樹木褐根病之PCR分子檢測標準 技術之建立

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，為掌握防治先機，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為多，病根組織為較佳之檢測樣本，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。此實驗成果可作為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。

Brown root rot disease is the most serious disease affecting trees, caused by the pathogenic fungus *Phellinus noxius*. Trees are difficult to diagnose using external symptoms during the early infection stage. To seize the opportunity for prevention and control, our research team developed a polymerase chain reaction (PCR)-based molecular detection method for the pathogen in 2009, and the findings of this study help improve the efficiency of disease diagnosis.

This study aims to further establish standard molecular detection techniques for different sample sources based on the established PCR diagnostic method, and we also designed a pair of specific primers to enhance detection sensitivity. In this study, various brown root rot disease samples were employed, including diseased root tissues and soil samples from infected trees, combined with three different nucleic acid extraction protocols: (1) Extracting pathogen nucleic acids using commercial nucleic acid extraction kits; (2) Extracting pathogen nucleic acids via traditional organic extraction methods; (3) First isolating the pathogen on a culture medium before conducting nucleic acid extraction, followed by PCR-based molecular detection, in an attempt to identify an optimal detection method for *Phellinus noxius*.

The results showed that all three nucleic acid extraction methods achieved satisfactory detection performance, with commercial kits yielding slightly better outcomes. *Phellinus noxius* was more abundant in diseased root tissues than in soil samples, making diseased root tissues the optimal test samples. However, if soil screening is necessary, duplex PCR can be applied to improve detection sensitivity and achieve the detection goal. Thanks to the high specificity of the designed primer pair, the tedious strain purification process can be omitted, greatly shortening the detection timeline. These experimental results can serve as a reference for establishing standard operating procedures (SOPs) for PCR diagnosis and sampling of diseased roots/soil for brown root rot disease in trees in future applications.