First Report of Leaf Spot of Salvia miltiorrhiza Caused by Nigrospora sphaerica in Zhejiang province, China

Salvia miltiorrhiza (Danshen), an economically important medicinal plant, is widely cultivated throughout China. In late summer of 2024, leaf spot symptoms were observed on S. miltiorrhiza in Hangzhou (30.04°N, 119.96°E), Zhejiang Province, China, with a disease incidence of approximately 35 to 45%. Initial symptoms on leaves were yellowish-brown, irregularly shaped spots, which later enlarged and turned necrotic blackish-brown. Ten plants exhibiting severe symptoms were sampled for pathogen isolation. Symptomatic leaf tissues (5×5 mm) from lesion margins were surface-sterilized in 75% ethanol for 30 s, 1% sodium hypochlorite for 1 min, rinsed three times in sterile distilled water, and plated on potato dextrose agar (PDA). A representative isolate, SM-B3, was purified by single-spore isolation. On PDA, colonies were initially white, turning dark gray with age. After 10 days at 28°C, the fungus produced black, spherical, aseptate conidia measuring 12.3-24.6 μm×15.1-26.5 μm (n=50), consistent with the morphology of Nigrospora sphaerica (Wang et al. 2017). For the molecular identification, genomic DNA of isolate SM-B3 was extracted. The internal transcribed spacer (ITS) region, translation elongation factor-1 alpha (TEF1-α), and beta-tubulin (TUB2) genes were amplified with primers ITS1/ITS4, EF-798F/EF-986R, and Bt2a/Bt2b (White et al. 1990; Lee et al. 2023), respectively. The resulting sequences were deposited in GenBank (accession nos. ITS: PV715904; TEF1-α: PV770253; TUB2: PV770254). BLAST analysis of the sequences from isolate SM-B3 showed high identity to reference isolates of N. sphaerica. Specifically, the ITS sequence shared 99.42% identity with isolate XYB-1 (GenBank: MN795544), the TEF1-α sequence shared 99.59% identity with isolate (GenBank: MN053315), and the TUB2 sequence shared 100.00% identity with isolate JZB 3230014 (GenBank: MN549391). A multi-locus phylogenetic analysis confirmed the placement of isolate SM-B3 within the N. sphaerica clade. Pathogenicity of the isolate was confirmed on healthy, four-week-old potted S. miltiorrhiza seedlings. In addition to the representative isolate SM-B3, two other isolates were subjected to ITS sequence analysis. Their sequences were over 99.9% identical to that of SM-B3, thus confirming their identity as N. sphaerica. A spore suspension (1×10 6 spores/mL), prepared from two-week-old cultures, was sprayed onto the leaves of ten plants until runoff. An additional five plants were sprayed with sterile distilled water to serve as controls. All plants were covered with plastic bags for 48 hours to maintain high humidity and then incubated in a greenhouse at 25°C. After 7 days, all inoculated plants developed leaf spots identical to those observed in the field, whereas control plants remained asymptomatic. The fungus was consistently re-isolated from the lesions of inoculated plants, fulfilling Koch's postulates. This entire experiment was repeated three times with similar results. It is reported that N. sphaerica has caused peach leaf spot disease (Li et al. 2024) and pumpkin leaf spot disease (Liu et al. 2024). To our knowledge, this is the first report of N. sphaerica causing leaf spot on S. miltiorrhiza in China. This finding is significant as the disease could potentially impact the yield and quality of this important medicinal plant.