First report of <i>Pestalotiopsis kenyana</i> causing needle blight of Japanese larch ( <i>Larix kaempferi</i> L.) seedlings in Korea

Larix kaempferi (Lamb.) Carr., is a deciduous conifer of high ecological, economic, and ornamental value but is highly susceptible to needle blight. In August 2024, pale lesions of needle blight were observed on three-year-old nursery seedlings at Chonnam National University, Korea (35°17′ N, 126°90′ E). Initial symptoms appeared as pale yellow to light brown needle lesions, which coalesced longitudinally, leading to severe necrosis, premature needle drop, and seedling dieback, consistent with Pestalotiopsis blight (Li et al. 2024). Disease incidence was 37% (74 of 200 trees) and the severity, scored and evaluated according to Chiang and Bock (2022), was 40.5%. For phytopathogenic isolation, samples were taken from symptomatic needles (brown lesions), surface sterilized in 5% sodium hypochlorite, and 5 × 5 mm pieces plated on potato dextrose agar supplemented with 100 mg L-1 streptomycin. The plates were incubated at 25 °C for 7 days, and hyphal-tipped to obtain pure colonies. Ten isolates showed colony morphologies typical of Pestalotiopsis sp., two were Diaporthe sp. and one Alternaria sp. Representative isolates Pk_JLD23, D_JLD23 and A_JLD23 for Pestalotiopsis, Diaporthe, and Alternaria genera, respectively, were tested for pathogenicity using twenty-four healthy three-year-old seedlings. Six seedlings were used for each isolate and the control. Each seedling was inoculated with a 2 mL suspension (106 spores/mL) or distilled water for controls, and conditions maintained between 25 to 35 °C, 70 to 80% relative humidity, with 10 to 11h natural light. After 2 weeks, only the seedlings inoculated with Pk_JLD23 began to develop pale yellow to light brown lesions that coalesced, resulting in needle necrosis, premature needle-drop, and dieback within 4 weeks, while seedlings in the control and the other fungal treatments remained healthy. The experiment was repeated twice to confirm the consistency of symptom development. The pathogen was reisolated from symptomatic needles and confirmed as P. kenyana based on morphological analysis. Morphologically, P. kenyana Pk_JLD23 produced fusiform, multi-celled conidia measuring 38.2 to 52.1 × 10.8 to 15.9 μm (45.5 ± 0.98 × 13.4 ± 0.88 μm, n=30). For molecular analysis, the multi-locus sequencing of internal transcribed spacer (ITS: ITS1/ITS4), translation elongation factor 1-alpha (TEF1-α: TEF1-983F/TEF1-1567R), and beta-tubulin (TUB2: BT2a/BT2b) genes based on Luo et al. (2024) were used. The pairwise alignment with Pestalotiopsis kenyana epitype (ITS: KM199302; TEF1-α: KM199502; and TUB2: KM199395) (Maharachchikumbura et al. 2014) revealed sequence similarities of 95.17% (138/145 bp) for ITS, 98.84% (171/173 bp) for TEF1-α, and 89.01% (162/182 bp) for TUB2. The phylogenetic analysis of concatenated sequences clustered Pk_JLD23 with P. kenyana strain CBS 442.67, confirmed the species as P. kenyana, fulfilling Koch’s postulate. In Korea, Pestalotiopsis theae, P. microspora, and P. maculans have causes blight on tea (Shin et al. 1999), Japanese yew (Jeon and Cheon 2014), and Quercus acutissima seedlings (Won et al. 2021), respectively. However, this is the first report of needle blight on L. kaempferi caused by P. kenyana, in Korea. The identification of P. kenyana and its symptom progression supports early detection and nursery monitoring, helping growers implement timely preventive and disease control strategies.