First Report of Heterophoma poolensis Causing Leaf Spot Disease on Mountain Spike Speedwell ( Pseudolysimachion rotundum var. subinte

Mountain spike speedwell (Pseudolysimachion rotundum var. subintegrum) is a perennial herb traditionally used as a medicinal plant for treating respiratory ailments and has recently gained importance as a nectar resource plant (Na et al. 2024; Song et al. 2015). In May 2024, leaf spot symptoms were observed on potted mountain spike speedwell plants in an approximately 4.0 × 2.5 m (10 m²) area at the Chonnam National University Arboretum in Gwangju, Korea (35°17′27″ N, 126°53′31″ E). Symptoms included brown to purplish irregular lesions that occasionally resulted in shot holes in the affected tissue. Disease incidence was 60% (18 of 30 plants), and six symptomatic leaves from different plants were collected for pathogen isolation. The leaves were cut into 1-cm segments, and all samples were surface-disinfected in 1% sodium hypochlorite for 1 min, followed by 70% ethanol for 30 s, and rinsed three times with sterile distilled water. Air-dried samples were placed on potato dextrose agar supplemented with 0.05% streptomycin and incubated at 25°C. The emerging fungal colonies initially appeared white and turned beige to green with age. Conidia were abundant, small, oval, and measured 3.23–4.47 × 1.44–2.05 μm (n = 50). Colony morphology and conidial characteristics were consistent with Phoma-like fungi, a group formerly classified in the genus Phoma (Aveskamp et al. 2010). One representative isolate (PH24) was selected for further molecular analysis. The rDNA internal transcribed spacer (ITS), RNA polymerase II second-largest subunit (RPB2), and β-tubulin (TUB2) regions were amplified and sequenced using the primer pairs V9G/ITS4, 7cF/11aR, and TUB2Fd/TUB4Rd, respectively (Aveskamp et al. 2009). The resulting sequences were deposited in GenBank (ITS: PZ253855, RPB2: LC924541, and TUB2: LC924542). Multilocus phylogenetic analysis based on concatenated ITS, RPB2, and TUB2 sequences placed isolate PH24 within the Heterophoma poolensis clade with 92% bootstrap support. Pathogenicity tests were conducted according to Koch’s postulates by inoculating leaves of six healthy mountain spike speedwell plants. Prior to inoculation, leaves were gently wiped with sterilized gauze moistened with 70% ethanol. Three wounds were made on each of three leaves per plant using a sterile scalpel, and each leaf was inoculated with 1 mL of conidial suspension (1 × 10⁶ conidia/mL). For the control plants, the conidial suspension was replaced with sterile distilled water. After inoculation, plants were covered with transparent plastic bags misted with sterile distilled water for 5 days. Approximately two months after inoculation, brown to purplish lesions developed on both wounded and unwounded tissues, which often turned necrotic and occasionally formed shot holes. All inoculated plants developed similar symptoms, and the same pathogen was consistently re-isolated from the lesions and confirmed as H. poolensis, whereas control plants remained symptomless. To our knowledge, this is the first report of leaf spot disease caused by H. poolensis on mountain spike speedwell in Korea. This report provides useful information on a fungal disease affecting mountain spike speedwell and may contribute to the development of effective disease management strategies for this species.