First Report of Fusarium liaoningense Causing Leaf Spot on Asarum heterotropoides in China

Asarum heterotropoides F. Schmidt is a popular and widely cultivated medicinal herb in China. In June 2024, irregularly shaped, light brown spots with white centers (2 to 10 mm wide) were observed on leaves of approximately 30% of plants of Asarum heterotropoides growing in a field (2.5 ha) in Fushun City (41.73°N, 123.03°E), Liaoning, China. About 40% of the leaf areas were affected. As the disease developed, the spots grew larger and developed necrotic centers, accompanied by widespread tissue collapse. Leaves with brown spot symptoms from five plants were collected from the field. Five leaf pieces (3 to 5 mm 2 ) were excised from spot margins, surface sterilized (Jiang et al. 2020), and incubated on potato dextrose agar (PDA) at 25°C. Five single-spore cultures were obtained. After 5 days of incubation, the colonies were white and pink with abundant aerial mycelia. Microconidia were ovate or ellipsoid, measuring 5.0 to 10.0 × 1.5 to 3.0 µm (n = 50). Macroconidia were falcate and slightly curved or straight, measuring 19.5 to 28.5 × 2.0 to 6.0 µm (n =50). Based on morphology, the pathogen was considered Fusarium sp. (Leslie et al. 2007). Two representative strains, WXXF and WXXF2, were selected for molecular identification, and genomic DNA was extracted to confirm morphological diagnosis. The translation elongation factor 1-alpha (TEF-lα) (O'Donnell et al. 1997) was amplified using the primers EF1/EF2, and RNA polymerase II second largest subunit (RPB2) (Liu et al. 1999) was amplified using the primers RPB2-5F2/ RPB2-7cR. The two isolates were identical based on comparisons of their TEF-1α and RPB2 sequences using MEGA 11. The TEF-1α (PQ654043) and RPB2 (PQ631073) sequences of isolate WXXF showed 100% identity to the Fusarium oxysporum species complex (FOSC) in the FUSARIUM-ID database. Phylogenetic analysis identified it as Fusarium liaoningense within the FOSC. The pathogenicity test was carried out using WXXF and WXXF2 isolates. Six healthy Asarum heterotropoides plants were inoculated with a spore suspension of 1 × 10 6 spores/mL. Two wells were prepared on one leaf of each plant by slightly wounding the leaf epidermis with a sterile syringe needle to create microwounds, and then 20 µL of the spore suspension was applied to each well. Sterile distilled water was used in place of the spore suspension for non-inoculated plants. Seedlings were incubated at 25°C, 12-h photoperiod with a relative humidity of 65 to 70% in a greenhouse. Five days after inoculation, all plants inoculated with the isolated pathogen showed the same symptoms as observed in the field, whereas the non-inoculated showed no symptoms. The pathogen was reisolated from infected tissues and confirmed using morphological and molecular methods, thus fulfilling Koch′s postulates (Gradmann 2014). The pathogenicity test was repeated three times, and similar results were obtained. Although this fungus is a common pathogen with a broad host range worldwide, to our knowledge, this is the first report of F. liaoningense causing leaf spot on Asarum heterotropoides in China. These findings provide a basis for developing management strategies for F. liaoningense infection.