- 期刊
樹木褐根病PCR快速檢測技術之建立
Establishment of PCR Rapid Detection Technique for Tree Brown Root Rot Disease
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，為掌握防治先機，針對病原菌應用聚合酶鏈鎖反應(PCR)進行分子檢測，有助於提高病害診斷效率。本研究以各林地採樣分離得到的褐根病菌為樣本，先以通用性引子對ITS1-F/ITS-4 進行PCR 增幅，產物經過定序與比對後重新設計出褐根病菌之專一性引子對G1F/G1R，引子對序列為G1F：5'-GCC CTT TCC TCC GCT TAT TG-3'；G1R：5'- CTT GAT GCT GGT GGG TCT CT-3'，可對褐根病菌增幅出653-bp的專一性片段。再利用改良後之快速程序抽取核酸，僅需菌絲洋菜塊0.16平方公尺或0.15克感病根部樣本，抽取時程僅需2小時，即可進行聚合酵素連鎖反應。由檢測的結果發現改良後之核酸抽取方法能大幅縮短作業時程及成本，也證實G1F/G1R引子對樹木褐根病菌的專一性極佳，且可針對極少量之樣本進行檢測，其靈敏度可達10pg。本研究所研發的樹木褐根病快速且靈敏的PCR診斷技術，應能廣泛應用於林木褐根病診斷及疫情監控。
並列摘要
Brown root rot caused by a fungi pathogen, Phellinus noxius, is the most severe disease of forest tree. This disease is not easy to be diagnosed by its incited symptoms especially in the early stage of infection. This study was dedicated to develop a rapid and accurate detection for P. noxius using the PCR assays to elevate the efficiency of diagnosis. Several isolates of P. noxius collected from different forest areas in Taiwan were used as the templates in the PCR amplification with the ITS1-F/ITS-4 common primer pair. Based on the results of sequencing and alignment of PCR products, a newly devised primer pair ”G1F/G1R” was developed for the more specific and sensitive detection of P. noxius. Primer pair G1F/G1R, composed of G1F: 5'- GCC CTT TCC TCC GCT TAT TG-3' and G1R: 5'- CTT GAT GCT GGT GGG TCT CT -3', were designed to amplify a specific 653-bp DNA fragment by PCR. A mini-preparation method of DNA extraction from the pathogens (0.16 cm^2 mycelia cultured on agar) or diseased root tissues (0.15 g) was also designed to prepare the DNA templates for PCR within 2 hours. The results demonstrated that the G1F/G1R primer pair has excellent sensitivity and specificity in the PCR detection of P. noxius. The pathogen could be detected even in the sample only containing 10 pg of DNA extract. This method can be further applied to the diagnosis of brown root rot disease and the monitoring of pathogen- spreading.