Abstract: A leaf spot disease was found on Orychophragums violaceus in Huanghua City, Hebei Province, during October of 2017. The infected leaves were initially black and brown with irregular small disease spots, which gradually developed into necrotic spots with concentric circles, affecting 96.2% of plants observed and 87.6% of leaves on each plant. In order to identify the pathogen, samples were isolated from infected leaves by tissue separation and cultured. After purification, three isolates were obtained (GS1-1, HH2-1, and HH3-1), and their pathogenicity tested according to Koch’s rule. Test results confirmed that the three isolates reproduced the original symptoms and were therefore the causative agent of the observed O. violaceus leaf spot disease. For the three isolates, DNA was extracted, amplified and sequenced by rDNA-ITS, and the results showed the three isolates had a very close relationship to Alternaria brassicicola (MN173824, MN173825, MN173823 and MF462311), and 100% similarity with A. brassicicola (MF462311). The results also showed a very close relationship of the three isolates to certain other A. brassicicola isolates (JX213350, KF889266, KT895946, KC584642, LC480212 and KT895946) through EF-1α gene sequencing, which was consistent with the results of morphological identification. The vegetative growth and sporulation conditions of isolate GS1-1 were studied by biological methods. It was found that the optimal medium for vegetative growth and spore production of isolated GS1-1 was PSA, and the optimal carbon and nitrogen sources for vegetative growth were maltose and peptone, respectively. The optimal carbon and nitrogen sources for sporulation were glucose and yeast extract, respectively. The optimal colony growth temperature was 25 ℃, with the temperature range over which colony growth occurred being 5-35 ℃, and the optimal spore production temperature was 28 ℃. Alternate 12 h light and dark periods promoted colony growth and sporulation.

Key words: Orychophragmus violaceus, leaf spot disease, Alternaria brassicicola, internal transcribed spacer (ITS), EF-1α, biological characteristics