台灣梨衰弱病二種植物菌質體之探討

Phytoplasmas of Two Different 16S rDNA Groups are Associated with Pear Decline in Taiwan

劉淑玲(Shu-Ling Liu)

指導教授 ： 林長平

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02979

摘要

本研究成功地利用semi-nested PCR由媒介昆蟲中國梨木蝨全DNA中增幅出1,761 bp及1,783 bp 植物菌質體rRNA基因序列之PCR產物，經由選殖定序及比對分析，顯示其分別為前人於研究台灣梨衰弱病 (pear decline-Taiwan, PDTW) 時所發現屬於group 16SrX之PDTW phytoplasma與另一屬於group 16SrII之PDTWII phytoplasma之16S rDNA, 16S-23S rDNA integenic spacer及部分之23S rDNA序列。根據上述PDTW與PDTWII植物菌質體之rRNA基因序列，在本研究中分別設計出PDTW phytoplasma之專一性引子對fPD2/ rPDs1配合APf3/ rPDs1，以及PDTWII phytoplasma之專一性引子對II-Pf1/ II-Pr1配合IIPf2/ IIPr1，作為後續探討田間發病生態及媒介昆蟲帶菌情形PCR或semi-nested PCR檢測之工具。本研究藉由中國梨木蝨咬食傳菌試驗，證實PDTW phytoplasma以及PDTWII phytoplasma皆能由中國梨木蝨傳播至梨株；此外，嫁接傳菌試驗亦由田間罹病梨株枝條將PDTW phytoplasma及PDTWII phytoplasma傳至日日春植株。於2006年在和平地區田間亦發現感染PDTWII phytoplasma之罹病梨株，並已出現梨樹葉片葉緣向內捲曲、葉尖向後翻捲而變形之病徵，其梨株內PDTWII phytoplasma之 rDNA序列與中國梨木蝨蟲體內及嫁接日日春植株內之PDTWII phytoplasma之rDNA序列完全相同。在本研究中於2005年8月份以PCR技術監測新竹尖石鄉地區之中國梨木蝨，發現其蟲體內同時帶有PDTW phytoplasma與PDTWII phytoplasma，隨後當地在2006年9月份始發現出現葉片紅化與捲葉病徵之疑似罹病株，即分別以 PDTW phytoplasma及PDTWII phytoplasma之專一性引子對進行偵測且均能獲得PCR片段，將該PCR產物作進一步之選殖、定序與比對後，發現梨株內同時存在了PDTW phytoplasma與 PDTWII phytoplasma。整合上述研究結果顯示台灣梨衰弱病之發病範圍已逐年擴大，目前田間數量較大且具族群優勢之中國梨木蝨可同時傳播二種梨衰弱病植物菌質體，而且此二種植物菌質體在田間梨樹能造成複合感染。本論文亦針對罹病梨樹、黔梨木蝨及中國梨木蝨進行TEM切片觀察，分別於植物之韌皮部薄壁細胞、篩管以及二種蟲體之腸壁組織中，皆觀察到植物菌質體。

關鍵字

中國梨木蝨 ； 黔梨木蝨 ； 媒介昆蟲 ； 聚合連鎖酵素反應(PCR) ； 台灣梨衰弱病 ； 植物菌質體

並列摘要

PCR-based detection strategies were applied in the etiology and vectorship studies of pear decline in Taiwan (PDTW). The 16S rDNA sequences were amplified from the total DNAs prepared from PDTW-affected pear trees and pear psylla Cacopsylla chinensis (Yang and Li) with PCR. Based on the sequence analyses, phytoplasmas of two different 16S rDNA groups, 16SrII and 16SrX, were revealed to be associated with PDTW-affected pear trees and carried by C. chinensis (Yang and Li). For detection purposes, specific primers IIPf1/ IIPr1 along with IIPf2/ IIPr1 for group 16SrII-PDTW phytoplasma, PDTWII phytoplasma, and fPD2/ rPDs1 along with APf3/ rPDs1 for group 16SrX-PDTW phytoplasma, PDTW phytoplasma, had been developed and applied in semi-nested PCR. The 16S rDNA sequences of PDTW and PDTWII phytoplasmas amplified from diseased pear tree and those amplified from C. chinensis were identical. Transmission trials of phytoplasmas associated with PDTW to healthy pear plants were carried out with C. chinensis. One of the 17 tested plants was affected with both PDTW and PDTWII phytoplasmas and ten of the 17 tested plants were affected with PDTWII phytoplasma. In grafting tests, PDTW and PDTWII phytoplasmas were transmitted from the same diseased pear to periwinkle plants (Catharanthus roseus (L.) G. Don). Phytoplasma bodies were examined in the sieve tube elements and phloem parenchyma cells of the diseased plants and in cells of intestine wall of C. chinensis and C. qianli by using transmission electron microscopy.

並列關鍵字

Cacopsylla chinensis ； C. qianli ； PCR ； pear decline ； insect vector ； phytoplasma

參考文獻

洪士程. 2006. 柑桔木蝨傳播黃龍病之生態研究. 國立台灣大學昆蟲學研究所博士論文。

廖萬正、王芳美、楊耀祥. 1992. 橫山梨及鳥梨莖頂組織培養之研究. 台中區農業改良場研究彙報 34: 1-9。

劉秀玲. 2007. 台灣梨衰弱病之病原與其媒介昆蟲之探討. 國立台灣大學植物病理與微生物學研究所博士論文。

Chou, L., and Fang, S. 1994. New record of Psylla qianli (Homoptera: Psyllidae) from Taiwan. J. Agric. Res. China 43: 467-468.

Yang, I. L. 1969. Studies on witches’ broom of sweet potato in Taiwan. Taiwan J. Agric. Res. 18: 50-60.