First report of Colletotrichum liaoningense causing anthracnose on winter squash in Korea.

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 F137a‒c) 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 F137a‒c 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.

First Report of Anthracnose on Jerusalem Cherry Caused by Colletotrichum liaoningense in Shandong, China.

Jerusalem cherry (Solanum pseudocapsicum), which belongs to the genus Solanum and the family Solanaceae, possesses high ornamental value and is widely cultivated as an indoor ornament due to its bright red berries at maturity (Xu et al., 2018). In September 2019, leaf spot was detected on jerusalem cherry plants in Yuxiu Park, Shizhong district, Jinan, Shandong Province. Field surveys were done in a 1/15 ha park. Disease incidence was estimated at approximately 18% across the survey area. Foliar symptoms began as small white spots. As the disease progressed, lesions expanded and merged, and developed into large irregular white spots, with pale grey edge. At last, lesions were densely distributed throughout the leaves. To isolate the pathogen, twenty leaf tissues (5 × 5 mm) were cut from the border between diseased and healthy tissue, surface disinfected in 75% alcohol for 15 s, soaked in 0.1% mercuric chloride for 1 min, washed with sterile distilled water three times, and cultured on potato dextrose agar (PDA) at 25°C. Nineteen fungal isolates were obtained and were single-spored to obtain pure cultures. The colony of LCL7, a representative isolate, on PDA was initially white to orange, but turned black after 3 to 4 days incubation with black conidial masses. Conidia were single-celled, hyaline, straight, cylindrical, apex obtuse, and ranged from 13.4 to 18.3 × 3.2 to 4.9 µm (n = 50) （Diao et al., 2017）. To validate the species identification, rDNA internal transcribed spacer (ITS) region (White et al., 1990), and the partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB2), and chitin synthase (CHS-1) (Damm et al., 2019; He et al., 2019), were amplified and sequenced. The ITS, GAPDH, ACT, TUB2, and CHS-1 sequences of isolate LCL7 were submitted to GenBank (MW221320, MW227217, MW227218, MW227219, and MW266988, respectively). ITS, ACT, TUB2, and CHS-1 BLAST showed 99-100% homology with sequences of Colletotrichum liaoningense (ITS, 100% to MH636504; ACT, 100% to MH622582; TUB2, 99.56% to MH622714, CHS-1, 99.33% to MH622446, respectively), although GAPDH showed 93.98% homology with sequence MH681383 (234/249bp). Neighbor-joining tree based on concatenated sequences of the five genes was constructed using MEGA7.0. The results showed the isolate was closely related to C. liaoningense. Based on morphological and molecular characteristics, the isolate LCL7 was identified as C. liaoningense. Pathogenicity tests were performed by spraying a conidial suspension (1 × 105 conidia/mL) on ten two-year-old healthy jerusalem cherry plants. Ten other plants with sterile water served as controls. All samples were incubated in a growth chamber at 25±2°C and transparent plastic bags to keep relative humidity high for 2 days. All inoculated plants showed symptoms similar to those observed in the field after 21 days, but no disease occurred on control plants. The same fungus was successfully reisolated from inoculated leaves and reidentified based on morphology and molecular characteristics, and the fungus was not isolated from the control plants, thus confirming Koch's postulates. Pathogenicity tests were repeated twice. C. liaoningense can cause anthracnose in chili and mango in China (Diao et al., 2017; Li et al., 2019).To our knowledge, this is the first report of anthracnose on jerusalem cherry caused by C. liaoningense in China, which influences ornamental value and reduces market value. Identification of the causes of the disease will help develop effective strategies for managing this disease.