First Report of Leaf Spot on Smilax glabra Caused by Neopestalotiopsis clavispora in China

Smilax glabra Roxb., commonly known as Tufuling, is a perennial liana of high economic and medicinal value in China. In July 2025, typical leaf spot symptoms were observed on one-year-old S. glabra seedlings in a commercial plantation in Yangxipu Town, Yunyang District, Shiyan City (32.83°N, 110.87°E). Disease incidence reached 28% via five-point field sampling. Initial symptoms were small, brown necrotic spots, which gradually expanded into irregular dark brown lesions. In severe cases, local tissue wilt and reduced photosynthetic efficiency occurred. For pathogen isolation, 10 symptomatic leaves were collected from 10 distinct seedlings, one lesion-bearing leaf per plant. Small tissue sections (5 × 5 mm) from the lesion margins were surface-sterilized in 75% ethanol for 30 s, followed by 2% NaClO for 1 min, and rinsed three times with sterile distilled water. Tissues were plated on potato dextrose agar (PDA) and incubated at 26°C under a 12-h light/dark cycle. After 5 days, 15 fungal isolates with consistent morphological characteristics were obtained via single-spore isolation. Isolate BSND was chosen as the representative strain owing to its high virulence in preliminary pathogenicity assays and was the only isolate used for multi‑locus molecular identification. Colonies of isolate BSND on PDA were white and cottony with undulate margins. Black acervuli formed after 8 days. Conidia were fusiform to clavate, five-celled and four-septate, 18.1-24.5 × 5.1-8.0 μm(n=30). The three median cells were versicolored with the central cell darkest; apical and basal cells were hyaline. Conidia had one basal appendage (3.7-8.6 μm, n=30) and two to three apical appendages (14.1-27.7 μm, n=30). These characteristics matched Neopestalotiopsis clavispora (Maharachchikumbura et al. 2014). Genomic DNA was extracted from the mycelium of isolate BSND. The internal transcribed spacer (ITS) region, β-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified using primer pairs ITS1/ITS4(White et al. 1990), T1/Bt2b(Glass et al. 1995), and EF1-728F/EF-2(Carbone et al. 1999), respectively. BLASTn identities were 99.8% (ITS), 100% (TUB2), and 99.6% (TEF1-α) against N. clavispora (MFLUCC 12-0281). Phylogenetic analysis based on the combined ITS-TUB2-TEF1-α sequences (concatenated sequences were aligned using DNAMAN, and a neighbor-joining phylogenetic tree was constructed in MEGA 7.0 with 1000 bootstrap replicates for branch support estimation) confirmed that isolate BSND belonged to the species N. clavispora. Sequences were deposited in GenBank under accession numbers PZ255410, PZ264047, and PZ264048.Pathogenicity tests were performed on healthy one-year-old S. glabra seedlings in a greenhouse (25±2°C, 80% RH). A 1×10⁶ conidia/mL suspension (sterile distilled water) was applied as 5 μL droplets onto wounded leaf halves; the opposite half of each leaf received equal sterile water as an internal control, separated by an intact uninjured gap to prevent liquid flow. Three biological replicates were established, with 3 seedlings per replicate and 2 mature leaves per seedling. To avoid inter-plant contamination, disposable gloves were replaced for each operation, and seedlings were independently moisturized daily. Seven days post-inoculation, typical leaf spots developed on leaves inoculated with the pathogen, whereas for the control group, the wounded leaf halves treated only with sterile water remained asymptomatic. The pathogen was reisolated from symptomatic tissues and confirmed through morphology and molecular biology as N. clavispora, which fulfilled Koch's postulates. N. clavispora is a cosmopolitan pathogen with a broad host range, previously reported to cause leaf spot on various hosts including Cocos nucifera (Chong et al. 2023), Garcinia mangostana (Zhu et al. 2023), Phoebe bournei (Xu et al. 2023), and Dendrobium officinale (Cao et al. 2022). To our knowledge, this is the first worldwide report of N. clavispora causing leaf spot on S. glabra. This report provides essential baseline data for accurate diagnosis and management of this emerging disease.