Zelkova serrata is a widely planted tree species valued both as a source of lumber and as an important street tree in the urban ecosystems of East Asian countries, including South Korea. In 2023 and 2024, severely infected leaves showing leaf spot symptoms were frequently observed on 35 Z. serrata trees in Daejeon (36°22'09.2"N 127°21'05.1"E and 36°22'19.9"N 127°20'36.2"E), with around 80% of leaves affected within 0.14 and 1.25 ha areas, respectively. Initial symptoms included pale- to dark-brown orbicular or irregular spots, which developed into larger blotches, leading to bleaching and leaf loss (Fig. 1). Stromata were large (35–40 μm), globular, olivaceous brown, and partly superficial or immersed. Conidiophores appeared in dense fascicles, cylindrical, 18–35 × 3.5–4.3 μm, smooth, usually with one septum. Conidia were guttulate, cylindrical to narrowly obclavate, 17–39 × 2.8–4.3 μm, with one to three septa. Fungal isolations were directly made by transferring spores from leaf spots onto 2% malt extract agar (MEA), followed by incubation at 25 ℃. On MEA, colonies were circular, raised, corrugated with smooth, grey. Of those successfully isolated, two representative isolates, CDH063 and CDH064, were selected from each of the two areas and were subjected to the phylogenetic analysis based on a maximum-likelihood. This was achieved based on partial sequences of the internal transcribed spacer (ITS) (Gardes and Bruns 1993; White et al. 1990), actin (Carbone and Kohn 1999; Groenewald et al. 2013), translation elongation factor-1 alpha (TEF-1α) (Nakashima et al. 2016; Carbone and Kohn 1999), and the second largest subunit of RNA polymerase II (rpb2) (Nakashima et al. 2016). The sequence data produced in this study were deposited in GenBank: (ITS: PQ469938–939; actin: PQ496835–836; TEF-1α: PQ496831–832; 40 rpb2: PQ496833–834). The multi-locus phylogenetic analysis revealed that the fungus isolated in this study was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Pseudocercospora, which is most closely related to P. zelkovae MUCC872 (ITS: GU269835, TEF-1α: GU384547, actin: GU320537, rpb2: KX462665) (Nakashima et al. 2016; Groenewald et al. 2024) (Fig. 2). Morphologically, the isolates obtained in this study differed from P. zelkovae in stromata size and conidial septation. In addition, sequence comparisons showed that this pathogen differed from P. zelkovae by 5 of 312 (~1.6%) in the TEF-1α, 1 of 187 (~1.0%) in the actin, and 29 to 671 (~4.3%) in the rpb2 sequences. These multi-locus differences, together with the morphological distinctions, suggest that the isolate represents a genetically distinct lineage. Pathogenicity tests were conducted twice, each using six seedlings of Z. serrata were used. For inoculation, the leaves of each seedling were sprayed with 10 ml of conidial suspension (1 × 10 6 conidia/ml of the isolate, CDH063), while three seedlings were treated with distilled water as controls. Leaf spot symptoms identical to those observed in the field developed on all inoculated seedlings one week after the inoculation, while no symptoms appeared on the controls. Re-isolations were successfully made from the treatments, thus fulfilling Koch’s postulates. In addition to P. zelkovae that was previously reported as a pathogen causing leaf spot disease on Z. serrata in South Korea (Farr et al. 2024), our work additionally discovered the tree pathogenic fungus on Z. serrata. Given the fact that Z. serrata is the most common street and protected tree in South Korea, providing important ecological and cultural functions in urban areas, the emergency of a new Pseudocercospora lineage causing leaf spot on this tree species is of particular concern to urban ecosystems in the country. Consequently, this indicates that further studies are required to gain a more detailed understanding of the distribution of the diseases and the intraspecies diversity of this species.
Kim et al. (Fri,) studied this question.