First Report of Nigrospora sphaerica Causing Leaf Spot on Tobacco ( Nicotiana tabacum ) in China

Tobacco (Nicotiana tabacum) as a major commercial crop in China is susceptible to multiple foliar pathogens, which severely decrease the grade of cured tobacco leaves and affect its economic value. In September 2024, the veins of tobacco leaves displayed leaf blight symptoms in a field located in Xuchang (34°8′8″N, 113°48′32″E), Henan Province, China. The incidence was estimated to approximately 30% in a 0.06-ha field. The initial symptom showed strip-shaped or irregular brown lesions with a size ranging from 0.1 to 1 cm. These spots developed to dark brown necrotic lesions. Leaves exhibiting brown or dark brown necrotic lesions along the veins were collected from 10 plants in the field. Then the leaves were put in sterilized paper bags, and transported to the laboratory for fungal isolation. In a clean bench, the junction of the diseased and healthy portions of a leaf sample was detached and disinfected with 1% NaClO solution for 10 s and then with 75% alcohol solution for 60 s. The disinfected leaf specimens were rinsed three times using sterile water, air-dried, and put on potato dextrose agar (PDA) plates in the bench. The PDA plates were incubated for 5 days at 28℃ in darkness subsequently. Seven isolates with similar colony morphology were obtained. Two representative isolates (XC0901 and XC0902) were used for identification of the pathogen. Colonies of the isolates exhibited cottony mycelia, and turn into light brown septate hyphae with sporulation. Conidia were smooth, black, spherical to subspherical and unicellular, being measured 14.7 ± 3.6 × 12.2 ± 3.8 µm (n=50), in width and length aligning with previous morphologic descriptions of N. sphaerica by previous studies (Wang et al. 2024; Liu et al. 2024). Molecular identification of the isolates XC0901 and XC0902 was conducted using internal transcript spacer (ITS) region (ITS1/ITS4), β-tubulin (TUB2) (Bt-2a/Bt-2b), and the translation elongation factor 1-alpha (TEF1-α) (EF1-728F/EF-2) primers for PCR amplification of genomic DNA (Khoo et al. 2023). Sequence alignment was performed with BLAST search tool in the GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The results of sequence alignment revealed that the ITS, TUB2, and TEF1-α sequences of the isolate XC0901 exhibited 99.59%, 100.00%, 99.56% identity with the N. sphaerica accessions KX985992.1 (527 bp), KY019516.1 (410 bp), and KY019333.1 (469 bp) respectively. While the isolate XC0902 showed 99.80%, 99.48%, and 99.77% sequence identity with these same accessions as mentioned above. Nucleotide sequences of the XC0901 and XC0902 isolates were deposited in the GenBank with accession numbers of PQ637527, PV750773 (ITS), PQ677888, PV785689 (TUB2), and PQ677889, PV785690 (TEF1-α), respectively. Based on the sequences of ITS region, TUB2 and TEF1-α genes, a phylogenetic tree was constructed with Maximum Parsimony, and indicated that both isolates were clustered in the same clade with N. sphaerica. To test pathogencity of the two isolates according to the Koch's postulates, PDA plugs with the fungal development were placed on five detached leaves and five tobacco plants (cv. Zhongyan100) without wounding, respectively. Clean PDA plugs were used as negative controls. Triple repeats were used in all tests. Tested detached leaves and tobacco plants were cultivated in an artificial climate incubator (28℃, 4000 Lx of light intensity, 16 h/d of illumination duration, 60% of relative humidity) and in a greenhouse at 28℃ respectively. At 3 to 5 days after inoculation, strip-shaped or irregular brown lesions appeared on detached leaves and tobacco plants leaves that were inoculated with isolates, but not on the controls. Approximately 85%-95% of inoculation sites were infected. Pure cultures were reisolated from diseased tissues and identified as N. sphaerica by morphological and molecular methods described above. To our knowledge, this is the first report of N. sphaerica-induced tobacco leaf spot in China, expanding its host range, which underscores the need to monitor and manage this emerging disease to mitigate its impact.