First report of Aspergillus tubingensis causing leaf spot on Pogostemon cablin in China

Patchouli (Pogostemon cablin (Blanco) Benth) is an important medicinal and aromatic plant widely cultivated in China, India, and other Southeast Asian countries. It is renowned for its diverse applications in traditional medicine and its detoxification, antibacterial, anti-inflammatory, and other pharmacological properties (Wu et al. 2016; Fang et al. 2022). In May 2023, a severe leaf spot disease was observed on Pogostemon cablin plants grown in most plantations in Yulin, Guangxi, China (22°26′N; 109°83′E), with over 50% incidence rate. Symptoms began as small, circular, brown spots on leaves, enlarging with yellow halos. Lesions expanded into irregular shapes with necrotic centers. Advanced stages showed extensive yellowing, browning, and leaf senescence. A total of 20 symptomatic plants were sampled from 5 different locations within the detected area, with 4 plants sampled per location. To isolate the pathogen, 20 affected leaves were collected from these plants and preliminarily washed with sterile distilled water (SDW). Five small tissue pieces (5×5 mm) were excised from the lesion edge of each leaf, surface-disinfected with 75% ethanol and 1% NaClO, rinsed thrice with SDW, and placed on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Out of these, 18 plants (90%) yield fungal isolate with recurrent and similar morphological characteristics. Four representative isolates (X5-1-1, X5-1-3, X5-1-5, and X5-1-7) were selected for further analysis. On PDA, colonies were initially white, gradually turning black on the surface, with light yellow on the reverse side of the plate. Conidia were brown to black, globose, rough-walled, and 2. 6 to 5. 2 µm in diameter. Conidial heads were brown-black, and conidiophores were smooth and hyaline. Morphological characteristics matched those of Aspergillus sp. (Guo et al. 2017). For molecular identification, the internal transcribed spacer (ITS) region and the β-tubulin (TUB) gene of all four isolates were sequenced (Lim et al. 2019). All four isolates (X5-1-1, X5-1-3, X5-1-5, and X5-1-7) showed consistent morphological characteristics and 100% identical ITS and TUB sequences. Representative sequences from isolate X5-1-5 were submitted to GenBank (ITS: PP789632; TUB: PP798205). The obtained ITS and TUB sequences showed 99% similarity to Aspergillus tubingensis (ITS: OP737633; TUB: MG991377). Based on morphological and molecular analyses, the fungus was identified as A. tubingensis (Palmer et al. 2019). For pathogenicity tests, a spore suspension (1 × 10⁶ conidia/mL) was prepared from 7-day-old cultures of A. tubingensis grown on PDA. The suspension was sprayed onto leaves of 10 healthy Pogostemon cablin plants until runoff. Control plants were sprayed with SDW. All plants were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, symptoms identical to those observed in the field developed on all pathogen inoculated plants, while control plants remained asymptomatic. The fungus was successfully re-isolated from infected leaves in three successive trials, fulfilling Koch's postulates. Notably, A. tubingensis has previously been reported causing field diseases on strawberry in California, Jatropha curcas and Helleborus species in China (Palmer et al. 2019; Guo et al. 2017, Liaquat et al. 2019), and vine canker on table grape in Italy (Vitale et al. 2012). To our knowledge, this is the first report of A. tubingensis causing leaf spot on Pogostemon cablin in China. This finding provides a foundation for further investigate into the biology, epidemiology, and management of this disease.