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First Report of Bacterial Leaf Spot of Witloof, Caused by Pseudomonas cichorii in Korea

In August 2011, bacterial leaf spot was observed on witloof (Cichorium intybus L. var. foliosum) grown in a commercial field with 15% incidence in Injae, Korea. Symptoms on leaves included irregular brown to reddish brown spots in the center. Bacterial streaming from the lesions was observed microscopically. Bacterial isolates (BC3286, BC3287, and BC3308-BC3310) were recovered on Trypticase soy agar from lesions surface-sterilized in 70% ethyl alcohol for 30 s. The isolates were gram negative, urease negative, fluorescent on King's B agar, and had aerobic rods with 2 to 6 polar flagella. Pathogenicity tests were separately performed in different greenhouses located in Suwon (National Academy of Agricultural Science) and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by spray inoculation of healthy, 10-day-old leaves of witloof plants (two plants/isolate) with a suspension of original field isolate (10⁶ CFU/ml). Sterile distilled water was used as negative control. The inoculated plants were incubated in a growth chamber (25°C and 95% relative humidity [RH]) overnight, then transferred to a greenhouse at 23 to 27°C and 60 to 70% RH. Characteristic leaf spot symptoms were observed on inoculated witloof plants 8 days after inoculation. No symptoms were observed on control plants. The bacterium reisolated from the inoculated leaves was confirmed by analyzing sequence of the gyrB gene with direct sequencing method of PCR products using primers gyr-F and gyr-R (2). The sequence of reisolated bacteria shared 100% similarity with inoculated ones. In LOPAT (1) tests, all isolates and the reference strain of Pseudomonas cichorii CFBP2101^T (=BC2595) were levan negative, oxidase positive, potato rot negative, arginine dihydrolase negative, and tobacco hypersensitivity positive, indicative of group III (–, +, –, –, +) of fluorescent pseudomonads. The 16S rRNA (1,408 bp), and gyrB (676 bp) regions were sequenced to aid in identification of the original field isolates as well as P. cichorii CFBP 2101^T (=BC2595) using reported sets of PCR primers, fD1/rP2 and gyr-F/gyr-R, respectively (2,4). Phylogenetic analyses based on partial sequences of the gyrB and the 16S rRNA of Psudomonas spp. available in GenBank, the reference strain of P. cichorii CFBP2101^T (=BC2595), and the witloof field isolates were conducted using the neighbor-joining method with Juke-Cantor model of distance calculation in MEGA version 5.1 (3). The isolates and the reference strain of P. cichorii CFBP2101^T (=BC2595) was clustered in one group with P. cichorii strains in both phylogenetic trees based on the two sequences. Sequences of the 16S rRNA region had a distance index value ranging from 0.000 to 0.001 between the reference strain of P. cichori CFBP2101^T (GenBank JX913784) and the field isolates (JX913785 to JX913789), and ranged from 0.000 to 0.001 within the field isolates. Sequences of the gyrB region had a distance index value ranging 0.029 to 0.033 between the reference strain (JX913790) and the field isolates (JX913791 to JX913795), and ranged from 0.000 to 0.041 within the field isolates. To our knowledge, this is the first report of bacterial leaf spot of witloof caused by P. cihorii in Korea. P. cichorii has a wide host range, and an important economic impact on vegetables. The disease is expected to result in a significant economic impact on root production of witloof in Korea.

References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) H. Sawada et al. J. Mol. Evol. 49:627, 1999. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) W. G. Weinsburg et al. J. Bacteriol. 173, 697, 1991.