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標題: 瓜類細菌性果斑病菌檢測技術之研發
The development of detection techniques of Acidovorax avenae subsp. citrulli
作者: 周盈甄
Chou, Ying-Chen
關鍵字: Acidovorax avenae subsp. citrulli;瓜類細菌性果斑病菌;semi-selective medium;real time PCR;半選擇性培養基;即時聚合酵素連鎖反應
出版社: 植物病理學系所
引用: Bahar, O., Efrat, M., Hadar, E., Dutta, B., Walcott, R. R., and Burdman, S. 2008. New subspecies-specific polymerase chain reaction-based assay for the detection of Acidovorax avenae subsp. citrulli. Plant Pathol. 57:754-763. Chen, M. S. 2003. Genetic diversity of Acidovorax avenae subsp. citrulli in Taiwan. Master thesis. Department of Plant Pathology, National Chung Hsing University. Taichung, Taiwan. Cheng, A. H. and Huang, T. C. 1998. Bacterial fruit blotch on melon, and bitter gourd caused by Acidovorax avenae subsp. citrulli. (Abstr.) Plant Pathol. Bull. 7:216. Cubero, J., and Graham, J. H. 2005. Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate Xanthomonas strains on citrus. Phytopathology 95:1333-1340. Cuppels, D. and Kelman, A. 1974. Evaluation of selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathology 64:468-475. DeBoer, S. H., Ward, L. 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瓜類細菌性果斑病是由果斑病菌 Acidovorax avenae subsp. citrulli 所引起的病害,可危害多種瓜類作物。為了避免此病害藉由帶菌種子廣泛傳播,必須要有良好且靈敏度高的方法來偵測種子的帶菌情形。使用半選擇性培養基直接觀察菌落的有無即為一果斑病菌常用的偵測方法。已知果斑病菌常用的半選擇性培養基有 WFB68、AacSM 及 M-WFB68。本實驗目的為比較 M-WFB68 培養基與 AacSM 培養基在篩選、分離果斑病菌效果之差異,以及配製成液態培養基後對於果斑病菌選擇性增量之情形,進一步改良培養基之配方,以找出更適合台灣地區果斑菌菌株使用之培養基,並將改良後的液態培養基加入分子檢測技術之流程,作為前置之果斑病菌增量配方,以求達到更佳檢測效果。實驗發現果斑菌從 M-WFB68 培養基的回收率不佳,需調整抗生素濃度及種類進行改良,而各果斑菌菌株於半選擇性 AacSM 培養基上生長情形不一,不如在 WFB68 培養基上型態特性均一,易影響檢測效果。為改良培養基使用效果,本實驗將 M-WFB68 培養基中所含抗生素調整為 cefoperazone 10 ppm 及 ceftriaxone 20 ppm,改良後的培養基 WFB68MA 可抑制非目標菌的生長,且對果斑菌 Aac33、Aac155 皆有 80% 以上的回收率。另外,果斑病菌菌株於 AacSM 培養基中生長速度較其他培養基緩慢,且原配方內含有的抗生素 penicillin B 台灣未販售,因此其配方調整主要為增加碳氮素源含量,並搭配適宜台灣地區菌株使用之抗生素作篩選。將 AacSM 培養基中酵母萃取物 (yeast extract) 的濃度提升至 50 ppm,可促進果斑菌生長速率。然而在抗生素篩選方面,發現添加抗生素後對非目標細菌抑制效果不佳,且影響果斑菌回收率降低至 10% 以下,因此後續結合至 real time PCR 便採用液態 WFB68MA 培養基進行。經過測試,將帶菌種子利用液態 WFB68MA 培養基清洗得到的種子洗出液,其中的雜菌量只有 102-103 CFU/ml 以下,遠低於使用液態 WFB68 培養基洗出的 106 CFU/ml 的雜菌量,且可回收 103-104 CFU/ml 的果斑病菌。利用即時聚合酵素連鎖反應 (real time PCR) 檢測帶菌種子洗出液時亦發現,使用液態 WFB68MA 培養基清洗得到的種子洗出液有較佳的檢測效果,顯示液態 WFB68MA 培養基可降低種子上的雜菌干擾,進而提升果斑菌的檢測效果。
其他識別: U0005-2008200910054700
Appears in Collections:植物病理學系

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