First Report of Colletotrichum theobromicola Causing Anthracnose on Euonymus japonicus in China.

Abstract

Euonymus japonicus, a broad-leaved evergreen tree, is widely planted in the parks and landscapes in the world (Huang et al. 2016). In 2019, anthracnose lesions were found on leaves of E. japonicus with 4~15% incidence from the Wufulinglong park (116°28' E, 39°94' N) in the Haidian District of Beijing, China. Irregular chlorotic spots appeared on the surface of the leaves, then larger necrotic spots with numerous acervuli gradually formed, and finally the infected leaves dried and dropped, detracting from the aesthetic of the landscape. To identify the pathogen, six representative leaves with typical symptoms in Wufulinglong park from three plants were collected and diseased tissue was cut into 2 mm × 2 mm pieces, disinfested in 75% ethanol for 20 s, rinsed twice in sterile water, plated onto potato dextrose agar (PDA), which were then incubated at 25℃ under 12 h photoperiod. A total of 24 morphologically identical isolates were obtained from the samples. Two of these isolates were randomly selected for further analysis to confirm their identity. The cultures (HDwfll1907HY and HDwfll1908HY) were deposited in the culture collection of the Beijing Academy of Agriculture and Forestry Sciences, China. Furthermore, genomic DNA was extracted and the sequences of internal transcribed spacer (ITS) regions, calmodulin (CAL), beta tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified using the primers ITS1/ITS4 (Gardes et al. 1993, White et al. 1990), CL1C/CL2C (Weir et al. 2012), T1/Bt2b (Glass et al. 1995, O'Donnell et al. 1997), GDF1/GDR1 (Guerber et al. 2003), ACT-512F/783R and CHS-79F/345R (Carbone et al. 1999), respectively. Newly obtained sequences were deposited into GenBank with accession numbers MZ229612~13 and MZ305422~31. Phylogenetic tree based on the assessed gene loci revealed that both strains clustered closely with C. theobromicola. The strain HDwfll1907HY was randomly selected for morphological description and pathogenicity assay. The colony was initially light gray with dense aerial mycelia on PDA, which later turn to gray with grayish green on the reverse side of plates. Conidiogenous cells were cylindrical, arising from swollen hyphae. Conidia were single-celled, hyaline, subcylindrical to clavate, often with broadly rounded ends, 13.78-21.99×3.47-5.44 µm (n=50). Both phylogenetic analysis and morphology support the identification of two strains as C. theobromicola. Pathogenicity tests were performed on three healthy one-year-old E. japonicus plants using the randomly selected isolate HDwfll1907HY. The leaves were sprayed with 20 mL of conidial suspension (107 conidia/mL), sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the incubator at 25°C and 90% relative humidity under 12 h photoperiod. After 18 d, all the leaves treated with the conidial suspension showed typical symptoms of anthracnose, similar to those in the field, whereas the control leaves remained symptomless. The disease assay was replicated three times for consistency. The same fungus was reisolated from the infected leaves and identified as C. theobromicola. This is the first report of C. theobromicola causing anthracnose on E. japonicus, which provides the foundation for the management of anthracnose on E. japonicus.