First Report of Leptosphaeria biglobosa Causing Black Leg on Brassica campestris ssp. chinensis var. purpurea in Central China
Authors: Cai X. , Yang L. , Zhang J. , Li G. Q. 
Published: March 2014

Purple cai-tai (Brassica campestris ssp. chinensis var. purpurea) is a traditional vegetable widely grown in southern China. In 2012 and 2013, black leg disease was observed on purple cai-tai in three surveyed cities (Jingzhou, Qianjiang, and Huanggang) in Hubei Province of China. Disease incidence ranged from 5 to 88% in eight surveyed fields. White cankers occurred on basal stems and numerous black pycnidia and pink conidia were present on the stem surface. Surface-sterilized (5% NaOCl for 90 s, rinsed in sterilized water three times) stem pieces were plated on potato dextrose agar (PDA) and incubated at 20°C and 12 h light/12 h dark for 7 days. A total of 22 isolates were obtained. All of the isolates appeared similar in colony morphology on PDA (20°C, 7 to 10 days), producing yellow pigment and black-brown, globose pycnidia containing cylindrical hyaline conidia (4 to 5 × 2 μm). These characteristics matched the description for Phoma lingam, the anamorph Leptosphaeria maculans and L. biglobosa (2). Species-specific primers LbigF, LmacF, and LmacR (1) were used in PCR-based identification of the isolates. A 444-bp DNA fragment characteristic of L. biglobosa was amplified from DNA extracted from all of the collected isolates. DNA amplification from the isolate UK-1 of L. maculans from B. napus in Hertfordshire of the United Kingdom yielded a 331-bp fragment. Two isolates, HGHCT2-1 and HGHCT2-2, were further identified by cloning and analysis of the ITS sequences and the partial sequences encoding β-tubulin and actin (3,4). The ITS sequences (586 bp, GenBank Accession. Nos. KF371660 and KF371661) were 100% identical to L. biglobosa ‘brassicae’ strain UK28 (DQ133893). The DNA sequences for β-tubulin (479 bp, KF307760 and KF307761) and actin (899 bp, KF307758 and KF307759) were 99 and 100% identical to the partial β-tubulin gene sequence (AY748997) and the partial actin gene sequence (AY748949) of the L. biglobosa ‘brassicae’ strain 2379-4, respectively. Pathogenicity of six randomly selected isolates was determined on two purple cai-tai cvs. Wanzi Qianhong and Jiu Yue Xian. Cotyledons of 10-day-old seedlings grown in potting mix in pots were pricked with a sterilized needle, and each wound was inoculated with 10 μl of conidial suspension (1 × 107 conidia/ml) of an isolate or 10 μl sterilized water (control). There were 12 cotyledons for each isolate and control. The experiment was repeated once. The treated seedlings were incubated at 20°C in an incubator under 12 h light/12 h dark for 12 days. The control cotyledons were healthy, but necrotic lesions were developed on the cotyledons that were inoculated with L. biglobosa and formation of pycnidia was observed on some lesions. Fungi re-isolated from the lesions were similar to the original L. biglobosa isolates both in colony morphology on PDA and in species-specific PCR testing. No fungi were isolated from the control cotyledons. This is the first report of L. biglobosa causing black leg on B. campestris ssp. chinensis var. purpurea in central China. The finding will be useful for understanding of the epidemiology of black leg on cruciferous crops and for management of this disease. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) R. A. Shoemaker and H. Burn. Can J Bot. 79:412, 2001. (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, San Diego, CA, 1990.