First Report of Root Rot of Ophiopogon japonicus Caused by Clonostachys chloroleuca in China

Ophiopogonis Radix, the dried root of Ophiopogon japonicus, is a widely cultivated traditional Chinese medicinal plant with diverse pharmacological activities, including cardiovascular protection, anti-inflammatory effects, and antidiabetic properties (Zhang et al. 2024). In July 2025, root-rot symptoms were observed in O. japonicus plants cultivated at Zhejiang Chinese Medical University (30.08°N, 119.88°E), China. Symptoms began as chlorosis along the leaf margins, progressed to brown lesions on the fibrous roots, and ultimately led to root necrosis and rot. Disease incidence reached 55% across an area of approximately 25 m2. Six symptomatic root-tuber pieces (5 × 5 mm) were surface-sterilized in 75% ethanol for 30 seconds, then in 2.5% NaClO for 1 min. After three rinses with sterile water and air-drying, the pieces were placed on potato dextrose agar (PDA) medium and incubated in the dark at 28°C for five days. Two fungal isolates (OJPF07 and OJPF08) were obtained by mycelial separation on PDA. On PDA, colonies produced green conidial masses with white, flocculent aerial mycelium and a yellowish-white reverse. Concentric rings were visible on both surfaces. Conidia were single-celled, aseptate, ellipsoidal to ovoid, measuring 3.8–8.2 × 3.1–7.0 μm (average 5.6 × 4.3 μm; n = 50), consistent with Clonostachys species (Barreras et al. 2022). The internal transcribed spacer (ITS), transcription elongation factor 1-alpha (TEF1-α), and ATP citrate lyase (ACL1) regions were amplified using primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), ACL1-230up/ACL1-1220low (Moreira et al. 2016). Sequences were deposited in GenBank: ITS (PX471650), TEF1-α (PX091868), ACL1 (PX060962). BLASTn searches showed 99–100% identity to C. chloroleuca type strain CML 1941. Specifically, ITS matched KC806286 (510/513 bp), TEF1-α matched KX184988 (436/437 bp), and ACL1 matched KX184857 (785/785 bp). A maximum-likelihood phylogeny (Tamura-Nei model and 1,000 bootstrap replicates) built in MEGA 11 clustered isolate OJPF07 with C. chloroleuca. Pathogenicity was tested using a soil-drench method. A spore suspension of isolate OJPF07 (1 × 10⁶ conidia/mL) was poured onto the soil surface around the base of three healthy O. japonicus plants (about 50 mL per plant to ensure contact with the root-zone). Three control plants received the same volume of sterile water. Plants were kept at 28°C, 85% relative humidity, and a 12-hour photoperiod. After 30 days, inoculated plants developed symptoms mirroring those in the field, with 100% incidence, while controls remained symptomless. C. chloroleuca was reisolated from symptomatic roots, fulfilling Koch’s postulates. No pathogens were recovered from the control plants. To our knowledge, this is the first report of C. chloroleuca causing root rot of O. japonicus in China. C. chloroleuca has previously been reported as the causal agent of soybean root rot in Heilongjiang Province (Zhao et al. 2024). These results provide important insights into C. chloroleuca-induced root rot in O. japonicus and underscore the need for effective detection and management strategies to protect medicinal herb cultivation.