| Summary: | 碩士 === 國立中興大學 === 植物病理學系 === 87 === Burkholderia caryophylli is the causal agent of bacterial wilt of carnation and baby''s breath. It also causes the leaf rot, crown rot and wilt of statice. In this study, strains of B. caryophylli were also isolated from wilting eustoma plants from fields at Tianwei, Changhua. Survival of B. caryophylli CO8r（a spontaneous mutant of CO8 with a rifampicin-resistant marker）in soil was studied. The results showed that B. caryophylli could not oversummer in field soil at Datsuen, Changhua. It suggested that the primary inoculum source of B. caryophylli might be from pathogen-infected or contaminated seedlings. Various techniques were developed and used to detect B. caryophylli in this study. Two antisera against whole cells and outermembrane protein（35kDa） of B. caryophylli CO8, respectively, were prepared and used for detection of B. caryophylli. In Ouchterlony double diffusion assays, strains of B. caryophylli from carnation, baby''s breath and eustoma formed complete identical bands with these two antisera. However, there was no any band formed when these two antisera were reacted with the other phytopathogenic bacteria. In addition, these two antisera only reacted with strains of B. caryophylli but not with other phytopathogenic bacteria in the DAS-ELISA tests. DAS-ELISA with these two antisera could rapidly and specifically detect the presence of B. caryophylli in the B. caryophylli infected eustoma plants from fields. Tissue blotting immunoassay（TBIA）with CO8 whole cell antibody could rapidly and specifically detect and locate the B. caryophylli in the B. caryophylli-infected carnation and baby''s breath tissues. It was found that tissues of statice have high concentration of alkaline phosphatase, thus TBIA with alkaline phosphatase labeled secondary antibody could not be used for detection of B. caryophylli in statice tissues. In addition, ultrathin sections of B. caryophylli infected carnation tissues treated with CO8 whole cell antibody IgG and gold-labeled secondary antibody, immunogold-labeled B. caryophylli cells could be observed in infected tissues with transmission electron microscope. The specificity and sensitivity of primer pairs 20L/21R and nL/nR were tested by polymerase chain reaction（PCR）for detection of B. caryophylli. The results showed that 20L/21R and nL/nR could specifically amplify a 500 bp and a 400 bp DNA fragment, respectively, from chromosomal DNAs or NaOH-treated cell suspensions of B. caryophylli strains isolated from carnation, baby''s breath and eustoma. And there was no any DNA fragment amplified with these two primer pairs from DNAs of other phytopathogenic bacteria tested. The sensitivity of PCR for detection of B. caryophylli with primer pairs 20L/21R and nL/nR was 20 cells. TBIA, DAS-ELISA and PCR techniques could detect B. caryophylli in symptomless tissues of carnation artificially inoculated with B. caryophylli. The SCA medium was formulated as a selective medium for B. caryophylli. It could inhibit the growth of most nontarget bacteria. However, the recovery rate of B. caryophylli on this medium was relative low when compared with that on potato dextrose agar medium. Cells of B. caryophylli formed translucent and domed colonies on SCA medium. The reverse side of colonies showed dew-drop like morphology which was quite unique. And the SCA medium might be used as a differential medium for B. caryophylli.
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