Plant Disease
Editor-in-Chief: Mark L. Gleason
Published by The American Phytopathological Society
ISSN: 0191-2917 Accepted for publication http://dx.doi.org/10.1094/PDIS-03-15-0251-PDN
First report of Cercospora armoraciae, causal agent of Cercospora leaf spot, on horseradish in Serbia Dr. Nenad Trkulja Institute for Plant Protection and Environment, Plant Disease, Teodora Drajzera 9, Belgrade, Serbia, 11000;
[email protected] Miss Anja Milosavljevic Institute for Plant Protection and Environment, Plant Disease, Belgrade, Serbia;
[email protected] Dr. Milana Mitrovic Institute for Plant Protection and Environment, Department of Plant Pests, Banatska 33, Zemun, Serbia, 11080;
[email protected] Dr. Jelena Jovic Institute for Plant Protection and Environment, Department of Plant Pests, Banatska 33, Belgrade, Serbia, 11080;
[email protected] Dr. Ivo Tosevski Institute for Plant Protection and Environment, Department of Plant Pests, Banatska 33, Zemun, Serbia, 11080;
[email protected]
Horseradish, Armoracia rusticana (P. Gaertn., B. Mey. & Scherb.), is a native or cultivated perennial plant that is grown worldwide and used for nutritional purposes or in alternative medicine (Agneta et al. 2013). Cercospora leaf spot, caused by Cercospora armoraciae (Sacc.), is a common fungal disease of horseradish (Walters and Wahle 2010). Production of horseradish in Serbia covers nearly 1,500 ha. Symptoms of Cercospora leaf spot were observed on leaves of horseradish in late autumn 2013 in the Braničevo district of Serbia. In total, 30 fields were inspected, with the incidence of symptoms ranging from 30 to 70%/field. Circular to subcircular, sunken lesions were observed with amphigenous
Plant Disease
caespituli and slight discoloration, from dark brown, tanned edges to a light brown zone in the center of each lesion. Conidiophores were cylindrical, solitary, or in loose to divergent fascicles, pale brown, simple and straight to strongly geniculate (60 to 120 μm × 4 to 6 μm). Conidia were solitary, hyaline, straight to mildly curved, cylindro-obclavate to acicular, distinctly thickened at the base, 4- to 9-septate, and obtuse at the apex (75 to 125 μm × 3 to 6 μm) (Groenewald et al. 2013). Single-spore isolates (n = 36) collected from affected fields, each derived from a germinated conidium from a different lesion, were established on potato dextrose agar (PDA). Isolates R5, R6, and R7 were chosen randomly to confirm pathogenicity using Koch's postulates. Colonized mycelial PDA plugs, each 5 mm in diameter, were placed mycelial side down in contact with the adaxial leaf surface of 5week-old horseradish seedlings. Two leaves per plant were inoculated and slightly curved using garden wire to prevent the agar plugs from dislodging. Six plants were inoculated with colonized agar plugs, and six plants were used as non-inoculated control plants treated similarly but with sterilized, non-colonized PDA plugs. Seedlings were kept in a moist chamber for 72 h and then transferred into a greenhouse set at 25 ± 2oC. After two weeks, symptoms resembling those observed on the original plants in the field developed on all of the inoculated plants, while the control plants remained asymptomatic. The same fungal pathogen was re-isolated from inoculated, symptomatic leaves and identified to species morphologically, but not from the control plants. DNA was extracted from R5, R6, and R7 isolates, and PCR assays used to amplify: 1) a portion of the internal transcribed spacer (ITS) region of ribosomal DNA, 2) the calmodulin gene (CAL), and 3) the histone gene (H3), using the primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013), and CYLH3F/CYLH3R (Crous et al. 2004), respectively. Sequences of the CAL, H3, and ITS regions of R5, R6, and R7 isolates were deposited in GenBank (Accession Nos. KP868780 to KP868788), respectively. BLAST analysis of the ITS sequences revealed 100% identity with ITS sequences of Cercospora armoraciae, C. nicotianae, C. malayensis, C. beticola, and C. citrullina. The CAL and H3 sequences were identical to reference sequences of C. armoraciae (CAL: JX142808, JX142809, and JX142810l; H3: JX142554, JX142556, and JX142560). This record, to our knowledge, represents the first report of C. armoraciae causing leaf spot on horseradish in Serbia.
