First Report of Gray Mold on Jinxianlian (Anoectochilus roxburghii) Caused by Botrytis cinerea in China

Anoectochilus roxburghii is one of the important herbal medicines in China. As a national rare and endangered plant, A. roxburghii is presently cultivated in bottles through greenhouse productions. The cultivation of A. roxburghii in Zhejiang Province accounts for a large proportion in China. Gray mold was found to severely affect the production of A. roxburghii. In 2018, the incidence of gray mold indicated by water-soaked gray mold symptoms with white to gray mycelia and conidiogenous apparatus on infected stems and leaves was observed for 6% of seedling bottles. When the conditions are suitable, gray mold spreads to the whole leaf and then gradually turns into grayish black and produces brown dead plaque, which causes the plant to wrinkle in severe cases. Thirty-one diseased leaves collected from different greenhouses in Jinhua City were picked with a needle on 2% potato dextrose agar (PDA) and cultured in the dark at 23°C. The colonies on the PDA were initially grayish white, followed by brownish black, and produced one-celled, round conidia (8.78 ± 0.4 × 12.60 ± 0.3 μm). These morphological features are consistent with Botrytis cinerea. In order to confirm the identification based on morphological features, DNA-dependent RNA polymerase subunit II (RPB2), heat-shock protein 60 (HSP60), and glyceraldehyde-3-phosphate (G3PDH) were amplified from the DNA extracted from the mycelium produced on PDA (Berbee et al. 1999; Liu et al. 1999; Smith 1989). The sequences were deposited in NCBI under accession numbers RPB2 (MN464111, MN464112, MN464113), HSP60 (MN464108, MN464109, MN464110), and G3PDH (MN464105, MN464106, MN464107). BLAST analysis of the RPB2 (1,106 bp) segment showed 100% identity to the RPB2 sequence of B. cinerea isolate S4GBR40 (GenBank accession no. MK3880531). The HSP60 (956 bp) segment showed 100% identity to the HSP60 sequence of B. cinerea isolate F2-17 (GenBank accession no. MK256903). And the G3PDH (994 bp) gene segment showed 100% identity to B. cinerea isolate GZFQ-1 (GenBank accession no. MH479930). Phylogenetic analysis based on RPB2, HSP60, and G3PDH showed that the pathogen was B. cinerea. Uninfected A. roxburghii healthy plants were sprayed with 2 × 10⁶ conidia/ml in distilled water as inoculum, and plants inoculated with only distilled water were adopted as the blank control. For each isolate, 16 intact plants were inoculated. Plants were then kept at 25°C and 95% relative humidity in a 12-h light/dark incubation. After 10 days, all three tested isolates caused gray mold symptoms on A. roxburghii, whereas no symptoms appeared in the controls. Cultures reisolated from lesions and cultured on PDA exhibited the same morphological characteristics as B. cinerea (Carmichael et al. 2019), confirming Koch’s postulates. The inoculations were performed twice. To the best of our knowledge, this is the first report of the gray mold in A. roxburghii, which is the main factor limiting its productions. Measures must be taken to manage the disease in a timely manner.