First report of <i>Alternaria alternata</i> causing leaf blight on <i>Corydalis yanhusuo</i> in Zhejiang Province, China

Corydalis yanhusuo W. T. Wang (commonly known as Yan-Hu-Suo or Yuan-Hu), a traditional Chinese medicinal herb belonging to the family Papaveraceae, has been domesticated and cultivated in China for centuries. In clinical practice, its dried tubers are widely utilized for their therapeutic effects in promoting blood circulation, enhancing energy, and alleviating pain (Feng et al. 2023). In March 2025, a novel leaf blight was discovered on C. yanhusuo plants in the primary cultivation regions of Jinhua, Hangzhou, and Lishui cities in Zhejiang Province, China (27.02°-31.11°N, 118.01°-123.10°E), with field disease incidences of 65%, 50%, and 40%, respectively. Early symptoms appeared as dark brown lesions on the tips or margins of the leaves. As the disease progressed, the lesions gradually expanded and coalesced, forming extensive necrotic areas that ultimately resulted in leaf curling and desiccation. To identify the causal agent, 12 symptomatic leaves were randomly collected from six diseased plants. Tissue samples (5×5 mm) excised from the margin between healthy and diseased regions were surface sterilized sequentially with 75% ethanol for 30 s and 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and subsequently transferred to potato dextrose agar (PDA). Following incubation at 28 °C in darkness for a week, seven fungal colonies with identical morphological characteristics were obtained, and a representative single-spore isolate (YH-7) was selected for further morphological and molecular characterization. The YH-7 colonies appeared cottony with gray-white aerial hyphae, which gradually turned brown and became more compact as they matured. Conidia were pale to dark brown, ovoid to ellipsoid, and measured 12.0 to 35.5 × 5.0 to 13.5 μm (n=50). They were typically arranged in short chains (3 to 8 conidia) with branches, possessed 1 to 4 transverse and 0 to 3 longitudinal septa, and were mostly beakless or featured a short conical beak. The morphological characteristic of YH-7 isolate is consistent with the description of Alternaria alternata (Simmons 2007). For molecular identification, genomic DNA was extracted with a Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China) from the pure culture of YH-7. The internal transcribed spacer (ITS) region, translation elongation factor 1-α gene (TEF1-α), and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A.alternata were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1α-F/EF-1α-R (Carbone and Kohn 1999), and GDF1/GDR1 (Berbee et al. 1999), respectively. The ITS, TEF1-α, and GAPDH gene sequences from isolate YH-7 were deposited in GenBank (PV760278, PV765638, and PV776884) and showed 100% sequence identity to the corresponding sequences of the reference isolates A. alternata (OQ589884 for ITS, 570/570 bp; LC132709 for TEF1-α, 270/270 bp; OP418452 for GAPDH, 618/618 bp). A maximum-likelihood phylogenetic tree was constructed with the combined sequences of ITS, TEF1-α, and GAPDH using IQ-TREE. The results showed that YH-7 clustered robustly within the A. alternata clade. Combined with its morphological characteristics, the isolate YH-7 was therefore identified as A. alternata. For pathogenicity testing, a conidial suspension (1×106 spores/ml) prepared from a 10-day-old culture of YH-7 was sprayed on leaves of healthy one-month-old C. yanhusuo seedlings. Control treatments were sprayed with distilled water. All test plants were placed in a greenhouse maintained at 25 °C and 90% relative humidity, under a 12-h light/12-h dark cycle. Eight plants were used for each treatment and the experiment was repeated three times. At 15 days post-inoculation, YH-7-inoculated plants exhibited typical symptoms of brown lesions similar to those observed in the fields, while leaves of control plants showed no symptoms. The fungal pathogen was reisolated from the inoculated leaves and confirmed as A. alternata based on morphological and molecular characteristics, satisfying Koch’s postulates. Although several pathogens have been documented to infect C. yanhusuo (Fang et al. 2022), this study represents the first report of A. alternata causing leaf blight on C. yanhusuo in China. Elucidating the etiology of this disease is crucial for developing effective control strategies to mitigate yield losses.