First Report of Tip Blight Caused by Nigrospora aurantiaca on Zanthoxylum nitidum in China

Zanthoxylum nitidum (Roxb.) DC., the botanical origin of the traditional Chinese medicine Liangmianzhen, is valued for its dried root's pharmacological properties (Chen et al. 2025). In November 2023, an emerging tip blight disease was observed on this species at a cultivation base in Fangchenggang City (108.3°E, 21.37°N), Guangxi, China, with an estimated incidence of 30 to 40%. Initial symptoms included black spots on terminal branches and lateral buds, which later elongated into irregular streaks. The affected tissues eventually blackened and withered. To isolate the causal pathogen, 20 segments of diseased apical bud tissue were randomly collected, cut into small pieces (5 × 5 mm), and surface-sterilized (75% ethanol for 30 s, followed by 2.5% NaClO for 2 min, and rinsed three times with sterile distilled water). The tissue pieces were then placed on potato dextrose agar (PDA) amended with rifampicin and incubated at 28°C under a 12 h light/dark cycle for 3 days. The hyphal tips were transferred to fresh PDA to obtain pure cultures. Among 41 isolates, 27 morphologically similar isolates were recovered from all symptomatic tissues and preliminarily identified as the putative pathogen. Representative isolate LB2 was selected for further study. The colony produced floccose aerial mycelia that were initially white, turning dark grey with age, accompanied by pale brown hyphae and a diffusible rose-red pigment. Globose pycnidia developed after 30 days. Conidia were ovoid to sub-circular, smooth-walled, black, and measured 11.6–15.8 × 12.6–16.9 µm in diameter (n = 30), consistent with Nigrospora spp. (Wang et al. 2017). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1), and beta-tubulin (TUB2) genes of isolate LB2 were amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences (GenBank accession nos. PP898108, PP915464 and PP915463, respectively) showed 99–100% similarity (551/554 bp for ITS, 290/291 bp for TEF1, and 401/401 bp for TUB2) to corresponding sequences of N. aurantiaca (accession nos. PP574342, OR725095, and OR670516, respectively). Phylogenetic analysis using maximum likelihood in MEGA 12 placed isolate LB2 within the N. aurantiaca clade. Pathogenicity tests were conducted by spraying a conidial suspension (1 × 10⁶ spores/mL) of LB2 onto the tips of six healthy 2-year-old Z. nitidum seedlings in vivo, with each seedling receiving approximately 5 mL of the suspension. Six control seedlings received sterile distilled water. All plants were maintained in a greenhouse at 25°C and 80% relative humidity. After 15 days, typical tip blight symptoms developed on inoculated shoots and apical buds, matching field observations. N. aurantiaca was re-isolated from symptomatic tissues but not from the controls, fulfilling Koch's postulates. N. aurantiaca has previously been reported to cause leaf spot on bayberry (Fu et al. 2025) and tobacco (Yu et al. 2021), as well as leaf tip necrosis on Camellia oleifera (Luo et al. 2025). To our knowledge, this is the first report of N. aurantiaca causing tip blight on Z. nitidum in China. This finding provides a theoretical foundation for the integrated management of diseases affecting this medicinal plant.