First Report of Target Spot Caused by <i>Corynespora cassiicola</i> on <i>Cucumis melo</i> in China

Melon (Cucumis melo L.) is an essential, fruit-based vegetable grown in a large agricultural area in China. In May 2024, target spot-like symptoms were observed on the leaves of Topmark (Cucumis melo L. ssp. melo) plants in a greenhouse with more than 60% incidence among a total of 1,000 plants. Additionally, field observations revealed an incidence rate of over 40% among a total of 3,000 plants in Qinhuangdao, Hebei Province, China (39.9°N, 119.5°E). A specimen (ME-CC20240525) was stored in the Hebei Key Laboratory of Horticultural Germplasm Excavation and Innovative Utilization. Initially, small, round, yellowish spots appeared on the leaves, merging into dark brown lesions surrounded by a yellow halo, eventually leading to leaf wilt. Leaves with typical symptoms were collected for pathogen investigation. Leaves were washed thoroughly with sterile water, and small tissue fragments (5×5 mm) were excised around the lesions. These fragments were surface disinfested with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and then incubated in the dark on potato dextrose agar (PDA) at 28°C for 7 days. Five isolates with similar morphological characteristics were obtained by single-spore isolation. After 7 d of incubation on PDA at 28°C in the dark, the upper surface of the colonies was white to gray with abundant mycelium, and the lower surface was dark black. Conidia were 31.3-129.8 × 5.5 -9.6 μm in diameter (n = 50). Morphological characters were like Corynespora sp. (Liu et al., 2019). For molecular identification, the genomic DNA of the isolate was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Shanghai, China). The internal transcribed spacer (ITS) region of rDNA and beta-tubulin (btub2) gene were amplified and sequenced with primers ITS4/ITS5 and Bt2a/Bt2b (Glass and Donaldson, 1995), respectively. The internal transcribed spacer sequences (ITS, 534bp, PQ454663) and beta-tubulin (btub2, 469bp, PQ465884) were deposited in GenBank. The homology of ITS to Corynespora cassiicola (MW426360) is 100%, and the homology of btub2 to C. cassiicola (PP419027) is 100%. Based on the ITS and btub2 sequences, phylogenetic analysis by the neighbor-joining method was performed using MEGA 7.0 to cluster the isolates with C. cassiicola. According to Koch's postulates, leaf pathogenicity tests on melon seedlings were lightly scratched and inoculated with 5- mm mycelial plugs. Control seedlings received PDA plugs. In addition, leaves were sprayed inoculated using spore solution and sterile water was used as control. Five leaves per plant and five plants per treatment were evaluated. All seedlings were kept in a greenhouse (12 h/12 h light/dark cycle, 28 ± 2°C, 80-90% humidity). After a 7- days incubation, all leaves inoculated with the fungus showed symptoms consistent with field observations, whereas control plants were asymptomatic. The pathogenic fungus was reisolated successfully from the diseased leaves and identified as C. cassiicola based on morphology, pathogenicity, and sequence results, a pathogen reported from many crops (Lu et al., 2021). To the best of our knowledge, this is the first reported of C. cassiicola on C. melo in China.