First Report of Colletotrichum karstii Causing Anthracnose on Stephania epigaea in China.

Abstract

The perennial herbaceous vine Stephania epigaea Lo, known as Di Burong in Chinese, is generally used as an anti-inflammatory and analgesic drug, as well as an ornamental plant (Dong et al., 2018). In May 2024, symptoms resembling anthracnose were observed on S. epigaea plants in an herbal nursery (33 m2, approximately 400 plants) in Kunming, Yunnan Province (24°33' 59.21" N, 99°55' 43.71" E), China, with an incidence rate of 75%. The disease initially occurred as small, round brown spots on the leaf margins, which later expanded into irregular lesions. The centers of these lesions were dark brown with yellow edges. To isolate and identify the pathogen, 0.5 × 0.5 mm sections of diseased leaf tissue were sterilized by dipping them in 75% ethanol for 30 s, then in 1% NaCIO for 180 s, followed by three rinses in sterile water. The samples were then cultured on potato dextrose agar (PDA) in the dark at 28 °C for 3 days. The fungal isolates were purified using the single-spore purification method, and 10 isolates with similar morphological characteristics were obtained. The colonies on PDA were initially white, changing to a grayish-white color over time. The ascomata were dark brown, spherical, and either on the surface or partially immersed in the PDA. The conidiophores were smooth, septate, branched, and hyaline to light brown. The conidia were cylindrical, aseptate, hyaline, and smooth, with dimensions of 10.0 to 16.7 × 5.2 to 6.9 μm (average 13.5 × 6.0 μm, n = 30). A 24-hour slide culture (Cai et al., 2009) of the conidia suspension revealed solitary appressoria that were light to dark brown, nearly spherical to oval, and smooth-walled to undulate, with dimensions of 6.8 to 10.5 × 4.7 to 10.0 μm (average 8.7 × 6.7 μm, n = 30). To further characterize the pathogen, isolate SeF03 was randomly selected for further testing. Total genomic DNA was extracted using the cetyl trimethylammonium bromide method, and the nuclear ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 3 (HIS3), actin (ACT), and calmodulin (CAL) genes were amplified using the primer pairs ITS1/ITS4 (White et al., 1990), gpd1/gpd2 (Berbee et al., 2019), CYLH3F/CYLH3R (Crous et al., 2006), ACT-512F/ACT-783R (Carbone and Kohn, 1999), and CL1C/CL2C (Weir et al., 2012), respectively. BLASTn homology search showed that the ITS (PQ963009), GAPDH (PV021394), HIS3 (PV021395), ACT (PV021396), and CAL (PV021397) sequences of SeF03 were 100, 98.81, 100, 98.15, and 100% identical to Colletotrichum karstii strain CBS 128500 (JQ005202, JQ005289, JQ005463, JQ005550, and JQ005723), respectively. A phylogenetic tree of Colletotrichum species was built based on concatenated nucleotide sequences of ITS, GAPDH, HIS3, ACT, and CAL using the maximum likelihood method with the Tamura-Nei model. SeF03 and C. karstii clustered on the same branch. Morphological and molecular characteristics also confirmed that SeF03 was identical to C. karstii. Pathogenicity was evaluated through foliar spray inoculation with a conidial suspension (1 × 10⁶ conidia/mL) applied until runoff, using sterile water-treated plants as controls. All sprayed plants were placed in an incubator at a constant temperature of 28 ℃ with alternating light and dark periods (L/D = 12 h/12 h, 80% humidity). Each treatment consisted of 3 plants, with 2 replicates. After 15 days, obvious brown spots appeared on leaves treated with the SeF03 spore solution, while the control plants sprayed with sterile water remained asymptomatic. The fungal strains isolated from the symptomatic leaves shared the same morphological characteristics as SeF03. C. karstii has been reported to cause anthracnose on Fatsia japonica, Dalbergia odorifera, and Piper nigrum. To our knowledge, this is the first report of anthracnose caused by C. karstii on S. epigaea in China. The disease significantly threatens S. epigaea's ornamental value, requiring urgent and targeted control measures.