RESUMO
Winter squash (Cucurbita maxima) is rich in vitamins C and B6 and is also a source of beta-carotene, a provitamin A carotenoid. About 13,000 tons have been produced annually in South Korea over the past 10 years. In the summer of 2022, severe rot was observed in winter squash for sale at a wholesale market in Jinju, South Korea, with approximately 10% of the 500 squashes observed affected. White fungal hyphae and dark orange spore masses were observed on the surface of the decayed squash. To isolate the causal agents, symptomatic tissues (3 × 3 mm) between diseased and healthy tissues per squash from 3 diseased squashes were excised, disinfested with 1% sodium hypochlorite for 20 s and 70% ethanol for 10 s, washed twice in sterilized distilled water, dried on sterilized filter paper, transferred to water agar, and incubated at 25°C for 2 days. Agar blocks (3 mm2) containing fungal colonies were transferred to potato dextrose agar (PDA) plates and incubated at 25°C until fungal colonies grew. Three isolates (GNU F137aâc) with similar morphology were subcultured using the single-spore method. In PDA, the colonies looked like gray cotton when viewed from the front, were pale orange from the back, and numerous small black sclerotia-like grains could be observed on both sides. Setae were pale to medium brown, verrucose, 40-120 µm long, and 3-6 septated. Conidiophores were hyaline to pale brown, smooth-walled, septate, branched, and up to 45 µm long. Conidia were hyaline, smooth walled, aseptate, straight, cylindrical, the apex and base rounded, and 14-18 × 5-7 µm (n = 30). Appressoria were single, brown, aseptate, ellipsoidal to irregular in outline, with crenate margins, and 3.5-5 × 3-5 µm (n = 30). The morphological features of the fungal isolates matched descriptions of Colletotrichum species. To confirm the identity of the isolated fungus, genomic DNA of all three isolates was extracted using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, Baltics, UAB). The internal transcribed spacers (ITS) of the ribosomal RNA gene region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone H3 (HIS3), actin (ACT), and beta-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4, GDF/GDR, CHS-79F/CHS-354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R, and T1/T2, respectively. The sequences were deposited in GenBank (acc. nos., PP504320 and PP555649-PP555653). Concatenated sequences of the six genes obtained from isolates GNU F137aâc and ex-types from each accepted taxon in previous studies were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11. The fungus isolated from winter squash was in the same clade as C. liaoningense. Therefore, the isolates were identified as C. liaoningense. For pathogenicity tests, three winter squash were wounded with a sterilized needle and inoculated with each isolate by injecting 100 µl conidial suspension (105 conidia/ml). Control squash were injected with sterilized distilled water. All treated squash were incubated at 25°C in the dark. The test was performed three times. All inoculated winter squash reproduced symptoms within 15 days, whereas the control squash were symptomless. The morphological characteristics and ITS sequence of the re-isolated strain matched those of the inoculated strain. To the best of our knowledge, this is the first report of fruit rot of winter squash in Korea and is even the first report on C. liaoningense in Korea. This disease is considered a post-harvest disease because no cases have yet been discovered in the field in Korea. This report will facilitate epidemiological research and the development of effective disease control strategies.