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標題: 利用核酸生物技術鑑識臺灣常見之黑穗病菌
Identification and Differentiation of Smut Fungi Commonly Observed in Taiwan by Nucleic Acid Biotechnology
作者: 陳松雪
Chen, Song-Sheue
關鍵字: Smut Fungi;黑穗病菌;Identification;Nucleic acid biotechnology;鑑識;核酸生物技術
出版社: 植物病理學研究所
本研究分析7個茭白黑穗病菌(Ustilago esculenta)分離株,5個玉米黑穗病菌(U. zeae)分離株及甘庶黑穗病菌(U. scitaminea)、狗牙根黑穗病菌(U. cynodontis)和紅燕麥黑穗病菌(U. avenae)各一分離株之電泳核型。供試菌之小生子細胞培養至對數生長期,以0.05MEDTA(pH 8.0)離心水洗兩次後,將之包埋於低熔點之瓊脂膠體(Bio-Rad),旋用Novozyme酵素和蛋白裂解酵素處理以抽取染色體。將包埋有染色體的膠塊使用Bio-Rad公司出廠的CHEF-DR II或CHEF Mapper電泳系統,進行電泳核型系統分析。發現供試的茭白黑穗病菌其染色體數目大致為12-18條,分子量大小的分佈在400到5400 Kbs之間,總量基因組大上則在22到26 mb之間。雖然這些供試菌株的染色體條帶分布型式並不一致,然而來自相同作物品種的分離株相較其他作物品種存在較高的相似性。而玉米黑病菌的5個供試分離株其染色體數目大小約16到19條,分子量大小分布為340至2290 Kbs,而總量基因組大小則在13到17mb之間。三個國內的玉米黑穗病菌其染色體條帶分佈型式顯然極為相似,而國外的兩分離株與國內分離株相比,可見有極大的不同;就整個條帶分佈的型式可明顯看出這5個黑穗病菌的種或菌系的鑑定上已頗具潛力。U. esculenta均含有三條大於4mb的染色體,而相反地,僅U. scitaminea, U. cynodontis和 U. avenae具有小於300 Kbs的染色體。來自不同茭白品種的U. esculenta分離株或於國內及國外之U. zeae分離菌株之間其條帶分布型式有極大的變異,進一步顯示以所建立的核型為菌系鑑別上之可能性。為了瞭解供試菌株染色體的多形性,共用了七個DNA選殖體(三個由彭氏自U. esculenta基因庫所構築,四個來處先前RAPD試驗)為核酸探針,以南方式雜合法偵測染色體DNA。其中p119A探針由U. esculenta基因庫次選殖而來,具某程度的鑑別反應,然而來自相同基因庫的p119B和p117探針則無法區別供試的真菌種類。相反地,四個來自RAPD的探針均可偵測到標的染色體DNA的多型性,其中又以探針p5-070B和p8-95A顯示對種間具專一性,即僅分別與U. esculenta和U. zeae染色體DNA有雜合反應。電泳的分子核型分析在病害診斷應用上和寄主與病原之間交互作用之分子基礎的研究上值得進一步去探討。

The electrophoretic karyotypes of 7 isolates of Ustilago esculenta 5 isolates of U. zeae, and 1 Isolate each for U. scitaminea, U. cynodontis and U. avenae were examined. Sporidia cells of test fungi were harvested from log phase Czapek broth cultures, spin washed twice with 0.05M EDTA, and embedded in low melting agarose (Bio-Rad) for chromosome extraction by Novozym and proteinase K treatment. For electrophoretic karyotype analysis, the obtained sample chromosomes in agar blocks were resolved by pulse field gel electrophoresis with CHEF-DRII or CHEF Mapper electrophoresis setup from BioRad. Estimated chromosome numbers of test U. esculenta were approximately 12 to 18; the size distribution appeared to range from 400 to 5400 kbs, and total genome size was estimated around 22 to 26 mbs. Although none of the chromosome banding pattern of the these test isolates were identical, a greater similarity of the banding pattern did exist among the isolates obtained from the same cultvar as compared to that from different cultivars. For the 5 test isolates of U. zeae, total numbers of chromosome were about 16 to 19; the size distribution ranged from 340 to 2290 kbs; and total genome size was around 13 to 17 mbs. The chromosome banding pattern of the 3 local isolates of U. zeae appeared to be quite similar; whereas the 2 compared U.S.A. isolates were quite distinct as compared to the local ones. The overall banding pattern clearly indicated the potential of species or strain differentiation among the five test smut fungi. The existence of at least 3 chromosomes larger than 4 mbs seems to be a distinct characteristics of U. esculenta. As a comparison, the existence of chromosomes smaller than 300 kbs seems to be true only for U. scitaminea, U. cynodontis and U. avenae. The greater diversity of banding pattern among U. esculenta isolates obtained from different Zizania cutivars or among local and U.S.A. U. zeae isolates, further suggested the possibility of using the established karyotypes as a basis for strain identification and differentiation. In order to understand the chromosome polymorphisms of test fungi, a total of 7 DNA clones (3 from the U. esculenta genomic library constructed by Peng, 1991; and 4 from the random amplified polymorphic DNAs from previous experiments) were used to probe the detected chromosomal DNAs by Southern hybridization. Among them, probe p119A which was subcloned from the U. esculenta genomic library, showed certain extent differential responses while reacted with the test chromosomes. However probes p119A and p117 obtained from the same genomic library both failed to differentiate the test fungal species. In constract to this, the 4 RAPD probes all performed well for the detection of polymorphisms among all target chromosome DNAs. Among them, probe p5-070B and p8-95A appeared to be species specific which reacted each with only respective chromosome DNAs of U. esculenta and U. zeae. Their potential application in disease diagnosis and in studies on the molecular basis of host parasite interaction worth great attention.
Appears in Collections:植物病理學系

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