First Report of Schizothyrium ramosum sp. nov. Causing Flyspeck on Apple Fruits in Korea

Apple fruit (Malus × domestica Borkh., cv. Fuji) showing flyspeck symptoms were collected from orchards in Cheongsong, Uiseong, and Pyeongchang, Korea (36°16'13.2"N 128°52'52.2"E, 36°25'12"N 128°45'35"E, and 37°25'52"N 128°20'31"E, respectively) from August to October in 2023–2024. Disease incidence was approximately 3–5% in the surveyed orchards. Fungal colonies appeared as shiny, black, and round to ovoid shapes devoid of mycelial mats, measuring 214–441 × 183–393 µm (n = 30, av. 298 × 277 µm). A total of 23 symptomatic apple fruit were collected. To isolate the causal agents, the fruit were disinfected using 70% ethanol, after which colonies were transferred to potato dextrose agar (PDA). Three fungal strains (KNUF-23-CS03, KNUF-24-US04, and KNUF-24-PC03) with similar morphology were isolated out of 17 strains. To observe morphological traits, strain KNUF-24-US04 was cultured on PDA and oatmeal agar (OA) for 2 weeks at 25℃. Colonies on OA were flat, circular, with smooth margins, and cream-colored, reaching 17–21 mm. Conidiophores were erect, scattered, and flexuous. Stipes of conidiophore were smooth, dark brown, measuring 18–42 × 4–7 μm. Conidiogenous cells were polyblastic, doliiform to ellipsoidal, and measured 6–11 × 4–7 μm. Conidia were smooth, hyaline, obtuse at the apex, and 1–5-septate. One-septate conidia was 11–19 × 6–9 µm. Two-septate conidia were straight or clover-shaped, measuring 11–39 × 6–10 μm. For 3–5-septate conidia, 1–3 branches were formed above the basal cell of conidia, measuring 18–32 × 7–9 μm. The morphology of KNUF-24-US04 was consistent with the genus Schizothyrium but distinguished from other species in the genus by possessing conidia borne on 1 to 3 branches (Batzer et al. 2008; Gao et al. 2014). For phylogenetic analysis, the internal transcribed spacer (ITS) region as well as translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB2) genes were amplified from three strains using primer pairs ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1993), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were deposited in GenBank (PV764650 to 55 and PV766719 to 21). A BLAST search revealed that the sequences of ITS regions, TEF1, and TUB2 showed similarity ≤ 98.8%, 85.5%, and 93.6%, respectively, with known Schizothyrium species. In maximum likelihood phylogeny (MEGA 11), the three strains were distinct from previously described Schizothyrium species. Based on morphological characteristics and phylogenetic analysis, the strains were proposed as a novel species, Schizothyrium ramosum J. W. Choi, S. Y. Lee & H. Y. Jung, sp. nov. (MycoBank number: MB 859610), and name “ramosum” refers to the branched shape of conidia. Strain KNUF-24-US04 was deposited in the Korean Agricultural Culture Collection (KACC 411058). The strain KNUF-24-US04 was inoculated onto apple fruit (cv. Fuji) following the method of Gao et al. to fulfill Koch’s postulates (2014). A suspension of mycelial fragments and conidia was prepared by homogenizing 4-week-old colonies in sterile distilled water (3 × 10 6 CFU/mL). Mature fruit (cv. Fuji) was surface sterilized using 70% ethanol, inoculated with the suspension, and incubated in a moist chamber at 25℃ in the dark. The experiment was repeated three times, and sterile distilled water served as the control. After 3 weeks, dark, circular, or irregular symptoms appeared on the inoculated apple, and no symptoms were observed in the controls. Morphology of strains isolated from inoculated apple fruits matched that of S. ramosum. This is the first report of flyspeck on apple fruit caused by S. ramosum sp. nov. in Korea. This study expands the diversity of the sooty blotch and flyspeck species complex and provides a foundation for the development of effective management strategies.