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|標題:||應用rDNA內轉錄區間 (ITS) 序列特性作為炭疽病菌鑑定與親緣關係研究之輔助依據
Application of rDNA internal transcribed spacer (ITS) sequence characteristics as an aid for the identification and phylogenetic study of Colletotrichum spp.
|關鍵字:||Colletotrichum;炭疽病菌;rDNA ITS regions;specific primer;PCR;heteroduplex mobility assay;HMA);heteroduplex pattern;HP);identification;phylogenetic study;核糖體去氧核糖核酸內轉錄區間;專一性引子;聚合連鎖反應;異質雙股泳動性分析;異質雙股型式圖譜;鑑定;親緣關係研究||出版社:||植物病理學系||摘要:||
炭疽病菌Colletotrichum gloeosporioides (Cg) 可危害多種作物，嚴重影響產品品質與儲架壽命。傳統Colletotrichum spp. (C-spp) 之鑑定主要依據型態特徵、病徵表現及寄主範圍特性等，然單就Cg而言，由於其寄主範圍甚廣，且形態特徵常因培養條件及變異等之影響而不易確認，加上不同種C-spp會有交叉感染情形，鑑定上常備受困擾且相當費時。為因應加入世界貿易組織後，農產品上此類病原快速檢測與鑑定之需，本研究嘗試利用rDNA之ITS核酸，以PCR增幅分析其不同種間核酸序列差異，進而並利用PCR增幅產物配合異質雙股泳動性法 (heteroduplex mobility assay，簡稱HMA)，瞭解其在鑑識與快速檢測C-sp上之應用潛力。利用共同性引子對ITS1與ITS4 (White et al., 1990) 針對29個C-sp菌株 (分別由CCRC購置、農藥所等相關研究單位提供及本研究室自行分離)及7個非C-spp菌株進行PCR，所有供試之C-spp菌株皆可獲得一約600bps之增幅產物，其他非C-spp菌株之增幅產物則大小分別在500-800bps範圍，與C-spp菌株迥異。逢機選取15個C-sp菌株之增幅產物，以Topo TA cloning 試劑組選殖後解序，可見其全長在535至555bps間，其中計含ITS 1區160至181bps，ITS 2區152至153bps，及5.8S rDNA部分160bps；經由序列比對發現ITS1區 (相同度53.9 ％) 種間歧異性遠較ITS 2 (相同度81.8 ％) 大。利用ITS區全長序列及ITS1區單獨序列，進而以distance matrix及parsimony (Felsenstein, J. et al., 1988) 法統計分析，發現可將這15個C-spp菌株分別以C. gloeosporioides (CG)、C. acutatum (CA)、C. musae (CM) 、C. lindemuthianum (CL) 、C. capsici (CC) 為代表之五群。另將此些ITS1及ITS2序列資料與GenBank / EMBL資料庫比對，發現在ITS1部份除CM及CL群外，皆可找到相同度達99% - 100%的同源性序列，而在ITS2部份則僅CA、CG群菌株可找到相同度達98%以上的同源性序列。有鑑於利用C-spp種專一性引子對 (Mills et al.,1992) 做為供試C-spp菌株種的鑑定工具，在應用上效果不如預期，本研究嘗試另將這15個C-spp菌株與其他供試C-spp菌株之rDNA增幅產物進行HMA分析，所得mobility值經換算成distance後，按上述方法統計分析發現，所有供試菌株可歸類成同上述5群，唯依異質雙股之條帶分佈，則進而可歸納出6種異質雙股型式圖譜 (Heteroduplex pattern，簡稱HP)，除上述CA、CC、CM、CL外，另可將CG分成CG1及CG2兩群。綜合上述核酸分析檢測結果，歸類為CC群之Cgr1、Cgr2、Cd1三個菌株，傳統方法鑑定結果分別屬於C. graminicola (Cgr1、Cgr2)或C. dematium (Cd1)，然其ITS區間序列卻與C. capsici同質性達98-100%，顯示此些菌株之鑑定結果有進一步探討確認之必要；同樣的歸屬CG群之Cg8原以傳統方法鑑定為C. musae；歸屬CA群之Cg2、Ca1、Cg9、C-spp1，原本均被鑑定為Cg；其中以Cc1菌株為例，其原由CCRC購置，孢子形態甫近經再次單孢培養已確定應為如Cg之橢圓形，並非如C. capsici之彎月形，原本之鑑定結果顯然有誤；綜合上述試驗結果，利用rDNA ITS區間序列確可將炭疽病菌區分至種階層，而利用PCR增幅並配合HMA及HP等分析方法，則可作為檢測鑑定炭疽病菌相當明確的輔助依據。
Colletotrichum gloeosporioides (Cg), the causal agent of anthracnose on various important crops, is the most notorious post-harvest pathogens greatly deteriorating the quality and shelf-life of agricultural products. The traditional way for identification of Colletotrichum spp. (C-spp) is quite often frustrating and time consuming mainly due to the wide host range of the pathogen, the great morphological variation; and what even worse was that cross infection might occur among different Colletotrichum spp. To cope with the increasing need of rapid detection and identification of this fungal pathogen from various agricultural products after the country become a member of the World Trade Organization (WTO), the potential application of rDNA ITS sequence characteristics as a biochemical tool was explored. The full length ITS rDNAs of a total of 29 C-spp isolates, and 7 non-C-sp isloates, were produced by polymerase chain reaction (PCR) amplification using the universal primer pair ITS1/ITS4 (White et al.1990). The Colletotrichum species tested included C. gloeosporioides (Cg), C. acutatum (Ca), C. musae (Cm), C. graminicola (Cgr), C. coccodes (Cco), C. capsici (Cc), C. dematium (Cd), C. lindemuthianum (Cl), and Colletotrichum sp. (C-sp) that species status awaited to be identified. The non- Colletotrichum species tested included Rhizopus oryzae, Penicillium digitatum, Pestalotia sp., Fusarium solani, Alternaria brassica, Ustilago esculenta, and Trichoderma reesici. The 600 bps amplicons were generated from all C-spp isolates tested as was expected; the amplicons from 7 non-C-spp isolates ranged from 500-800bps. Fifteen of the C-spp amplicons which include 7 from Cg; 2 each from Cc, Cgr; and one each from Cm, Cd, Cl and C-sp, were cloned and sequenced. The full length of these amplicons ranged from 535 to 555 bps; in which ITS1 region was from 160 to 181bps, ITS2 region was from 152 to 153bps; whereas the 5.8S rDNAs all appeared to be 160bps. Comparison of the inter-specific sequence identity indicated the existence of much greater divergence among ITS1 (identity reached only 53.9%) as compared to that among ITS2 region (identity was approximately 81.8%). Comparative analysis of the ITS full length and the ITS1 sequence data by the distance matrix method and the parsimony method both concluded these 15 tested isolates into 5 distinctive species groups namely, CG (C. gloeosporioides), CA (C. acutatum), CM (C. musae), CL (C. lindemuthianum), and CC (C. capsici). The sequence data also revealed that the ITS1 sequence of tested CG, CA and CC isolates were 99-100% identical to each compared isolate available in the GenBank/EMBL data bank; whereas for ITS2 region, only those from CA and CG isolates were greater than 98% in identity. For the rapid detection and identification of Colletotrichum spp. by PCR, the efficacy of the species specific primer pairs developed by Mills et al. (1992) were examined; the results appeared to be non-satisfactory. To resolve the problem, the heteroduplex mobility assay (HMA) was attempted using the ITS-rDNA amplicons of all 29 tested C-spp isolates as the targets. The analysis by the above described distance matrix method using the distance value estimated from the mobility changes concluded the 29 isolates into 5 distinctive species groups same as that by sequence data analysis. Moreover, from the repeatable band distribution characteristics, a total of 6 heteroduplex patterns (HP) were established. By the established HP fingerprint, the species group CG can be further divided into CG1 and CG2; while the other 4 species groups remain unchanged. In the established species group system, Cgr1, Cgr2 and Cd1 isolates, originally identified as C. graminicola and C. dematium respectively, were reclassified as member of CC since their ITS sequences were 98-100% identical to that of C. capsici. Likewise, the species status of Cg8 isolate was changed from C. musae to a member of CG; Cg2, Ca1, Cg9 and C-sp1 isolates were changed from C. gloeosporioides to be members of CA; and Cc1 isolate was changed from C. capsici to a member of CG2. Among these isolates that original identification were disputable, the species status of Cc1 was reexamined by the original author lately and proved the previous assignment as C. capsici might be a misinterpretation. The results discussed in this investigation, although only limited numbers of fungal isolates had been explored, appeared to provide evidence that sequence analysis of ITS rDNAs a dependable tool in the species level identification of Colletotrichum spp. Also the combination of the genus- or species-specific PCR together with HMA and HP analysis is easy to process, not time consuming, and provides repeatable and distinct data bases useful for species identification. The use of the established techniques for practical application in rapid detection and pathogen identification of anthracnose, and even other fungal diseases, are thus recommended.
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