First Report of Leptosphaeria biglobosa Causing Black Leg on Raphanus sativus in Central China

Chinese radish (Raphanus sativus) is an important vegetable grown widely in China. In 2010 to 2013, surveys for Leptosphaeria spp. on cruciferous vegetables were conducted in 17 counties in Hubei Province, China. Black leg symptoms on tuber roots and basal stems of radish were observed in Hanchuan, Jingmen, and Macheng counties. Disease incidence ranged from 2 to 25% in 10 surveyed radish fields. Five fungal isolates were obtained from diseased radish plants by surface-sterilizing radish tissue (5% NaOCl for 90 s, and then rinsed in sterilized water three times) and plating onto potato dextrose agar (PDA) plates incubated at 20°C. The isolations produced fluffy white colonies with a yellow pigment, and black-brown, globose pycnidia with pink conidial ooze formed after 10 days. Pycnidia were 150 to 200 × 80 to 100 μm. Conidia were hyaline, cylindrical, and 4 to 5 × 2 μm. The cultural and morphological characteristics of the isolates matched the description for Phoma lingam, anamorph of Leptosphaeria maculans and L. biglobosa (3). All five isolates were identified by PCR assay using the species-specific primers LbigF, LmacF, and LmacR (1), and isolate HCLB-1 was identified further by PCR cloning and analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (3,4). Genomic DNA was extracted from mycelium of each isolate harvested from 7-day-old cultures in V8 broth using the CTAB method (5). A 444-bp DNA fragment was detected by PCR assay, suggesting that all five isolates belonged to L. biglobosa rather than L. maculans as the latter generates a 331-bp DNA fragment (1). The HCLB-1 sequences for ITS (587 bp, GenBank Accession. No. KC880981), actin (899 bp, KF307762), and β-tubulin (432 bp, KF220296) genes were 99 to 100% identical to those of L. biglobosa isolates in GenBank. All five isolates were tested for pathogenicity on R. sativus cultivars Duan Ye 13 and Qi Ye Hong. Cotyledons of 10-day-old radish seedlings and post-harvest mature roots were wounded using a sterilized needle, and 10 μl of a conidial suspension (1 × 10⁷ conidia/ml) of each isolate was pipetted onto the wounded area on each cotyledon or root, respectively, with 12 cotyledons (= 24 wounded sites) and 1 root (= 6 wounded sites) inoculated/isolate. One wounded root and 12 wounded cotyledons inoculated with water were used as control treatments. Treated roots and seedlings were incubated at 20°C and 100% RH in the dark for 7 days, and under a 12 h light/12 h dark cycle for 12 days, respectively. While the control roots and cotyledons remained asymptomatic, the roots and cotyledons inoculated with all five test isolates formed black cankers and necrotic lesions, respectively, in the inoculated wounds. A fungus re-isolated from symptomatic roots and cotyledons resembled the original isolates in colony morphology and the 444-bp DNA fragment detected by PCR assay. No fungus was isolated from control seedlings or roots. Thus, L. biglobosa appears to be the causal agent of black leg observed on radish in Hubei, China. L. biglobosa was reported to infect wild radish (R. raphanistrum) (2). To our knowledge, this is the first report of L. biglobosa causing black leg on R. sativus. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990. (5) J. Zhang et al. Mycologia 102:1114, 2010.