樹木靈芝病害快速PCR分子檢測技術建立

ESTABLISHMENT OF PCR DETECTION TECHNIQUE FOR TREE GANODERMA ROOT ROT DISEASE

黃裕星(Yue-Hsing Huang) ； 劉則言(Tse-Yen Liu) ； 莊鈴木(Ling-Mu Jaung) ； 洪挺軒(Ting-Hsuan Hung) ； 陳昭翰(Chao-Han Chen) ； 張東柱(Tun-Tschu Chang) ； 吳孟玲(Meng-Ling Wu)

《中華林學季刊》 50卷4期 (2017/12) Pp. 393-407

摘要

靈芝根莖腐病是台灣常見的樹木病害，感染後會造成樹木輸導組織及樹體結構受損，導致樹木衰弱甚至死亡，最終也可能造成樹木倒伏而造成公安危險。本病在感染初期不容易被發現，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。為了提升病害檢測效率，本研究建立靈芝根腐病菌之快速分子檢測技術，經由序列比對設計可進行聚合酶鏈鎖反應(PCR)之引子對，並萃取五種靈芝，包括南方靈芝、赤芝、狹長孢靈芝、熱帶靈芝及韋伯靈芝等的核酸，以及另外五種常見植物病原菌核酸作為對照組，經由PCR增幅反應測試設計之引子對的專一性及靈敏度，篩選出三對良好引子組，能呈現良好增幅反應，同時具有良好專一性及靈敏度。其中一組為靈芝通用性(屬專一性)引子對(命名為Gsp-comF與Gsp-comR)，可針對病原菌核酸增幅出172 bp的片段，其靈敏度可達約2 pg。另外兩組則為可檢測靈芝病原菌至種的種專一性引子對，能快速準確的判別危害林木或造成木材腐朽之靈芝是南方靈芝或熱帶靈芝；其中南方靈芝的種專一性引子對(命名為Ga-F2與Ga-R2)，可成功增幅南方靈芝病原菌核酸，得到大小為395 bp的單一片段，靈敏度可達約20 pg；而熱帶靈芝的種專一性引子對(命名為Gt-F1與Gt-R3)，則可成功增幅熱帶靈芝病原菌核酸，得到大小為298 bp的單一片段，靈敏度可達約2 pg。本研究所發展的三組靈芝病原菌快速檢測引子對，可應用於日後靈芝根莖腐病的快速檢測鑑定及病原監測。

關鍵字

南方靈芝 ； 熱帶靈芝 ； 聚合酶連鎖反應(PCR)

並列摘要

Ganoderma root rot disease caused by various Ganoderma spp. is a widespread tree disease in Taiwan. These pathogens cause damage to the structure and conducting tissue of trees, thereby causing infected trees to get weaker or even death, and may finally leading to collapse and causing public security risk. The disease is not easy to be diagnosed in the early infection stage or in trees with latent infection. Therefore, it is difficult to prevent and control in the early infection stages. Furthermore, the disease may be spread in the case of unknowingly infecting by afforestation or transplantation. In this study, we developed a rapid and accurate detection method for Ganoderma root rot using the PCR assays to improve the efficiency of diagnosis. We successfully obtained three primer pairs for rapid detection. One is specific for the Ganoderma genus (named Gsp-comFand Gsp-comR), which can amplify a 172-bp DNA fragment through PCR and show a sensitivity of about 2 pg. The other two primer pairs are species-specific for fast identification of G. austral and G. tropicum, respectively. The primer pair Ga-F2 and Ga-R2 for the identification of G. austral can amplify a 395-bp DNA fragment and show a sensitivity of about 20 pg. The primer pair Gt-F1 and Gt-R3 for the identification of G. tropicum can amplify a 298-bp DNA fragment show a sensitivity of about 2 pg. After the reaction test, three primer pairs showed good amplification results and have good specificity and sensitivity. This method can be applied to the fast diagnosis of Ganoderma root rot and pathogen-spreading monitoring in the future.

並列關鍵字

Ganoderma australe ； Ganoderma tropicum ； Polymerase chain reaction (PCR)