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Development and Application of Techniques for Detection of Burkholderia caryophylli
|關鍵字:||Burkholderia caryophylli;細菌性萎凋病;tissue blotting immunoassay;polymerase chain reaction;selective medium;DAS-ELISA;組織轉漬免疫分析;聚合酵素連鎖反應;選擇性培養基;雙抗體包夾酵素連結抗體免疫反應||出版社:||植物病理學系||摘要:||
Burkholderia caryophylli可引起康乃馨與滿天星之細菌性萎凋及星辰花之葉腐、冠腐及萎凋等病徵，本研究中則發現此菌亦可引起洋桔梗之細菌性萎凋。測試B. caryophylli CO8r（具抗rifampicin標識之菌株）於彰化縣大村鄉田間土壤中之存活情形，結果顯示B. caryophylli無法於土壤中越夏存活。B. caryophylli之最初感染源可能係來自帶菌之種苗。本研究主要探討B. caryophylli之偵測技術之發展與應用。以B. caryophylli CO8全細胞與其外膜蛋白（35kDa）分別製備抗血清，應用不同之血清技術進行B. caryophylli之偵測與鑑定。以雙向擴散反應測試分離自康乃馨、滿天星與洋桔梗之B. caryophylli菌株，得悉此些不同寄主來源之菌株與此兩種抗血清所形成之反應帶，菌株間完全融合，且此兩種抗血清具專一性，以DAS-ELISA測試結果亦顯示此兩種抗血清除B. caryophylli之菌株外，並不與其他測試之植物病原細菌有反應。自田間取回之洋桔梗罹病組織以DAS-ELISA可快速地偵測到B. caryophylli之存在情形。應用組織轉漬免疫法可偵測B. caryophylli在接種後康乃馨與滿天星罹病組織內之分佈與進展情形。本研究發現星辰花組織內含有鹼性磷酸酵素活性，因此以鹼性磷酸酵素為標識之系統不適合應用組織轉漬免疫法偵測星辰花組織內B. caryophylli存在之情形。此外接種B. caryophylli後之康乃馨罹病組織之超薄切片經免疫金標識處理後，於電子顯微鏡下可觀察到罹病組織內B. caryophylli菌體為免疫金標識之情形。以引子對20L/21R與nL/nR對康乃馨、滿天星及洋桔梗等不同寄主來源之B. caryophylli菌株之染色體DNA或經NaOH處理之細胞懸浮液進行PCR偵測，皆能分別增幅出500 bp或400 bp左右之專一性片段，而以其他供試之植物病原細菌DNA為模版時則無法增幅出B. caryophylli專一性之DNA片段，此兩組引子對偵測B. caryophylli之靈敏度皆為20個細胞。應用組織轉漬免疫法、DAS-ELISA及PCR等三種技術皆可在測試之康乃馨植株尚未出現萎凋病徵前偵測到B. caryophylli。本研究製備之SCA培養基具選擇性，能抑制大部份非標的細菌之生長，然對B. caryophylli之回收率低，B. caryophylli在此培養基上可形成透明隆起之菌落，菌落背面呈露珠狀，可做為鑑別B. caryophylli用。
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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