First Report of Leaf Anthracnose Caused by <i>Colletotrichum fructicola</i> on <i>Tetrastigma hemsleyanum</i> in China

Tetrastigma hemsleyanum in the family Vitaceae, is a rare and endangered medicinal plant endemic in China (Ji et al. 2021). In October 2024, leaf anthracnose was observed in Lishui city (118°96'E, 28°13'N), Zhejiang, affecting T. hemsleyanum plantings over an area of 5.3 × 103 m2. Disease incidence ranged from approximately 30 to 60%. Early symptoms were small circular or irregular brown spots of foliage, gradually expanding in size, then coalescing to form large irregular dark brown spots with grayish white centers causing leaves to senesce and resulting in plant death in severe cases. Leaf pieces (5×5 mm) from nine symptomatic leaves were surface disinfected with 75% ethanol for 30 s, 2.5% NaClO for 1 min, rinsed in sterile water three times, dried, placed on potato dextrose agar medium, and cultured in darkness at 28°C for 5 days. Five isolates (THP10 to THP14) were obtained by the hyphal-tip method from the nine leaves. The colonies were olivaceous to dark gray with white margins and cottony mycelium; reverse sides had black centers. Conidia were single, colorless, cylindrical, 13.37 to 17.89 × 3.91 to 5.73 μm (average 15.42 × 5.11 μm; n=50). The morphological characteristics of the isolates overlapped with those of Colletotrichum species within the C. gloeosporioides complex, including C. fiucticola (Weir et al. 2012). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS)，calmodulin (CAL), actin (ACT), and beta-tubulin 2 (TUB2) genes were amplified using ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, CL1C/CL2C, ACT-512F/ACT-783R, and T1/Bt2b primer sets, respectively (Weir et al. 2012). Sequences were deposited in GenBank with accession Nos. ITS: PQ571715 - PQ571719; GAPDH: PQ593912 - PQ593916; CHS: PQ593906 - PQ593910; CAL: PQ593900 - PQ593904; ACT: PQ593894 - PQ593898; TUB2: PQ593918 - PQ593922. BLASTn analysis of THP10 sequences had highest matches to the type strain of C. fructicola ICMP 18581 with ITS sequences 100% identical (JX010165; 549/549 bp), GAPDH sequences 99% identical (JX010033; 267/269 bp), CHS sequences 100.00% identical (JX009866; 274/274 bp), CAL sequences 100% identical (JX009676; 731/731 bp), ACT sequences 99% identical (JX009501; 270/272 bp), and TUB2 sequences 100% identical (JX010405; 699/699 bp). A maximum likelihood phylogenetic tree was constructed with the combined sequences data sets using MEGA 11, and the five isolates clustered with C. fructicola (Weir et al. 2012). To test pathogenicity, five isolates of C. fructicola were evaluated, leaves on three healthy 6-month-old potted T. hemsleyanum seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelial plugs. Sterile PDA plugs served as controls. After inoculation, the plants were incubated at 28°C, 85% relative humidity, with a 12 h photoperiod. The experiment was repeated three times. Symptoms similar to those from the field were observed 21 days after inoculation, whereas control leaves remained asymptomatic. The pathogen was successfully re-isolated from the symptomatic leaves and identified by morphology and ITS, GAPDH, CHS, CAL, ACT, TUB2 genes, completing Koch's postulates. No pathogens were isolated from the control plants. C. fructicola has been reported to cause anthracnose in many medicinal plants (Hou et al. 2024; Kang et al. 2023). This is the first report of C. fructicola causing leaf anthracnose on T. hemsleyanum in China. The accurate identification of the pathogen will provide a basis for the prevention and control of leaf anthracnose on T. hemsleyanum in the future.