First Report of Brown Leaf Spot of Elymus nutans Caused by Bipolaris sorokiniana in Northwestern China.

Abstract

Elymus nutans is an important feed and forage resource in alpine region of northwestern China, and helps to maintain the ecological balance on Qinghai-Tibetan plateau (Fei et al. 2010). In September 2021, brown leaf spots were observed in Shandan Horse Farm (38.3061°N, 101.2453°E), Gansu province of China.Disease severity of leaves was estimated to be 38% based on a random sample of 100 leaves from each of 10 arbitrarily placed quadrats on the farm.The typical symptoms on leaves appeared as brown to dark brown spots with grayish white in the center, surrounded by a yellow halo. A total of twenty diseased leaves were collected from five different plants in the field. The lesions (about 5 mm) from these samples were initially soaked in 75% ethanol for 30 s followed by soaking in 1% NaClO solution for 1 min, repeatedly rinsed with sterilized water, air dried, and put on potato dextrose agar (PDA). After 7 days of incubation at 25°C in the dark, the same fungus was consistently isolated; the pure isolates were obtained by single-spore cultures (Cai et al. 2009). The colonies were irregular in shape and black in color with white margins. Conidia were spindle-shaped, dark brown, 5 to 9 septa (7 septa in general), and a size range of 21.74 to 78.97 × 7.95 to 21.12 µm (avg. 57.25 × 17.76 µm, n = 50),morphologically similar to B. sorokiniana reported on Avena nuda in China (Li et al. 2019). The ITS region of rDNA and partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of the representative isolate PJC5 were amplified using the primers ITS1/ITS4 and gpd1/gpd2, respectively (Manamgoda et al. 2014). After sequencing sequences were deposited into GenBank with accession numbers OM419194 (ITS) and OM718009 (GAPDH). BLAST analysis showed 99% and 100% identity with the corresponding sequences (accession numbers MH855159.1 and HG779088.1) of known strain CBS 140.31 of B. sorokiniana (Manamgoda et al. 2014). For pathogenicity tests, ten 8-week-old healthy plants obtained by growing E. nutans in pots were spray-inoculated separately with a conidia suspension of 1 × 106 conidia/ml of isolate PJC5. Ten plants were also sprayed with sterilized distilled water as controls. Then all plants were individually covered with transparent polyethylene bags for three days to maintain high relative humidity and placed in greenhouse at 25°C. Seven days after inoculation, symptoms of leaf infection were similar to those observed in the field, while no symptoms appeared on leaves of control plants. The experiments were conducted three times and the pathogen was re-isolated from inoculated leaves and was confirmed as B. sorokiniana based on morphological and molecular analyses.B. sorokiniana has been reported to cause leaf spot of E. riparius in the United States (Roane et al. 2005) and E. breviaristatus (Zhuang, 2005) and A. nuda (Li et al. 2019) in China. To our knowledge, this is thefirstreportofB.sorokinianacausing leaf spot onE. nutansin China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.