Taehyun Chang

First Report of Anthracnose of Persimmon (Diospyros kaki L. f.) Caused by Colletotrichum siamense in Korea

Sweet persimmon (Diospyros kaki L. f.) is one of the most popular fruits in the world, owing to its fabulous taste. It is the most cultivated fruit crop in almost all the states of South Korea. Anthracnose of persimmon caused by Colletotrichum species results in both pre- and postharvest fruit decay with reduced yield and quality. The Colletotrichum species causing persimmon anthracnose reported from Korea include Colletotrichum gloeosporioides, C. acutatum, and C. horii (Kwon et al. 2011, 2013). In October 2015, persimmon fruits showing anthracnose disease symptoms were collected from an orchard in Cheongdo-gun, north Gyeongsang province, South Korea. The causal agents were isolated from the necrotic lesions. A small piece of necrotic tissue was cut from inside of the lesion of the diseased fruits, surface disinfected with 0.5% sodium hypochlorite solution for 2 min followed by rinsing with sterile distilled water, and placed onto Petri plates containing potato dextrose agar (PDA) amended with streptomycin (0.05 g/liter). Plates were incubated at 25°C for 4 days in the dark, and a pure culture was obtained by subculturing the hyphal tip of each isolate on fresh PDA plates. Seven-day-old colonies had white aerial mycelium with olivaceous gray mycelium below. The colonies were creamy white when observed from the reverse side. Hyphae were multinucleated and septate. Conidia were hyaline, cylindrical, straight or slightly curved, nonseptate, 12.3 to 18.6 × 3.9 to 6.6 μm (n = 25) (average 15.8 × 5.3 μm), and obtuse at both ends. Appressoria were ovate, globose, or ellipsoidal, 11.7 to 19.3 × 5.4 to 12.1 μm (n = 25) (average 14.5 × 7.6 μm), and dark brown in color. The morphological characteristics of the present isolate overlap with those of other species within the C. gloeosporioides complex including C. siamense (Weir et al. 2012). For accurate identification, the genomic DNA sequences were obtained from internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) genes and deposited in GenBank. A BLAST search (National Center for Biotechnology Information) of the resulting sequence of ITS (LC208833) showed 611/619 (99%) similarity with the sequence from C. gloeosporioides strain 16-412 (GenBank accession no. LC194223). GAPDH (LC208834) showed 280/280 (100%) similarity with the sequence from C. siamense strain Xt-18-1 (KM053171), and CAL (LC208835) showed 748/756 (99%) similarity with the sequence from C. siamense strain LF177 (KJ954645). Separation of C. siamense from other Colletotrichum species based on ITS is not reliable (Weir et al. 2012). Phylogenetic analysis using MEGA6, based on the GAPDH, CAL, and ApMat (LC307174) gene, showed that the present isolate clustered with the C. siamense strain, with high bootstrap support (>75%). Based on cultural characteristics and sequences similarity data, the current isolate associated with anthracnose disease of D. kaki was identified as C. siamense. Koch’s postulates were performed by spraying the conidial suspensions (10⁵ conidia/ml) on healthy and ripe fruits. Tiny (pin-headed) black spots were observed on the surface of the inoculated fruits after 3 to 5 days of inoculation. The fungus was successfully reisolated and identified as C. siamense according to the methods described above. C. siamense has been reported as a causal agent of anthracnose of Perseaamericana, Pistaciavera, Sarcandraglabra, Hymenocallis sp., Corchorus sp., Prunus persica, and Capsicum sp. (De Silva et al. 2017; Hu et al. 2015; Weir et al. 2012). To our knowledge, this is the first report of C. siamense causing anthracnose of persimmon in South Korea.

Molecular and Morphological Characterization of Colletotrichum species in the Colletotrichum gloeosporioides Complex Associated with Persimmon Anthracnose in South Korea

Anthracnose is a major disease of persimmon in the pre- and postharvest phase. Several species of Colletotrichum (Colletotrichum gloeosporioides, C. acutatum, and C. horii) have been reported as causal agents of persimmon anthracnose in South Korea. In this study, a collection of 50 isolates associated with persimmon anthracnose were collected from Sangju (n = 25) and Cheongdo-gun (n = 25), South Korea. The morphological characteristics of all 50 Colletotrichum isolates were similar, and it was difficult to identify the isolates to the species level. A subsample of eight isolates was characterized phylogenetically to ascertain species. BLAST search and phylogenetic analysis of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) genes revealed two species: C. horii as well as a previously unreported persimmon anthracnose causal agent C. siamense. C. siamense isolates were confirmed again by phylogenetic analysis of the ITS, ACT, GAPDH, calmodulin, and Apn2-Mat1-2 intergenic spacer partial mating type genes. Kochs postulates for C. horii and C. siamense were fulfilled, confirming the pathogenicity of the two species in persimmon fruit. Morphological characteristics (colony morphology and size and shape of conidia and appressoria) from two representative isolates support results of the phylogenetic analysis and match those of previous descriptions of C. horii and C. siamense.