First Report of Colletotrichum nymphaeae Causing Anthracnose on Ampelopsis grossedentata in China.

Abstract

Ampelopsis grossedentata, known as vine tea, is a perennial woody vine traditionally used for making herbal tea from its tender leaves and stems in southern China (Wu et al. 2023). In recent years, its plantations have been devastated by pathogens such as Pestalotiopsis and Diaporthe, significantly impacting the quality of its products (Yuan et al. 2022; Liu et al. 2024). In May 2024, 135 symptomatic samples exhibiting shoot blight, branch dieback, canker, and leaf spot were collected from four tea plantations (approximately 130 acres) in Yongshun County, Tujia and Miao Autonomous Prefecture, Xiangxi, Hunan Province, China. Disease incidence in the tea plantation was almost 100%, with yield reductions varying from 80 to 100%. Samples from the diseased-healthy tissue junction were treated with 75% ethanol for 30 s and then washed three times in sterile water. They were placed on PDA at 25°C in the dark. After 5 d, 34 isolates out of 135 samples with the same morphology were obtained (isolation frequency was 5.9%, 14.3%, 17.2%, and 23.4% from four tea plantations, respectively), with a colony morphology different from Pestalotiopsis and Diaporthe, and three isolates (AG-CN-4, AG-CN-5, and AG-CN-6) were selected for further analysis. After 7 d of incubation on PDA, fungal colonies were white to gray, and pale gray to pale orange with black spots on the lower surface. On the 25th day, the surface view showed gelatinous orange conidial masses. The conidia were unicellular, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with both ends rounded or one end acute, and measuring 6.4 to 13.1 × 2.6 to 5.4 μm (mean 8.3 ± 1.6 × 3.6 ± 0.5 μm, n = 40). Culture and conidial morphology were in concordance with published descriptions of Colletotrichum acutatum sensu lato (Damm et al. 2012). Sequences of the internal transcribed spacer, β-tubulin, chitin synthase, and glyceraldehyde 3-phosphate dehydrogenase genes from the three isolates were then amplified (Weir et al. 2012) and deposited in GenBank (accession nos. PP922606, PP987161-PP987163, PQ813639-PQ813640, PQ815057-PQ815062). A Maximum Likelihood phylogenetic tree was generated by combining all sequenced loci in MEGA7 (Kumar et al. 2016). Three isolates were classified in the C. nymphaeae clade with 99% bootstrap support. Both morphological and molecular characteristics confirmed that our isolates were highly similar to C. nymphaeae. We used Koch's postulates to verify these results. Four two-year-old potted plants were inoculated with spore suspensions (1 × 10⁶ conidia/ml) from each isolate using an atomizer, while control plants were sprayed with distilled water. The plants were maintained in an incubator at 25 ± 1°C, 95 ± 5% relative humidity, and a 14/10 h light/dark cycle. After 7 d, the twigs and leaves inoculated with C. nymphaeae withered, and some leaves exhibited sunken necrotic lesions with brown centers, enclosed by dark brown margins. The morphology of the reisolated cultures was consistent with the test isolate, fulfilling Koch's postulates. In contrast, the controls were asymptomatic and no pathogens were isolated. The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut (Ma et al. 2022) and peaches (Tan et al. 2022) in China. To our knowledge, this is the first report of C. nymphaeae infection on A. grossedentata in China, signaling a potential new threat that growers should monitor vigilantly.