First Report of Choanephora cucurbitarum Causing Leaf Blight on Cynanchum wilfordii in China

Cynanchum wilfordii is a valuable medicinal herb with multiple pharmacological properties in China. In July 2024, a severe leaf blight outbreak was observed on C. wilfordii in an outdoor field located in Chadian Town, Yunyang District, Shiyan City, China (32°46′13′′N, 110°49′27′′E). The disease incidence reached approximately 20% across a 0.02-hectare cultivation area, with disease severity ranging from 30% to 50% on symptomatic plants. Typical symptomatic features included the emergence of dark brown necrotic lesions on leaves, which were subsequently covered with white floccose mycelia and dark masses of sporangiophores bearing brown to black sporangia and sporangiola. Ten diseased leaf segments collected from five plants were surface sterilized in 75% ethanol for 30 s and 1.5% sodium hypochlorite (NaClO) for 60 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) plates. Following dark incubation at 28°C for 3-5 days, pure fungal cultures were acquired by single-spore isolation, with nine isolates identified and designated WD1 to WD9. All isolates exhibited consistent morphological characteristics on PDA, colonies grew rapidly, covering the entire 90-mm Petri dish within 48 h. The colonies were white and cottony, bearing scattered sporangiola on the surface, with a pale-yellow reverse. Sporangiola were brown, ellipsoid to broadly ellipsoid (10.99 to 19.12 × 7.51 to 11.55 μm, n = 50), each subtended by a short cylindrical pedicel and characterized by distinct longitudinal coarse striations. Sporangia, which are apically borne on the sporangiophores, developed into brown to dark brown structures with a globose to subglobose shape upon maturation. Sporangiophores were erect, hyaline to brown, aseptate, and unbranched. Sporangiospores released from sporangia were ellipsoidal to ovoid (14.41 to 23.94 × 8.75 to 13.44 μm, n = 50), dark brown, and possessed hyaline appendages at both polar ends. These morphological traits were highly consistent with the descriptions of Choanephora cucurbitarum (Park et al. 2016). For molecular identification, the internal transcribed spacer (ITS) region and the large subunit (LSU) of rDNA from the representative isolate WD2 were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and LROR/LR7 (Vilgalys and Hester 1990), respectively. The sequences were deposited in GenBank (accession nos. PX664330 for ITS and PX664331 for LSU). BLASTn analysis confirmed that the ITS sequence of WD2 (599 bp) shared 100% identity with that of C. cucurbitarum strain LMJM-2 (OR002157, 611 bp), and the LSU sequence of WD2 (1,438 bp) shared 100% identity with that of LMJM-2 (OR002181, 1,456 bp), with high query coverage in both comparisons. Phylogenetic analysis using the neighbor-joining method in MEGA 7.0 based on concatenated ITS-LSU sequences from 11 reference strains placed WD2 within a well-supported clade of C. cucurbitarum (98% bootstrap) together with the ex-type strain CBS 178.76T. Based on morphological and phylogenetic analysis, the isolate WD2 was identified as C. cucurbitarum. For pathogenicity tests, six healthy C. wilfordii plants were spray-inoculated with a sporangiospore suspension (1×10 5 spores/mL) of isolate WD2 in sterile distilled water until runoff (approximately 4 mL per plant). Control plants were inoculated in an identical manner with sterile distilled water at the same time. To prevent cross-contamination, inoculated and control plants were maintained in separate greenhouse compartments. All plants were kept in a greenhouse at 28°C with 85–95% relative humidity. The pathogenicity test was repeated three times. After 7 days, sporulation of C. cucurbitarum was present on all symptomatic plants similar to that observed in the field, while control plants were asymptomatic. Fungi consistently reisolated from the induced lesions were morphologically and molecularly identical to the original inoculated isolate WD2, fulfilling Koch’s postulates. C. cucurbitarum has been reported on a broad range of hosts including fresh pea (Deng et al. 2025), passion fruit (Qin et al. 2025), and lettuce (Ryu et al. 2023). However, to our knowledge, this is the first report of C. cucurbitarum causing leaf blight on C. wilfordii in China. This study highlights an emerging threat to this specific medicinal crop and necessitates the formulation of management strategies.