First Report of Colletotrichum aenigma Causing Anthracnose on Vicia amoena in China

Vicia amoena is renowned for its high protein content and nutritional value, making it significant in animal production and traditional Chinese medicine production. In July 2023, typical anthracnose symptoms were observed on V. amoena leaves in Suihua City (125°82'E, 46°22'N), Heilongjiang Province, China, affecting approximately 40% of the plants (a total of 200 plants were surveyed). Symptoms initially appeared as black-purple spots, gradually developing into larger, sometimes irregular reddish-brown lesions surrounded by yellow margins, with easily perforated necrotic lesions. Samples were collected from 20 leaves exhibiting distinct symptoms. Each leaf was cut into 5 x 5 mm sections at the junction of diseased and healthy tissues. The sections were surface sterilized: first, in 75% ethanol for 30 s, then in 1% NaClO for 2 mins, followed by three rinses with sterile distilled water. After air-drying, the samples were placed onto potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d. Subsequently, 15 isolates were obtained using the single-spore method, with nine (SYW1 to SYW9) showing similar morphological characteristics. On PDA, colonies appeared cottony and pale yellow from above, with a black-brown center on the reverse side. The conidia were hyaline, aseptate, subcylindrical with broadly rounded ends, measuring 13.8 to 18.6 μm in length and 4.8 to 6.4 μm in width (n = 50). Appressoria were subcylindrical or irregular, with a few lobes, brown to dark brown, and measured 9.2 to 12.3 μm in length and 5.1 to 7.2 μm in width (n = 50). Morphological analysis suggested a close resemblance to Colletotrichum aenigma (Weir et al. 2012). Genomic DNA was extracted from three representative isolates (SYW1 to SYW3) for molecular identification. The ITS, GAPDH, TUB2, CHS-1, and ACT genes were amplified and sequenced using primers previously described by Weir et al. (2012). Sequences were submitted to GenBank (ITS: PP949783 to PP949785; GAPDH: PP952315 to PP952317; TUB2: PP952318 to PP952320; CHS-1: PP952321 to PP952323; and ACT: PP952324 to PP952326). BLAST analysis revealed 100% identity with C. aenigma (GenBank accessions: OM663726, KY820889, MW387032, MN075525, and LC815246). Phylogenetic analysis placed the three isolates and C. aenigma in the same clade. Pathogenicity tests were conducted twice on 4-week-old V. amoena seedlings in a greenhouse. A conidial suspension of the SYW1 isolate (1 × 106 conidia/ml) was sprayed onto five pots, each containing four seedlings, while sterile distilled water was sprayed on the control pots. All plants were grown in a light incubator with a 12-h photoperiod, 100% relative humidity, and 26°C temperature. Two weeks post-inoculation, the inoculated seedlings showed reddish-brown spots and typical anthracnose lesions on inoculated leaves, with C. aenigma successfully reisolated from symptomatic tissues. To the best of our knowledge, this is the first report of C. aenigma causing anthracnose leaf spot on V. amoena in China. Although C. aenigma has previously been associated with anthracnose in crops such as grapes (Kim et al. 2021), mulberry (Zhu et al. 2023), and pecan (Zhao et al. 2024), its identification as a pathogen of V. amoena highlights a significant threat to this plant species in China. This finding underscores the urgent need for targeted disease management strategies to prevent further spread and damage, particularly in regions where V. amoena is economically or ecologically important.