top diameter × 5 cm bottom diameter, three replicates for the isolate HH2) were seeded with creeping bentgrass and inoculated with a spore suspension (1×105 conidia/mL) after two months of plant growths. Healthy creeping bentgrass inoculated with distilled water served as controls. All pots were covered with plastic bags, placed in a growth chamber with a 12-h day/night cycle at 30/25°C and 90% relative humidity. Disease symptoms (yellowing and melting out leaf) were noted after 7 days. B. sorokiniana was recovered from the diseased leaves and identified morphologically and molecularly as described above. To our knowledge, this is the first report of melting out on creeping bentgrass caused by B. sorokiniana in China. The report here will provide a scientific basis for developing management strategies on this disease in the future. Additional study is needed to investigate the prevalence of the disease on putting greens from golf courses in larger regions of China.