树木褐根病PCR快速检测技术之建立

Establishment of PCR Rapid Detection Technique for Tree Brown Root Rot Disease

褐根病(brown root rot disease)是林木最严重的病害，由病原真菌Phellinus noxius所引起。树木罹病初期不易以外部病徵来诊断，为掌握防治先机，针对病原菌应用聚合酶链锁反应(PCR)进行分子检测，有助於提高病害诊断效率。本研究以各林地采样分离得到的褐根病菌为样本，先以通用性引子对ITS1-F/ITS-4 进行PCR 增幅，产物经过定序与比对後重新设计出褐根病菌之专一性引子对G1F/G1R，引子对序列为G1F：5'-GCC CTT TCC TCC GCT TAT TG-3'；G1R：5'- CTT GAT GCT GGT GGG TCT CT-3'，可对褐根病菌增幅出653-bp的专一性片段。再利用改良後之快速程序抽取核酸，仅需菌丝洋菜块0.16平方公尺或0.15克感病根部样本，抽取时程仅需2小时，即可进行聚合酵素连锁反应。由检测的结果发现改良後之核酸抽取方法能大幅缩短作业时程及成本，也证实G1F/G1R引子对树木褐根病菌的专一性极佳，且可针对极少量之样本进行检测，其灵敏度可达10pg。本研究所研发的树木褐根病快速且灵敏的PCR诊断技术，应能广泛应用於林木褐根病诊断及疫情监控。

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.