First Report of Colletotrichum aenigma Causing Anthracnose of Grape in Korea.

Grape (cv. Kyoho) is one of the most popular dessert fruits in South Korea. Anthracnose caused by Colletotrichum species is a common and very destructive disease of grape in the country. In 2019, severe outbreaks of anthracnose was observed in different grape orchards in Gimcheon (36º09´N, 128º00´ E), South Korea. The disease incidence on fruit was up to 50% in the orchards with most severe outbreaks and infected fruit displayed typical anthracnose symptoms including sunken necrotic lesions with orange-like conidial mass. For isolation of putative causal agents, nine diseased fruits were collected from three commercial orchards. A total of nineisolates were made from nine of the infected fruit by spreading spore masses (1x106 conidia mL-1) from each fruit on water agar and collecting single germinated spores after incubation at 25 ºC overnigh. The single germinated spores were transferred on to fresh potato dextrose agar (PDA) (Difco, Becton Dickinson) and incubated at 25ºC in the dark. Seven day old colonies were cottony white on the upper side and gray at the center on the reverse side. Conidia were cylindrical with round ends and measured 13.9 - 20.1 × 5.4 - 8.1 μm (mean = 16.5 × 6.6 μm, n = 30). Appressoria were brownish, sub-cylindrical with a few lobes and 10.3 -16.7 × 6.6 - 10.9 μm (mean = 13.1 × 8.1 μm, n = 30). The morphological characteristics of the solates resembled those of Colletotrichum species within the C. gloeosporioides complex (Weir et al. 2012). DNA was amplified using the following primer pairs: ITS1/ITS4, GDF / GDR, ACT-512F / ACT-783R, Bt2a/ Bt2b, and CHS79-F/CHS-354R (Weir et al. 2012). Accession numbers, LC586811 to LC586825 were obtained after depositing all the resulting sequences in GenBank. A 50% majority rules phylogenetic tree (Bayesian phylogenic analysis) was constructed based on concatenated sequences of ITS, GAPDH, ACT, TUB, and CHS using MrBayes 3.2.10. The present isolates formed a single clade with the reference isolates of C. aenigma (isolate ICMP 18608 and ICMP 18686). For a pathogenicity test, healthy grapefruits were collected from an orchards, surface sterilized by dipping in 1% sodium hypochlorite, rinsed with sterilized water and dried by blotting. A conidial suspension (1×106 conidia mL-1) in sterilized water were prepared from one week old colonies of isolates GRAP10 and GRAP12. A small wound was made on sterilized detached fruit by punching with a sterile pin. A drop of the conidial suspension was placed on the wound, while the control fruit received a drop of sterile water. Similarly, unwounded fruit were also inoculated with a single droplet of conidial suspension. For each isolate and method (wounded and unwounded), ten fruit were inoculated, and ten non-inoculated fruit were used as control. All the treated fruit were kept in a plastic box containing moist tissue and incubated at 25º C in the dark. Typical anthracnose lesions appeared on all inoculated wounded fruit while non-inoculated and inoculated unwounded fruits remained asymptotic. Koch postulates were fulfilled by re-isolating and re-identifying the causal agent from inoculated fruit. Colletotrichum aenigma has been reported as the causal agent of anthracnose on Juglans regia, Camellia sinensis and Actinidia arguta in China (Weir et al. 2012; Wang et al. 2016; Wang et al. 2018). Previous studies reported four Colletotrichum species (C. acutatum, C. gloeosporioides, C. fructicola, and C. viniferum) to cause this disease on grapes in South Korea (Oo and Oh 2017; Lim et al. 2020). To the best of our knowledge, this is the first report on grape anthracnose caused by C. aenigma in South Korea. This finding may help to take effective control measures of this disease.