First Report of Botrytis cinerea Causing Leaf Spot on Ranunculus ternatus Thunb. in China

Ranunculus ternatus Thunb. (RT), a species in the Ranunculaceae family, has been officially recognized in traditional Chinese medicine and included in the Chinese Pharmacopoeia since 1977. Its dried root has been widely used in Chinese medicine to treat a variety of ailments, including stomatitis, tuberculosis, and other inflammatory conditions. More recent research has expanded its potential, showing promise in the treatment of modern diseases such as diabetes, gastric carcinoma, and ovarian cancer. It is widely cultivated in several Chinese provinces. In February 2025, leaf spot disease was observed in a 2-ha field in Funan County, Anhui Province (115°16'32''E, 32°24'25''N), with a disease incidence of 30%. Early symptoms included brown spots or streaks on the leaves. As the disease progressed, large, irregular brown spots merged, eventually covering the entire leaf and causing leaf death. A layer of grey mold was observed on affected leaves. Leaf tissue segments (1 cm²) were surface-sterilized with 75% ethanol for 30 s, followed by 5% NaClO for 1 min, rinsed three times with sterile water, and cultured on potato dextrose agar (PDA). After mycelial growth, hyphal tips were transferred to fresh PDA plates, purified through subculturing, and preserved. Five pathogenic strains with similar morphological features were isolated, and strain fy11-5 was selected for further characterization. After growing on PDA at 25 °C for 6 days, the colony produced white, fluffy mycelia that turned grey-brown. Microscopic examination revealed ellipsoidal to spherical, hyaline conidia (9.65–14.15 μm × 4.93–5.74 μm). Conidiophores were clustered, greyish, and branched, with subglobose conidiogenous cells, resembling a grape-like structure. Based on these features, the isolate was identified as Botrytis cinerea (Ellis 1971). Pathogenicity testing was conducted by inoculating R. ternatus Thunb. leaves with a 5 × 5 mm mycelial plug from an actively growing colony; control leaves received a sterile PDA plug. Disease progression was assessed at 3, 5, and 10 days post-inoculation in a controlled environment at 25 °C, 75% relative humidity, under a 12-h light/12-h dark cycle. Grey lesions developed on inoculated leaves, leading to wilting, while control leaves remained asymptomatic. The fungus was re-isolated from symptomatic tissue, and its morphology matched the original isolate, fulfilling Koch’s postulates. Genomic DNA of strain fy11-5 was PCR-amplified for the ITS, HSP60, RPB2, and G3PDH genes, yielding sequences of 519 bp, 1117 bp, 882 bp, and 1018 bp, respectively. The primers used were ITS1/4 (White et al. 1990), HSP60-F/HSP60-R, RPB2-F/RPB2-R, and G3PDH-F/G3PDH-R (Staats et al. 2005). BLAST analysis of the NCBI GenBank showed 99-100% identity with B. cinerea strains (GenBank accession nos. PV776292, PV791144, PV791145, PV791146 for ITS, G3PDH, HSP60, and RPB2, respectively). Phylogenetic analysis of the combined sequences confirmed that fy11-5 is B. cinerea. Thus, both morphological and molecular evidence identified strain fy11-5 as B. cinerea, responsible for leaf spot disease on R. ternatus Thunb. This is the first report of B. cinerea causing leaf spots on R. ternatus in Anhui Province, China, providing a basis for controlling the disease and its economic impact.