First Report of <i>Colletotrichum liriopes</i> Causing Anthracnose on <i>Viola tricolor</i> in China

In June 2025, typical anthracnose symptoms were observed on V. tricolor grown on the campus of Jiangxi Agricultural University (Nanchang, Jiangxi, China), with a disease incidence of approximately 60%. Symptoms began as water-soaked white spots with light-yellow margins, expanding into sunken, necrotic lesions that caused defoliation. Under high humidity, lesions developed black acervuli producing pink, mucilaginous conidial masses. For pathogen isolation, symptomatic leaf pieces (5 × 5 mm) were surface-sterilized with 70% ethanol for 30 s and 1% sodium hypochlorite for 30 s, followed by three rinses with sterile distilled water, then cultured on potato dextrose agar (PDA) at 28 °C for 3–4 days. Hyphal tips were subcultured for purification. One isolate (JFRL 03-5504), representative of ten morphologically consistent Colletotrichum spp. isolates, was selected for further study. On PDA, colonies were white to pale greenish-gray with dense aerial mycelium on the front, and dark gray on the reverse. Conidia were hyaline, aseptate, curved or slightly curved, measuring 13.8-23.4 μm × 2.4-6.1 μm (mean 17.5 × 4.1 μm, n=40). To confirm the taxonomic identity of the pathogen, DNA sequencing was conducted for partial sequences of the internal transcribed spacer (ITS) region and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT), and β-tubulin 2 (TUB2) genes as described previously (Damm et al. 2009). The generated sequences were deposited in GenBank with the following accession numbers: ITS, LC899029; GAPDH, LC901570; CHS-1, LC901567; HIS3, LC901568; ACT, LC901566; TUB2, LC901569. BLASTn analysis of these sequences showed 99-100% identity to corresponding genes of the C. liriopes ex-type strain CBS 119444. Maximum-likelihood (ML) phylogenetic analysis was conducted using PhyloSuite v1.2.2 software, based on the concatenated ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2 dataset. The analysis placed isolate JFRL 03-5504 in a well-supported clade (bootstrap value = 100%) alongside C. liriopes reference strains CBS 119444 and CBS 122747. Based on combined morphological and molecular data, the isolate was conclusively identified as C. liriopes (Damm et al. 2009). For the pathogenicity assay, six healthy leaves of two 1-year-old potted V. tricolor were gently wounded with a sterile needle and inoculated with 5-mm-diameter mycelial plugs from 7-day-old PDA cultures. Six additional leaves were inoculated with sterile PDA plugs as controls. All inoculated plants were incubated at 25 °C under 90% relative humidity (RH) with a 12-h light/12-h dark photoperiod. After 6 days, the inoculated leaves developed water-soaked, necrotic lesions, while the control leaves remained asymptomatic. The fungus was successfully reisolated from the symptomatic lesions, and the reisolated strain was confirmed as C. liriopes by morphological and phylogenetic analyses, thereby fulfilling Koch’s postulates. C. liriopes has been previously reported to infect Ophiopogon japonicus (Yi et al. 2022), Rohdea japonica (Zhu et al. 2023), Liriope spicata (Chen et al. 2019), and Fagopyrum esculentum (Chen et al. 2021). To our knowledge, this is the first report of C. liriopes causing anthracnose on V. tricolor in China, providing a basis for disease diagnosis and management.