First Report of <i>Curvularia lunata</i> Causing Leaf Spot on <i>Elymus ciliaris</i> in China

Elymus ciliaris is a perennial grass widely distributed in northern China, valued for its adaptability, cold resistance, drought tolerance, and persistence in poor soils. It is characterized by July senescence, September re-greening, and winter evergreen foliage. This species serves as high-quality cool-season turf and as an ecological restoration grass, commonly used in mixed sowing systems with warm-season turfgrasses in the middle and lower Yangtze River basin (Zhang et al. 2024). In April and May 2025, leaf spot symptoms were observed on E. ciliaris growing in the artificial turf experimental plots at Anqing Normal University (30.62°N, 117.00°E), Anhui Province, China. Yellow to yellow-brown necrotic spots first appeared on lower leaf tips and margins, later expanding to cover entire leaves (incidence rates of 15 to 25%). Twenty diseased leaves were collected from the field. The margins of diseased tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol (30 s) and 2% sodium hypochlorite (60 s), rinsed three times with sterile water before isolation. Ten fungal isolates were obtained by plating pieces onto potato dextrose agar (PDA) and incubating at 25℃ for 4 days. Colonies were brownish-black to light gray. The medium turned orange to dark brown. Conidia (n=50) were fusiform, smooth-walled, melanized, brown, with three transverse septa, 16.1 to 26.9 × 7.1 to 11.2 μm, with the second cell distinctly curved and enlarged, consistent with Curvularia lunata (Wakker) Boedijn (Ellis 1971). Three representative isolates (ECP-3, ECP-14, ECP-21) were subjected to molecular identification based on ITS, TEF1, and GAPDH sequences amplified with primers ITS1/ITS4, EF-1/EF-2 (O′Donnell and Cigelnik 1997), and gpd1/gpd2 (Berbee et al. 1999). Sequences were deposited in GenBank under accession numbers PX925100–PX925102 (ITS), PX931761–PX931763 (TEF1), and PX931764–PX931766 (GAPDH), and showed 100%, &gt;99.8%, and &gt;99.6% sequence identity, respectively, to C. lunata (accession numbers PX555854, PX063961, and PX063958) based on BLAST analysis. Maximum likelihood phylogenetic analysis of concatenated sequences placed isolates within a highly supported clade containing a reference strain of C. lunata. For pathogenicity testing, five pots (each containing one plant) were used for each isolate treatment and the control. 110-day-old healthy E. ciliaris plants with 30–35 tillers were sprayed separately with a conidial suspension (1 × 10⁵ conidia/mL) of ECP‑3, ECP‑14, or ECP‑21; control plants received sterile water. Plants were maintained at 25°C and 90% relative humidity under a 12‑h light/12‑h dark photoperiod. After ten days, inoculated plants developed spots coalescing into disease symptoms similar to field observations, while controls remained asymptomatic. The tests were repeated three times with similar results. C. lunata was reisolated only from symptomatic tissues and confirmed by morphology and sequencing, fulfilling Koch’s postulates. This is the first report of C. lunata causing leaf spot on E. ciliaris in China. This finding reveals the susceptibility of this species to fungal infection in spring, potentially compromising its aesthetic quality and persistence in mixed turfgrass systems and ecological restoration projects (Zhang et al. 2017).