First Report of Neopestalotiopsis rosae Causing Leaf Spot on Tobacco in China

Tobacco (Nicotiana tabacum L.) is a globally important cash crop, and leaf quality is essential for cigarette production. In May 2026, leaf spot symptoms were observed on tobacco in Shixing County, Guangdong, China (24°31’-25°06’N, 113°54’-114°22’E; alt. 401 m). Early symptoms were necrotic spots with yellow halos; severe lesions caused perforation and complete necrosis. About 15% of 3,000 plants surveyed showed symptoms and growth inhibition, reducing yield, leaf quality, and growers’ income. Leaf samples were collected from symptomatic plants of the cultivar Yueyan 97 for pathogen isolation. Twenty diseased tobacco leaves were collected, and a total of 60 lesion margin segments were excised for isolation. Leaf pieces (approx. 0.5 cm) from lesion margins were surface-disinfected with 75% ethanol for 45 s and 0.1% HgCl2 for 1 min, rinsed three to four times with sterile distilled water, dried on sterile filter paper, and placed on potato dextrose agar (PDA). Plates were incubated at 26°C in the dark for 5 to 7 days (Fan et al. 2021). Four single-spore isolates were obtained. Neopestalotiopsis rosae was recovered from 68.3% (41/60) of the tested leaf pieces. A detached-leaf pathogenicity test was performed, and the most virulent isolate, designated ZB494, was selected for further study. On PDA, the fungus produced white, cottony colonies, and black conidiomata developed after 9 days. The conidia were fusiform, four-septate, measured 5.52-8.16 × 17.21-25.05 μm, mean = 7.72 × 23.07 μm (n = 50), and were morphologically similar to Neopestalotiopsis rosae. The apical and basal cells were hyaline, the apical cell conical and the basal cell obconic, while the three median cells were pale brown to brown. Apical cell bore 2-3 filiform appendages (mean 21.73 μm), basal cell one (mean 9.42 μm) (n = 50). ITS, TUB, and EF-1α were amplified and sequenced (Zheng et al. 2023). The sequences were deposited in GenBank under accession numbers PZ899444 (ITS), PZ903514 (TUB), and PZ903515 (EF-1α). BLAST analysis showed that the ITS, TUB, and EF-1α sequences exhibited more than 99% identity with the corresponding sequences of Neopestalotiopsis rosae strain CBS 101057* in GenBank. A neighbor-joining phylogenetic tree was constructed based on the concatenated sequences of the three genes using 12 reference isolates from GenBank (Saitou, N., et al. 1987). The results showed that isolate ZB494 clustered with Neopestalotiopsis rosae on the same clade. For pathogenicity tests, three healthy potted tobacco seedlings (at the 6- to 8-leaf stage) were selected, with three leaves per plant used for inoculation. Using a wound inoculation method, four mycelial plugs (5 mm in diameter) were placed on each wounded leaves of the cultivar Yueyan 97. Sterile PDA plugs served as controls. Inoculated plants were kept in a greenhouse at 28°C and 90% relative humidity. After 7 days, all pathogen-inoculated leaves developed necrotic lesions surronded by yellow halos, whereas control leaves inoculated with sterile PDA plugs remained symptomless. The conidial morphology observed from the lesions was consistent with that of N. rosae (Maharachchikumbura et al. 2014). The same pathogen was re-isolated from the inoculated leaves and confirmed as N. rosae based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of N. rosae causing leaf spot on tobacco in China. Identification of the causal agent is fundamental for developing effective disease management strategies.