Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89346
標題: 種傳十字花科黑腐病菌檢測流程之改良
Development of a modified procedure for detecting seed-borne Xanthomonas campestris pv. campestris
作者: Pin-Ru Chen
陳品儒
關鍵字: 種子檢測
半選擇性培養基
生物性擴增核酸連鎖反應
seed testing
semi-selective media
bio-PCR
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摘要: 由十字花科黑腐病菌 (Xanthomonas campestris pv. campestris, Xcc) 所引起之黑 腐病 (Black rot) 為十字花科作物重要病害之一。此病原菌可藉種子傳播,在高溫高 濕氣候條件,當種子帶菌率高於 0.03% 即可在田間造成嚴重病害,因此,種植無 病原菌種子為防治十字花科黑腐病之首要策略。本研究乃修改國際種子檢查協會 (International Seed Testing Association, ISTA) 檢測種傳 Xcc 之流程,將樣品以食鹽 水洗滌液 (0.85% NaCl, 0.01% Tween20) 於 25℃ 強烈震盪 1 分鐘後,吸取部分懸 浮液經序列稀釋後塗佈於半選擇性培養基,另取部分懸浮液經離心並水浴煮沸後進 行聚合酶連鎖反應 (polymerase chain reaction, PCR)。在平板分析部分,為選擇合適 培養基計算活菌數,比較 SM、Xan-D 及 mCS20ABN 三種半選擇性培養基對 Xcc 之鑑別效果,結果顯示可辨識目標菌之培養時間分別為 SM 培養基約 6 - 7 天、 Xan-D 培養基約 4 - 5 天、mCS20ABN 培養基約 2 - 3 天,Xcc 在 mCS20ABN 培 養基上呈現淡黃色、微黏稠之菌落型態,周圍有澱粉分解透化圈;此外,在 mCS20ABN 培養基上,Xcc 之回收率及篩選率皆高於 Xan-D 與 SM 培養基,因 此本研究以 mCS20ABN 作為檢測之培養基。另為增加樣品中目標菌的活菌數,利 用發芽兩天之芽體進行固態增量,以插入子 (Insertion sequence) IS1478 序列設計之 PCR 引子對 (BP254 / BP255) 進行生物性擴增核酸連鎖反應 (bio-PCR),增幅產物 大小為 650 bp。利用此引子對進行 PCR 可檢測最低核酸濃度為 1 pg,最低細菌濃 度為 3 CFU/µl,本研究開發之 bio-PCR 檢測流程可檢測種子帶菌率為 0.006%,相 較於 ISTA 檢測 Xcc 靈敏度有提升之效果。本研究採用之引子對 BP254 / BP255 具高靈敏特性並可應用於種子檢測,另以發芽作為生物性增量策略進行 bio-PCR 為 首次應用於種傳 Xcc 檢測。根據本研究結果,未來可採用 mCS20ABN 培養基計 量活菌數並配合本研究開發之 bio-PCR 流程檢測種傳十字花科黑腐病菌。
Black rot, a destructive systemic disease of Brassicaceae that usually appears with V-shaped necrosis, is caused by the seed-borne Xanthomonas campestris pv. campestris (Xcc). Black rot disease occurred when the percentage of seed infestation was higher than 0.03% under field conditions. Hence, planting pathogen-free seeds is the primary disease management strategy. Modified from the detection protocol of seed-borne Xcc by the International Seed Testing Association (ISTA), bacteria from seed lots were extracted by 1-min vortex at 25°C with 0.85% sterilized saline supplemented with 0.01% Tween20, followed by dilution plating onto semi-selective media and PCR. In this study, three semi-selective media, SM, Xan-D, and mCS20ABN were used for comparing the efficiency of recovery and selection for Xcc. The inoculation time for observing typical colony morphology of Xcc is 6 - 7 days on SM medium, 4 - 5 days on Xan-D, and 2 - 3 days on mCS20ABN. Typical Xcc colonies were pale yellow, mucoid, and surrounded by a zone of starch hydrolysis on mCS20ABN medium, which also had the best recovery and selection efficiency for Xcc from seed-washings and was chosen as the semi-selective medium in this this study. In addition, to increase the viable Xcc cells in seed lots, 2-day post germinating cabbage sprouts were used as an tactic for biological polymerase chain reaction amplification (bio-PCR), and a 650 bp DNA fragment was amplified with the primers of BP254 and BP255 that were derived from the insertion sequence IS1478. Using the designed primers, the detection limits of PCR were determined to be 1 pg purified Xcc DNA, and 3 CFU/µl bacterial cells. Bio-PCR protocol was subsequently used to investigate seed infestation by mixing artificially infested cabbage seeds, and the limit for detecting seed infestation is 0.006%. In comparison with the standard Xcc-detection method approved by ISTA, the bio-PCR protocol developed in this study has higher efficiency in detecting seed-borne Xcc, which can be integrated into the current protocol for future applications.
URI: http://hdl.handle.net/11455/89346
文章公開時間: 2015-07-16
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