蝴蝶蘭細菌性褐斑病菌Acidovorax avenae subsp. cattleyae專一性引子之開發

A Specific Primer Pair for Diagnosis and Detection of Bacterial Brown Spot Pathogen: Acidovorax avenae subsp. cattleyae on Phalaenopsis Orchid.

許秀惠(S. H Hseu) ； 宋嘉仁(C. J. Sung) ； 申屠萱(H. Shentu)

《植物病理學會刊》 20卷1&2期 (2011/06) Pp. 52-64

https://doi.org/10.6649/PPB.201106_20(1_2).0006

摘要

以80個逢機引子應用隨機增幅多型性核酸技術（RAPD）增幅並篩選出對蝴蝶蘭褐斑病菌Acidovorax avenae subsp. cattleyae （AAC）共有之567 bp的專一性片段，進一步將該片段接入TOPO選殖載體 pCR® II-TOPO得到選殖株，並分析其核酸序列，再根據其序列設計出對AAC具專一性之引子組Ac46f/Ac46r。應用此引子組進行聚合酵素連鎖反應（polymerase chain reaction, PCR），供試144株褐斑病菌皆能增幅出463 bp的片段，但對供試之8屬20種共119株其他非標的之病原細菌及腐生細菌則不會產生任何片段。以Ac46f/Ac46r引子組測試細菌性褐斑病菌DNA，其靈敏度可達10~50pg，菌落偵測之靈敏度則為5.8~7.5×10^1 cfu/ml個活菌數。將10^6 cfu/ml的AAC菌液分別與其他細菌之菌液10^5~10^8 cfu/ml等比率混合後再進行聚合酵素連鎖反應並不影響AAC之偵測結果。應用單一菌落快速檢定法可於3-4小時內鑑定蝴蝶蘭細菌性褐斑病菌。將所設計之引子組應用於人工接種褐斑病菌之偵測以及田間蝴蝶蘭褐斑病樣品中褐斑病菌之偵測均具準確性，顯示本研究所設計之引子組Ac46f/Ac46r可用於蝴蝶蘭細菌性褐斑病菌快速診斷鑑定及檢測。

關鍵字

蝴蝶蘭 ； 細菌性褐斑病菌 ； 引子 ； 診斷 ； 鑑定 ； 偵測

並列摘要

Phalaenopsis cultivation is a very important floral industry in Taiwan since the early 1990s. Several bacterial diseases may occur on the orchids and cause serious economic loss during orchid cultivation. Among these diseases, the bacterial brown spot, caused by Acidovorax avenae subsp. cattleyae (AAC), is recognized as the most important bacterial disease in the Phalaenopsis cultivation in Taiwan. In this study, a highly specific primer pair was developed for diagnosis and detection of the pathogen. Totally eighty random primers were used to find specific DNA fragments of AAC, and a specific DNA fragment of 567 bp amplified by the primer OPR-02 was identified and cloned into the pCR® II-TOPO vector. The cloned DNA fragment was further sequenced and used for designing the specific primer pair Ac46f/Ac46r for AAC identification and detection. In order to elucidate the specificity of the primer pair, totally 119 isolates, collected from eight genera and twenty species of bacteria, were subjected for PCR, and the results showed that only AAC could produce a unique 463 bp fragment. Sensitivity of AAC using PCR was between 10~50 pg for purified DNA and 5.8-7.5 x 10^1 cfu/ml for bacterial cells. The results were not affected when AAC was mixed with other non-target bacteria. The time for PCR detection of AAC strains using primers Ac46f/Ac46r only took 3 to 4 hours. The primer pair was further used for detecting AAC on the leaves of infested Phalaenopsis orchids and was proved to be equally effective.

並列關鍵字

Phalaenopsis ； Acidovorax avenae subsp. cattleyae ； PCR ； primer ； diagnosis ； identification ； detection