Hubei Normal University

Atractylodes lancea, listed as one of the “Ten Great Medicinal Herbs of Chu” in Hubei Province, China, is used in traditional medicine for its dried rhizome. Approximately 75% of A. lancea plants exhibited leaf spot symptoms in April 2025 in a cultivated field in Yingshan County, Hubei Province. Initial symptoms appeared as black necrotic lesions with brown margins at leaf tips or centers, which expanded over time and often led to leaf abscission. To identify the causal agent, symptomatic leaves were surface-sterilized and placed on potato dextrose agar (PDA). After incubation at 28℃ in the dark, hyphal tips from emerging colonies were transferred to fresh PDA for purification. Ten fungal strains were isolated, two of which—named MCZ7 and MCZ14—were selected for further study. On PDA, isolate MCZ7 formed vigorous colonies that were initially white and later turned grayish-brown, with a cottony texture, dense grayish-white aerial mycelium, and concentric rings in later stages. Conidia were mostly ovoid to short-cylindrical, pale olive-green, smooth-walled, with 3–4 transverse septa and 0–2 longitudinal septa, measuring (17.95 ± 7.64) × (9.14 ± 2.09) μm on average. Isolate MCZ14 produced brown colonies with irregular white margins on PDA. Aerial mycelium became sparse in later stages, and dark brown punctuate structures developed. Its growth rate was notably slower than MCZ7. Conidia were ovoid to obclavate, light brown, smooth-walled, with 2–5 transverse septa and 0–1 longitudinal septum, averaging (18.79 ± 6.20) × (7.49 ± 1.00) μm. For molecular identification, the internal transcribed spacer (ITS), β-tubulin (TUB2), and RNA polymerase II second largest subunit (RPB2) were amplified and sequenced (Romain et al. 2022; Zheng et al. 2023). Sequences were deposited in GenBank under the following accession numbers PX915538, PX929565, PX929567 for MCZ7; PX915539, PX929566, PX929568 for MCZ14. Phylogenetic analysis using MEGA11 and Phylosuite placed MCZ7 in the Alternaria alternata clade and MCZ14 with Alternaria tenuissima. Pathogenicity was tested following Koch’s postulates. Healthy A. lancea leaves were surface-sterilized with 75% ethanol (15–30 s) and 1.5% NaClO (90 s), rinsed three times in sterile water, and dried. Leaves were wounded and inoculated with 5-mm mycelial plugs from active PDA cultures; controls received sterile PDA plugs. Each treatment had three technical and three biological replicates. After 3 days at 28°C under dark, humid conditions, leaves inoculated with MCZ7 or MCZ14 developed typical leaf-spot symptoms, while controls remained healthy. The same fungi were re-isolated from lesions and confirmed morphologically and molecularly. Previous studies have reported A. alternata, Alternaria gaisen, Fusarium acuminatum, and Epicoccum sorghinum as leaf spot pathogens of A. lancea (Du et al. 2020; Hu et al. 2024; Kawashimo et al. 2024; Zheng et al. 2023). However, A. tenuissima has not previously been reported to cause leaf spot on this host. Currently, chemical pesticides remain the primary control method for A. lancea diseases. This report has refined the diagnostic methodologies for A. lancea leaf spot disease, and future studies should focus on its control.