First Report of Colletotrichum fructicola Causing Fruit Anthracnose on Akebia trifoliata in Sichuan Province, China

Bāyuèguā (Akebia trifoliata) is a recently domesticated horticultural crop in China, with nutrient-rich edible fruits containing sugar, vitamin C, and amino acids (Wang et al. 2022). In July 2025, anthracnose symptoms were observed on leaves and fruits of cultivated A. trifoliata ‵Shusen 17′ in Dazhou city (31.33°N, 107.54°E), Sichuan province, China, affecting a 10-hectare area with an incidence of 20%. Symptomatic samples were collected from 18 randomly selected plants across six sites (three plants per site). Leaf and fruit tissues adjacent to and including lesions were excised, superficially disinfected sequentially with 70% ethanol for 10 s and then 1% NaClO for 30–60 s, and washed six times with sterile dH2O. Sterilized tissues (7–8 pieces) were cultured on PDA containing 50 mg/L streptomycin sulfate in the dark at 25 ℃. After three days, three to five fungal colonies were observed on each PDA plate. Transferring hyphal tips from growing colonies' edges to fresh PDA yielded 30 purified isolates. Six isolates (Y8, Y9, Y10, Y11, H2-5 and B2-2) showed identical morphology. After 14-day culture on SNA, the upper surface and reverse side of the mycelia were white. Setae were pale to dark brown, aseptate, and 64.7 to 145.8 µm long, showing inflated or cylindrical base and acute tip. Asci were 68.1 ± 7.6 × 11.9 ± 1.9 µm in size (n = 20) in size, eight-spored, fasciculate and clavate. Meanwhile, colonies produced abundant conidia, rounded at the ends, measuring 19.4 ± 1.6 × 5.7 ± 1.3 µm (n = 30). Their morphology was consistent with that of Colletotrichum species (Liu et al. 2022; Xue et al. 2020). The other 24 isolates showed morphological differences, belonging to different genera. All the isolates were preserved in 20% (v/v) glycerol at -70 ℃. For the six isolates, rDNA internal transcribed spacer (ITS) regions, Actin (ACT), Beta-tubulin (TUB2), Chitin synthase (CHS), Histone3 (HIS), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) gene were amplified using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, T1/Bt2b, CHS-79F/CHS-345R, CYLH3F/CYLH3R and GDF1/GDR1 (Liu et al. 2022), respectively. The ITS (PX589098-PX589103), ACT (PX610115-PX610120), TUB2 (PX610139-PX610144), CHS (PX610121-PX610126), HIS (PX610133-PX610138) and GAPDH (PX610127-PX610132) sequences were deposited in GenBank. In BLASTn analyses, our ITS, ACT, TUB2, CHS and GAPDH sequences showed 98.81–100% identity to the ex-type strain of C. fructicola ICMP 18581. Based on the six DNA sequences, multi-locus phylogenetic analyses confirmed our isolates as C. fructicola. In pathogenicity test, healthy fruits were inoculated with conidial suspension (1 × 106 conidia/mL) of isolates B2-2, H2-5, and Y11, respectively. The control fruits were treated with sterile dH2O. Each treatment was incubated in a greenhouse (at 25 °C and 90% RH, with a 16/8 h light/dark cycle). The experiment was repeated three times. At five days post-inoculation, inoculated fruits exhibited anthracnose symptoms, unlike the controls. The reisolated pathogens from diseased fruits were morphologically and genetically identical to C. fructicola, fulfilling Koch's postulates. C. gloeosporioides and C. acutatum were reported as causal agents of anthracnose on A. trifoliata (Pan et al. 2021; Kobayashi et al. 2004). To our knowledge, this is the first report of C. fructicola causing fruit anthracnose on A. trifoliata worldwide. Our study will assist in developing target disease management strategies in A. trifoliata.