瓜類細菌性果斑病菌血清偵測技術之研發

Development of Serological Detedtion Technique for Bacterial Fruit Blotch Acidovorax avenae subsp. citrulli in Cucurbit Crops

王惠亮( H. L Wang) ； 鄭安秀( A. H Cheng)

《植物病理學會刊》 10卷3期 (2001/09) Pp. 129-138

https://doi.org/10.6649/PPB.200109_10(3).0005

摘要

瓜類細菌性果斑病菌（Acidovorax avenae subsp. citrulli ）供試菌株分別分離自台中、台南、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，結果顯示宋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，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 ，lgG）和酵素結合免疫球蛋白 (conjugate IgG) ，在DAS-ELISA檢定法中，以IgG濃度1 μg/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1 μg/ml 為最適宜。ELISA為最宜。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２２１kDa。西方黑點分析結果，有二種蛋白質可和製備的免疫球蛋白反應，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。

關鍵字

瓜類作物 ； 細菌性果斑病菌 ； 血清學

並列摘要

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 Ilan 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 (lgG) and enzyme conjugate immunoglobulin G (conjugate IgG) were 1 μg/ml and 1/l200 dilution in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), respectively. The optimum concentration of IgG was 1 μg/ml in indirect ELISA. The minimum concentrations that resulted in Positive reaction DAS-ELISA and indirect ELISA were 105 and 104 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、Erwil1ia chrysal1themi 、 E. carotovora subsp. carotovora and X. can1pestris 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.

並列關鍵字

cucurbit crops ； bacterial fruit blotch ； serology