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Development and Application of Techniques for Detection of Acidovorax avenae subsp. citrulli in Watermelon and Muskmelon Seeds
Acidovorax avenae subsp. citrulli引起的瓜類細菌性果斑病為西瓜及洋香瓜的重要病害，最近才在台灣發生。本病菌為種子傳播，檢測種子以確認種子不帶菌是防治本病害最重要的措施。目前國外應用長出試驗法 (grow-out test) 來檢測種子，此法費時費力，又需大空間，因此快速的檢測技術有待發展。先前的研究計畫已設計出用於PCR的專一性引子對，此引子對 (SL1/SR1) 可快速且專一地鑑定及偵測培養基、罹病葉片及果實上的果斑病菌，但用於檢測種子時，其靈敏度不足。因此本年度計劃之目的主要探討增量培養、免疫磁珠分離及PCR三者結合下對提高靈敏度的效果。本研究將先改進培養基的選擇性，利於種子上病菌的增量，再製備免疫磁珠，用於分離增量後的病菌，最後將免疫磁珠分離的病菌，以PCR偵測之。本研究預期可發展出PCR配合其他處理的技術，以應用於瓜類種子上果斑病菌之檢測，並建立檢測種子的流程，利於未來防檢疫之執行。
Bacterial fruit blotch of watermelon and muskmelon caused by Acidovorax avenae subsp. citrulli (Aac) is a destructive disease, and was found recently in Taiwan. The pathogen is seed-borne. Seed testing to ensure absence of the pathogen is the most important control measure. Currently, the only reliable method for watermelon seed testing is the grow-out test, which is time-consuming, and requires a large space for testing. Therefore, a rapid, specific and sensitive technique needs to be developed. In our previous study, a specific primer pair (SL I/SRI) for use in PCR has been developed. The PCR technique is useful for rapid identification and detection of Aac in cultures, diseased leaves and fruits. However, the sensitivity of the PCR technique for detection of Aac in seeds is not sufficient. The objective of the proposed study is to improve the efficiency of the detection by using immunomagnetic separation in combination with bio-PCR. The selectivity of a semi-selective medium will be improved for use in the enrichment of Aac in seed samples. Aac cells from the enrichment culture of seeds will then be captured by the immunomagnetic separation method. Finally, template DNA can be prepared from the immunomagnetic bead-bound Aac cells and detected by the PCR. The study aims to develop a protocol of the PCR-based technique for detection of Aac in seeds and can be used in seed health test and in plant quarantine inspection.
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