First Report of Leaf Blight on <i>Alpinia zerumbet</i> Caused by <i>Nigrospora osmanthi</i> in China

Shell ginger (Alpinia zerumbet [Pers.] B.L.Burtt & R.M.Sm) is a perennial ornamental plant used for flavoring and in traditional medicine in China. In September 2024, leaf blight symptoms were observed on this plant in a field in Shenzhen (22°36'09"N, 113°59'15"E), Guangdong Province, China. In an 800 m² area survey, about 70% plants showed disease symptoms, with each plant exhibiting 30 to 60% leaf necrosis. Initial symptoms appeared as brown spots on the leaves. Spots gradually enlarged, becoming elongated, irregular, and brown, each surrounded by a yellow halo, and 0.1 to 10.5 cm × 0.1 to 2.5 cm. Eventually, the affected leaves became blighted and desiccated. Ten symptomatic leaves from different plants were collected for pathogen isolation using the tissue transplanting method. Three morphologically similar fungal strains (MBSZU 25-034 to MBSZU 25-036) were obtained, with an isolation frequency of 75%. Colonies on PDA reached a diameter of 90 mm after one week at 25 °C, were flat with entire margins, displayed gray centers and white margins, had dense aerial mycelia, and showed a similar gray center and white margin on the reverse side. Colonies on synthetic nutrient-poor agar were 60-65 mm in diameter, flat with entire margins, and exhibited a white surface and reverse. Hyphae were branched, septate, smooth, and hyaline to pale brown. Conidiophores mostly reduced to conidiogenous cells. Conidiogenous cells were solitary, hyaline to pale brown, subspherical, ampulliform to cylindrical, 4.4 to 11.7 × 4.9 to 7.4 µm. Conidia were solitary, light brown to black, globose to subglobose, aseptate, smooth-walled, 11 to 14.6 × 8 to 13.3 µm (n = 50). Morphologically, the strains resembled Nigrospora species (Wang et al. 2017). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1-α), and β-tubulin (tub2) genes were amplified using the primer pairs ITS5/ITS4 (White et al. 1990), EF1-728F/EF-2 (Carbone and Kohn 1999; O’Donnell et al. 1998) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of ITS (PX061797 to PX061799), tef1-α (PX070317 to PX070319), and tub2 (PX070320 to PX070322) were deposited in GenBank. BLAST results indicated that the ITS, tef1-α, and tub2 sequences showed 99.62, 97.70, and 99.76% similarity to N. osmanthi (CGMCC3.18126), respectively. Phylogenetic analyses based on maximum likelihood and Bayesian inference of three concatenated gene regions confirmed all strains as N. osmanthi. Pathogenicity tests were performed on unwounded healthy plants using an attached leaf assay. All leaves were surface-disinfested with 0.1% NaClO and rinsed three times with sterile water. A conidial suspension (106 conidia/ml) was obtained from 2-week-old cultures in sterile water, and 15 µl was inoculated onto each leaf. Control leaves were mock-inoculated with sterile distilled water. Each treatment included ten replicates and was performed twice. Plants were kept at 25 °C and a relative humidity of 80 to 85%. Seven days post-inoculation, brown lesions appeared on all inoculated leaves, while the control leaves showed no symptoms. Nigrospora osmanthi was reisolated from the lesions and confirmed through morphological examination and DNA sequencing, thereby fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf blight caused by N. osmanthi on A. zerumbet in China and worldwide. These findings will provide insights into epidemiological investigations and future approaches to managing this disease.