First Report of Neopestalotiopsis rosae Causing Leaf Spot on Stephania epigaea in China.

Stephania epigaea Lo is an herb of significant medicinal and ornamental value in China. In May 2024, leaf spot disease was observed on S. epigaea in the medicinal plant nursery base of Yunnan University of Chinese Medicine, Kunming, Yunnan Province, China (24°33'59.21"N, 99°55'43.71"E), with an incidence rate of 75%. The symptomatic leaves exhibited irregular brown lesions along the margins, surrounded by a yellow halo. To determine the causal agents, symptomatic leaf tissues (5 × 5 mm) were excised, surface-sterilized in 75% ethanol for 30 s, followed by immersion in 1% sodium hypochlorite (NaClO) for 180 s, and rinsed three times with sterile distilled water. The sterilized tissues were transferred onto potato dextrose agar (PDA) and incubated at 28°C in the dark for 3 d. Pure cultures were obtained using the single-spore purification method. A total of five single-spore isolates were obtained. The isolates exhibited white, cottony aerial mycelia with undulated colony margins. Conidia were fusiform and four-septate, with three versicolor median cells and hyaline apical and basal cells (17.3-26.8 µm long × 3.7-6.2 µm wide; n = 30). The apical cells bore two to three appendages (19.6-28. 6 μm long; n = 30), while the basal cells bore a single unbranched appendage. To further identify the fungus, isolate SeF01 was randomly selected for sequencing. The nuclear ribosomal internal transcribed spacers (ITS), large subunit ribosomal RNA gene (LSU), β-tubulin gene (TUB2), and translation elongation factor-1α (TEF-1α) regions were amplified and sequenced using primer pairs ITS1/ITS4 (White et al., 1990), LR0R/LR5 (Schoch et al., 2012), T1/T2 (O'Donnell and Cigelnik, 1997), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. BLASTn homology analysis revealed that the ITS (GenBank accession no. PV363391), LSU (PV363539), TUB2 (PV384116), and TEF-1α (PV384117) sequences of isolate SeF01 exhibited 99.44-100% identity with Neopestalotiopsis rosae strain CBS 101057 (KM199359, KM116245, KM199429, and KM199523). A phylogenetic tree of Neopestalotiopsis species was built based on concatenated nucleotide sequences of ITS, LSU, TUB2, and TEF-1α using the maximum likelihood method. Isolate SeF01 clustered with N. rosae on the same branch. Morphological and molecular analyses confirmed that SeF01 was identical to N. rosae. To confirm pathogenicity of the isolate, conidial suspension with 1 × 106 conidia/mL was sprayed onto S. epigaea plants until runoff, while the control plants were sprayed with sterile distilled water. Two replicates (6 plants/replicates) were performed. All plants were incubated at 28℃ and 80% relative humidity with a 12 h light/12 h dark photoperiod. After 15 d, inoculated plants developed leaf spot symptoms, whereas control plants remained symptom-free. Fungal strains re-isolated from symptomatic leaves displayed morphological characteristics identical to SeF01. N. rosae has been reported to cause leaf spot on Fragaria × ananassa, Punica granatum, and Lonicera caerulea. To our knowledge, this is the first report of leaf spot caused by N. rosae on S. epigaea in China. The disease significantly reduces the value of S. epigaea, and pathogen-specific control measures are recommended.