First Report of Colletotrichum gloeosporioides Causing Anthracnose on Grapevine (Vitis vinifera) in Shaanxi Province, China

Grape anthracnose caused by Colletotrichum spp. is an economically important disease in most vineyards during rainy and humid weather. In June 2021, widespread anthracnose was observed in leaves of Vitis vinifera cv. Red Globe in Yangling city (latitude = 34.28, longitude = 108.07), Shaanxi Province. Diseased plants exhibited circular brown necrotic lesions with yellow borders in young leaves (Fig. 1A). Approximately 80% of the 75 grape plants showed disease symptoms that were consistent with those previously reported for anthracnose on grape caused by C. acutatum, C. gloeosporioides, and C. viniferum (Hong et al. 2008; Oo and Oh 2017). To isolate the pathogen, ten diseased leaves were collected. Small pieces (0.5 mm × 0.5 mm) of symptomatic tissue were surface sterilized with 75% alcohol for 30 s, transferred to 4% NaClO for 2 min, and then washed twice with sterile distilled water. The pathogen was isolated and cultured on potato dextrose agar (PDA) at 25 °C with a 12 h light/dark photoperiod. Hyphal tips of the isolate were transferred to fresh PDA after 1 day. Acervuli were formed after 15 days on PDA. The colonies were single-spored to obtain pure cultures. Pure cultures on PDA appear white to grey, and the reverse of the colonies was olive gray or yellowish white (Fig. 1B, C). Conidia were single-celled, hyaline, straight cylindrical with ends rounded, and measured 12.50 to 17.78 μm (length) (mean=15.19 μm, n=50) × 3.66 to 5.82 μm (width) (mean=4.48 μm, n=50) (Fig. 1D). The morphological characteristics matched the previous descriptions of C. gloeosporioides by Sawant et al. (2012). For molecular identification, genomic DNA was extracted from isolate YLRG13, and the actin (ACT), internal transcribed spacer (ITS), and beta-tubulin (TUB2) were amplified using the primer pairs ACT-512F/ACT-783R (Carbone and Kohn 1999), ITS1F/ITS4 (White et al. 1990), and T1/T2 (O' Donnell and Cigelnik 1997), respectively. The ACT (OQ031183 and MZ686707), ITS (OP999326 and MZ669974), and TUB2 (OQ031184 and MZ686708) nucleotide sequences of isolates YLRG5 and YLRG13 were deposited in GenBank. BLASTn showed 99 to 100% similarity with C. gloeosporioides. Neighbor-joining trees based on the three genes was constructed using PAUP version 4.0b10. The results showed the isolated YLRG5 and YLRG13 from V. vinifera was closely related to C. gloeosporioides with high bootstrap values (Fig. S2). Pathogenicity testing was performed with isolate YLRG13 by 10 μL conidial suspension (4×108 conidia/ml) on the surfaces of five wounded detached leaves of six-year-old Vitis vinifera cv. Red Globe. Sterile distilled water was used as a control. Inoculated plants were placed in a humid chamber at 25 °C. Conidial germination was observed at 24 hours post-inoculation (Fig. S1E). Four days after inoculation, typical anthracnose symptoms developed on inoculated grape leaves (Fig. S1F). No symptoms were observed on the controls. The fungus reisolated from the symptomatic grape leaves showed the same morphological characteristics of the inoculated isolate, fulfilling Koch's postulates. To our knowledge, this is the first report of grape anthracnose caused by C. gloeosporioides in Northwestern of China. C. gloeosporioides was reported causing anthracnose on a wide variety of horticultural crops, such as almond, avocado, apple, banana, cashew, citrus, cucumber, grape, guava, mango, onion, papaya, passion fruit, pepper, strawberry, tomato, and watermelon (Sharma and Kulshrestha 2015), suggesting the potential cross-infection between grape and other horticultural crops.