First Report of Colletotrichum fructicola Causing Anthracnose on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Abstract

Epimedium sagittatum (Sieb.et Zucc.) Maxim., belonging to the family Berberidaceae and genus Epimedium, is a perennial herb widely studied for its anti-osteoporosis, anti-cancer, and anti-sexual-dysfunction effects in Asian countries (Tan et al. 2016; Zhang et al. 2016). High levels of bioactive chemicals in Epimedium spp. has endowed it with important clinical and commercial values (Liu et al. 2013). In September 2021, a leaf disease was found in Zhumadian City, China (32°58'12" N, 114°37'48" E). Survey statistics indicated that disease prevalence in a 266-ha planting area was approximately 29.6%. The lesions appeared at the leaf tips, gradually enlarged, and were brown with a yellow halo. Further, the lesions were dry with distributed black spots. Thirty infected leaves collected from five sites within the planting base . The collected leaves were cut into 5×5 mm pieces , surface-sterilized in 75% alcohol for 15 s, triple washed with sterile ddH2O, disinfested with 0.1% HgCl2 solution for 30 s (Liu et al. 2021), triple washed again with sterilized ddH2O, and then placed onto PDA and incubated in the dark for 3 d at 28°C. Subsequently, five fungal strains were purified; among them, only the isolate HY3-2 infected the host plant and was selected for further morphological characterization. The colonies of HY3-2 initially appeared white, their mycelia became gray at the center after 4 d, and orange-red conidial clumps appeared in them after 7 d. Conidia (10.0-19.5 μm × 4.5-5.6 μm, n=50) were single celled, nearly spherical or stick-shaped and colorless. Morphological characteristics of the isolate were consistent with those of Colletotrichum species. Additionally, glycerol-3-phosphate dehydrogenase (gapdh), actin (act), calmodulin (cal), β-tubulin 2 (tub2), and chitin synthase-1 (chs-1), (Weir et al. 2012) were amplified and sequenced using the primers GDF/GDR, ACT-512F/783R, CL1C/CL2C, T1/Bt2b, and CHS-79F/354R, respectively for molecular identification. The resulting sequences were deposited in GenBank: gapdh (ON351609), act (ON351608), tub2 (ON351610), chs-1 (ON532788), and cal (ON532787). Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method (Zhang et al. 2020). The phylogenetic tree showed that the isolate belongs to C. fructicola clade with a credibility value of 85%.To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the isolate HY3-2 were prepared with sterile ddH2O to infect the leaves. Ninety healthy leaves from 30 plants in pots were punctured using a sterilized needle (Huang et al. 2022), and inoculated by spraying the conidial suspension on the leaves in a greenhouse at 25°C and 80% relative humidity. In the control plants, the suspension was replaced with water. After 7 d, the inoculated plants showed symptoms similar to those of the original infected plant, whereas the control showed no symptoms. C. fructicola was isolated and identified again as previously described. A pathogenicity test was also conducted in the field using the same method as that used in the greenhouse in July 2022, the results of which were consistent with those of the greenhouse. In China, C. fructicola has been reported on Walnut (Wang et al. 2022), Punica granatum (Hu et al. 2023) and others. To our knowledge, this is the first report of C. fructicola causing anthracnose in E. sagittatum in China. This report provides an important basis for further disease control research.