First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Abstract

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 μm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.