First Report of Downy Mildew Caused by Hyaloperonospora parasitica on Cardamine violifolia in China

Cardamine violifolia is a selenium-enriched plant native to the Wuling Mountain region spanning western Hubei and Hunan provinces. In Enshi City, Hubei Province, it is cultivated on ~2,000 hectares as a critical economic plant for selenium protein extraction. In January 2025, downy mildew-like symptoms were observed on C. violifolia leaves at a cultivation base in Enshi (30°19'20.15''N; 109°28'20.25''E), Hubei Province, China, affecting an area of 1,800 hectares. The symptoms included light green to yellow spots on the adaxial leaf surface, accompanied by sparse white to grayish-white mycelial growth on the abaxial surface under humid conditions. The pathogen was characterized morphologically using a light microscope. Sporangiophores emerged from stomata, either singly or in bundles of 2-4, were colorless, aseptate, slightly swollen at the base, and exhibited dichotomous branching 2-5 times, and a total length of 154.5-415.5 μm. The central axis and branches formed an acute angle. Apical branches were sharp, inwardly curved slightly like forceps, bearing one sporangium at each tip. Sporangia were colorless, unicellular, oblong to ovoid, measuring 19.8-30.9 μm × 18.4-28.3 μm, and germinated via lateral germ tubes without forming zoospores. Oospores were yellowish-brown, unicellular, spherical, with diameters ranging from 27.9-45.3 μm and sleek surfaces. The oosphere diameter was 12.4-27.5 μm, and the cell wall was thick, exhibiting either wrinkled or smooth textures. Oospores demonstrated strong resistance to adverse conditions and could directly produce germ tubes for infection under favorable environments as described previously (Saharan et al., 2017). For pathogen identification, DNA was extracted from sporulating lesions using the CTAB method. Fragments of the ribosomal internal transcribed spacer (ITS) region (White et al., 1990) and the mitochondrial cytochrome B oxidase subunit 2 (cox2) gene (Hudspeth et al., 2000) were amplified by PCR and sequenced bidirectionally. The sequences were deposited in GenBank under accession numbers PV395549 (ITS) and PV573477 (cox2). BLAST analysis revealed 99.88% and 99.51% sequence identity to Hyaloperonospora parasitica (EU049244 and DQ365708, respectively). Pathogenicity tests were conducted by rinsing sporangia from infected leaves and spray-inoculating ten 4-week-old C. violifolia plants with a suspension of 1 × 105 sporangia/ml until runoff. Five control plants were sprayed with distilled water. All plants were maintained in a growth chamber at 22°C/18°C (day/night) with 80% relative humidity for 24 hours. Seven days post-inoculation, characteristic symptoms, including chlorotic spots and sporulation, were observed on inoculated plants, while control plants remained asymptomatic. The pathogen was successfully reisolated from inoculated leaves onto detached C. violifolia leaves, and its identity was confirmed morphologically and molecularly through PCR amplification and sequencing of the ITS and cox2 genes. To our knowledge, this is the first report of downy mildew caused by H. parasitica on C. violifolia in China. Given the broad host range of H. parasitica and the economic importance of C. violifolia, this disease poses a significant threat to the cultivation of this valuable crop. Therefore, it is imperative to develop effective management strategies to prevent further spread and mitigate potential economic losses.