Anthracnose of persimmon (Diospyros kaki) caused by Colletotrichum horii in Sangju, Korea

Sangju, South Korea, is famous worldwide for dried persimmon (gotgam) and contributes 60% to the nation’s total production of persimmon. However, one of the main problems in cultivating persimmon is anthracnose disease, which results in huge economic losses. Colletotrichum species such as Colletotrichum gloeosporioides, C. acutatum, and C. horii are implicated in causing persimmon anthracnose in Korea (Kwon et al. 2011, 2013). In 2013, diseased fruits showing typical anthracnose symptoms such as circular, sunken, and dark black spots were collected from an orchard in Sangju, and the presumed causative agents were isolated from them. Diseased tissue (1 cm²) was cut aseptically from inside of each lesion, surface-disinfected in 0.5% sodium hypochlorite solution for 2 min followed by three washes with sterile distilled water, subsequently placed onto potato dextrose agar (PDA) amended with streptomycin (0.05 g/liter), and then incubated at 25°C in the dark for 3 days. To obtain pure cultures, hyphal tips were transferred onto a fresh PDA plate. Morphological characteristics were monitored to identify the causal agent. Seven-day-old colonies were white to gray in color with conidia developing across colonies. The mycelia were hyaline and septate. Conidia were cylindrical, straight or slightly curved with round ends, single celled, and 14.5 to 22.5 × 4 to 6.5 μm (avg. 17 × 4.5 μm) (n = 25). Appressoria were short-cylindric, 9.3 to 13.8 × 5.0 to 8.6 μm (avg. 11.0 × 6.8 μm), and brown. The morphological characteristics of our isolates were mostly similar to those of Colletotrichum species in C. gloeosporioides species complex including C. horii (Weir and Johnston 2010; Weir et al. 2012). Genomic DNA was extracted from the isolates for molecular analysis. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) regions of genomic DNA were amplified using primer pairs ITS-1F/ITS4, GDF/GDR, and ACT-512F/ACT-783R, respectively. BLAST search analyses based on ITS gene sequence of the present isolates (LC186039-42) showed 100% similarity with the sequence from C. horii isolate OGaC1 (GenBank KU239182), ACT (LC186031-34) showed 100% similarity with the sequence from C. horii strain CPC 20992 (KC566947), and GAPDH (LC186035-38) showed 100% similarity with the sequence from C. horii strain C1069 (JX010001). Based on the combined dataset of ITS, ACT, and GAPDH, a molecular phylogenic analysis was performed using the neighbor-joining method in MEGA6. The studied isolates formed a clade with reliable C. horii strain relatives from GenBank with 100% bootstrap support. To confirm the pathogenicity of our isolates, pathogenicity tests were performed on mature persimmon fruits. Conidial suspension (10⁵ conidia/ml of ICK-111) was sprayed on the fruits (disinfected) until the suspension ran off. The fruits were placed in a sterile plastic box and incubated at 25°C in the dark. After 3 to 5 days of inoculation, black pin-headed spots were observed on the surface of inoculated fruits. The fungus was successfully reisolated and identified as C. horii (confirming Koch’s postulates). C. horii has been reported to cause anthracnose of persimmon in Gyeongnam Province of southern Korea, New Zealand, and Brazil (Kwon et al. 2011, 2013; May de Mio et al. 2015; Weir et al. 2012). To our knowledge, this is the first report of C. horii causing anthracnose of persimmon (Diospyros kaki L. f.) in Gyeongsangbuk-do Province and the second in South Korea.