利用real-time PCR進行二種台灣梨衰弱病菌質體之多重檢測及定量分析

Multiplex Detection and Quantification of Two Pear Decline Phytoplasmas in Taiwan by Real-Time PCR

李芷芸(C. Y. Lee) ； 林長平(C. P. Lin)

《植物病理學會刊》 20卷1&2期 (2011/06) Pp. 11-20

https://doi.org/10.6649/PPB.201106_20(1_2).0002

摘要

本研究以同步聚合酶鏈鎖反應（real-time PCR） 針對台灣梨衰弱病菌質體（pear decline-Taiwan phytoplasma, PDTW phytoplasma）及第二群台灣梨衰弱病菌質體（PDTWII phytoplasma）設計專一性引子對，進行台灣梨衰弱病之多重檢測及定量分析。台灣梨衰弱病最早在1994年六月台灣中部東勢、和平等梨樹栽種地發現，本實驗室研究之初先證實該病害之病原為分類地位屬於第十群（group 16SrX）之PDTW phytoplasma，隨後證實其可藉由黔梨木蝨（Cacopsylla qianli）與中國梨木蝨（C. chinensis）傳播，並於2005年在中國梨木蝨蟲體內發現第二群（group 16SrII）之PDTWII phytoplasma，至2006年則發現此兩群植物菌質體可複合感染（coinfection）梨樹，為檢測並追蹤此二群植物菌質體在罹病梨樹及媒介昆蟲體內消長的情形，在本研究中針對二者之16S rDNA序列設計出多重同步聚合酵素連鎖反應（multiplex real-time PCR）引子對，配合以real-time PCR為基礎之定量分析，發現在先前以PCR檢測確認有第十群PDTW phytoplasma感染之梨樹病株檢體DNA中，亦可偵測到第二群之PDTWII phytoplasma之存在，確認其為複合感染，顯示田間罹病植株受兩群植物菌質體複合感染之情形相當普遍。經靈敏度測試則發現real-time PCR優於PCR，而multiplex real-time PCR亦優於multiplex PCR。此定量技術之建立將可應用於追蹤兩群台灣梨衰弱病菌質體於梨樹及媒介昆蟲體內之細胞數量及二者間族群消長情形之觀察。

關鍵字

台灣梨衰弱病 ； 同步聚合酶鏈鎖反應

並列摘要

Pear decline (PD) was first reported in 1994 in pear orchards near central Taiwan and was previously proved to be caused by pear decline-Taiwan (PDTW) phytoplasma (Liu et al., 2007. Eur. J. Plant Pathlo. 117:281-291). PDTW phytoplasma can be transmitted by two pear psyllas, Cacopsylla qianli and C. chinensis. Besides PDTW phytoplasma, PDTWII phytoplasma of 16SrII group was first identified in pear psyllas since 2005. In 2006, co-infection of PDTW phytoplasma and PDTWII phytoplasma in pear trees was later identified by PCR. To detect these two phytoplasmas in pear plants and insect vectors, specific primer pairs for multiplex real-time PCR and real-time PCR were designed and applied effectively in this study. The copy number of 16S rDNA of PDTW phytoplasma, which is relatively easier to be cloned from phytoplasma than other genes, was used as a reference sequence to quantify the number of phytoplasma cell in plant tissues. DNA samples previously prepared from PDTW phytoplasma-infected pears that had been confirmed by PCR were reexamed in this study using real-time PCR, and many of them were found to harbor the DNA of PDTWII phytoplasma as well. The results indicated that the co-infection of PDTW and PDTWII phytoplasmas in pear trees and insect vectors may be common in fields. The detection of PD phytoplasmas in pear trees and insect vectors based on the multiplex real-time PCR and real-time PCR showed higher sensitivity than those based on multiplex PCR and conventional PCR, respectively. The real-time PCR-based quantification of PDTW and PDTWII phytoplasmas described in this study can be applied with a great potential to monitor the population fluctuations of these two phytoplasmas in pear trees and insect vectors.

並列關鍵字

pear decline ； real-time PCR