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學校名稱	輔仁大學
系所名稱	生命科學系
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學號	492546094
研究生(中)	劉慈芬
研究生(英)	Liu, Tzu-Fen
論文名稱(中)	Xanthomonas屬植物病原細菌之檢測及分子類型區分方法之研究
論文名稱(英)	Studies on detection and molecular typing methods of plant pathogenic Xanthomonas spp.
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指導教授(中)	李永安
指導教授(英)	Lee, Yung-An
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畢業學年度	94
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關鍵字(中)	植物病原菌種子鑑別性培養基脈衝氏電泳香菜種子分子類型區分插入序列上菌落數靈敏度抗生素淡綠色萃取液
關鍵字(英)	plant pathogen seed differential medium PFGE coriander seeds molecular typing insertion sequence taiwan after
摘要(中)	本章的實驗目的為研發Xanthomonas屬植物病原細菌的種子檢測法。在研發此屬病原細菌的鑑別性培養基方面，首先以mTBM選擇性培養基做測試，並加以改良研發成Xanthomonas之鑑別性培養基，命名為mTBM-42。以Xanthomonas屬病原細菌共8個種19個病原小種作測試，結果顯示在28℃培養3-5天，菌株均呈現淡綠色至深綠色、水亮、上突、菌株周圍有透明與白霧狀兩種暈圈。由植物葉片與種子分離出來的黃色而非Xanthomonas菌株，如Pantoea sp.、Pseudomonas sp.、Erwinia sp.、Arthrobacter sp.，在鑑別性培養基上，菌株型態均為黃色且扁平，除屬於Arthrobacter sp.菌屬的菌株，周圍只含有透明暈圈外，其餘均無透明與白霧狀兩種型態的暈圈。將此研發出的鑑別性培養基，應用於罹病甘藍葉片病原菌的分離，在培養3-4天即可分離出具明顯特徵的Xanthomonas屬病原細菌，可避免選到黃色而非Xanthomonas屬的細菌。另外，應用此鑑別性培養基在種子檢測上，以X. campestris pv. campestris XCC1-1菌株在此培養基與LA培養基上菌落數之比值，顯示不含抗生素的mTBM-42(A)培養基與LA培養基上菌落數之比值為128%，含抗生素cephalexin (65 ppm) 與5-fluorocil (12 ppm) 的mTBM-42(B)與LA培養基上菌落數之比值之回收率為120%，外加抗生素cycloheximide mTBM-42(C)與LA培養基上菌落數之比值為109%。以香菜葉枯細菌X. campestris pv. coriandri NCPPB 1457在此培養基與LA培養基上菌落數之比值，顯示在mTBM-42(A)其比值為109%，在mTBM-42(B)之比值為101%，在mTBM-42(C)之比值為95%。由此結果得知XCC1-1或NCPPB1457在此選擇性培養基上生長比LA培養基好，培養基所加的抗生素也對這兩個菌株只有輕微抑制作用。此實驗同時觀察到這兩個菌株在不含抗生素的培養基上，約4天可變綠色，在含抗生素的培養基上，約5天可變綠色。將X. campestris pv. campestris XCC1-1菌株混合青江菜 (Brassica chinensis) 種子，或X. campestris pv. coriandri NCPPB 1457混合香菜種子，再使用mTBM-42、mTBM-42A與mTBM-42B培養基分離。結果顯示，雖然此培養基不能抑制所有腐生菌生長，但在28℃培養3-4天仍可發現具明顯特徵的Xanthomonas屬病原細菌。再利用此鑑別性培養基檢測進口香菜種子之Xanthomonas屬細菌。結果顯示只有代號為高雄-1的種子，可發現到少數具明顯特徵的Xanthomonas屬細菌。挑選2個代表性菌株K1-A-27與K1-A-37進行16S rDNA序列，結果顯示與X. campestris有高達99 %的相似性，對煙草也會產生過敏反應，證實分離菌株為Xanthomonas屬植物病原細菌。再將以上4個代表性菌株, K1-A-4、K1-A-9、K1-A-27與K1-A-37，與香菜葉片分離菌株C2-2-1、C4-3、B3-7、B5-5及X campestris pv. coriandri NCPPB 1457，以PFGE進行分子類型區分分析，結果顯示進口香菜種子 (高雄-1) 所分離之菌株與香菜葉片分離菌株呈現不同的PFGE pattern。除利用鑑別性培養基檢測外，亦研發PCR的檢測法。使用DNeasy? Tissue Kit抽取種子萃取液的total DNA，再以hrpG-F+hrpG-R進行PCR反應，結果顯示PCR靈敏度約為104-105 cell/ml。本論文的實驗目的是比較使用IS-RFLP (IS-restriction fragement length polymorphism analysis) 及PFGE (pulsed field gel electrophoresis) 兩種方法，在台灣分離之Xanthomonas campestris pv. campestris (XCC) 菌株的分型之結果。利用IS1404 (IS3 family)、IS1479 (IS5 family) 以及 ISXac3做probe，進行IS-RFLP分析。結果顯示使用IS1404-RFLP方法，在所測試的XCC菌株中有8個分子類型。使用IS1479-RFLP與ISXac3-RFLP方法，也是分成8個分子類型，此結果顯示不管使用那種IS進行IS-RFLP分析，在XCC菌株的分型上，所得到的結果是相同的。再將此些菌株的total DNA，以限制酵素 SpeI 處理後，再以PFGE進行分子類型區分分析，也可將其分成8個分子類型。進一步使用PFGE在香菜 (Coriandrum sativum) 葉枯病原菌株的分型上。因為推測2000年夏天在台灣首先發現的病原菌，可能藉由進口種子引入。所以比較在台灣香菜病葉所分離之14株疑似的X. campestris pv. coriandri菌株，以及最早在印度發現的葉枯病原菌X. campestris pv. coriandri NCPPB 1457的PFGE pattern。結果顯示台灣分離的菌株可分成五種PFGE pattern，而其中一種type與X. campestris pv. coriandriNCPPB 1457有高度相似性。此結果顯示台灣分離的菌株可能來自包括印度的不同國家，而印度為台灣香菜種子最主要的進口國家。這些菌株進一步使用hrpG和virD4基因做探針，進行Southern hybridization。結果顯示大多數的菌株都含有這兩個基因，但有一些菌株則沒有，而含有hrpG和virD4的菌株呈現多種RFLP patterns。
摘要(英)	The purpose of this chapter was to develop methods for detection of the plant pathogenic Xanthomonas spp. in plant seeds. The mTBM semi-selective media was chosen to develop a differential medium of Xanthomonas spp., mTBM-42. The nineteen pathovars of eight species of Xanthomonas were tested on mTBM-42. Colonies of Xanthomonas spp. were light green and deep green, glistening, convex with entire margins, and surrounded by a large clear zone and a smaller milky zone after 3-5 days of incubation at 28oC. Some yellow-pigmented non-xanthomonads isolated from plant leaves and seeds, such as the Pantoea sp., Pseudomonas sp., Erwinia sp., Arthrobacter sp., grew on the differential media as yellow, flat, and without clear and milky zones, except Arthrobacter sp., which was surrounded by a large clear zone only. The mTBM-42 differential medium was used to isolate plant pathogenic Xanthomonas spp. from infected cabbage leaves. The characteristic colonies of Xanthomonas sp. were observed after 3-4 days of incubation at 28oC. The medium could differentiate Xanthomonas spp. from yellow-pigmented non-xanthomonads and avoid isolation of non-xanthomonads. Furthermore, the differential medium was used to detect Xanthomonas spp. in plant seeds. The recovery of X. campestris pv. campestris XCC1-1 on mTBM-42 medium was 128%, compared with LA medium. The recovery was 120% on mTBM-42 medium supplemented with cephalexin (65 ppm) and 5-fluorocil (12 ppm) (mTBM-42A), and 109% on mTBM-42A medium with further addition of cycloheximide (75 ppm) (mTBM-42B). The recovery of X. campestris pv. coriandri NCPPB 1457 was 109% on mTBM-42, 101% on mTBM-42 A, and 95% on mTBM-42B. Thus, the addition of antibiotics had a slight inhibitive effect on the growth of Xanthomonas spp. The Xanthomonas spp. tested developed the characteristic colonies in 4 days on mediua without antibiotics and 5 days on mediua with antibiotics. The seeds of Brassica chinensis and coriander were artifically inoculated with X. campestris pv. campestris XCC1-1 and X. campestris pv. coriandri NCPPB 1457, respectively. The seed extracts were plated on mTBM-42, mTBM-42A, and mTBM-42B. Although the media did not eliminate growth of all saprophytes, the characteristic colonies of Xanthomonas spp. could still be observed after 4-5 days of incubation at 28oC. The differential media were further used for the detection of Xanthomonas sp. in imported coriander seeds. A few characteristic colonies of Xanthomonas spp. were found in the coriander seeds of Kaohsiung-1. Two isolates, which K1-A-27 and K1-A-37, were chosen for 16S rDNA sequence analysis. The sequences were highly identical (99%) to that of X. campestris. The hypersensitive reactions were observed in Nicotiana benthamiana incoulated with K1-A-27 and K1-A-37. The results indicated that the characteristic colonies isolated on the media were plant pathogenic Xanthomonas sp. The molecular types of K1-A-4, K1-A-9, K1-A-27, and K1-A-37 isolates, and strains C2-2-1, C4-3, B3-7, B5-5, NCPPB 1457 of X. campestris pv. coriandri which were isolated from coriander leaves were determined by using pulsed field gel electrophoresis (PFGE). There were two PFGE patterns among isolates from imported seeds of Kaohsiung-1, which were different from the patterns of strains isolated from coriander leaves. PCR assays for seed detection of Xanthomonas spp. was also developed. The total DNA of seed extracts was isolated by DNeasy? Tissue Kit and used in PCR amplification using primer set, hrpG-F/hrpG-R. The sensitivity of PCR assay was approximately 104-105 cell/ml. The purpose of this study is to compare the insertion sequence-restriction fragments length polymorphism (IS-RFLP) and pulsed field gel electrophoresis (PFGE) methods in the molecular typing of Xanthomonas campestris pv. campestris (XCC) strains in Taiwan. Three insertion sequences, IS1404 (IS3 family), IS1479 (IS5 family), and ISXac3, were used as probes for IS-RFLP analyses. There were at least eight molecular types in IS1404-RFLP analysis. The number of molecular types was almost the same by using IS1479-RFLP and ISXac3-RFLP methods, indicating that the results of molecular typing would be the same regardless of what IS used in IS-RFLP analysis. PFGE analysis also obtained the same number of molecular types using SpeI for digestion of total DNAs. PFGE was further used to characterize the suspected strains of coriander (Coriandrum sativum) leaf blight pathogen. The pathogen was first found during the summer of 2000 in Taiwan and might be introduced from imported seeds. PFGE patterns of fourteen isolates of X. campestris pv. coriandri from diseased coriander leaves in Taiwan were compare with that of X. campestris pv. coriandri NCPPB 1457, which was isolated in India where coriander leaf blight first occurred. There were five PFGE pattern types in the strains isolated in Taiwan, and one type is highly similar to that of X. campestris pv. coriandri NCPPB 1457. The results indicated that the strains in Taiwan might be from different foreign countries including India, which is the major exported country of coriander seeds. The strains were further characterized by Southern hybridization using the hrpG and virD4 as probes. The result showed that most strains contained both genes but some did not, and various RFLP patterns were found in strains with hrpG and virD4.
論文目次	目錄第一章 Xanthomonas屬植物病原細菌之種子檢測法之研究中文摘要------------------------------------------------------------------------------------------1-1 英文摘要------------------------------------------------------------------------------------------1-3 前言------------------------------------------------------------------------------------------------1-5 材料與方法-------------------------------------------------------------------------------------1-13 1.鑑別性培養基之研發--------------------------------------------------------------------1-13 1-1.鑑別性培養基配方之挑選與改良------------------------------------------------1-13 1-2.Xanthomonas屬病原細菌在鑑別性培養基上之測試-------------------------1-14 1-3.黃色非Xanthomonas屬病原細菌在鑑別性培養基上之測試---------------1-14 2.鑑別性培養基之應用--------------------------------------------------------------------1-15 2-1.自罹病植物組織分離Xanthomonas屬植物病原菌---------------------------1-15 2-2.自種子分離Xanthomonas屬植物病原菌---------------------------------------1-15 2-2-1.Xanthomonas屬病原細菌在鑑別性培養基與LA培養基上菌落數之比值測試-----------------------------------------------------------------------------1-1 2-2-2.Xanthomonas屬病原細菌在鑑別性培養基上單一菌落型態之觀察-1-16 2-2-3.種媒病原菌之萃取與使用鑑別性培養基分離之流程------------------1-17 2-2-4.混合Xanthomonas屬病原細菌之種子以鑑別性培養基之分離測試-1-17 2-2-5.進口香菜種子之檢測---------------------------------------------------------1-18 2-2-5-1.香菜種子高雄-1分離菌株的鑑定-------------------------------------1-18 2-2-5-2.香菜種子高雄-1分離菌株之分子檢測-------------------------------1-18 2-2-5-3.香菜種子高雄-1分離菌株分子類型區分----------------------------1-18 3.以PCR檢測種子中之種媒病原菌-----------------------------------------------------1-20 3-1.所使用的引子對之篩選------------------------------------------------------------1-20 3-2.香菜病葉分離菌株Xanthomonas. campestris pv. coriandri Tc3與種子混合後之PCR敏感度測試----------------------------------------------------------------1-20 4.基礎分生技術-----------------------------------------------------------------------------1-22 結果----------------------------------------------------------------------------------------------1-26 1.鑑別性培養基之研發--------------------------------------------------------------------1-26 1-1.鑑別性培養基配方之挑選與改良結果------------------------------------------1-26 1-2. Xanthomonas屬病原細菌在鑑別性培養基上之測試結果------------------1-29 1-3.黃色非Xanthomonas屬病原細菌在鑑別性培養基上之測試結果---------1-29 2.鑑別性培養基之應用--------------------------------------------------------------------1-30 2-1.自罹病植物組織分離Xanthomonas屬植物病原菌之結果------------------1-30 2-2.自種子分離Xanthomonas屬植物病原菌---------------------------------------1-31 2-2-1.Xanthomonas屬病原細菌在鑑別性培養基與LA培養基上菌落數之比值測試-----------------------------------------------------------------------------1-3 2-2-2. Xanthomonas屬病原細菌在鑑別性培養基上單一菌落型態之觀察-結 2-2-4.混合Xanthomonas屬病原細菌之種子以鑑別性培養基之分離測試結果 2-2-5.進口香菜種子之檢測結果---------------------------------------------------1-33 2-2-5-1.種媒病原菌之萃取與使用鑑別性培養基分離之結果-------------1-33 2-2-5-2.香菜種子高雄-1分離菌株的鑑定結果-------------------------------1-35 2-2-5-3.香菜種子高雄-1分離菌株之分子檢測結果-------------------------1-36 2-2-5-4.香菜種子高雄-1分離菌株之分子類型區分結果-------------------1-36 3.以PCR檢測種子中之種媒病原菌-----------------------------------------------------1-36 3-1.所使用的引子對之篩選結果------------------------------------------------------1-36 3-2.香菜病葉分離菌株Xanthomonas. campestris pv. coriandri Tc3與種子混合後合之PCR敏感度測試結果-------------------------------------------------------------1-37 討論----------------------------------------------------------------------------------------------1-39 參考文獻----------------------------------------------------------------------------------------1-46 圖表----------------------------------------------------------------------------------------------1-51 目錄第二章 Xanthomonas屬植物病原細菌之分子類型區分方法之研究中文摘要------------------------------------------------------------------------------------------2-1 英文摘要------------------------------------------------------------------------------------------2-2 前言------------------------------------------------------------------------------------------------2-3 材料與方法---------------------------------------------------------------------------------------2-9 1.Xanthomonas屬植物病原細菌之分子類型區分之研究-----------------------------2-9 1-1.菌種的取得、培養條件、儲存的方法----------------------------------------------2-9 1-2.IS種類、引子對設計與選殖---------------------------------------------------------2-9 1-3.病原菌的分子類型 (molecular typing)------------------------------------------2-11 1-3-1.IS–RFLP(IS-restriction fragement length polymorphism)----------------2-11 1-3-2.脈衝式電泳(pulsed-field gel electrophoresis；PFGE)---------------------2-11 2.Xanthomonas屬植物病原細菌之分子類型區分之應用---------------------------2-12 2-1.香菜葉片分離菌株的取得、培養條件、儲存的方法---------------------------2-12 2-2.香菜病葉分離之菌株鑑定---------------------------------------------------------2-13 2-2-1.生理生化測試------------------------------------------------------------------2-13 2-2-2.16S rDNA基因選殖、定序及比對------------------------------------------2-15 2-2-3.對菸草之過敏性測試---------------------------------------------------------2-15 2-2-4.分子檢測------------------------------------------------------------------------2-15 2-3.香菜病葉分離之菌株的分子類型(molecular typing)區分-------------------2-15 2-3-1.脈衝式電泳---------------------------------------------------------------------2-15 2-3-2.RFLP-----------------------------------------------------------------------------2-16 2-3-3.PCR-RFLP----------------------------------------------------------------------2-16 3.基礎分生技術-----------------------------------------------------------------------------2-17 結果----------------------------------------------------------------------------------------------2-25 1.Xanthomonas屬植物病原細菌之分子類型區分之研究---------------------------2-25 1-3.病原菌的分子類型 (molecular typing)------------------------------------------2-25 1-3-1.IS–RFLP(IS-restriction fragement length polymorphism)---------------2-25 1-3-2.脈衝式電泳(pulsed-field gel electrophoresis；PFGE)---------------------2-26 2.Xanthomonas屬植物病原細菌之分子類型區分之應用---------------------------2-27 2-1.自香菜細菌性葉枯病的香菜分離菌株------------------------------------------2-27 2-2.香菜病葉分離之菌株鑑定結果---------------------------------------------------2-27 2-2-1.生理生化測試------------------------------------------------------------------2-27 2-2-2.16S rDNA基因選殖、定序及比對------------------------------------------2-28 2-2-3.對菸草之過敏性測試---------------------------------------------------------2-28 2-2-4.分子檢測------------------------------------------------------------------------2-28 2-3.香菜病葉分離之菌株的分子類型(molecular typing)區分-------------------2-29 2-3-1.脈衝式電泳---------------------------------------------------------------------2-29 2-3-2.RFLP-----------------------------------------------------------------------------2-30 2-3-3.PCR-RFLP----------------------------------------------------------------------2-31 討論----------------------------------------------------------------------------------------------2-33 參考文獻----------------------------------------------------------------------------------------2-36 表-------------------------------------------------------------------------------------------------2-41 圖-------------------------------------------------------------------------------------------------2-58
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