Anthracnose Leaf Spot on <i>Hymenocallis littoralis</i> Caused by <i>Colletotrichum endophyticum</i> in Guangxi, China

Hymenocallis littoralis (Jacq.) Salisb. is a secondary protected plant in China with high ornamental value (Nadaf et al. 2018) and widely distributed in Guangxi. In March 2023, 100 % of H. littoralis plants and over 80 % leaves of each plant showed leaf spot in three parks located in Nanning (N22°80'-22°82', E108°30'-109°33'), Guangxi, China. In the early stages, the edges and central areas of the lesions were yellowish and green, respectively. Later, the lesions became reddish-brown to brown in central areas and yellowish at the edges. A pathogen was isolated from three diseased leaf samples that were collected from the three parks, respectively. Tissues were cut from infected margins, sterilized, and placed on potato dextrose agar (PDA) at 25 °C. After 5 days, twenty-four isolates with similar morphology were retained. Three strains BJB7-1, BJH5-1, and BJH3-1, one from each of three parks, were selected for further studies. On PDA, the isolates exhibited white colonies, with single-celled, hyaline, and cylindrical conidia which measured 12.3 to 14.0×2.0 to 3.8 μm (n = 90). The appressoria were single, brown or black, and irregular in shape, and measured 7.6 to 8.6×6.1 to 6.9 μm (n = 90). These morphological characteristics were similar to Colletotrichum spp. (Damm et al. 2019). The ITS region, and ACT, CAL, CHS-1, GAPDH, and TUB2 genes were sequenced for further identification (Silva et al. 2019). All sequences were deposited in GenBank (ITS: PQ218080 to PQ218082, ACT: PQ219629 to PQ219631, CAL: PQ219632 to PQ219634, CHS-1: PQ219635 to PQ219637, GAPDH: PQ219638 to PQ219640, TUB2: PQ219641 to PQ219643). BLASTn analysis of sequences of three strains exhibited 99% similarity with C. endophyticum (strains LC 0324) NR160814.1 of ITS (over 99%), KF306258.1 of ACT (97-99%), KC832854.1 of GAPDH (over 98%), and MZ673954.1 of TUB2 (over 99%). Based on the concatenated sequence ITS-ACT-CAL-CHS-1-GAPDH-TUB2, a phylogenetic tree was constructed using RAxML version 8.2.10, and the three strains clustered with the type strain C. endophyticum LC 0324. Pathogenicity was performed on healthy, tender, and attached leaves of five-month H. littoralis plants with and without wounds by inoculating the leaves with 20 μL (106 conidia/mL), and blank control was inoculated with 0.05% Tween-20. The plants were kept in a greenhouse at 25°C and covered with plastic bags to maintain humidity. After 9 days, all wounded and inoculated leaves showed symptoms, while the unwounded and control remained asymptomatic. The pathogenicity test was repeated three times with similar results. Koch's postulates were completed by reisolating from inoculated and non-inoculated tissues. C. endophyticum was only recovered from inoculated tissues, and identity of the fungus was confirmed based on morphology and multigene sequences. C. siamese (Huang et al., 2021) and C. gloeosporioides (Zhao et al., 2019) were reported on H. littoralis in China. C. endophyticum could cause anthracnose leaf spot on Capsicum annuum (Noor et al., 2018), Camellia sinensis (Wang et al., 2016), and coffee plant (Cao et al., 2019). To the best of our knowledge, this is the first report of anthracnose leaf spot caused by C. endophyticum on H. littoralis. The latter is a very important garden plant, but may be susceptible to leaf spots, thus affecting its ornamental value. Therefore, this investigation can contribute to effective management of the disease.