| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 97 === Xanthomonas spp. is an seed-borne bacterial pathogen, can be spread by seeds. It will cause a serious damage of important economic crops, so ensuring absence of the pathogen is an important measures for preventing the bacterial disease. We have developed the differential medium of Xanthomonas spp., mTBM-42. We can separat alive strains of Xanthomonas from seeds or infected plants by mTBM-42. Xanthomonas spp. appeared as glistening, raised, and green colonies in mTBM-42. Colonies of Xanthomonas will surround by a smaller milky zone and a larger clear zone. We have known that Xanthomonas spp. appeared milky zone, it is associated with hydrolysis of Tween 80. We found an esterase gene from X. campestris pv. campestris XCC1-1 by gene cloning, XCC-estA, and designed a pair of specific primers of Xanthomonas spp. (Xc-lip-F2 + R2) by sequencing and alignment. The purpose of this study was to study on if there is any other bacterium can interfere application of differential medium, to improve sensitivity of mTBM-42 by immunomagnetic separation, and develop PCR and DNA microarray to characterize Xanthomonas spp. to species and to pathovars. At first, when we isolated pathogen from seeds, we found Strenotrophomonas maltophilia (original name was Xanthomonas maltophilia) had same characterization with Xanthomonas spp. in mTBM-42 medium. GYCA medium could distinguish S. maltophilia and Xanthomonas spp. S. maltophilia had no pathogenicity to leaves of cabbage, and could not amplify target fragment by Xanthomonas spp. estA primers (Xc-lip-F2 + R2). We isolated Xanthomonas spp. strains and S. maltophilia from cauliflower seeds, used Xanthomonas spp. estA primer to PCR, the Xanthomonas strains could amplify target fragment, and had pathogenicity to leaves of cabbage, S. maltophilia could not amplify target fragment by Xanthomonas spp. estA primer, and had no pathogenicity to leaves of cabbage. By using XCC-EstA antibody in ELISA test, XCC-EstA antibody had specificity to Xanthomonas spp. We used Xanthomonas spp. in western blot, it revealed that Xanthomonas spp. could produce EstA without Tween 80 induction. EstA could secret to extracellular, and most of all are in cell membrane. Using XCC-EstA antibody in immunomagnetic separation (IMS) separation could isolate Xanthomonas spp. from samples effectively, by using IMS followed by plating on mTBM-42 medium and PCR. We prepared XCC1-1 antibody by X. campestris pv. campestris XCC1-1 antigen. We used XCC1-1 antibody and XCC-EstA antibody in ELISA test. They had specificity to XCC1-1 and S. maltophilia, but S. maltophilia was higher than Xanthomonas spp.. We used XCC1-1 antibody after eliminating the antibody which could bind with S. maltophilia (named XCC1-1Ab-[Sm]) from XCC1-1 antibody in ELISA test. The result indicated the absorbent of XCC1-1Ab-[Sm] antibody, which could decrease in 0.5 with S. maltophilia, but it still had higher absorbent with XCC1-1; the antibody had the same specificity to other Xanthomonas spp. strains. We designed XD4B8S-s primers from Xanthomonas spp. virD4-virB8 intergenic sequences in DNA microarray. We used eight species, thirteen strains in gene cloning and constructing plasmid. Furthermore, to create phylogenetic relationship by XD4B8S-s fragment in these thirteen strains. It could categorize to eight groups, and selected one strain from each group to hybridize with which target DNA. There were six strains could be separated apparently. We could characterize by PCR, except DNA microarray. To combine three pairs of specific primers to species designed by predecessors, and design specific primers to another three different strains. After all, we can characterize six strains. To summarize above, we can create a method and process for plant pathogenic Xanthomonas spp. from plant seeds.
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