Oilseed rape (Brassica napus L.) is one of the most important oilseed crops in China. It is widely cultivated in China, with winter oilseed rape in Yangtze River basin and in southern China, and spring oilseed rape in northern China. In August 2017, a survey for Leptosphaeria spp. on spring oilseed rape was conducted in Minle county, Zhangye city, Gansu Province, China. The symptoms typical of blackleg on basal stems of oilseed rape were observed in the field. A large number of black fruiting bodies (pycnidia) were present on the lesions (Fig. 1A). The disease incidence of basal stem infection in the surveyed field was 19%. A total of 19 diseased stems were collected to isolate the pathogen. After surface sterilizing (75% ethanol for 30 s, 5% NaOCl for 60 s, followed by rinsing in sterilized water three times), diseased tissues were cultured on acidified potato dextrose agar (PDA) plates at 20°C for 7 days. Twelve fungal isolates were obtained. All fungal isolates produced typical tan pigment on PDA medium, and produced pycnidia after two weeks (Fig. 1B). Colony morphological characteristics indicated that these isolates might belong to Leptosphaeria biglobosa. To confirm identification, multiple PCR was conducted using the species-specific primers LmacF, LbigF, LmacR (Liu et al. 2006). Genomic DNA of each isolate was extracted using the cetyltrimethylammonium bromide (CTAB) method. DNA samples of L. maculans isolate UK-1 and L. biglobosa isolate W10 (Cai et al. 2015) were used as references. Only a 444-bp DNA band was detected in all 12 isolates and W10, whereas a 333-bp DNA band was detected only in the UK-1 isolate (Fig. 1C). PCR results suggested that these 12 isolates all belong to L. biglobosa. In addition, the internal transcribed spacer (ITS) region of these 12 isolates was analyzed for subspecies identification (Vincenot et al. 2008). Phylogenetic analysis based on ITS sequence showed that five isolates (Lb1134, Lb1136, Lb1138, Lb1139 and Lb1143) belonged to L. biglobosa ‘brassicae’ (Lbb) with 78% bootstrap support, and the other seven isolates (Lb1135, Lb1137, Lb1140, Lb1141, Lb1142, Lb1144 and Lb1145) belonged to L. biglobosa ‘canadensis’ (Lbc) with 95% bootstrap support (Fig. 1D). Two Lbb isolates (Lb1134 and Lb1136) and two Lbc isolates (Lb1142 and Lb1144) were randomly selected for pathogenicity testing on B. napus cultivar Zhongshuang No. 9 (Wang et al. 2002). Conidial suspensions (10 μL, 1 × 107 conidia mL-1) of these four isolates were inoculated on needle-wounded cotyledons (14-day-old seedling), with 10 cotyledons (20 wounded sites) per isolate. A further 10 wounded cotyledons were inoculated with water and served as controls. Seedlings were maintained in a growth chamber at 20°C with 100% relative humidity and a 12-h photoperiod. After 7 days, cotyledons inoculated with the four isolates showed necrotic lesions in the inoculated wounds. Control cotyledons had no symptoms (Fig. 2). Fungi re-isolated from the infected cotyledons showed similar colony morphology as the original isolates. Therefore, L. biglobosa ‘brassicae’ and L. biglobosa ‘canadensis’ appear to be the pathogens causing the observed blackleg symptoms on spring oilseed rape in Gansu, China. In previous studies, L. biglobosa ‘brassicae’ has been found in many crops in China, including oilseed rape (Liu et al. 2014; Cai et al. 2015), Chinese radish (Raphanus sativus) (Cai et al. 2014a), B. campestris ssp. chinensis var. purpurea (Cai et al. 2014b), broccoli (B. oleracea var. italica) (Luo et al. 2018), ornamental kale (B. oleracea var. acephala) (Zhou et al. 2019a), B. juncea var. multiceps (Zhou et al. 2019b), B. juncea var. tumida (Deng et al. 2020) and Chinese cabbage (B. rapa subsp. pekinensis) (Yu et al. 2021 accepted). To the best of our knowledge, this is the first report of L. biglobosa ‘canadensis’ causing blackleg on B. napus in China.
Genome-Wide Analysis Reveals Genetic Structure and Selective Sweeps in Chinese Indigenous Pig Breeds
