以高效率bio-PCR檢測種傳十字花科黑腐病菌

High-efficiency detection of seed-borne Xanthomonas campestris pv. campestris by bio-PCR

"由十字花科黑腐病菌（Xanthomonas campestri s p v . campestris, Xcc）所引起之黑腐病（Black rot disease）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下，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% Tween 20）於25℃高速震盪1分鐘後，吸取部分懸浮液經系列稀釋後塗佈於半選擇性培養基，另取部分懸浮液經離心與沸水破菌後進行聚合酶連鎖反應（polymerase chain reaction, PCR）。在平板培養部分，本研究比較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6-7天、Xan-D培養基4-5天、mCS20ABN培養基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作為種子檢測之培養基。此外，為了增加樣品中目標菌的活菌數及PCR檢測靈敏度，以溼室培養2天之甘藍種子芽體進行固態增量，再以插入子（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），其增幅產物大小為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2.2 CFU/μl。本研究開發之bio-PCR檢測流程可成功檢測污染率0.006%之甘藍種子，其靈敏度高於ISTA檢測方法。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流程提高種傳十字花科黑腐病菌檢測效率。"

"Black rot of crucifers, a destructive systemic disease of Brassicaceae showing V-shaped necrotic and chlorotic symptom, is caused by the seed-borne Xanthomonas campestris pv. campestris (Xcc). Under field conditions, seeds with 0.03% and higher infection rate can lead to serious epidermics. Hence, planting pathogen-free seeds is the primary strategy for disease management. In this study, we modified the detection protocol of seed-borne Xcc approved by the International Seed Testing Association (ISTA) by replacing the seed wash solution with 0.85% sterilized saline supplemented with 0.01% (v/v) Tween 20, vortexing the seed washings at 25°C for 1 min, and PCR testing using primers BP254 and BP255 that were derived from the insertion sequence IS1478. For bacterial cultivation and enumeration, three semi-selective media, SM, Xan-D, and mCS20ABN, were tested for the efficiency of Xcc recovery and selection. The results indicated that mCS20ABN medium had the highest recovery and selection efficiency for Xcc, and it is ideal for cultivating Xcc cells from seed-washings. In order to increase the number of viable Xcc cells in testing seed lots, 2-day post germinating cabbage sprouts were used as an tactic for biological polymerase chain reaction (bio-PCR), and a 650-bp DNA fragment was amplified with the primers BP254 and BP255. Using the designed primers, the detection limits of PCR were determined to be 1 pg purified Xcc DNA, and 2.2 CFU/μl bacterial cells. The bio-PCR protocol developed in this study successfully detected Xcc when only 0.006% of seed was infested. In comparison with the ISTA-approved Xcc-detection method, the bio-PCR protocol has higher efficency in detecting seed-borne Xcc, which can be integrated into the current protocol for future applications."