First Report of Bacterial Canker of Tomato Caused by Clavibacter michiganensis subsp. michiganensis in Korea.

Abstract

In 2007, a new bacterial disease was observed in greenhouse-cultivated cherry tomatoes in Cheorwon and Iksan provinces, Korea. The disease caused severe wilt of tomatoes (Solanum lycopersicum cv. Koko). Infected young petioles were curled downward. Margins of the leaves rolled upward and whole leaves were distorted. Stem cankers had reddish or dark brown cavities. Vascular tissues in stems cut longitudinally were brown to deep brown, but no bird's eye lesions were observed. Eight bacterial strains recovered from the stems of wilted tomatoes produced yellow colonies on nutrient broth-yeast extract agar and pink colonies on triphenyl tetrazolium chloride. Pathogenicity of the strains (three plants per strain) on 18-day-old tomatoes (cv. Koko) was confirmed by clip inoculation of petioles of second leaves and spray inoculation with bacterial suspensions (1 × 108 CFU/ml) in sterile distilled water. Wilt and canker symptoms were observed 2 weeks after inoculation. Symptoms produced by both inoculation methods were systemic and localized. Clip inoculation of tomatoes resulted in wilt, defoliation, and open stem cankers, whereas small, white spots (2 to 3 mm in diameter) and sometimes water-soaked, dark brown-to-black lesions on the leaf margins were observed with spray inoculation. Bacteria were reisolated from stems and leaves of the inoculated plants and their identities confirmed by direct PCR using specific primer set CMM5/CMM6 (1). No symptoms were observed on negative control plants inoculated with sterile water. All strains were gram-positive aerobic rods with no polar flagella. Strains were positive for esculin hydrolysis, gelatin liquefaction, H2S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)-melezitose and d(-)sobitol, and utilization of acetate, formate, lactate, propionate, and ribose. Identification as C. michiganensis subsp. michiganensis was confirmed using 16S rDNA universal primers fD1 and rP2 (4) and internal primers (3). The 1,439-bp PCR fragment of strain BC2643 was sequenced (GenBank Accession No. EU685335) and compared with reference C. michiganensis subspecies strains in GenBank: AM410696 (C. michiganensis subsp. michiganensis), AM410693 (C. michiganensis subsp. tessellarius), AM410697 (C. michiganensis subsp. nebraskensis), AM410694 (C. michiganensis subsp. sepedonicus), and AM410695 (C. michiganensis subsp. insidiosus). The sequence had a similarity index of 0.999 calculated by Juke-Cantor model (2) with the 16S rRNA sequence of C. michiganensis subsp. michiganensis (AM410696). The fragment size of eight strains amplified by PCR using CMM5/CMM6 (1) was identical to that of the C. michiganensis subsp. michiganensis reference strain KACC20122. On the basis of the physiological, genetic, and pathological characteristics, all strains were identified as C. michiganensis subsp. michiganenesis. To our knowledge, this is the first report of C. michiganensis subsp. michiganenesis causing bacterial canker on tomato in Korea.