First Report of Leptosphaeria biglobosa Causing Blackleg of Ornamental Kale (Brassica oleracea var. acephala) in China

Ornamental kale (Brassica oleracea var. acephala), a biennial herbaceous cabbage, is widely grown for its ornamental leaves over the world. In May 2018, necrotic lesions on lower leaf petioles and blackleg symptoms on basal stems of flowering-stage ornamental kale were observed in Yangzhou, Jiangsu Province. The disease incidence was relatively low with approximately 5% in surveyed fields. To determine the causal agent, diseased leaf petioles were cut into pieces, surface sterilized (Zhou et al. 2019), and placed on potato dextrose agar plates at 20°C for 7 days. Four fungal isolates were obtained, and all fungal colonies formed fluffy white aerial mycelia with a yellow pigment. In addition, black-brown and globose pycnidia of 159 × 143 μm in size (74 to 370 × 74 to 333 μm, n = 50) were also observed on the fungal colonies after 10 days. On a V8 juice agar plate, pink pycnidiospore ooze formed on top of pycnidia after 17 days of cultivation. Conidia were cylindrical, hyaline with the size of 5.5 × 2.3 μm (5 to 7.5 × 1.7 to 2.5 μm, n = 100). The cultural and morphological characteristics of the isolates matched the description for Leptosphaeria biglobosa rather than L. maculans, because the later produces no yellow pigment (Williams and Fitt 1999). For molecular identification, genomic DNA of all four isolates was extracted using the cetyltrimethylammonium bromide method and then, together with DNA samples of L. maculans isolate UK-1 and L. biglobosa isolate B2003, used for polymerase chain reaction assay with the species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006), respectively. In addition, the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (Vincenot et al. 2008) were also cloned and used for further identification of isolate NY8-1-2. Because only a 444-bp DNA band was detected in all four isolates, they should belong to L. biglobosa rather than L. maculans, as the latter generates a 331-bp DNA band. Sequences of ITS (496 bp, GenBank accession no. MH472567), actin (900 bp, MH475334), and β-tubulin (948 bp, MH475335) for isolate NY8-1-2 were 100% identical to those of L. biglobosa strains HCLB-1 (ITS, KC880981.1), 2379-4 (actin, AY748949.1), and B3.6 (β-tubulin, AY748995.1) in GenBank, respectively. Cotyledons of six ornamental kale cultivars (Jingyu No. 1, Jingyu No. 2, Jingguanbai No. 1, Jingguanhong No. 7, Jinglianhong No. 3, and Fenhong No. 1), an oilseed rape cultivar (Zhongshuang No. 9), and a cabbage cultivar (Jingfeng No. 1) were used to test the pathogenicity. Pycnidiospore suspension (10 μl, 1 × 10⁷ conidia/ml) of isolate NY8-1-2 was wound inoculated on cotyledons of 14-day-old seedlings, with 12 cotyledons (= 24 wounded sites) per isolate, and 12 wounded cotyledons inoculated with water served as a control group. Treated seedlings were incubated at 20°C and 100% relative humidity under 12 h of light/12 h of dark for 7 days. Whereas the control group remained healthy, necrotic lesions around the inoculated sites were observed for all cultivars of ornamental kale, oilseed rape, and cabbage. The fungus reisolated from the diseased cotyledons resembled colony morphology to the original isolate. Therefore, L. biglobosa was determined to be the causal agent of blackleg on ornamental kale in China. Although L. biglobosa can cause blackleg on cabbage (B. oleracea) in Mexico (Dilmaghani et al. 2010), to our knowledge this is the first report of L. biglobosa causing blackleg on B. oleracea var. acephala in China.