First Report of Anthracnose of Japanese Plum (Prunus salicina) Caused by Colletotrichum nymphaeae in Korea

Japanese Plum (Prunus salicina) is an important stone fruit, rich in sucrose, fructose, glucose, anthocyanins, and vitamins. Yield and quality of Japanese plum fruits can be negatively impacted by various factors including anthracnose, which causes pre- and postharvest fruit decay. In October 2017, Japanese plum fruits showing anthracnose were collected from Daegu Market, South Korea. Lesions on the surface of affected fruits were brown, round, sunken, and around 16 mm in diameter. The causal agents were isolated by a single spore isolation technique (Cai et al. 2009) from the necrotic lesions. The spore suspension was prepared by transferring spore masses from lesion to sterilized water with a sterilized wire loop. The spore suspension, after diluting to a reasonable concentration (10⁴ conidia/ml) was spread onto the surface of PDA, followed by an incubation overnight at 25°C. Single germinating spores were picked up with a sterilized needle and placed on to fresh PDA plates. Plates were incubated at 25°C in the dark. The 1-week-old culture color was gray on the upper side with whitish aerial mycelium and interspersed areas of off-white and dark brown on the reverse side with black dots. Conidia were hyaline, smooth-walled, one-celled, cylindrical, 10.34 to 18.6 × 3.8 to 6.9 µm (n = 35) (avg. 15.8 ± 2.08 × 5.3 ± 0.72 µm), with one end round and one end slightly acute or both ends round. Appressoria single or multiple in chain, oval, medium brown, and 8.12 to 15.2 × 4.5 to 9.3 µm (n = 25) (avg. 11.5 ± 2.1 × 7.2 ± 1.2 µm). The morphological characteristics of the present isolate match with some Colletotrichum spp. within the Colletotrichum acutatum complex including C. nymphaeae (Damm et al. 2012). Phylogenetic relationships were determined for the representative isolates (PAM-4) of collected Colletotrichum isolates. DNA of the PAM-4 isolate was extracted and five gene regions including the ITS, TUB2, GAPDH, CHS-1, and ACT were amplified using primers ITS1/ITS4, Bt2a/Bt-2b, GDF/GDR, CHS-79F/CHS-354R, and ACT512F/ACT783R, respectively (Damm et al. 2012; Weir et al. 2012). Sequencing of the purified PCR products were accomplished by Macrogen Inc., Korea, and deposited in GenBank (accession nos. LC348957–LC348961). Neighbor-joining analysis (NJ) using MEGA6, based on the multilocus alignment (ITS, GAPDH, CHS-1, ACT, and TUB2), showed that the present isolate clustered with the C. nymphaeae strain, with high bootstrap support (>99%), as also suggested by the BLAST results. Based on cultural characteristics and sequence similarity data, PAM-4 was identified as C. nymphaeae (part of the C. acutatum species complex). In order to confirm pathogenicity, five fresh and healthy plum fruits were wound inoculated with a 10-µl droplet of conidial suspension (10⁵ conidia/ml); five fruits inoculated with sterile water were used as negative controls. Brown lesions (≥14.19 mm in diameter) were observed on the surface of all fruits inoculated with conidial suspension after 3 to 5 days of incubation at 25°C in the dark under moist conditions. No symptoms were observed on the negative control. The fungus was successfully reisolated and identified as C. nymphaeae according to the methods described above. C. nymphaeae was previously associated with anthracnose on Eriobotrya japonica, Nuphar luteum subsp. Malus domestica, polysepalum (Damm et al. 2012; Velho et al. 2014; Wu et al. 2018). To our knowledge, this is the first report of C. nymphaeae causing anthracnose of plum in South Korea.