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題名 | 瓜類細菌性果斑病菌血清偵側技術之研發=Development of Serological Detection Technique for Bacterial Fruit Blotch Acidovorax Avenae Subsp. Citrulli in Cucurbit Crops |
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作者 | 王惠亮; 鄭安秀; Wang, H. L.; Cheng, A. H.; |
期刊 | 植物病理學會刊 |
出版日期 | 20010900 |
卷期 | 10:3 2001.09[民90.09] |
頁次 | 頁129-138 |
分類號 | 435.26 |
語文 | chi |
關鍵詞 | 瓜類作物; 細菌性果斑病菌; 血清學; Cucurbit crops; Bacterial fruit blotch; Serology; |
中文摘要 | 瓜類細菌性果斑病菌(Acidovorax avenae subsp. citrulli)供試菌株分別分離自臺中、臺南、高雄及宜蘭地區洋香瓜、西瓜、甜瓜、苦瓜罹病葉片或果實。由甜瓜分離菌株Aa 31細菌全細胞製備抗血清。利用SDS免疫擴散反應進行抗血清力價測定,結果顯示未稀釋和稀釋二倍後的抗血清均能與細菌性果斑病病原細菌(A. avenae subsp. citrulli)形成2條明顯弧形反應帶。所有由洋香瓜、西瓜、甜瓜、苦瓜分離之細菌性果斑病菌株均與所獲得之抗血清反應,且形成兩條明顯弧形反應帶,而此抗血清並不與其他五種不同病原細菌菌株形成任何反應帶;在間接和雙層夾心式酵素連結抗體檢測法【double antibody sandwich (DAS) ELISA】試驗中,亦可順利偵測到分離之所有細菌性果斑病菌株,而在兩種ELISA試驗中,其他五種細菌均無反應。所製備免疫球蛋白(immunoglobulin G,IgG)和酵素結合免疫球蛋白(conjugate IgG),在DAS-ELISA檢定法中,以IgG濃度1μg/ml和conjugate IgG稀釋1200倍之組合為最佳。在間接ELISA檢測法中,以IgG濃度1μg/ml為最適宜。ELISA檢測法對細菌性果斑病病原細菌之靈敏度測定結果顯示,在間接ELISA和DAS-ELISA試驗中,分別可以偵測到104和105 cfu/ml濃度之細菌性果斑病病原細菌。A. avenae subsp. citrulli Aa 31萃取之全量蛋白質,分子大小由15kDa至大於221 kDa。西方墨點分析結果,有二種蛋白質可和製備的免痠球蛋白反應,其分子量均大於221kDa,顯示本試驗所製備之抗血清,是由此兩種蛋白質反應製備而得。 |
英文摘要 | Acidovorax avenae subsp. citrulli which infected fruits and leaves of cucurbit crops including muskmelon、watermelon、melon and bitter gourd were collected from Taichung、Tainan、Kaohsiung and Ilam in Taiwan. The strain designated as Aa 31 of A. avenae subsp. citrulli isolated from melon was used for producing antiserum. The titer of antiserum was 1/2 dilution of crude antiserum in SDS-double diffusion test with two visible reaction bands. The optimum concentrations of immunoglobulin G(IgG)and enzyme conjugate immunoglobulin G(conjugate IgG)were 1μg/ml and 1/1200 dilution in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA),respectively. The optimum concentration of IgG was 1μg/ml in indirect ELISA. The minimum concentrations that resulted in positive reaction in DAS- ELISA and indirect ELISA were 105 and 10 4 cfu/ml of Aa 31,respectively. All strains of A. avenae subsp. citrulli from different cucurbit crops also reacted with antiserum resulting in two precipitation bands in SDS-double diffusion test. and reacted With IgG in indirect ELISA and DAS-ELISA However the antiserum did not react with other five species of bacteria including Xanthomonas axonopodis pv. citri、X. campestris pv. campestris、Erwinia chrysanthemi、E. carotovora Subsp. carotovora and X. campestris pv. mangiferaeindicae in SDS double diffusion test, indirect ELISA and DAS-ELISA. The result of SDS-polyacrylamide gel electrophoresis and western bloting showed that two reacting bands with protein molecular Weight over 221 kDa were observed. |
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