First Report of <i>Clonostachys rosea</i> Causing Root Rot of <i>Polygonatum cyrtonema</i> in China

Polygonatum cyrtonema Hua, commonly known as Huangjing, is a traditional Chinese medicinal and edible plant prized for its roots. In recent years, P. cyrtonema Hua root rot disease has frequently occurred in China, significantly affecting both yield and medicinal quality. In October 2024, a severe outbreak of root rot was observed in P. cyrtonema Hua cultivation fields located in Hecheng District, Huaihua City, China (N27°40’22”, E109°55’9”), where the cultivation area over 200 hectares. The disease incidence reached approximately 42.5%, posing a significant threat to local production. Symptoms on the infected roots appeared brown, water-soaked lesions, causing soft rot and eventually leaf yellowing and wilting to death. (Fig.1-B, C). To isolate the causal organism, rotted root tissues were surface-sterilized with 75% ethanol (1 min), 2% NaClO (3 min), rinsed four times with sterile distilled water, blotted dry on sterile filter paper. Tissue fragments (1×1 cm) from lesion margins were plated on potato dextrose agar (PDA), then incubated at 28°C in darkness for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. From a total of 25 diseased root samples, 80 tissue segments were plated. This process resulted in the isolation and purification of 42 fungal isolates.The isolate, HJBK 4-9 was selected for further characterization. The colonies exhibited concentric rings, white mycelium with a slight yellow pigment on the reverse side. Sporulation was induced by culturing on carnation leaf agar (CLA) at 25°C in darkness (Yang et al. 2024). These microspores were oval, colorless, aseptate and slightly curved, with (5.8−12.5) μm× (2.2−3.8) μm in size. Fungal mycelia were used for PCR, using the following primer sets: ITS1/ITS4 (Hao et al. 2023), EF1/EF2 (O'Donnell et al. 1998), and β 2a/2b (Glass and Donaldson.1995) respectively. PCR products of the isolate were sequenced. Sequences were deposited in GenBank (ITS, PV249395; TUB2, PX130413; EF-1α, PX130414). BLASTn searches revealed that the strain was 99.81% (KP689232.1) for ITS, 99.36% (ON209102.1) for TUB2 and 99.28% (PP394350.1) for EF-1α. The isolate was identified as C. rosea based on the morphological characteristics and molecular phylogeny. To test pathogenicity, 6 pieces of healthy roots of P. cyrtonema Hua were pin-pricked with a sterile needle and spray-inoculated with 50μL of spore suspension per root (2×107conidia/mL). Another 6 pieces of healthy roots were pin-pricked with a sterile needle and inoculated with sterile water, and served as controls. All roots were kept in an artificial climate chamber controlled conditions (26°C and 60% relative humidity). After 15 days, most inoculated roots turned brown and partially rotten (Fig.1-D), similar to the field symptoms, whereas the control remained symptomless. The pathogen was reisolated from all inoculated roots only and confirmed as C. rosea based on morphology and sequence analysis. To our knowledge, this is the first report of C. rosea causing root rot of P. cyrtonema Hua in China. The finding may be helpful to the control and prevention of root rot disease of P. cyrtonema Hua.