First report of wheat leaf spot disease caused by Curvularia inaequalis in China.

Wheat (Triticum aestivum) is an economically important crop widely cultivated in China. In August 2022, brown oval leaf spots with yellow halos were observed on approximately 10% wheat seedlings over an area of about 1 hectare in Xining City, Qinghai Province, which adversely affected wheat growth and production. Six diseased leaves were collected from the field in Huangyuan county (101°69' E, 37°04' N). The 0.5 cm × 0.5 cm pieces were cut from the border between healthy and diseased regions of the sampled leaves, surface sterilized for 10 s in 75% ethanol, followed by a 1% NaClO for 90 s, and rinsed three times with distilled sterile water. The pieces of leaf tissue were dried with sterile tissue, and plated on potato dextrose agar (PDA) amended with streptomycin (0.02 g/L) and ampicillin sulfate (0.05 g/L) to eliminate bacterial contamination. The dishes were placed in an incubator at 25°C for 72 h in dark. Three isolates, WGC201, WGC202 and WGC203, were obtained by a single-spore culture method. Fungal colonies on PDA media were dark green (Fig. 1A and 1B). Conidiophores were septate and geniculate terminals, while conidia exhibited straight or slightly curved forms with four transverse septa, the central cell being notably longer and wider than the others. The size of such conidia were 27.34 µm to 40.62 µm× 11.61 µm to 15.97 µm (number = 50) (av. 32.71 µm× 13.11 µm) (Fig. 1C and 1D) (Moubasher et al. 2010). The internal transcribed spacer (ITS) region of nuclear ribosomal DNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced using universal primers ITS1/ITS4 and GPDF/GPDR (White et al. 1990; Berbee et al. 1999). DNA sequences were deposited into the NCBI database (ITS, PP789629, PP801333, and PP801574; GAPDH, PP849124, PP849125, and PP849126). Phylogenetic analysis with a neighbor-joining method based on the concatenated sequences of ITS and GAPDH genes showed that the three isolates clustered within a C. inaequalis branch (Fig. 2). Based on morphological and molecular identification, the fungal isolates were identified as C. inaequalis. The pathogenicity test was conducted in a greenhouse at 25°C using a spore suspension method and three isolates were used. Conidia were produced on PDA media (25℃) for 14 days. Plates were washed with sterilized distilled water and filtered with cheese cloth. Conidial suspension was adjusted to a concentration of 1×107 conidia/mL. Fifteen healthy seedlings of a wheat cultivar Xiaoyan-6 at a 3-4 leaf stage were inoculated by evenly spraying a 100mL spore suspension. Plants inoculated with sterile water served as a control. All plants were covered with plastic bags for 3 days. At 7 days after inoculation, all pathogen-inoculated plants showed similar symptoms (brown leaf oval spots with yellow halos) with those observed in the field, while all plants inoculated with sterile water showed no symptoms (Fig. 1E and 1F). The pathogen was reisolated from the symptomatic leaves and proved to be C. inaequalis. Morphological, molecular and pathogenic results indicated that C. inaequalis is the pathogen causing wheat leaf disease in China. The results are consistent with a previous report in Azerbaijan (Özer et al. 2020). To our knowledge, this is the first report of C. inaequalis causing spot disease on wheat in China. The occurrence, spread and economic importance to different wheat cultivars of the emerging disease in China will be further investigated and evaluated.