First Report of Alternaria alternata Causing Leaf Spot on Leymus chinensis in China

Leymus chinensis is a dominant plant in the Eurasian steppe and plays a crucial role in ecological protection and restoration due to its functions in sand fixation, soil conservation, and ecosystem stabilization (Liu et al. 2022). In August 2024, leaf spot disease was first observed on L. chinensis in a grassland of Abaga Banner, Xilinhot City, Inner Mongolia, China (43°54′47″N, 115°34′1″E). Investigations covering 0.25 hectares revealed that the disease incidence stood at 15%. Initial symptoms appeared as elliptical spots with brown edges and grayish-white centers. These spots gradually expanded into elongated lesions at later stages, eventually causing leaf wilting and, in severe cases, plant death. Thirty leaves displaying typical leaf spot symptoms were collected for pathogen isolation. Tissue pieces (5 mm×5 mm) from lesion margins were surface-sterilized (70% ethanol, 20 s; 1% NaClO, 2 min), rinsed three times with sterile distilled water, and placed on PDA. The plates were incubated at 25 ℃ in the dark for 5 days. Hyphal tips from actively growing colonies were picked and transferred onto fresh potato dextrose agar (PDA) plates for purification, yielding 10 isolates with consistent morphological characteristics. All isolates formed gray, circular colonies with gray edges, and the reverse sides of the culture plates showed gray pigmentation.Two representative isolates, YH1 and YH2, were selected for detailed characterization. Conidia were clavate to drum-shaped, measuring 10.85-34.96 × 5.86-13.76 μm (n=30), with 3-5 transverse septa, 0-2 longitudinal septa. These morphological characteristics were consistent with descriptions of Alternaria species (Simmons 2007). For molecular identification, genomic DNA of isolates YH1 and YH2 was extracted using a commercial kit (Aidlab Novel Plant Genomic DNA Extraction Kit). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1-α) gene, and Alternaria major allergen (Alt a 1) gene were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990), Ef728M/Tef1R (Stępień et al. 2012), and Alt-for/Alt-rev (Woudenberg et al. 2015), respectively. The obtained sequences were deposited in GenBank under accession numbers: ITS (PX644766, PX644767), TEF1-α (PX653172, PX653174), and Alt a 1 (PX653171, PX653173). BLASTn analysis revealed 100% (ITS), 99.73% (TEF1-α), and 100% (Alt a 1) sequence similarity with Alternaria alternata. A phylogenetic analysis based on the combined dataset of the three genes placed isolates YH1 and YH2 within the A. alternata clade. Pathogenicity tests were conducted by spraying leaves of fifteen healthy L. chinensis plants (three plants per pot, five pots total) with a conidial suspension (1×10⁶ conidia/mL, containing 0.2% Tween 20) until runoff. Fifteen control plants were sprayed with sterile water containing 0.2% Tween 20. All plants were maintained in a greenhouse at 25 ℃, 80% relative humidity, under a 12 h light/dark cycle. The experiment was repeated twice. Ten days post-inoculation, symptoms identical to those observed in the field developed on inoculated leaves, while control plants remained symptomless. The fungus re-isolated from the lesions was confirmed as A. alternata based on morphology and molecular analysis, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata infecting L. chinensis in China. This study provides essential information for the diagnosis, understanding of disease epidemiology, and development of control strategies for this newly identified disease.