First report of Didymella glomerata Causing Didymellaceae leaf spot of Elymus nutans in Northwestern China

Elymus nutans, a perennial grass in the family Gramineae widely distributed across the Tibetan Plateau, is a key forage species commonly used in alpine grassland restoration and livestock production (Li et al. 2020; Lu et al. 2025). Previous research by our team has revealed the diversity and complexity of Elymus spp. pathogens (Liu et al. 2025). On this basis, the present study identifies a novel foliar fungal pathogen previously unreported in E. nutans. In September 2023, reddish brown leaf spots were observed in Maqu County (34.5667°N, 103.7°E), Gansu province of China. Disease severity of sampled leaves was estimated at 29%, determined by assessing 100 leaves within each of 10 randomly positioned quadrats on the farm. The typical symptoms on leaves appeared as black to reddish brown spots with grayish white in the center. A total of thirty diseased leaves were collected from six different plants in the field. For isolation of the causal agent, three to five tissue pieces (approximately 1 cm × 0.5 cm) of each sample were cut with a sterilized blade at the junction of diseased and healthy tissue. The tissue pieces were surface sterilized with 75% ethanol for 60 s, followed by 1% NaClO for 1 min, rinsed with sterile water, and then transferred to potato dextrose agar (PDA) supplemented with 50 mg/L ampicillin sodium salt. Plates were incubated at 20 °C in the dark. After 7 days of incubation at 20 °C in the dark, the same fungus was consistently isolated; pure cultures were obtained using the single hyphal tip isolation method (Xue et al. 2023). Twelve fungal isolates with similar colony characteristics were obtained, and three representative isolates (EPJC29, EPJC105, and EPJC110) were selected for further study. Colonies grown on PDA for 12 days reached 81.16 - 87.47 mm in diameter with regular margins, exhibiting an olive green center surrounded by white to gray mycelia on the surface and a dark green to brown reverse. To induce sporulation, representative isolates were transferred to oatmeal agar (OA) and incubated under near-ultraviolet (NUV) light at 20 °C. After inoculated in OA under NUV light for 10 to 15 days, the conidia were induced: ellipsoid with obtuse ends, hyaline, and a size range of 4.31 to 9.52 × 2.10 to 5.17 µm (avg. 7.28 × 4.65 µm, n = 35). Primers detailed by Chen et al. (2017) were used to amplify the ITS-rDNA and LSU-rDNA regions, and the RPB2 and TUB2 protein-coding genes. The generated sequences have been deposited in GenBank, with accession numbers PP343074, PV124844, and PV124845 (ITS); PV111930, PV124856, and PV124857 (LSU); PV111777, PV129906, and PV129907 (RPB2); and PV107117, PV129908, and PV129909 (TUB2). Phylogenetic analyses using maximum likelihood (RA × ML) and Bayesian inference (MrBayes) of the combined ITS, LSU, RPB2, and TUB2 alignment consistently placed the three newly sequenced isolates within a subclade containing three previously reported Didymella glomerata isolates, with strong support (100% bootstrap / 1.0 Bayesian posterior probability). Following the protocol of Xue et al. (2020), ten 7-week-old E. nutans plants were spray-inoculated with a mycelial suspension (1.5 × 10⁴ CFU/mL), while ten control plants received a sterile distilled water spray. All plants were then covered with transparent polyethylene bags to maintain high humidity for three days at 25 °C in a greenhouse. Fourteen 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 D. glomerata based on morphological and molecular analyses. D. glomerata is a member of the family Didymellaceae, which includes several known foliar pathogens responsible for leaf spot diseases in grasses and other hosts (Valenzuela-Lopez et al. 2018). To our knowledge, this is the first report of D. glomerata causing leaf spot on E. nutans in China.