Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31056
標題: Burkholderia caryophylli 之特性及其血清偵測
Characterization and serological detection of Burkholderia caryophylli
作者: 黃冬青
Huang, Tung-chin
關鍵字: carnation
康乃馨
baby's breath
outer membrane protein
serological detection
滿天星
外膜蛋白
血清偵測
出版社: 植物病理學系
摘要: 自南投埔里康乃馨及滿天星細菌性萎凋病罹病植株, 共計分離到 31 個 Burkholderia caryophylli 菌株,此些菌株為革蘭氏陰性菌呈桿 狀, 具多條極 生鞭毛, 在馬鈴薯葡萄糖瓊脂培養基 ( PDA ) 平板上, 可產生可溶性褐色素, 然在 KB 培養基上則不產生螢光色素,可引起煙 草葉片過敏性反應,不引起馬鈴 薯切片之軟腐, 可形成 Kovacs' 氧化 酵素、精氨酸二水解酵素,可在 41 ℃下 生長。以 Biolog GN microplate 測試對 95 種碳源之利用情形,於 24 小時之 反應結果顯示 ,此 31 個菌株可利用其中 57 種碳源,而有 15 種碳源之利用菌 株間 有差異, 其餘 23 種碳源則所有菌株皆不能利用。 以 SDS-PAGE 分 析 Burkholderia caryophylli 之外膜蛋白圖譜, 結果顯示 B. caryophylli 與測 試之其他病原細菌 Acidovorax avenae subsp. citrulli, B. andropogonis, Erwinia carotovora subsp. carotovora,Erwinia chrysanthemi, Ralstonia solanacearum 與 Pseudomonas aeruginosa 菌株間有明顯差異, B. caryophylli 菌株在 41 kDa, 35 kDa 與 27 kDa 附近有明顯之條帶 (band)。 病原 性之測定結果顯示,對康乃馨與滿天星之致病力在 B. caryophylli 菌株 間 有差異。土壤中之存活測試結果顯示,低溫有利於 B. caryophylli 在土壤中之 存活, 於 20-25 ℃時 B. caryophylli 於土壤中可存活 16 週,而在 35 ℃時 則只存活 7 週。 在血清試驗中,以 B. caryophylli CO8 菌株全細胞與外膜蛋 白 ( 35 kDa ) 分別製備之抗血清, 在 SDS- 瓊脂雙向擴散反應中, 此 31 個 B. caryophylli 菌株與此兩種製備之 抗血清所形成之反應帶, 菌株間可完全融 合,並無叉狀反應發生。此兩 種抗血清對其他測試之植物病原細菌則無明顯之反 應帶形成。 以 DAS-ELISA 反應測試結果亦顯示, 此兩種抗血清與 B. caryophylli 及其他測試之植物病原細菌有明顯之差異。 以 B. caryophylli CO8 全細胞製備之抗血清 ( 免疫球蛋白濃度 1 ug/ ml, 酵素連結抗體稀釋 1000 倍 ),可偵測到之 B. caryophylli 菌量最低 為 2.4 X 105 cells/ well ,而以其外膜蛋白 ( 35 kDa ) 所製備之抗 血清 ( 免疫球蛋白濃度 2 ug/ ml , 酵素連結抗體稀釋 500 倍 ), 可偵測到之菌量最低為 2.4 X 106 cells/ well。 應用此兩種抗血清以 DAS-ELISA 方法, 可快速偵測到 B. caryophylli 於罹病植株之存在情 形。
A total of 31 strains of Burkholderia caryophylli were isolated from carnation and baby's breath grown at Puli, Nantou. They were Gram- negative rods with lophotrichous polar flagella. They produced diffusible brown pigment on potato dextrose agar medium, and did not form fluorescent pigment on King's medium. They could induce hypersensitive reaction on tobacco leaves but did not cause soft rot on potato slices. They produced Kovacs' oxidase, arginine dihydrolase and could grow at 41 ℃. Utilization of carbon sources by these strains was examined in the Biolog GN microplate. The results ( 24 hr reaction time ) revealed that among the 95 carbon sources tested, all strains of B. caryophylli could utilize 57 carbon sources, and varied in utilization of 15 carbon sources; in addition, they all did not utilize the other 23 carbon sources. Outer membrane profiles of B. caryophylli, Acidovorax avenae subsp. citrulli, Burkholderia andropogonis, Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi, Ralstonia solanacearum and Pseudomonas aeruginosa were analyzed by SDS-PAGE, the results indicated that all strains of B. caryophylli usually had three distinct bands at the size of 41 kDa, 35 kDa and 27 kDa. The outer membrane protein profiles of B. caryophylli and other phytopathogenic bacteria tested were significant different. In the pathogenicity tests, the virulence of B. caryophylli to carnation and baby's breath was varied among strains tested. The survivability of B. caryophylli in soil was much better at low temperature ( 20 ℃ ) than that at high temperature ( 35 ℃ ). They could survive 16 weeks at 20-25 ℃, whereas they could only survive 7 weeks at 35 ℃. Two antisera against whole cells and outer membrane ( 35 kDa ) of B. caryophylli CO8, respectively were prepared. In SDS- immunodiffusion assays, all these 31 strains of B. caryophylli formed completely identical bands with the two antisersa. They were complete identity among the strains tested. However, there was no distinct band formed when these two antisera were tested with the other phytopathogenic bacteria. In addition, these two antisera also did not react with other phytopathogenic bacteria in the DAS-ELISA tests. The sensitivities of DAS-ELISA with these two antisera for detection of B. caryophylli were examined. When antiserum against whole cells of B. caryophylli CO8 was used ( IgG 1ug/ ml, conjugate IgG 1000x ), the lowest concentration of B. caryophylli detected was 2.4 x 105 cells/ well; whereas the lowest detection level was 2.4 x 106 cells/ well when antiserum against outer membrane ( 35 kDa ) was used. ( IgG 2 ug/ ml, conjugate IgG 500x ). Furthermore, DAS-ELISA with these two antisera could rapidly detect the presence of B. caryophylli in the B. caryophylli infected plant tissues.
URI: http://hdl.handle.net/11455/31056
Appears in Collections:植物病理學系

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