A simple method to differentiate Xanthomonas campestris pv. campestris and X. campestris pv. raphani isolated from cruciferous seeds.

• Main Authors: CHEN,HUI-CHEN, 陳慧真
• Other Authors: LEE,YUNG-AN
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/84295466207542339557

碩士 === 輔仁大學 === 生命科學系碩士班 === 104 === The Xan-D differential medium was used to detect Xanthomonas spp. from cruciferous seeds. Some strains of isolated Xanthomonas spp. were black rot pathogens (Xanthomonas campestris pv campestris; XCC), and some were leaf spot pathogens (X. campestris pv raphani; XCR) determined by the plant inoculation tests on cabbages. Because XCC and XCR strains cannot be distinguished on the Xan-D medium, the purpose of this research is to develop a medium for differentiation of XCC and XCR. It was found that XCC strains were yellow, but XCR strains were light green yellow on the Luria-Bertani agar medium with X-gal (called LA-X medium).When glucose was added to the LA-X medium (LAG-X), XCC strains remained yellow, but XCR strains turned yellow green. Therefore, the XCC and XCR strains can be distinguishable by the color development on the LAG-X medium. Three media, TY-X (tryptone, yeast extract, X-gal), TYG-X (containing glucose) and the TYGNa-X (TYG-X containing NaCl), were prepared based on the chemical components of LA medium. It was found that XCC and XCR strains were yellow on the TY-X medium, but both XCC and XCR strains turned yellow green on the TYG-X medium, indicating that glucose can enhance both strains to degrade X-gal. When NaCl was added into the TYG-X medium (TYGNa-X), XCC strains became yellow but XCR strains remained yellow blue. The results show that NaCl could inhibit the X-gal degradation ability of XCC strains, but did not affect that of XCR. More strains of XCC and XCR isolated from cruciferous seeds, and other Xanthomonas spp. were also tested on the TYG-X and TYGNa-X media. All tested strains of XCC, XCR, and Xanthomonas spp. turned yellow blue on the TYG-X medium. However, on the TYGNa-X medium, XCR, and Xanthomonas spp. remained yellow blue, but all XCC strains tested became yellow. The results show that Xanthomonas spp. have a capacity to degrade X-gal, but only that of XCC can be inhibited by NaCl. Two bacteria, Stenotrophomonas maltophilia and S. rhizophila, which were often isolated from cruciferous seeds and developed yellow green color on Xan-D medium, were flat and yellow on both TYG-X and TYGNa-X media. Accordingly, TYGNa-X is a chromogenic differential medium for XCC, and can be used to distinguish XCC and XCR strains after the Xanthomonas spp. are isolated on the Xan-D medium from cruciferous seeds. The TYG-X medium was modified to become a selective and differential medium, SYG-X (CF24) with cycloheximide and 5-fluorouracil, for the isolation of Xanthomonas spp. The colonies of XCC and XCR strains on the SYG-X (CF24) medium were round, convex, and yellow blue surrounded by the transparent mucoid, which are easily distinguishable from other bacterial colonies. In addition to NaCl, other chemicals containing metal ions, such as KCl, MgCl2 and MgSO4, can inhibit the X-gal degradation ability of XCC strains. The X-gal degradation ability was reduced in the cytosol of XCC strains cultured on the TYGNa medium, compared with that on the TYG medium. The results indicate that metal ions would at least reduce the amount or activity of X-gal degradation enzymes of XCC. The further mechanisms of inhibitory effects of metal ions on XCC are still understudy.