劉瑞芬臺灣大學：植物病理與微生物學研究所蔡惠玲Tsai, Hui-LingHui-LingTsai2007-11-272018-06-292007-11-272018-06-292005http://ntur.lib.ntu.edu.tw//handle/246246/58006台灣地處亞熱帶及熱帶地區，氣候及環境皆適於作物生產，但是病害的發生，也相對較為嚴重。就芒果而言，由Colletotrichum gloeosporioides所引起之炭疽病(anthracnose)最為嚴重，會使果實之櫥架壽命與經濟價值降低，為芒果外銷的最主要限制因子。由於C. gloeosporioides寄主範圍廣泛，再加上形態特徵常因遺傳特性及後天培養條件等之影響而有變異，以致診斷鑑定上常備受困擾且費時耗力。C. gloeosporioides感染芒果時，常在未成熟果實進行潛伏感染，而至果實後熟時期才造成明顯病徵，是芒果產銷過程最為棘手的問題。為開發潛伏感染檢測技術，本研究建立以低溫處理法，提早顯現潛伏感染於芒果葉片或果實炭疽病菌的存在情形。分析結果顯示，低溫處理法之檢測效率，與先前已發表之巴拉刈及益收生長素處理法相當，但提供了更為安全與方便之檢測技術。此外，本研究根據核醣體內轉錄間隔區(ribosomal RNA internal transcribed spacer, ITS)設計引子對，開發以Nested polymerase chain reaction (Nested PCR)檢測C. gloeosporioides之技術，但由於C. gloeosporioides與C. musae之ITS序列具高度相同性，無法利用此技術加以區分。為進一步開發C. gloeosporioides專一性檢測技術，另針對炭疽病菌粒線體 cytochrome c oxidase subunitⅠ(COXⅠ)及subunit Ⅱ (COXⅡ)進行序列分析，以便據以設計核酸引子對。利用所設計之degenerate primers所進行之PCR顯示，自C. acutatum菌株可增幅出預期之核酸片段，序列分析顯示其序列亦與其他真菌之COXⅠ及COXⅡ具顯著保守性，但在C. gloeosporioides及C. musae則無法增幅出來任何預期片段。另外，本研究亦嘗試應用Amplified fragment length polymorphism (AFLP)分析C. gloeosporioides及C. musae菌株，並針對部分差異性片段進行序列分析，期能開發出能有效區分這兩種炭疽病菌之PCR檢測技術。In Taiwan, Colletotrichum gloeosporioides are important plant pathogens which cause leaf spotting, fruit staining and rot on Mangifera indica (mango). The detection and diagnosis of Colletotrichum spp. are usually difficult, because of the latent infection and highly morphological variations in the culture conditions. To improve the efficiency of detection and identification of Colletotrichum, we used freezing process, chemical treatment and molecular techniques. In this study, a more safe and convenient of freezing treatment process was investigated and developed as a substitute for Paraquat and Ethephon in the detection of latent infection of Colletotrichum spp. on mango plants. Besides, we designed two primer pairs within ribosomal RNA internal transcribed spacer (ITS) to detect C. gloeosporioides by Nested polymerase chain reaction (Nested PCR). However, due to the sequence conservation of the ITS region between C. gloeosporioides and C. musae, we failed to discriminate by Nested PCR. To improve the species-specificity detection techniques, degenerate primers were designed bared on the mitochondrial cytochrome c oxidase subunitⅠ(COXⅠ) and subunit Ⅱ (COXⅡ) of Colletotrichum spp. The results indicated that an expected fragment genes in C. actatum, but not C. gloeosporioides and C. musae was amplified only. Furthermore, the sequences of the fragments from C. actatum were similar to those COXⅠand COXⅡ reported in Ascomycota. In addition, Amplified fragment length polymorphism (AFLP) was utilized to analyze the difference between C. gloeosporioides and C. musae at the molecular level. Sequence analyses of the differentially amplified fragment are needed to further develop more efficient PCR detection technique for C. gloeosporioides and C. musae.中文摘要-------------------------------------------------------------------------------Ⅰ Abstract --------------------------------------------------------------------------------Ⅱ 壹、前言---------------------------------------------------------------------------------1 貳、前人研究---------------------------------------------------------------------------3 一、芒果炭疽病之病害史------------------------------------------------------3 二、炭疽病菌之分類地位、形態特性---------------------------------------3 三、炭疽病菌之侵入機制與病徵---------------------------------------------5 四、炭疽病菌潛伏感染機制之探討------------------------------------------6 五、炭疽病菌之鑑定與檢測技術---------------------------------------------8 （一）組織分離培養---------------------------------------------------------8 （二）顯微鏡鏡檢------------------------------------------------------------8 （三）化學藥劑處理誘發潛伏感染---------------------------------------8 （四）分生技術應用於炭疽病檢測---------------------------------------9 參、材料與方法-----------------------------------------------------------------------13 一、芒果果樹及供試菌株之來源--------------------------------------------13 二、菌株分離培養與保存-----------------------------------------------------13 三、孢子懸浮液之製備與接種方式-----------------------------------------13 四、分生孢子於植物組織上的觀察-----------------------------------------15 五、以化學藥劑及低溫冷凍處理法偵測芒果炭疽病菌-----------------15 六、核酸製備及濃度測定-----------------------------------------------------16 七、核酸定序--------------------------------------------------------------------17 （一）rDNA ITS核酸片段增幅-------------------------------------------17 （二）PCR擴增產物選殖至T-vector (TA-cloning) --------------------18 （三）選殖株篩選-----------------------------------------------------------18 （四）定序反應--------------------------------------------------------------19 （五）酒精沈澱--------------------------------------------------------------20 （六）序列分析--------------------------------------------------------------20 八、限制片段長度多形性分析-----------------------------------------------20 九、引子對選試-----------------------------------------------------------------23 十、聚合酵素連鎖反應--------------------------------------------------------24 （一）Nested PCR----------------------------------------------------------24 （二）SSR-PCR-------------------------------------------------------------24 十一、增幅產物之瓊脂電泳分析-----------------------------------------------25 肆、結果---------------------------------------------------------------------------------26 一、芒果炭疽病菌的分離與培養--------------------------------------------26 二、接種濃度與病害發生之關係--------------------------------------------26 三、以化學藥劑及冷凍處理法偵測潛伏感染之芒果炭疽病-----------27 （一）檢測人工接種之組織---------------------------------------------27 （二）檢測田間潛伏感染之組織---------------------------------------29 四、ITS region 之分析結果---------------------------------------------------31 （一）rDNA序列分析----------------------------------------------------31 （二）選試引子對之專一性及靈敏度測試---------------------------32 （三）Nested PCR 偵測結果--------------------------------------------33 五、Cytochrome c oxidase gene之分析結果---------------------------------34 六、限制片段長度多形性分析之分析結果----------------------------------37 （一）AFLP分子標誌分析----------------------------------------------37 （二）差異性DNA片段的回收與增幅-------------------------------37 （三）SSR-PCR偵測結果-----------------------------------------------38 伍、討論---------------------------------------------------------------------------------40 陸、參考文獻---------------------------------------------------------------------------46 柒、表------------------------------------------------------------------------------------55 捌、圖------------------------------------------------------------------------------------61 玖、附錄---------------------------------------------------------------------------------891633338 bytesapplication/pdfen-US芒果炭疽病潛伏感染內轉錄間隔區粒線體mangoanthracnoselatent infectionITSmitochondrialotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58006/1/ntu-94-R91633015-1.pdf