First Report of Colletotrichum incanum Causing Anthracnose on Epimedium sagittatum in China

Epimedium sagittatum Maxim., an important perennial medicinal herb in China, contains bioactive compounds with various pharmacological properties(Wang et al. 2023a; Wang et al. 2023b). In June 2024, a severe anthracnose-like disease was observed in an understory plantation of E. sagittatum in Shennongjia Forestry District (31°45′N, 110°40′E), Hubei Province, China. Disease incidence reached 30% to 45% across surveyed fields. Initial symptoms included chlorotic along leaf margins, which subsequently developed into brown necrotic lesions. Symptomatic leaf samples were collected from four locations—Zhaibao, Daping, Mawan, and Baishaliangzi—with six plants sampled from each site, yielding a total of 24 samples. Tissue segments (4 × 4 mm) were surface-sterilized with 75% ethanol (30 s) followed by 0.1% (w/v) HgCl₂ (1 min), rinsed thoroughly with sterile water, and plated on potato dextrose agar (PDA). After incubation at 28 °C in darkness for 72 h, hyphal tips from colony margins were repeatedly transferred to obtain pure cultures. A total of 10 suspected pathogenic isolates were obtained from diseased tissues, all identified as the same fungal species and designated as Fungi 3. When cultured on PDA, it exhibited rapid radial growth, with colony diameters ranging from 6.5 - 7.2 cm after 7 days at 28°C. For molecular identification, genomic DNA was extracted from the mycelia of isolate Fungi 3. The internal transcribed spacer (ITS) region and actin (ACT) gene were amplified and sequenced using specific primer pairs (Liu et al. 2026). The resulting sequences were deposited in GenBank (accession Nos. pending). BLASTn analysis of the ITS sequence (580 bp total) showed 99% identity (576/580 bp) to the reference sequence of Colletotrichum incanum (PQ686302.1). The ACT sequence (281 bp total) showed 95% identity (277/281 bp) to the reference sequence of C. incanum (ON189039.1). To further resolve its phylogenetic position, multilocus phylogenetic analysis was performed using maximum likelihood and maximum parsimony methods. In both analyses, isolate Fungi 3 clustered consistently with C. incanum reference strains with high bootstrap support (>95%) and Bayesian posterior probability (>0.95). Pathogenicity tests were performed following Koch's postulates(Gao et al. 2026). A conidial suspension (1 × 10⁶ conidia/ml) prepared from isolate Fungi 3 cultured on PDA at 25°C for 7 days was inoculated onto intact leaves of potted E. sagittatum seedlings. Control leaves were treated with sterile water. The experiment included three replicates per treatment and was conducted twice. Inoculated plants were immediately transferred to a growth chamber at 25°C. Leaves were observed daily for symptom development, which was recorded and photographed. At 10 days post-inoculation, plants inoculated with Fungi 3 developed typical disease symptoms consistent with those observed in the field, while control plants remained asymptomatic. C. incanum has been previously reported to infect soybean (Yang et al. 2014) and Arabidopsis thaliana (Gan et al. 2016); however, this is the first global report of C. incanum causing anthracnose on E. sagittatum. This finding expands the known host range of C. incanum and provides a basis for developing targeted management strategies for this emerging disease in medicinal Epimedium cultivation.