First Report of Gray Mold Caused by <i>Botrytis pseudocinerea</i> on <i>Helleborus thibetanus</i> in China

Helleborus thibetanus is a species in the family Ranunculaceae endemic to China. In China, it is appreciated for its medicinal and ornamental value (Li et al. 2019). In November 2024, symptoms of gray mold were found on approximately 60% of H. thibetanus plants in a 0.3-hectare commercial greenhouse planting located in Kunming, Yunnan Province (24.32°N, 102.49°E). Initial symptoms manifested as small brown spots (1-3 mm in diameter) on the leaves, followed by leaf withering and the development of gray mycelium on the affected tissues. Affected leaves were collected following a five-point sampling method, with five leaves sampled per point, for causal pathogen identification. Small tissue sections from lesion margins were surface-sterilized by sequential immersion in 1% NaOCl (30 s) and 75% ethanol (30 s), rinsed thrice with sterile distilled water, blotted dry on sterile filter paper, transferred to potato dextrose agar (PDA), and incubated at 28°C for 3 days. Subsequently, distinct fungal colonies were purified onto fresh PDA plates, yielding 13 isolates from the original tissue. During 7 days of incubation on PDA at 28°C, all 13 fungal strains rapidly grew to cover the entire 90-mm-diameter Petri dish. Colonies on PDA were gray. Conidiophores were simple and erect. Conidia were unicellular, hyaline, elliptical to ovoid, and borne in grapelike clusters. This morphological characteristic is very similar to that of the genus Botrytis. For further analysis, three representative fungal strains (TKZ2024111201, TKZ2024111203, and TKZ2024111209) were selected. Genomic DNA was extracted from pure PDA cultures using the Plant DNA Extraction Kit, followed by PCR amplification of the internal transcribed spacer (ITS) region and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 60 (HSP60), and RNA polymerase II second largest subunit (RPB2) (Abbey et al. 2024). The sequences obtained from strains TKZ2024111201, TKZ2024111203, and TKZ2024111209 were deposited in GenBank under the following accessions: ITS (PV459679–PV459681), G3PDH (PV490906–PV490908), HSP60 (PV490909–PV490911), and RPB2 (PV490912–PV490914). Following BLAST analysis of the homologous sequences of the ITS, G3PDH, HSP60, and RPB2 genes with GenBank, these sequences were aligned and concatenated, and a phylogenetic tree was produced (Tamura et al. 2021). According to the data, three fungal sequences were 100% identical to Botrytis pseudocinerea. The morphological and molecular traits of isolates TKZ2024111201, TKZ2024111203, and TKZ2024111209 were identical to the type strain B. pseudocinerea YC-7 (Walker et al. 2011). Pathogenicity of the three isolates was confirmed through inoculation assays. Three experiments were conducted per isolate, each using 12 H. thibetanus plants per treatment group. Test plants were inoculated on their leaves with mycelial plugs from 8-day-old PDA cultures. Control plants received plugs of sterile PDA. For five days, all plants were maintained in a growth chamber at a constant 24°C with 12 hours of light, 12 hours of darkness, and 80% relative humidity. As shown in Fig.S1 E, F, G, and H, control plants remained healthy, whereas inoculated plants displayed leaf browning, severe withering, and leaf abscission, followed by the formation of gray mycelium on lesions. The pathogen's morphology was found to be identical to that of the inoculated pathogen when it was recovered from symptomatic leaflets. B. pseudocinerea has been reported to infect fruits and vegetables in Germany (Plesken et al. 2015), but no reports of B. pseudocinerea infecting H. thibetanus in China were found. This is the first report of gray mold on H. thibetanus that we are aware of. This study will be useful to the control of gray mold in H. thibetanus in China.