First Report of Leaf Spot Disease Caused by Alternaria japonica on Orychophragmus violaceus in China

Orychophragmus violaceus (L.) O.E. Schulz (Chinese violet cress) belongs to the Brassicaceae family and is widely distributed in China as a garden plant. In April 2016, severe leaf spot disease was observed on cultivated O. violaceus in Xuanwu Lake Park, Nanjing, Jiangsu province, China. The initial symptoms were small brown spots (1 to 2 mm in diameter) on the leaves. The spots enlarged to 10 to 15 mm in diameter, light brown to black, sometimes comprising concentric rings surrounded by a yellow halo. Severely infected leaves became black to gray and dry. More than 10 symptomatic leaves were randomly collected, and tissues (2 mm) were incubated on moistened filter papers at 25°C for 1 day. Three pure cultures (YZU 161008, YZU 161009, and YZU 161010) were obtained by single-spore isolation and deposited in the Culture Collection of Yangtze University. Each isolate was cultured on potato dextrose agar (PDA) at 25°C in darkness. After 7 days, the upper sides of colonies were cottony and white, and the reverse sides were buff, 37 to 38 mm in diameter, with irregular margins. For morphological observation, the fungus was cultured on potato carrot agar at 22°C with a light/dark period of 8 h/16 h according to the method of Simmons (2007). After 7 days, conidiophores arose directly from the agar surface. Conidia were 10 to 42 × 8 to 19 μm in size, short- to long-ovoid, smooth-walled, medium tawny in color, middle and basal parts of several cells were markedly swollen, beakless, with one to five transverse septa. They formed short chains of one to four conidia. Numerous microchlamydospores were observed. Individual microchlamydospores were 6 to 13 × 8 to 19 μm in size, irregular in shape, multicellular, cells swollen, thick-walled, and heavily pigmented. Branching groups were around 15 to 136 μm in length. The characteristics in culture and conidial morphology were identical to Alternaria japonica (Simmons 2007). Genomic DNA was extracted from fresh mycelia of two selected isolates (YZU 161009 and YZU 161010) according to the method of Cenis (1992). Three gene regions including internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial translation elongation factor 1 alpha (TEF1) were amplified using the primer pairs ITS4/ITS5 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. PCR amplifications were conducted using the following conditions: an initial denaturation at 94°C for 2 min; 30 amplification cycles of 98°C for 5 s, 50°C (ITS and TEF1)/57°C (GAPDH) for 15 s, and 72°C for 5 s; and a final extension phase at 72°C for 10 min. The DNA sequences of two isolates were deposited in the GenBank nucleotide database with accession numbers MK940391 (ITS, 597 bp), MK940389 (GAPDH, 611 bp), and MK940387 (TEF1, 367 bp) for YZU 161009 and MK940392 (ITS, 599 bp), MK940390 (GAPDH, 596 bp), and MK940388 (TEF1, 283 bp) for YZU 161010. All the sequences were compared using the BLASTn tool, and the results showed that they were 100% identical to A. japonica (CBS 118390) and A. nepalensis (CBS 118700). Simmons (2007) reported microchlamydospore production by A. japonica and not A. nepalensis, which supports the diagnosis of these isolates as A. japonica. Pathogenicity tests were carried out with YZU 161009 and YZU 161010 using two methods on 2-month-old healthy plants (six plants for each method). A 20-μl conidial suspension (10⁵ conidia/ml) was applied on five to six points per leaf with sterilized ddH₂O used as the negative control. Mycelia plugs grown on PDA (2 to 3 mm in diameter) were also applied to one point per leaf, with pure PDA plugs as the negative control. After 7 days at 25°C in a greenhouse, brown necrotic lesions developed on leaves for each inoculated with conidial suspension (1 to 5 mm in diameter) and mycelia plugs (10 to 20 mm in diameter). Control plants were symptomless. The fungus reisolated from diseased tissues was confirmed as A. japonica based on the morphology of conidia and microchlamydospores, thus fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of A. japonica causing leaf spot on O. violaceus in China. The identification of the pathogen is important for improving management strategies and control of this disease.