臺灣博碩士論文加值系統

從1994年六月起，台灣中部梨樹栽培區發生台灣梨衰弱病(pear decline-Taiwan, PDTW)，其病徵表現與國外發表之梨衰弱病(pear decline)相似，皆會引起罹病植株出現紅葉、衰弱及萎凋等病徵，嚴重時造成植株死亡，本論文針對該病原菌之核醣體RNA基因序列(rDNA)進行研究分析，證實此病害係由屬於apple proliferation group (group 16SrX)之group 16SrX-PDTW phytoplasma (簡稱PDTW phytoplasma)所引起。本研究利用南方雜配法，證實台灣梨衰弱病菌質體之核醣體RNA基因於基因體中應具二重複套組，適合做為檢測用之標的序列。研究中針對此PDTW phytoplasma之rDNA陸續開發出多項聚合酵素連鎖反應(polymerase chain reaction, PCR)之檢測技術，其中包括專一性放大式聚合酵素連鎖反應(specific booster PCR)、多引子聚合酵素連鎖反應(multiplex PCR)、巢式聚合酵素連鎖反應(nested PCR)與RFLP-PCR (restriction fragment length polymorphism of PCR product)等，可供田間罹病梨株、媒介昆蟲及健康梨接穗之病原菌檢測。本研究並由2002年至2004年歷時三年完成各月份罹病梨樹中PDTW phytoplasma之PCR偵測調查，以瞭解罹病梨樹中PDTW phytoplasma含量之季節性變化，結果顯示罹病梨樹中PDTW phytoplasma之檢出率於春季三至五月間開始上升，夏季六至九月份間病原菌檢出率最高，而冬季落葉期之檢出率則降為零。本論文亦針對田間不同種類之昆蟲如木蝨、蚜蟲等進行其體內PDTW phytoplasma之PCR檢測實驗，並於2003年五月間東勢慶福里大量發生之中國梨木蝨(Cacopsylla chinensis)檢體中，增幅到植物菌質體之專一性PCR片段，經由選殖定序後獲得到中國梨木蝨體內植物菌質體之16S rDNA部分序列及16S-23S rDNA intergenic spacer region全長序列，經由比對分析顯示其與PDTW phytoplasma之rDNA序列相同；再者，於2005年一月間和平詹園大量發生之黔梨木蝨(C. qianli)檢體中以植物菌質體廣用型引子對P1/ P7成功增幅出PDTW phytoplasma之16S rDNA及16S-23S rDNA intergenic spacer region全長序列。經由上述結果顯示，中國梨木蝨與黔梨木蝨之檢體內皆可攜帶PDTW phytoplasma，然而PCR增幅實驗結果顯示，黔梨木蝨檢體內所攜帶之PDTW phytoplasma之含量明顯較中國梨木蝨為高。

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting typical symptoms of PD disease were observed in orchards of central Taiwan. The sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the causative agent of PDTW, group 16SrX-PDTW phytoplasma (PDTW phytoplasma), was closely related to the phytoplasmas of the apple proliferation group (group 16SrX) that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S-23S rDNA intergenic spacer region and putative restriction site analyses of 16S rDNA and 16S-23S rDNA intergenic spacer region sequences provided the further support for the view that the PDTW phytoplasma causing PDTW may be a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, various polymerase chain reaction (PCR) primers and PCR-based strategies including specific booster PCR, multiplex PCR, nested PCR and PCR-RFLP were developed and applied in this study for the detection of the causative agent in pear trees and insect vectors. The study of the seasonal variation in the detection of the PDTW phytoplasma was conducted in four pear orchards in Dungshr and Heping. Samples collected from 7-20 infected trees were detected by booster PCR monthly in three consecutive years from 2002 to 2004. Unless there is no leaf sample can be collected from the pear trees in the winter, the PDTW phytoplasma in the pear trees can be detected readily. And the maximum detection rates of PDTW phytoplasma were obtained in the summer. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.

目錄
壹、前言………………………………………………………………....1
貳、前人研究………………………………………………………3
一、植物菌質體之發現及特性分析…………………………………..…………..3
二、植物菌質體之分類…………………………………………………..………..4
四、梨衰弱病(pear decline)病理學之研究…………..…………………..………..8
五、梨衰弱病分子生物學之研究……………………………………..…………..12
六、台灣梨衰弱病之發生…………………………………………..…………..15
參、材料與方法………………………………………………..……………………..18
一、試驗植物材料來源………………….……………………………..……..…...18
二、植物全DNA (total DNA)之純化………..……………………..…………….18
三、聚合酵素連鎖反應偵測台灣疑似梨衰弱病罹病株中之植物菌質體……21
（一）以植物菌質體廣用型PCR引子偵測台灣疑似梨衰弱病罹病株之植物菌質體………………………………………………………………...…21
（二）以國外梨衰弱病專一型PCR引子偵測台灣疑似梨衰弱病罹病株之植物菌質體…………………………………………………………...……22
四、聚合酵素連鎖反應產物之純化與選殖……………………………………22
（一）聚合酵素連鎖反應產物之純化……………………….......................22
（二）聚合酵素連鎖反應產物之選殖(cloning)………………. ...................23
五、聚合酵素連鎖反應產物轉型株之特性分析…………………………………25
（一）微量抽取質體DNA (plasmid DNA mini preparation)…. ....................25
（二）嵌入片段大小之分析……………………………….….......................26
（三）重組質體DNA之PCR反應…………………………....................……26
（四）準轉型株所帶重組質體嵌入DNA之核苷酸定序(sequencing)與其序列分析……………………..........................................…………………..26
六、台灣梨衰弱病原菌質體之鑑定及親緣系統之建立與其rDNA準限制酵素切位(putative restriction site)之分析………………...……..................27
七、第十群台灣梨衰弱病菌質體專一性聚合酵素連鎖反應引子對之設計…………………………………………………………………….………29
八、以南方氏轉漬(Southern blotting)及雜配反應(hybridization)確定第十群台灣梨衰弱病菌質體16S rDNA基因之套數(copy number)……………29
（一）DNA探針之標識(labeling)……………………………........................…..30
（二）南方氏轉漬………………………………………………....................…..31
（三）雜配(hybridization reaction)及呈色反應………………............………..32
九、第十群台灣梨衰弱病菌質體聚合酵素連鎖反應檢測技術之研發…….33
（一）第十群台灣梨衰弱病菌質體專一性聚合酵素連鎖反應(PDTW phytoplasma-specific PCR)…...…………….………............…............33
（二）第十群台灣梨衰弱病菌質體多引子聚合酵素連鎖反應 (multiplex PCR)………………………………………………............................…..34
（三）第十群台灣梨衰弱病菌質體巢式聚合酵素連鎖反應(nested PCR)…………………………………………………………………….34
（四）第十群台灣梨衰弱病菌質體PCR-RFLP分析(PCR-restriction fragment length polymorphism)………….…….........….....................................35
十、第十群台灣梨衰弱病可能之田間媒介昆蟲之偵測……………………36
（一）昆蟲全DNA之純化…………………………............…..………………37
（二）以第十群台灣梨衰弱病菌質體專一型PCR引子偵測台灣梨衰弱病之可能媒介昆蟲…………………………...........…………............…......38
（三）台灣梨衰弱病媒介昆蟲體內病原菌質體16S rDNA全長之增幅與選殖………………………………………............……............………..39
（四）第十群台灣梨衰弱病菌質體可能媒介昆蟲帶菌率之調查.......…...40
十一、各月份罹病梨樹中第十群台灣梨衰弱病菌質體之偵測調查…......….40
肆、結果………………………………………………………………………………41
一、試驗植物研究材料來源…………………………………............…..……..41
二、植物全DNA純化………………………………………............…………41
三、聚合酵素連鎖反應偵測台灣梨衰弱病罹病株中之植物菌質體………42
（一）以廣用型PCR引子偵測台灣梨衰弱病罹病株之植物菌質體……42
（二）以國外梨衰弱病專一型PCR引子對fPD/ rPDS偵測台灣梨衰弱病罹病株之植物菌質體………………………..................................…..43
四、聚合酵素連鎖反應產物之選殖及選殖株之特性分析………..............…….43
五、重組質體pPDL1800嵌入DNA片段之特性分析……………..............…….44
六、第十群台灣梨衰弱病原菌質體之鑑定及親緣系統之建立………………..45
（一）以植物菌質體16S rDNA序列全長為依據構築之親緣樹狀圖…….45
（二）以apple proliferation group各植物菌質體16S-23S rDNA intergenic spacer region核苷酸序列為依據構築之親緣樹狀圖………….……..47
（三）第十群台灣梨衰弱病菌質體具可辨識性之16S rDNA片段….…..48
（四）第十群台灣梨衰弱病菌質體及其鄰近相似物種之rDNA準限制酵素切位(putative restriction site)分析…………………………..…….49
七、第十群台灣梨衰弱病菌質體專一性聚合酵素連鎖反應引子對之設計………………………………………………………………………….…50
八、以南方氏轉漬及雜配反應確定第十群台灣梨衰弱病菌質體16S rDNA基因之套數……………………………………….………...........………….….51
九、第十群台灣梨衰弱病菌質體聚合酵素連鎖反應檢測技術之研發……52
（一）第十群台灣梨衰弱病菌質體專一性聚合酵素連鎖反應........…..…52
（二）第十群台灣梨衰弱病菌質體多引子合酵素連鎖反應…...........…...52
（三）第十群台灣梨衰弱病菌質體巢式聚合酵素連鎖反應.......…..….53
（四）第十群台灣梨衰弱病菌質體PCR-RFLP分析技術……...........…..…..53
十、台灣梨衰弱病可能之田間媒介昆蟲之偵測……………………………54
（一）昆蟲全DNA之純化……………………………...………............…..….54
（二）以放大式聚合酵素連鎖反應(booster PCR)進行台灣梨衰弱病可能媒介昆蟲之偵測………………………………............………………….54
（三）台灣梨衰弱病媒介昆蟲體內病原菌質體16S rDNA全長之增幅與選殖…………………………………………………………...……..…..55
（四）第十群台灣梨衰弱病菌質體可能媒介昆蟲帶菌率之調查.......….......56
十一、各月份罹病梨樹中第十群台灣梨樹衰弱菌質體之偵測調查……..........57
伍、討論……………………………………………………………………………59
一、台灣梨衰弱病之病因探討……………………………............…..……………59
二、第十群台灣梨衰弱病菌質體之分類地位……………............…..……………62
三、第十群台灣梨衰弱病菌質體PCR檢測技術之研發與罹病梨株病原菌之偵測調查…………..………………………………………………............…..………66
四、台灣梨衰弱病田間媒介昆蟲之偵測………………………………............…..69
五、台灣梨衰弱病防治策略之探討……………………………………............72
六、台灣梨衰弱病之未來研究方向………………………………............…….75
陸、參考文獻………………………………………………………...................….77
柒、中文摘要…………………………………………………………………………...88
捌、英文摘要…………………………………………………………………………..90
玖、圖表………………………………………………………………………………...93

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