First Report of Anthracnose on Euonymus japonicus Thunb. Caused by a Provisionally Novel Species of Colletotrichum in the Magnum Complex in South Korea.

Euonymus japonicus Thunb., also known as the evergreen spindle tree, is an evergreen tree, which is widely planted as a hedge plant along streets in South Korea. In April 2022, severe anthracnose symptoms were observed on the leaves of this tree in Jangsu in the Jeonbuk Province of the country (35°43'49.44″N, 127°34'53.7″E). About 80% of the leaves of each affected tree within a 0.03-ha area showed incidence of the disease on approximately 30 trees were planted along the roadside (~30 m). These symptoms typically included circular or irregularly shaped whitish-gray lesions with a diameter of 2.0 to 3.0 cm. In cases where some leaves were severely affected, larger blotches formed. To isolate the pathogen, about ten leaves showing anthracnose symptoms on each tree were randomly selected and brought to the laboratory. Fungal isolations were made from acervuli filled with conidial masses on infected evergreen tissues, followed by plating onto 2% potato dextrose agar (PDA) as well as incubated at 25℃. On the PDA, colonies were circular, raised, green-grey or dark grey, and had a distinct white margin. The conidia were single-celled, transparent, cylindrical with rounded ends, had smooth walls, with a length ranging from 12 μm to 16.7 μm and a width raging from 4 μm to 6.5 μm (av. = 14.1 X 5.0 μm, n=40). Of those that were successfully recovered with approximately 90% frequency, two monoconidial isolates were deposited to the culture collection at Chungnam National University in South Korea (Accession number: CDH059-060). To ensure the identity of the fungus, genomic DNAs were extracted from the selected isolates, CDH059-060, and were sequenced. This was achieved based on partial sequences of the internal transcribed spacer (ITS), actin and beta-tubulin (TUB2) gene regions which were amplified using ITS1F / ITS4 (Gardes and Bruns 1993; White et al. 1990), ACT-512F / ACT-783R (Carbone and Kohn 1999), and T1 / Bt2b (O'Donnell and Cigelnik 1997; Glass and Donaldson 1995) primer pairs, respectively. The resulting sequences were deposited to GenBank (OR984424-425) for ITS, (OR996289-290) for actin, and (OR996291-292) for TUB2. For a phylogenetic analysis, sequences from different gene regions (ITS, actin and TUB2) retrieved from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. The phylogenetic result revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Colletotrichum , which is most closely related to Colletotrichum liaoningense (Y.Z. Diao, C. Zhang, L. Cai & X.L. Liu, CGMCC3.17616 (KP890104 for ITS, KP890097 for actin, and KP890111 for TUB, Diao et al. 2017). Sequence comparisons revealed that this pathogen differed from C. liaoningense at 20 of 494 characters (∼4.0%) in the ITS and 2 of 251 (∼1.0%) in the actin sequences. For pathogenicity tests, three seedlings of E. japonicus were used. The leaves for each tree were treated with 10 ml of a conidial suspension by spraying (1x10⁶ conidia ml-1 of the isolate, CDH059), while the three seedlings were treated with distilled water as control. After sprayed, the treated areas were sealed with plastic bags for a day to maintain humidity. Anthracnose symptoms identical to those observed in the field appeared seven days after inoculations, while no symptoms were observed in the control. Re-isolations were successfully achieved from the treatments, fulfilling Koch's postulates. Anthracnose associated with the provisionally novel species of Colletotrichum sp. on E. japonicus has not been recorded elsewhere, and in this regard, this is the first report of anthracnose caused by Colletotrichum sp. on E. japonicus in Korea. To effectively control the disease, more attention should be paid to the host range of the pathogen and other regions where the disease caused by the pathogen might occur in the country.