LAUSR

Orychophragmus violaceus (February orchid) is an annual or perennial cruciferous herbaceous plant that is widely cultivated and used as an ornamental plant or as a green manure in China. In September 2017, a new leaf blight disease was observed on O. violaceus plants with 80 to 100% disease incidence in a field located in Huanghua County, Cangzhou City, Hebei Province, in northern China. Symptoms on affected leaves began as pinpoint spots that enlarged to form irregular to circular black spots with an obvious wheel striatum, approximately 12 mm in diameter, after 14 days. After 7 days lesions became necrotic and occupied 30 to 70% of the leaf surface. Approximately 5- × 5-mm pieces from the margin of necrotic leaves were surface disinfested with 75% ethanol and 10% sodium hypochlorite, rinsed three times with sterile distilled water, and placed onto potato dextrose agar (PDA). After 2 days of incubation at 25°C in the dark, newly grown-out mycelia were transferred onto fresh PDA and purified by the single-spore method. On PDA, olive-green mycelium developed after 2 days of incubation at 25°C, turned dark green, and covered the Petri dish 10 days later. Primary conidiophores were mostly simple with a single conidiogenous site. Conidia formed long, branched chains 12 to 125 μm in length. The youngest conidia in a chain were ovoid, 8.7 to 26.1 × 4.4 to 6.2 μm. Older conidia were larger, ellipsoid to ovoid with dimensions 25.8 to 35.2 × 10.9 to 11.8 μm. Conidia had 0 to 8 transverse septa, usually without longitudinal septa. A very small conidial beak or no beak was observed on each conidium. On the basis of these morphological characteristics the fungus was identified as Alternaria brassicicola (Wiltshire 1947). For molecular identification, the translation elongation factor-1α (TEF-1α) and internal transcribed spacer (ITS) region of ribosomal RNA were amplified and sequenced from four isolates (GS1-1, GS1-2, HH2-1, and HH3-1) (Carbone and Kohn 1999, White et al. 1990). Partial TEF-1α gene sequences (EF1-986R/EF1-728F) and ITS (ITS1/ITS2) of isolates GS1-1, GS1-2, HH2-1, and HH3-1 were generated (GenBank accession nos. MH424919, MK249721, MK249722, MK249723; MH424918, MK249724, MK257127, MK257128). BLASTn analysis revealed 100% sequence identity to A. brassicicola (KT895946, KF889266, JX213350, KF889266; MH102059, MF462311, MF462313, JF439458). Pathogenicity testing with four single-conidium isolates (GS1-1, GS1-2, HH2-1, HH3-1) were conducted by inoculating leaves with a conidial suspension of the individual isolates (diluted with distilled water, approximately 1 × 10⁶ conidia/ml) on 20 plants at the three-leaf stage and placing them in a humidity chamber at 25°C for 24 h. The 20 plants at the three-leaf stage were mock inoculated with distilled water as a control. Initial symptoms developed after 3 days and the typical symptoms 5 days after inoculation with A. brassicicola and were similar to those observed in the field. The pathogen was reisolated and confirmed to be A. brassicicola, completing Koch’s postulates. No symptoms were observed on control leaves. To our knowledge, this is the first report of A. brassicicola causing leaf blight on O. violaceus in northern China. The summer in Hebei Province is hot and wet with an average temperature and precipitation in July of 26°C and 627 mm, respectively. Because of the suitable climatic conditions for leaf blight, strategies for managing A. brassicicola on O. violaceus in this region should be developed.