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First Report of Anthracnose of Apple Caused by Colletotrichum fructicola in Korea

Kim, C., Hassan, O., Lee, D., Chang, T.
Plant disease 2018 v.102 no.12 pp. 2653
Colletotrichum fructicola, DNA, Glomerella cingulata, agar, anthracnose, apples, appressoria, conidia, crop production, cultivars, culture media, data collection, fruits, genes, hyphae, internal transcribed spacers, mycelium, orchards, pathogenicity, photoperiod, phylogeny, polymerase chain reaction, sodium hypochlorite, streptomycin, Korean Peninsula
In August 2017, a severe outbreak of anthracnose disease was observed in commercial apple orchards of cultivar Fuji in Sangju, Korea (Republic of). Fifteen apple fruits (from three commercial orchards) showing dark, circular, and necrotic lesions were collected from the field and brought to the laboratory. Small pieces (2 mm²) of necrotic tissue were cut with a sterile scalpel from the center of the lesion, surface sterilized with a 0.5% NaOCl for 2 min followed by three washes in distilled water, dried by blotting, placed on water agar Petri plates amended with streptomycin (0.05 g/liter), and incubated at 25°C in the dark. Hyphal tips from colonies emerging from the edge of the tissue were transferred onto fresh potato dextrose agar (PDA) Petri plates and incubated at 25°C in the dark. Morphological characteristics after 7 days of incubation on PDA showed light-gray to whitish aerial mycelium. The underside of the colony was gray with black zonation. Conidia were cylindrical, aseptate, hyaline, rounded at both ends, and 14.6 to 24.4 × 5.4 to 7.5 μm (average ± SD = 18.1 ± 2.2 × 6.4 ± 0.6 μm). Appressoria were ovoid, lobed margin, 8.6 to 17.8 × 5.6 to 14.33 μm (average ± SD = 12.3 ± 2.16 × 8.6 ± 1.8 μm), and brown. The morphological characteristics were consistent with descriptions of Colletotrichum species in the C. gloeosporioides species complex, including C. fructicola (Weir et al. 2012). Molecular identification was conducted by amplifying the ITS1-5.8S-ITS2 gene region and partial sequences of four gene regions (TUB2, ACT, CHS-1, and CAL) from genomic DNA of isolate ICKA 15. Primers used for DNA amplification of the different regions were ITS1/ITS4, BT2a/BT2b, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and CL1C/CL2C (Weir et al. 2012). Amplified polymerase chain reaction products were sequenced and deposited in GenBank (accession nos. LC381301 to LC381305). A neighbor-joining and maximum likelihood tree was constructed using MEGA version 6.0 (Tamura et al. 2013) based on a combined dataset of the ITS, TUB2, ACT, CHS-1, and CAL sequences of the present isolate (ICKA 15) and Colletotrichum spp. sequences previously deposited in GenBank. Phylogenetic analysis delineated the Korean isolate from apple as C. fructicola, thus complementing the morphological identification. For the pathogenicity test, eight surface-sterilized apple fruits were wounded at three equidistant positions with a sterile syringe needle, and then five fruits were inoculated with a drop of 10 µl of 1 × 10⁶ conidia/ml suspension of C. fructicola isolate (ICKA 15) and three fruits with sterile water as controls. Inoculated fruits were incubated in a moist chamber at 25°C under 16-h photoperiods. After 10 days of incubation, all fruits inoculated with C. fructicola conidia showed typical anthracnose symptoms. Fruits inoculated with distilled water remained symptomless. C. fructicola was reisolated and identified according to the above description, confirming Koch’s postulates. C. fructicola is known to cause anthracnose of coffee berries, apple, and pears in Thailand, Brazil, and China, respectively (Bragança et al. 2016; Jiang et al. 2014; Prihastuti et al. 2009). To the best of our knowledge this is the first report of C. fructicola anthracnose of fuji apple in Korea (Republic of). Fuji apple is the main cultivar produced in Korea, representing the of 70% of the total apple production area in 2016 to 2017.