林木重要根莖腐病原PCR快速檢測技術之建立 (2/3)The establishment of PCR rapid detection technology for major root and stem rot pathogens of forest trees(2/3)

林木根莖腐病害在發生初期不容易被發現，甚至有潛伏感染的現象，難以在發生初期進行防治工作，甚至在不知已感染的狀況之下，藉由造林或移植將病原傳播出去。本年度計畫目標在建立熱帶靈芝根腐病之快速分子檢測技術，本年度已完成熱帶靈芝子實體收集，樹舌靈芝、狹長孢靈芝、南方靈芝及韋伯靈芝等病原菌樣本純化分離培養及菌種保存，，各菌種分別利用通用性引子對ITS4/ITS5進行聚合酶鏈鎖增幅反應後將核酸產物定序，利用軟體比對找出南方靈芝序列之保守性片段設計三組專一性PCR引子對，經過專一性及靈敏度測試後，有一個組合得到良好增幅反應，可應用於日後熱帶靈芝根腐病的檢測鑑定。 Root and basal stem rot diseases are not easy to be found at the initial infection stage and some latent infection also develop, which cause the difficulty of disease control and may spread out the pathogen unintentionally by afforestation or transplantation. The project purpose in this year is to establish rapid molecular detection technology for Ganoderma root and butt rot. The pure cultures and preservations of G. tropicum, G. applanatum, G. boninense, Ganoderma austral and G. weberianum are accomplished. The five fungal DNA are subjected to PCR reaction with the common primer set, ITS4/ITS5, and the products are sequenced respectively. Then the conservative fragments of G. tropicum are discovered by aid of alignment software to design 3 primer combinations. After specificity and sensitivity tests, 3 combinations showed good amplification results and can be applied in the examination and determination of Ganoderma root and butt rot in the future.林木根茎腐病在侵染初期往往难以被察觉，部分病原菌还存在潜伏侵染现象，这使得病害早期防控难度大幅提升，甚至可能在未察觉植株已感染的情况下，通过造林或移栽过程将病原菌无意扩散。本年度项目目标为建立热带灵芝（Ganoderma tropicum）根腐病的快速分子检测技术。本年度已完成热带灵芝子实体的采集工作，对树舌灵芝（Ganoderma applanatum）、狹長孢靈芝（Ganoderma boninense）、南方靈芝（Ganoderma austral）及韋伯靈芝（Ganoderma weberianum）等病原菌样本完成了纯化分离培养与菌种保藏。各菌株的DNA分别以通用引物对ITS4/ITS5进行聚合酶链式反应（Polymerase Chain Reaction, PCR）扩增，将所得核酸产物进行测序；借助序列比对软件，从热带灵芝的序列中筛选出保守性片段，据此设计了3组特异性PCR引物对。经特异性与灵敏度验证后，3组引物组合均可获得良好的扩增效果，可应用于后续热带灵芝根腐病的检测与鉴定工作。