First Report of <i>Botrytis cinerea</i> Causing Gray Mold on <i>Cremastra appendiculata</i> in China

Cremastra appendiculata (D. Don) Makino is a perennial rare medicinal plant in the family Orchidaceae. The dry pseudobulb is used as the medicinal part, exhibiting a range of pharmacological activities, including anti-tumor, anti-inflammatory, and antioxidant effects, among others (Liu et al., 2021; Jiang et al., 2022). In March 2024, a gray mold disease was observed in C. appendiculata fields planted in Dafang County (27°23'17.18″ N, 105°73'95.94″ E, 1774.6 m), Guizhou Province, China. The disease started from the leaf tip or edge and gradually formed large, irregular disease patches. Leaves curled and withered, with a dense gray mold layer on the surface, approximately 30% to 70% of the plants were affected. Twenty symptomatic leaf segments (5 × 5 mm2) were aseptically dissected from lesion margins, surface-sterilized with 75% ethanol for 30 s, triple-rinsed in distilled water, and maintained on PDA medium at 25℃ for three days. Twenty-one morphologically-identical isolates were single-spored. Initially, the mycelium appeared as sparse, white, radiating in growth, which later developed into gray, velvety colonies. Irregular black sclerotia formed at later stages of development. The conidiophores frequently branch and produce numerous conidia, forming grape spikes. The conidia are colorless single spores, oval, ranging from 8.15-12.59 μm × 6.78-9.83 μm (av.= 8.32 × 10.70, n = 50). The morphological characteristics closely resembled the description of Botrytis cinerea (Li et al., 2022). In addition, the internal transcribed spacer region (ITS), heat shock protein (HSP60), RNA polymerase II (RPB2), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were amplified by PCR and sequenced (Staats et al., 2005; White et al., 1990). The sequences were analyzed using RAxML-HPC BlackBox v.8.2.12, MrBayes v.3.2.7a and PAUP on ACCESS4.a168 on the CIPRES website for maximum likelihood (ML), maximum parsimony (MP) and Bayesian (BI) analyses. Two representative strains (DJS 01 and DJF 003) were registered in GenBank. BLASTn analysis showed >99% identity with Botrytis cinerea in GenBank: ITS: 100% (PP859488 versus KY817366 = 519/519 bp; PQ870055 versus KY817366 = 519/519 bp); RPB2: 99% (PP892380 versus AJ745676 = 1043/1044 bp; PQ776262 versus AJ745676 = 1089/1098 bp); G3PDH: 99-100% (PP868420 versus AJ705004 = 886/887 bp; PQ776260 versus AJ705004 = 886/886 bp); HSP60: 99-100% (PP892379 versus AJ716065 = 886/886 bp; PQ776261 versus AJ716065 = 975/976 bp). The multigene phylogenetic tree indicated that the two strains clustered with B. cinerea in a single clade. Pathogenicity tests were performed by spraying 20 C. appendiculata plants with conidial suspension (1×10⁵ spores/mL) of the DJS 01 and DJF 003 strains, while a control group of 6 plants was inoculated with distilled water. The plants were cultured under controlled conditions of 20°C and 90% relative humidity. After 10 days, spore-treated plants developed gray mold-like lesions, while controls remained asymptomatic. The pathogen was reisolated from lesions and identified as B. cinerea through morphological and molecular analysis. To our knowledge, this is the first report of B. cinerea causing gray mold on C. appendiculata in China. This finding provides crucial guidance for combating the devastating gray mold disease that severely threatens C. appendiculata cultivation.