樹木褐根病之PCR分子檢測標準技術之建立

Establishment of the SOP of PCR Detection Techniques for Brown Root Rot Disease

吳孟玲(Meng-Ling Wu) ； 陳昭翰(Chao-Han Chen) ； 張東柱(Tun-Tschu Chang) ； 莊鈴木(Ling-Mu Jaung) ； 洪挺軒(Ting-Hsuan Hung)

《中華林學季刊》 44卷1期 (2011/03) Pp. 7-17

https://doi.org/10.30064/QJCF.201103.0002

摘要

褐根病(brown root rot disease)是林木最嚴重的病害，由病原真菌Phellinus noxius所引起。樹木罹病初期不易以外部病徵來診斷，爲掌握防治先機，2009年本研究團隊已針對病原菌發展聚合酶鏈鎖反應(PCR)技術進行分子檢測，本研究成果有助於提高病害診斷效率。本研究主要是針對已建立PCR診斷技術，進一步對於不同檢測樣本來源，建立分子檢測標準技術，另外，我們還設計一組專一性引子對，以提高檢測靈敏度。本研究選用不同之褐根病樣本，包括褐根病之病根組織及土壤樣本，並搭配以不同之核酸抽取方法：(1)商品化核酸抽取套組抽取病原核酸；(2)以傳統之有機萃取法萃取病原核酸；(3)先以培養基分離病原再進行核酸抽取，之後進行PCR分子技術檢測，試圖尋得一套較佳的褐根病菌檢測法。結果證明不同之核酸抽取方法皆可獲得良好檢測效果，但商品化套組效果稍佳；褐根病菌存在於病根組織較病土樣本爲多，病根組織爲較佳之檢測樣本，但若必須進行土壤篩檢時，可利用雙重PCR提高檢測之靈敏度，亦可達到檢測之目的。因引子對之專一性佳，可省去繁瑣的菌種純化流程，大幅節省檢測時程。此實驗成果可作爲未來應用於林木褐根病PCR診斷，病根／土壤採樣SOP建立之參考依據。

關鍵字

褐根病 ； 褐根病菌 ； 聚合酶鏈鎖反應

並列摘要

Brown root rot disease caused by a fungi pathogen, Phellinus noxius, is one of the most severe diseases of forest trees. This disease is not easy to be diagnosed by its incited symptoms especially in the early stage of infection. In 2009, we have the rapid detection technique to accurate detection for P. noxius using the PCR assays to elevate the efficiency of diagnosis. In this study, we further establish a standard operating procedure with the PCR assay using root tissues and soil samples. In addition, we designed another pair of PCR primer to improve the sensitivity. We made the comparative effects in PCR assays with different DNA extraction methods from either diseased root tissues or soil samples. Three experimental treatments were used in this study for the comparative efficiency among them including (1) DNA extraction from root tissue and soil samples using commercialized DNA extraction kit; (2) DNA extraction from root tissues and soil samples using traditional organic extraction method; (3) DNA extraction from the mycelia grown on the culture medium inoculated by diseased root tissues and soil samples using traditional organic extraction method. The data showed that all DNA extraction methods could obtain good results for the detection of P. noxius, but the commercialized DNA extraction kit seemed to be somewhat more sensitive than the other methods. Besides, the results indicated that the populations of P. noxius in the diseased root tissues are more than those in the soil samples. Therefore, the diseased root tissues are better for the examination of P. noxius than soil samples. For the tests of soil samples, double PCR assays were better to obtain sensitive results of detection. With high specificity of primer sets, designed in this study, examination could be much simplified since the isolation and purification are not necessary. This study aids the establishment of the standard operating procedure (SOP) for detection of P. noxius including the entire PCR procedure and sampling either from root tissues or soil.

並列關鍵字

Brown root rot disease ； Phellinus noxius ； Polymerase chain reaction