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First Report of Leptosphaeria biglobosa ‘brassicae’ Causing Black Leg on Brassica juncea var. multiceps in China

Zhou, K., Yu, C., Cai, X., Wu, M. D., Li, G. Q.
Plant disease 2019 v.103 no.1 pp. 160
Brassica juncea var. multiceps, Brassica napus, DNA fragmentation, Plenodomus biglobosus, Plenodomus lingam, actin, cetyltrimethylammonium bromide, conidia, cotyledons, cultivars, culture media, disease surveys, fungi, internal transcribed spacers, mycelium, pathogenicity, plant tissues, polymerase chain reaction, pycnidia, relative humidity, ribosomal DNA, sauerkraut, seedlings, sodium hypochlorite, stems, tubulin, vegetable crops, vegetables, China
Red-in-snow mustard (Brassica juncea var. multiceps) is a widely grown vegetable crop and also an important source for making Chinese sauerkraut in China. In May 2018, black leg symptoms on basal stems of ripening-stage red-in-snow mustard were observed at two locations (Sinan, Guizhou Province; Wuhan, Hubei Province) of China during disease surveys on oilseed rape and cruciferous vegetables. Disease incidences at the Sinan and Wuhan locations were approximately 11 and 5% in surveyed fields, respectively. Five fungal isolates were obtained from surface-sterilized diseased plant tissues (75% alcohol for 30 s, 5% NaOCl for 60 s, followed by rinsing in sterilized water three times) cultured on potato dextrose agar (PDA) plates at 20°C for 4 days. All fungal colonies formed fluffy white aerial mycelia with a yellow pigment on PDA, and pink pycnidiospore ooze formed on top of black-brown, globose pycnidia on V8 juice agar postinoculation after17 days. Pycnidia were 75 to 450 × 75 to 200 μm in size (n = 50), and conidia were cylindrical, hyaline, and 5 to 7.5 × 1.7 to 2.5 μm in size (n = 100). The cultural and morphological characteristics of the isolates matched the description for Leptosphaeria maculans and L. biglobosa (Fitt et al. 2006). Moreover, genomic DNA of each isolate was extracted using the cetyltrimethylammonium bromide method and then used for molecular identification. All five isolates were identified through polymerase chain reaction assay using the species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006), and the DNA samples of L. maculans isolate UK-1 and L. biglobosa isolate B2003 served as references (Cai et al. 2014). Moreover, isolate QJ3-1 was identified further by analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (Vincenot et al. 2008). A 444-bp DNA fragment was detected in all five isolates, indicating that they belonged to L. biglobosa ‘brassicae’ instead of L. maculans owing to the latter, which generates a 331-bp DNA fragment. Sequences of ITS (496 bp, GenBank accession no. MH454482), actin (900 bp, MH459178), and β-tubulin (948 bp, MH459179) for strain QJ3-1 were 100% identical to the corresponding region for ITS (strain HCLB-1, KC880981), actin (strain 2379-4, AY748949), and β-tubulin (strain B3.6, AY748995) of L. biglobosa ‘brassicae’ isolates in GenBank. Pathogenicity assays of all five isolates were conducted on B. juncea var. multiceps cultivars Da Ye and Xiao Ye. Cotyledons of 14-day-old red-in-snow mustard seedlings were wound inoculated with a 10-μl pycnidiospore suspension (1 × 10⁷ conidia/ml) of each isolate, respectively, 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 light/12-h dark. After 4 days of incubation, whereas the control group remained asymptomatic, the inoculated cotyledons for isolates QJ3-1 and QJ-4 formed necrotic lesions around the wounds. A fungus showing colony morphology similar to the original isolates was reisolated from the diseased cotyledons. Therefore, L. biglobosa ‘brassicae’ was determined to be the causal agent of blackleg on red-in-snow mustard in China. To our knowledge, this is the first record of L. biglobosa ‘brassicae’ causing black leg on B. juncea var. multiceps in China.