First Report of Venturia inaequalis Causing Leaf Spot on Euonymus alatus in South Korea

Euonymus alatus (Celastraceae), known as burning bush, is widely cultivated in South Korea as an ornamental shrub in urban environments, because of low maintenance and striking colors. In October 2023, leaf spots were observed on leaves of E. alatus in Jecheon (37°11'29.3"N 128°11'56.3"E) and Seoul (37°35'35.5"N 127°02'35.4"E), South Korea. Symptoms included circular spots on leaves, which spread through the canopy as temperature dropped. Initially small spots were observed that subsequently expanded from the center to the periphery of leaves. Spots were typically olive green, gray, and dark brown, often forming concentric rings. To isolate the pathogen, we collected 10 symptomatic leaves from E. alatus in both locations and stored them in a moist chamber. Fungal hyphae grown from leaf lesions were isolated in potato dextrose agar (PDA). The isolates were cultured for 14 days at 21°C under a 16-hour light/8-hour dark photoperiod using white fluorescent light. We obtained fungal isolates with similar morphological characteristics from all symptomatic leaves. On PDA, colonies were gray-black, circular, and had a floccose texture. Conidia were oval-pyriform, truncated at the base, elongated at the apex, 1-septate, occasionally 2-septate, and often had a slightly constricted septum. Length measured 11.8 to 21.7 μm × 5.5 to 12.0 μm (n=15), similar with previously described morphological characters of Venturia inaequalis (Turan et al., 2019; Buonaurio et al., 2023). Genomic DNA was extracted from two isolates (C17, C20), and the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA regions were amplified using the primer sets of ITS1F/ITS4 (White et al. 1990) and LR0R/LR5 (Vilgalys and Hester, 1990), respectively. The sequences were deposited in GenBank (PV717320, PV717321 for ITS, PV717322, PV717323 for LSU). Phylogenetic analysis based on concatenated sequences of ITS and LSU regions were conducted with maximum-likelihood analysis using RAxML-NG v.1.2.2 (Kozolov et al., 2019) with 1,000 bootstraps. Isolates obtained in this study clustered in a clade with representative strains of V. inaequalis. Based on phylogenetic analysis and morphology, we identified our isolates as V. inaequalis. A total of nine branches on three living trees were selected for pathogenicity tests, with three branches inoculated per tree. Each branch had approximately 5-6 leaves. In September 2024, a conidial suspension (5 × 10⁵ conidia/mL) was sprayed evenly on the adaxial and abaxial surfaces of leaves until runoff. The leaves in three branches were sprayed with sterile distilled water. The inoculated leaves were enclosed in plastic bags for 48 hours to maintain high humidity. The inoculated plants were then maintained in outdoor conditions. After eight weeks, leaf spot symptoms similar to those observed in the field appeared on the leaves of the inoculated plants, while the control leaves remained asymptomatic. Isolates recovered from inoculated leaves were identified as V. inaequalis based on conidial morphology and phylogenetic analysis, fulfilling Koch’s postulates. V. inaequalis is known to cause apple scab (Bowen et al. 2011) and infect other woody hosts. However, to our knowledge, there are no recorded cases of plant diseases in Euonymus species caused by Venturia spp. Our findings underscore the need for the development of monitoring and control strategies for host range of pathogenic species, for effective control of plant diseases.