First report of Mochi tree (Ilex integra) Anthracnose caused by Colletotrichum fioriniae in Korea.

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Ilex integra, also called Mochi tree, is an woody ornamental common in Asia, particularly in Korea, China, Japan, and Taiwan. Anthracnose, caused by Colletotrichum spp., is an economically important disease worldwide, affecting both fruit and seed quality. In April 2019, symptoms of Anthracnose were observed on leaves from several Mochi trees in an urban planting in Wando-gun, South Korea. Irregularly shaped, light-to-dark brown spots of 1-4mm were observed on young leaves. The lesions coalesced as each spot enlarged, flat and black fruiting bodies (acervuli) occurred on the brown lesions. Four symptomatic leaves were collected; fractions were cut from symptomatic tissue, including healthy tissue, then were disinfected with 1% sodium hypochlorite and 70% ethanol, and placed on potato dextrose agar (PDA). After dark-incubation at 25℃ for 7 days two isolates were obtained, the fungal colonies appeared as white to light gray mycelium, then becoming dark and orange to pink on the underside. After acervuli were produced on the plate, orange-red conidial masses erupted. Conidia observed from two isolates were hyaline, 1-celled, and oblong with round to acute apices, and measured 7 to 12 × 2 to 5 μm (mean ± SD: 9.29 ±2.26 × 3.68± 1.31 μm) (n=30). Genomic DNA was extracted and multi-locus sequencing was performed with one representative isolate using the internal transcribed spacer (ITS) (White et al. 1990), actin (ACT) genes, chitin synthase 1 gene (CHS-1) (Carbone and Kohn 1999), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Calmodulin (CAL) (Weir et al. 2012) and submitted. Blast search results showed that the isolate had 100%, 98.45%, 99.74%, 100%, and 100% nucleotide sequence identity with those of C. fioriniae (MT607651, MH717601, MG666441, MN895544, MN974144) respectively (Jamin and Mateu 2008). The five sequences were deposited in NCBI GenBank (Accession No: MT457472, MT465884, MT465885, MT465886, MT465887), which were assigned to ITS, ACT, CHS-1, GAPDH, and CAL regions, respectively. Based on the morphology (Shivas and Tan 2009) and molecular characterization (Guerber et al. 2003), the isolate was identified as C. fioriniae. To confirm pathogenicity, a conidial suspension (10⁶ conidia/ml) of the sequenced isolate was used to inoculated, young and mature leaves of a 4-year-old Mochi tree. Ten leaves of the seedling were disinfected with 70% ethanol, then were wounded with a toothpick. The conidial suspension (20 µl) was placed on the wound. The inoculated plant and control plants were tested with sterilized water and incubated at 25℃ in a moist chamber. The pathogenicity test was repeated three times. Typical spots were observed on the young leaves 2 days after inoculation, whereas they were observed on the mature leaves 7 days after inoculation. Acervuli developed on both young and mature leaves 5 and 20 days after treatment, respectively. The control plants did not show symptoms, and the fungus was re-isolated from the inoculated plant; thus, fulfilling Koch's Postulates. In Korea, C. fioriniae has been recorded as a pathogen of fruit (apple, eggplant and peach), but this is the first report of the fungus causing anthracnose on Mochi tree. The pathogen has been reported on leaves of a different Ilex species in the eastern USA (Farr and Rossman 2020). Although this new disease of I. integra is limited occurrence, C. fioriniae may be able to infect other plant species in South Korea.