First Report of <i>Fusarium graminearum</i> Causing Twig Blight on <i>Taxus cuspidata</i> Siebold &amp; Zucc. in Korea

Taxus cuspidata Siebold &amp; Zucc. is an evergreen conifer with outstanding ornamental and bioactive properties. However, in Korea, T. cuspidata is classified as an endangered species, sparsely distributed in subalpine ecosystems due to environmental stress associated with climate change. In June 2024, twig blight symptoms characterized by wilting and browning of leaves and twigs were observed in a natural forest of T. cuspidata on Mt. Bangtae, Inje, Korea (37°53′05.2″ N, 128°24′19.1″ E, 1,354 m), with a disease incidence of 30% (6/20 trees). Three stem samples from each of six symptomatic trees were surface-disinfected with 1% sodium hypochlorite for 1 min and 70% ethanol for 30 s, and rinsed three times in sterile distilled water. Air-dried samples were placed on potato dextrose agar (PDA) amended with 0.05% streptomycin and incubated for 5 days at 25°C. The emerging fungal colonies were subcultured onto fresh PDA for pure isolation. Colonies were initially white to pale pink and gradually turned deep pink with yellowish aerial mycelia. The macroconidia on synthetic nutrient-poor agar (SNA), prepared following Nirenberg (1976), were slightly curved and 4–6-septate, with tapering apical and blunt to foot-shaped basal cells. The conidia measured 55.67 ± 8.40 × 4.86 ± 0.49 μm, consistent with previous descriptions of Fusarium graminearum (Leslie and Summerell, 2006). Three gene regions were amplified using primer pairs ITS4/ITS5 for the internal transcribed spacer (ITS), EF1/EF2 for translation elongation factor 1-alpha (TEF1), and 7cf/11ar for the RNA polymerase II second-largest subunit (RPB2). The PCR products were sequenced using both amplification primers and additional internal primers (EF3/EF22U for TEF1 and 5f2/7cr for RPB2), and consensus sequences were assembled from contigs (O’Donnell et al., 2022). The resulting sequences were deposited in GenBank under accession numbers PZ072235 (ITS), LC920517 (TEF1), and LC920518 (RPB2). Based on multilocus sequence comparison using FUSARIOID-ID database (https://www.fusarium.org), the isolate was identified as F. graminearum, showing &gt;99.5% similarity to reference strains such as F. graminearum CBS 797.20 and CBS 130953. Pathogenicity was assessed by inoculating five healthy 2-year-old T. cuspidata seedlings. The stem surface was slightly wounded with a scalpel, and half of a 4 mm-diameter mycelial plug was placed on the wound and sealed with Parafilm. As a control, five seedlings were inoculated using PDA plugs without fungal inoculum. Seedlings were maintained at 25°C with 80% relative humidity and a 12 h photoperiod. After three weeks, wilting and browning of leaves and twigs developed above the inoculation site, with browning initiating at the base. F. graminearum was re-isolated from symptomatic twigs and leaves, whereas control seedlings remained asymptomatic, thereby fulfilling Koch’s postulates. To our knowledge, this is the first report of F. graminearum causing disease on T. cuspidata in Korea. Fungal diseases of T. cuspidata have rarely been reported in Korea, with only a few cases involving Pestalotiopsis sp. and Phomopsis sp. (The Korean Society of Plant Pathology and National Institute of Agricultural Sciences, 2024). These findings provide new insights into fungal pathogens affecting T. cuspidata and may contribute to the development of effective disease management strategies.