First Report of Botrytis cinerea Causing Gray Mold on Cardamine violifolia in China

Cardamine violifolia, also known as Cardamine hupingshanensis, is a perennial herbaceous plant belonging to the Brassicaceae family with wide distribution in several provinces in China, including Hubei, Gansu, Hunan, Sichuan, Yunan, Guangxi, and Guangdong. As a newly discovered selenium (Se)-enriched plant, C. violifolia is rich in MeSeCys and SeCys, contributing to its potential antioxidant properties (Wei et al. 2022). Se is an essential trace element for humans as a vital component of at least 25 selenoproteins involved in various physiological processes, including immune function and maintenance of antioxidant defense to avoid tissue damage (Gao et al. 2011). In April 2024, we observed an outbreak of gray mold disease on C. violifolia plants in Enshi (30°19'20.15''N; 109°28'20.25''E), Hubei Province, China, with an infection rate of 75%. The disease manifested initially as small, water-soaked spots on the leaves, which progressed to grayish-brown lesions, often forming a V-shaped pattern. Accompanied by developing a gray fungal coating, this disease leads to leaf withering and loss, significantly impacting plant health. Diseased leaves were collected from a plantation in Enshi, Hubei province to identify the causal agent. Tissues at the junction between healthy and diseased areas were excised, disinfected with 75% ethanol for 30 seconds, followed by a 60-second immersion in a 0.1% mercuric chloride solution, and subsequently rinsed three times with sterile water for one minute each. Excess water was blotted away using sterilized filter paper before inoculating the samples onto the PDA medium and incubating them at 25°C. After three days, the edge hyphae of the growing fungal colonies were transferred to fresh PDA plates. This procedure was repeated every five days, transferring the mycelium from the growth edge to a fresh PDA medium until a pure strain was obtained. Three pathogenic bacteria strains were isolated by the tissue separation method, all displaying consistent morphological traits. One representative strain, designated CVES1-8, was chosen for further characterization. This strain was grown on a PDA solid medium at 28°C for six days. Initially, the mycelium was white and fluffy, gradually turning grayish-brown. Under microscopic examination, the mycelium was septate and branching irregularly. Conidiophores were observed at the apex, bearing clusters of conidia reminiscent of grapes. The conidia (n = 40) were colorless, monosporous, and ellipsoidal or oval, measuring 8-14 μm × 5-10 μm. For molecular identification, genomic DNA was extracted from CVES1-8. Polymerase chain reaction (PCR) amplification targeted the internal transcribed spacer (ITS), heat shock protein (HSP60), DNA-dependent RNA polymerase subunit (RPB2), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes using specific primers, the primers ITS1/4 (White et al. 1990), HSP60-F/HSP60-R, RPB2-F/RPB2-R, and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. BLAST search of the NCBI GenBank showed CVES1-8 (GenBank nos. PQ220130, PQ201101, PQ201100, and PQ201102 for ITS, G3PDH, HSP60, and RPB2, respectively) shared 99 to 100% identity with B. cinerea (OQ789962.1, ON009466.1, PP731566.1, and MH732872.1, respectively). Based on combined DNA sequence data of G3PDH, HSP60, and RPB2, phylogenetic analysis was constructed， and the result showed that the isolate CVES1-8 belongs to B. cinerea. Based on morphological and molecular evidence, strain CVES1-8 was identified as B. cinerea. To fulfill Koch's postulates, spore suspension (approximately 106 CFU/ml) of CVES1-8 was sprayed onto the leaves of four-week-old C. violifolia seedlings (n = 15) with sterile water as a negative control. After seven days of incubation under controlled conditions (28°C, 80% humidity), symptoms were observed that were previously described, while control plants were asymptomatic. The causal pathogen was then reisolated frm the lesions, and the reisolated pathogen showed the same colony and spore morphology as CVES1-8, confirming Koch's postulates. To our knowledge, this is the first report of B. cinerea causing gray mold on C. violifolia in China, providing essential insights for growers and agricultural technicians to diagnose and manage this disease.