林木幼苗猝倒病之PCR檢測技術建立

Establishment of PCR Detection Technique for Seedling Damping-Off Disease

吳孟玲(Meng-Ling Wu) ； 洪挺軒(Ting-Hsuan Hung) ； 蔡佳欣(Chia-Hsin Tsai) ； 劉亭君(Ting-Chun Liu) ； 黃子毓(Tzu-Yu Huang)

《中華林學季刊》 43卷2期 (2010/06) Pp. 339-353

https://doi.org/10.30064/QJCF.201006.0012

摘要

幼苗猝倒病為林木苗圃最嚴重的病害，危害幼苗根部並造成植株猝倒死亡，此病害主要由四個屬的真菌所引起，分別為立枯絲核菌(Rhizoctonia solani)、腐霉病菌(Pythium spp.)、疫菌(Phytophthora spp.)及鐮孢菌(Fusarium spp.)。本實驗為能同時且快速檢測以上4種幼苗猝倒病之真菌病原，設計4組專一性引子對並配合聚合酶鏈鎖反應(Polymerase chain reaction, PCR)，使4種病原真菌之PCR檢測流程能於相同之反應條件下進行。其中，腐霉病菌、疫菌、鐮孢菌三屬真菌從已發表的文獻中搜尋適當的引子對，經過測試分析後分別選用可於黏合溫度56℃進行PCR之引子對，腐霉病菌選用Pa3與ITS4為引子對，增幅片段為700bp；疫菌以FMPh-8b及FMPh-10b為引子對，增幅片段為457bp；鐮孢菌以ITS-Fu-f與ITS-Fu-r為引子對，增幅片段為389bp；而立枯絲核菌則選擇保守性高之基因片段Succinyl-CoA設計適合上述PCR條件下反應之新引子對R.S-F與R.S-R，經測試可對立枯絲核菌增幅出385bp之特異性PCR片段。此4對引子經專一性測試，每對引子皆能準確針對目標菌種進行增幅，且不對非目標病原反應，具高度專一性。由於此4對專一性引子對，其黏合溫度相同，且所增幅之PCR片段長度接近，故可同時於一臺PCR 機器進行增幅，使PCR 偵測更為省時便利。此技術目前已應用在林木幼苗根部及土壤病原檢測，每對引子皆可專一的增幅出特定片段。由於此4種病原菌為幼苗常見之病害，在未來推展健康林苗檢疫制度時，可應用在林苗猝倒病簡易、快速及高敏感之檢測。

關鍵字

林木幼苗猝倒病 ； 聚合酶鏈鎖反應

並列摘要

Damping-off disease is the most serious disease that attacks roots of plants and kills young seedlings. This disease is caused mainly by 4 genera of pathogenic fungi, Rhizoctonia solani, Pythium spp., Phytophthora spp. and Fusarium spp.. In this study, in order to detect 4 pathogens of damping-off disease at the same time, we searched and designed the primer pairs specific to these pathogens, and these primers could act on the same annealing temperature, 56℃, in polymerase chain reaction (PCR). The primer pairs of Pythium spp., Pa3 and ITS4, amplifies 700-bp DNA fragment by PCR. The primer pairs of Phytophthora spp., FMPh-8b and FMPh-10b, amplifies 457-bp DNA fragment. ITS-Fu-f and ITS-Fu-r amplifying 389-bp DNA fragment were selected as the primer pairs of Fusarium spp. The highly conserved region, Succinyl-CoA of Rhizoctonia solani, was used to design the primer pairs, R.S-F and R.S-R, amplifying 385-bp in the same PCR condition. In specific tests, each primer pairs could amplify correctly DNA fragment of target pathogen and not to other fungi. Because 4 primer pairs act on the same annealing temperature and amplify DNA fragments of a similar size, detection of 4 pathogens could perform conveniently in one PCR machine. In the present, this technique has already used to detect pathogens in seedling roots and soil. Because of the damping-off disease is the most common disease of the seedlings, this method could be applied to the diagnosis of damping-off disease and produce healthy forest seed and seedling.

並列關鍵字

Seedling damping-off ； Polymerase chain reaction PCR