First Report of Leaf Spot Caused by <i>Nigrospora coryli</i> on Tobacco in China

As a globally cultivated economic crop, tobacco (Nicotiana tabacum) is known for its addictive properties, which arise from the mildly irritating and psychoactive compounds it contains (Hu et al. 2010). Tobacco leaves are susceptible to a range of fungal and bacterial diseases during production and curing, including target spots, brown spots, wildfire, and powdery mildew (Guo et al. 2024). During a survey conducted in June 2025 in Zhengan (107.43° N, 28.55° E), Guizhou Province, China, tobacco (cv. Yunyan 87) plants were found affected by a leaf spot disease, with an incidence rate ranging from 41% to 47%. Initially, symptomatic leaves developed irregular, yellowish-brown spots that gradually expanded and turned necrotic, eventually acquiring a whitish appearance. To investigate the disease, six severely symptomatic plants were selected for pathogen isolation using the tissue transplanting method. From each plant, pieces (5 × 5 mm) of leaf tissue taken from the border between diseased and healthy tissue were surface-sterilized with 75% ethanol for 30 s, followed by 1% sodium hypochlorite for 1 min, and then rinsed three times with sterile distilled water before being placed on potato dextrose agar (PDA) medium. After incubating at 25°C in the dark for 7 days, a total of nine fungal isolates with similar morphology were obtained. One representative isolate, designated YB13, was selected for further identification (Fig. S1). The fungal colonies on PDA exhibited abundant aerial mycelia and were white in color, and covered the whole plates (90 mm in diameter) in seven days. After 10 days of incubation at 28°C, the fungus produced black, ovoid, smooth, and aseptate conidia with 12-15 μm in diameter. For molecular identification, genomic DNA was extracted from isolate YB13. The internal transcribed spacer (ITS) region, along with the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified using primers ITS1/ITS4 (White 1990), gpd1/gpd2 (Berbee et al. 1999), BT2Fd/BT4Rd (Li et al. 2017), and EF1-728F/EF1-986R (Carbone and Kohn 2019) respectively. The resulting sequences have been deposited in GenBank under the following accession numbers: ITS: PX736263; GAPDH: PX556631; TUB2: PX556632; and TEF1-α: PX711191. BLAST analysis of the sequences from isolate YB13 revealed high identity with those of Nigrospora coryli isolate W18. Specifically, the ITS sequence shared 99.14% identity with isolate W18 (GenBank: PP218065), the TUB2 sequence shared 99.71% identity with isolate W18 (GenBank: PP320372), and the TEF1-α sequence shared 100.00% identity with isolate W18 (GenBank: PP461302). A multilocus phylogenetic analysis based on a concatenated dataset of ITS, TEF1-α, and TUB2 genes further confirmed that isolate YB13 clusters within the N. coryli clade (Fig. S2). Pathogenicity of the isolate YB13 was confirmed on five healthy tobacco plants (cv. Yunyan 87) at seedling stage (four to five leaves). To wound the leaves, a 4 mm² area on each was lightly scratched with a sterile needle, after which a 5-mm diameter mycelial plug was placed on the wound. Control leaves were inoculated with PDA-only plugs. Following inoculation, leaves were maintained under high humidity by enclosing the treated plants in transparent plastic bags containing sterile water-soaked cotton at the base to maintain approximately 80% relative humidity. Plants were incubated in a greenhouse at 25°C. All experiments were performed in triplicate. The leaf disease development was observed and recorded daily. After 7 days, all inoculated leaves developed leaf spots consistent with symptoms observed in the field. Lesions appeared as irregular to circular spots, 5–12 mm in diameter, with a yellowish-brown color and often a chlorotic halo. As symptoms progressed, the lesions turned necrotic, developing dry, whitish centers surrounded by a darker margin and a yellow halo. In contrast, control plants remained completely asymptomatic. The pathogen was re-isolated from lesion margins and confirmed to be identical to the original inoculated strain based on colony morphology and DNA sequencing, thereby fulfilling Koch’s postulates. N. coryli has previously been reported as an endophyte within the stem of Corylus heterophylla at Mycorrhizal Seedling Cultivation Center in Guizhou, China (Wang et al. 2024). To our best of knowledge, this is the first report of N. coryli causing leaf spot on tobacco in China. These findings underscore the importance of continued pathogen surveillance and provide a basis for epidemiological studies and the development of management strategies for this emerging disease.