First report of dieback in Japanese larch ( <i>Larix kaempferi</i> L.) seedlings caused by <i>Botryosphaeria dothidea</i> in South Korea

Japanese larch (Larix kaempferi L.) is a significant conifer in South Korea, valued for its durable timber and ecological contribution to forest soils. In July 2024, dieback symptoms were observed in four-year-old L. kaempferi seedlings at a nursery of Chonnam National University, Gwangju, South Korea. Initial needle chlorosis and wilting progressed to necrosis, defoliation, and stem lesions, resulting in branch dieback and, in severe cases, seedling mortality. A survey of 210 seedlings revealed a disease incidence of 42%. For phytopathogenic isolation, stems from symptomatic seedlings were collected. After surface sterilization in 1% (v/v) sodium hypochlorite for 3 min, small tissue segments were plated on potato dextrose agar (PDA) supplemented with 100 µg/mL streptomycin and incubated at 25 °C. Morphologically consistent fungal colonies were obtained and purified by hyphal tipping. Colonies were white and fluffy with irregular margins, gradually turning olivaceous gray upon maturation. Conidiomata were semi-immersed to superficial, unilocular, and possessed a central ostiole. Hyaline conidia were thin-walled, aseptate, narrowly fusiform, and contained granular cytoplasm, consistent with characteristics of Botryosphaeria spp. (Jia et al. 2019). The mean conidia dimensions were 23.3 ± 0.8 × 7.7 ± 0.5 μm (n = 30). Molecular identification of a representative isolate Bd_JLD23, was performed using three gene regions: the internal transcribed spacer (ITS: ITS1/ITS4), translation elongation factor 1-alpha (TEF1-α: TEF1-728F/EF1-986R), and beta-tubulin (TUB2: Bt2a/Bt2b) (White et al. 1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The sequences were deposited in GenBank under accession numbers LC853241, LC853239, and LC853240, respectively. Pairwise sequence alignment revealed similarities of 100% (165/165 bp) for ITS, 99% (191/193 bp) for TEF1-α, and 100% (253/253 bp) for TUB2 with the Botryosphaeria dothidea epitype (ITS: MT367874, TEF1-α: MT385087, and TUB2: MT374083) (Marsberg et al. 2017). Phylogenetic analysis of concatenated sequences clustered isolate Bd_JLD23 with B. dothidea ZBW1. Pathogenicity testing was performed on twelve healthy seedlings by wounding stems with a sterile scalpel and inoculating with 5 mm mycelial plugs from 7-day-old cultures or sterile PDA (control). Six seedlings were inoculated with mycelia of Bd_JLD23, and six served as controls. All seedlings were maintained at 25-35 °C and 70-80% relative humidity under 10-11h of natural light. Within three to four weeks, inoculated seedlings developed needle chlorosis and tip browning, followed by defoliation and reduced vigor. After six weeks, needles became necrotic, and shoots exhibited dieback, while control seedlings remained healthy. The pathogen was re-isolated from diseased seedlings and identified as B. dothidea based on morphological and molecular analyses, thereby fulfilling Koch’s postulates. The experiment was repeated twice to confirm the consistency of symptom development. Botryosphaeria species are widely recognized as vital pathogens of fruit and woody trees in South Korea (Back et al. 2024). This is the first report of B. dothidea causing dieback in L. kaempferi seedlings in South Korea. These findings provide essential baseline information for early detection, surveillance, and the development of effective management strategies for this economically crucial conifer species.