In December 2012, symptoms of typical bacterial leaf blight were observed on carrot plants (Daucus carota L. subsp. sativus) cultivated in commercial fields in Kujwa, Jeju, Korea. The disease was detected in 40% of 50 fields surveyed with an incidence of 10% on average. The bacterial leaf blight lesions on leaf blades were elongated, dark brown to black with water-soaked edges and chlorotic halos. Lesions were also crescent-shaped to V-shaped on leaflets. Four bacterial isolates were recovered on trypticase soy agar from leaf lesions that were surface-sterilized in 70% ethyl alcohol for 20 s. Identity of the isolates was confirmed by PCR product (1,266-bp) using a specific primer set for Xanthomonas hortorum pv. carotae (Kendrick 1934) Vauterin et al. 1995, XhcPP03 (1). All isolates were gram-negative, aerobic rods with a single polar flagellum. Isolates were positive for catalase and negative for oxidase. In phenotypic tests for differentiation of Xanthomonas (2), the isolates positive for mucoid growth on yeast extract-dextrose-calcium carbonate agar, growth at 35°C, hydrolysis of esculin, protein digestion, alkaline in litmus milk, acid production from arabitol, and utilization of glycerol and melibiose. The isolates were negative for growth on SX medium, hydrolysis of starch, and ice nucleation. The gyrB gene (863 bp) and the rpoD gene (870 bp) were sequenced to aid identification of the original isolates using published PCR primer sets, Xgyr1BF/Xgyr1BR and XrpoD1F/XrpoD1R (4), respectively. Sequences of the gyrB gene (GenBank accessions KC920729 to KC920732) from the carrot isolates shared 100% sequence identity with that of the X. hortorum pv. carotae strain NCPPB 425 (EU285243). In phylogenetic analyses based on the partial sequences of the gyrB and the rpoD genes for Xanthomonas spp. available at NCBI (4), and sequences of the carrot isolates (KC920734 to KC920737 for rpoD gene) using the Neighbor-joining method in MEGA Version 5.1 (3), the isolates were clustered in the X. hortorum-cynarae-garnderi group. Pathogenicity of the isolates was tested by spray inoculation with a bacterial suspension (106 CFU/ml) prepared in sterile distilled water at 6 to 7 true-leaf stage (three plants per isolate). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) for 15 hr, and then transferred to a greenhouse at 24 to 28°C and 65% RH. Characteristic leaf blight symptoms developed on inoculated carrot plants, while no symptoms were observed on the negative control plants 14 days after inoculation. The bacterium was re-isolated from symptomatic tissue and the identity confirmed through gyrB gene sequence analysis (4). Based on PCR, morphological and phenotypic tests, sequence analysis, and pathogenicity assays, the isolates were identified as X. hortorum pv. carotae. To our knowledge, this is the first report of bacterial leaf blight of carrot caused by X. hortorum pv. carotae in Korea. The detection of this pathogen could have a significant economic impact due to yield losses from disease development. Consolidation of quarantine inspection on imported carrot seeds needs to control an outbreak of the disease. Crop rotation and plowing are recommended to reduce incidence of the disease in the infested fields.
References: (1) J. A. Kimbrel et al. Mol. Plant Pathol. 12:580, 2011. (2) N. W. Schaad et al. Page 189 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.
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