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|標題:||應用rDNA內轉錄區間 (ITS) 序列特性探討萎凋病菌之親緣關係及利用聚合酵素連鎖反應技術檢測番茄萎凋病菌
Application of rDNA internal transcribed spacer (ITS) sequence characteristics in phylogenetic study of Fusarium oxysporum and the use of Random Amplified Polymorphic DNA technique for detection of Fusarium oxysporum f. sp. lycopersici
|關鍵字:||Fusarium oxysporum f. sp. lycopersici;番茄萎凋病菌;rDNA ITS;RAPD;HMA;specific primers;rDNA 內轉錄區間;隨機增幅核酸多形性圖譜;異質雙股泳動性分析法;專一性引子對||出版社:||植物病理學系||摘要:||
造成萎凋病害之病原真菌Fusarium oxysporum，具有廣泛的寄主範圍，其中番茄萎凋病病菌Fusarium oxysporum f. sp. lycopersici (FoLy)則主為台灣番茄產量與品質之限制因子。傳統鑑別Fusarium spp.主要依據形態特徵、病原性測定或營養體親和性測定 (vegetative compatibility) 等，然其孢子形態大小差異大，且孢子堆的形成受制於各項環境因子，對於初學者之鑑別尤感困擾。為發展快速檢測鑑定Fusarium oxysporum之法，本研究嘗試利用rDNA序列作為標的，發展PCR檢測相關技術與檢測流程期能供本病原實際快速鑑定檢測之應用。本研究總共利用62個Fusarium spp.為測試材料，其中包括32個FoLy分離株、20個其他亞種 (fomae speciales) 及10個Fusarium其他種 (species)。利用共同性引子對ITS1/ITS4針對此62個Fusarium spp.菌株，所有供試菌株皆可獲得一約550 bps之增幅產物。選取13個具地區代表性之FoLy菌株與其他供試菌株，共計41個Fusarium spp.經選殖後解序，可見ITS序列全長度範圍為504至530 bps，於Fusarium oxysporum亞種間大致而言其相同度 (identity) 均達99.41-100%。唯其中F. o. f. sp. pisi (CCRC35290)為一例外，其與其他亞種相較，序列相同度只有93.27-93.66%，然與F. roseum及F. verticillioides菌株相較，其相同度則分別達99.8%及100%。與其他Fusarium oxysporum亞種相較序列相同度偏低之特性顯示此一購置CCRC之菌株，其種的特性或有重新檢討之必要。進而利用Maximum-likelihood (ML) 及Neighbor-joining (NJ) (Felsenstein, 1988) 統計方法來瞭解其親緣關係，證實可將13個FoLy菌株歸為同一群，唯其bootstrap值僅66%。進一步將41個供試菌株之ITS全長序列連同GenBank/EMBL資料庫中已知的193條Fusarium spp.之ITS序列，分別利用ML及NJ法分析比對。經由ML方法分析所得之結果，發現可將所有菌株區分為七大群，以NJ方法分析則可見四大群。於ML分析法中，FoLy菌株歸屬於第Ⅳ群，而F. o. f. sp. pisi、F. roseum及F. verticillioides則歸在第Ⅲ群；另值得一提是，以NJ方法分析時，所有供試Fusarium oxysporum (除F. o. f. sp. pisi)菌株之不同亞種皆歸為第iii群。為達快速檢測之目的，本研究另嘗試應用異質雙股泳動性分析 (Heteroduplex mobility assay, HMA) 及隨機增幅核酸多形性 (Random amplified polymorphic DNA analysis, RAPD) 來鑑別Fusarium spp.菌株。於HMA分析中，以編號FoLy-14為標準菌株與所有供試菌株之rDNA ITS增幅產物等量混合進行試驗，依異質雙股條帶分佈可得5種異質雙股形式圖譜 (Heteroduplex pattern, HP)。此外，利用RAPD技術探求核酸層次上相關遺傳訊息，所有供試菌株之總量DNA作為模板經隨機引子S111分析所得之電泳圖譜，皆可得到一致性的條帶，顯示取自本省不同地理位置之FoLy分離株其親緣關係應極為相近。此外，利用隨機引子S115可於供試FoLy菌株增幅出一條約900 bps之專一性DNA片段，此片段經載體選殖定序後設計專一性引子對FoLyF/FoLyR，已知可增幅出約150 bps之PCR產物，經非放射性標定後製備為核酸探針pFoLy。經PCR反應及南方氏漬染分析 (Southern blotting analysis) 試驗所有供試Fusarium spp.菌株，核酸探針pFoLy於所有FoLy菌株皆可得專一性核酸雜合訊號，而其他供試Fusarium spp.菌株則無此訊號產生。此專一性引子對FoLyF/FoLyR利用PCR技術來檢測FoLy基因體核酸時，其靈敏度可達1 ng，輔以pFoLy核酸探針進行南方漬染分析，則可達100 pg。上述證據充分顯示利用本試驗所設計之專一性引子對FoLyF/FoLyR輔以核酸探針pFoLy之FoLy菌株快速檢測鑑別技術，確為極具應用價值之分子檢測輔助工具。而至於HMA分析方法，則於Fusarium spp.鑑別應用上或只能適用至species階層，而無法用於formae speciales之區別上。
As a major fungal pathogen causing vascular wilt disease, Fusarium oxysporum (F. o.) has fairly wide spectrum of host range. The Fusarium wilt of tomato, caused by F. o. f. sp. lycopersici (FoLy) is one of the limiting factors of tomato cultivation in Taiwan. In tradition, the identification of Fusarium spp. is mainly based on the morphological characteristics, the pathogenicity, and the vegetative compatibility. However, as the spore morphology of the fungus varies greatly and the sporulation were greatly affected by various environmental factors, the identification of Fusarium wilt pathogen remains quite often frustrating and discouraging especially for the beginners. In order to develop methodology and protocol practically useful for fast identification of members of F. o., the polymerase chain reaction (PCR) approach with rDNAs as explored target was initiated. A total of 62 Fusarium isolates which including 32 of FoLys, 20 of other formae speciales of F. o., and 10 of other different species of Fusarium, were used for this investigation. By PCR amplification using the universal primer pair ITS1 and ITS4, an amplicon around 550 bps in size was consistently obtained from the genomic DNA preparations of these 62 Fusarium isolates. Among them, 41 which including 13 FoLys of representative collected locations were cloned and sequenced. The 41 cloned fragments have molecular weight ranging from 504 to 530 bps. Analysis of the data obtained indicated the existence of extremely high sequence identity among different formae species of F. o. tested. That of F. o. f. sp. pisi (CCRC35290) was an exception, however. Its sequence data showed only 93.27 to 93.66% identities with other formae speciales of F. o. tested, whereas a 99.8% and 100% identity was found as comparing to that of F. roseum and F. verticillioides respectively. The exceptional low sequence identity comparising to that of other formae speciales of F. o. indicated the need of reexamination on the taxonomical status of their purchase isolate. A followed phylogenetic study by Maximum-likelihood (ML) and Neighbor-joining (NJ) approaches, respectively, indicate that all these 13 FoLy isolates tested may be classified in the same group, although the bootstrap value was only 66%. A further comparison of the full-length sequence data of these 41 isolates with 193 Fusarium spp. ITS sequences available on GenBank/EMBL was proceeded by the ML method and the NJ method respectively. The phylogram constructed by ML method concluded the covered isolates into 7 different groups, whereas that constructed by NJ method classified them into only 4 groups. In the former classification, all the FoLys tested were classified in group-IV, and F. o. f. sp. pisi, F. roseum and F. verticillioides were in group-III. Also noted in the phylogram data was that all F. o. formae speciales isolates tested were concluded in the same group (iii) by the NJ method. For the fast identification of the studied Fusarium spp., both heteroduplex mobility assay (HMA) and random amplified polymorphic DNA analysis were applied. The results of HMA in which ITS rDNA amplicon of FoLy-14 isolate was used as a standard to react with that of all other tested Fusarium isolates concluded 5 characteristic heteroduplex patterns (HP). In followed RAPD analysis using total DNA as a template, the electrophoretogram obtained from an attempt using S111 as random primer demonstrated a nearly identical banding pattern of FoLys, indicating the existence of evolutionary close-relatedness among isolates of different geographical origins. Moreover, a 900 bps fragment specific to FoLy isolates was identified from PCR using S115 as a random primer. The specific fragment was cloned, sequenced; and a FoLy-specific primer pair FoLyF/FoLyR was adapted. The FoLy-specific DNA probe (pFoLy) was prepared by nick-translation using the 150 bps PCR products obtained from FoLyF/FoLyR specific amplification. The specificity of pFoLy was demonstrated by southern blot analysis and as well by PCR, using genomic DNAs obtained from all tested Fusarium isolates as templates. The detection limit by PCR using FoLyF/FoLyR primer pair reached 1 ng (genomic DNA); a follow-up southern blotting by Dig-labelled pFoLy application brought the detection limit further down to 100 pg level. It seems evident that for the fast identification and detection of FoLy, the specific primer pair FoLyF/FoLyR with the aid of specific probe DNA pFoLy would serve as valuable molecular tools. As for HP analysis, the methodology appeared to be useful for species level rather than for the formae speciales level differentiation.
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