Bacterial Leaf Spot of Iceberg Lettuce Caused by Xanthomonas campestris pv. vitians Type B, a New Disease in South Korea

In 2008 and 2009, a leaf spot of iceberg lettuce (Lactuca sativa var. capitata) was observed in two fields of Pyeongchang District and Jecheon City in South Korea, respectively. Disease incidence averaged 3.5% in the two fields. Symptoms on leaves included black, water-soaked, angular lesions with halos. Two bacterial isolates, BC2932 and BC3095, were recovered on trypticase soy agar (TSA) from lesions surface sterilized in 70% ethyl alcohol for 1 min. Both isolates had gram-negative, aerobic rods each with a single flagellum. Colonies on peptone sucrose agar were yellow and raised with smooth margins. Pathogenicity was evaluated on 3-week-old lettuce plants (cv. Avi). Bacteria were grown on TSA for 48 h at 28°C. A bacterial suspension in sterile distilled water (100 ml at 1 × 105 CFU/ml) was sprayed onto three plants for each isolate. Plants were maintained in a growth chamber at 28°C and 90% relative humidity. Isolates induced identical symptoms 3 days after inoculation as those originally observed in the fields. Pathogenicity of bacteria reisolated 10 days after inoculation from lesions surface sterilized in 70% ethyl alcohol was confirmed by inoculation as described above. No symptoms were observed on two control plants treated with sterile distilled water. Identity of bacteria reisolated from inoculated leaves was confirmed by PCR with specific primer set B162 (1). DNA of the original two isolates and 12 reisolates (two per inoculated plant) was amplified by PCR assay using Xanthomonas campestris pv. vitians Type B LMG938 (= BC2575) as a positive control treatment and X. axonopodis pv. vitians strain CFBP2538 (= BC2610) as a negative control treatment. The PCR amplicon for each of the 14 test isolates was identical in size to that of X. campestris pv. vitians Type B LMG938. No fragment of X. axonopodis pv. vitians CFBP2538 was amplified. Patterns of metabolic fingerprinting of the original two isolates were more similar to those of X. campestris pv. vitians Type B LMG938 than X. axonopodis pv. vitians CFBP2538 using Biolog Microbial Identification System Version 4.2 (Biolog Inc., Hayward, CA). X. campestris pv. vitians Type B LMG938, BC2932, and BC3095 were identified as X. campestris pv. pelargonii with a Biolog similarity index of 0.68, 0.45, and 0.78, respectively. Strain X. axonopodis pv. vitians CFBP2538 was identified as X. campestris pv. juglandis with an index of 0.48. The dnaK (958 bp), gyrB (859 bp), and rpoD (884 bp) regions were partially sequenced to aid in identification of the two original field isolates as well as X. campestris pv. vitians Type B LMG 938 and X. axonopodis pv. vitians CFBP2538 using reported PCR primers (3). Sequences were compared with those of reference strains of Xanthomonas in GenBank. Sequences of the three genes from the two lettuce field isolates shared 100% similarity to those of the genes of X. campestris pv. vitians Type B LMG938 and had a distance index value of 0.040, 0.099, and 0.046, respectively, with the reference strain of X. axonopodis pv. vitians CFBP2538 determined by p-distance modeling using MEGA Version 4.1 (2). Based on the pathogenicity test and sequence analyses, the isolates were identified as X. campestris pv. vitians Type B. To our knowledge, this is the first report of bacterial leaf spot of iceberg lettuce caused by X. campestris pv. vitians Type B in South Korea. References: (1) J. D. Barak et al. Plant Dis. 85:169, 2001. (2) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (3) J. M. Young et al. Syst. Appl. Microbiol. 31:366, 2008.