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Tobacco (Nicotiana tabacum L.), a broad-leaved annual in the Solanaceae family, is commercially cultivated for its valuable leaves (Chen et al. 2020). In a 0.5-hectare field of tobacco (cv. Yunyan 87) planted in May 2023 in Guizhou Province, China, leaf spot disease was observed with an incidence of 29–37%, based on an assessment of 500 randomly selected plants. Initial symptoms appeared as small, circular or oval white spots (1-2 mm) with distinct brown margins on leaves. As the disease advanced, spots enlarged to 5–8 mm, became sunken, and turned into necrotic lesions with central tissue often collapsing to form irregular holes. A total of seven symptomatic leaves, each collected from different tobacco plants (cv. Yunyan 87), were used for pathogen isolation following standard protocols of the tissue transplanting method. Bits (5 × 5 mm) of leaf tissue cut from the lesion-healthy tissue junctions were surface-sterilized with 75% ethanol for 30 s and 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and then transferred onto potato dextrose agar (PDA) medium. Following 7 days of incubation at 25°C in the dark, nine isolates with uniform morphology were recovered. On PDA, the colonies were initially white but became dense and developed velvety aerial hyphae within the 7-day period. Subsequently, one isolate, designated wz, was chosen for further study for its representative morphology among all recovered isolates as well as its stable and consistent growth on PDA, ensuring experimental reproducibility (Fig. S1). Genomic DNA of the isolate wz was extracted for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), large subunit (LSU), beta-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 (White 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), LROR/LR5 (Rehner and Samuels 1994), and BT2Fd/BT4Rd (Li et al. 2017), respectively. The resulting sequences were deposited in GenBank (accession nos. ITS: PX724618; RBP2: PX753868; LSU: PX745185; TUB2: PX761597). BLAST analysis confirmed that the sequences from isolate wz shared high nucleotide identity with those of Xylaria sp. ZS-2021c isolate 138. The ITS (99.14%), RBP2 (100%), and TUB2 (99.33%) sequences all showed near-complete to complete identity with their respective homologs in isolate 138 (GenBank accessions: MZ648853, PP320372, and MZ695792). Phylogenetic reconstruction based on the concatenated ITS-RBP2-TUB2 dataset further supported the placement of isolate wz within the Xylaria sp. ZS-2021c clad (Zhu et al. 2024) (Fig. S2, Table S1). The pathogenicity of isolate wz was assessed using six healthy tobacco seedlings (cv. Yunyan 87) of five- to six-leaf stage. The leaves were inoculated using a 5-mm mycelial plug. Control plants were treated similarly but with PDA-only plugs. Each treatment was replicated three times. Plants were grown in a high-humidity environment maintained with sterile moistened cotton and incubated in a greenhouse set at 25°C and 80% relative humidity. Disease development was monitored daily. After seven days, all inoculated leaves exhibited leaf spots consistent with field observations, characterized by irregular to circular white spots often surrounded by brown margin. As symptoms advanced, the lesions progressed to necrosis, with central tissue disintegration ultimately resulting in the formation of holes. Control plants remained completely asymptomatic throughout the study. The pathogen was re-isolated from the margins of lesions and confirmed to be morphologically and genetically identical to the original inoculated strain, thereby fulfilling Koch’s postulates. In addition, no asexual or sexual spores were observed despite multiple induction methods, including ultraviolet radiation, host inoculation, temperature variation, and mycelial washing, consistent with some sterile Xylaria isolates (Liu et al. 2008). Xylaria arbuscula has previously been reported as a pathogen causing leaf spot on flue-cured tobacco in China (Xie et al. 2021). To our best of knowledge, this is the first report of Xylaria sp. ZS-2021c causing leaf spot on tobacco in China. This work establishes a critical foundation for subsequent epidemiological investigations, studies of pathogen diversity, and the development of targeted management strategies to safeguard tobacco production.