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標題: 十字花科蔬菜黑腐病菌專一性核酸探針之製備及病菌偵測應用
Development and Application of a Specific DNA Probe for the Detection of Xanthomonas Campertris PV. Campertris
作者: 石信德
Shih, S.D.
關鍵字: 十字花科;蔬菜黑腐病茵
出版社: 植物病理學研究所
十字花科蔬菜黑腐病菌(Xanthomonas campestris pv. campestris, 簡稱Xcc)Xcc70菌株之全DNA,以pBluescriptⅡKS-為載體,在E.coli DH10B 構築之基因庫中,隨機選取10 個選殖株,將具有insert DNA的重組質體,以非放射性Digoxigenin-11-dUTP標幟後得到的探針,在點漬雜配分析法與南方式雜配分析法上, 和Xcc70菌株之DNA均有明顯之雜配訊號。由其中一個選殖株pXcc70-8之insertDNA以限制□EcoRⅠ切割後含有2.7kb、1.6kb及0.6kb片斷,將0.6kb片斷藉由次選殖後所製備的探針pXcc70-8-1與所有供試51個Xcc菌株均有雜配訊號,而與其它55株非Xcc 菌包括Xanthomonas campestris pv. begoniae (Xcb), X. campestris pv. citri(Xcci), X. campestris pv. difenbachiae (Xcd), X. campestris pv. glycines (Xcg), X. campestris pv. mangiferaeindicae (Xcm), X campestris pv. oryzae (Xco), X. campestris pv. phaseoli (Xcp), X. campestris pv. pruni (Xcpr ), X. campestris pv uppalii (Xcu), X. campestris pv. vesicatoria (Xcv), X. albilineans (Xa), X.fragariae (Xf), Pseudomonas cichorii (Pci), P. glumae (Pg) ,P. solanacearum (Ps), P. syringae pv. syring (pssi), Erwinia carotovora subsp. carotovora(Ecc), E. chrysanthemi (Ech), Escherichiai coli(E. coli), P. fluorescens (Pf), P. putida (Pp)及由甘藍葉面上分離未經鑑定之細菌沒有雜配訊號。又由pXcc70-30中所攜帶的1.5Kb EcoRⅠ片斷,經次選殖後所製備之探針pXcc70-30-1亦與所有的Xcc供試菌株有訊號反應,但是與少數非Xcc供試菌株如Xcb(6.5kb),Xcci(6.5kb), Xcg(6.5kb), Xco(7.8kb),Xcpr(1.8kb)及Xcu(7kb)在註明的DNA 片斷處有雜配訊號產生。此外,由pXcc70-25經限制沒BamHⅠ酵解後所得1.1Kb大小片斷,經次選殖後所製備的探針pXcc70-25-2與所有的Xcc供試菌株亦有訊號反應,然而與在多數X. campestris pathovars, Pci, Pf 及Pd 菌株有著不同DNA片斷處的訊號。此三個探針以點漬雜配法測定其敏感性時顯示 Xcc70全DNA經稀釋到0.19ng 時仍可被pXcc70-8-1探針所偵測到,而pXcc70-25-2及pXcc70-30-1可偵測之最低濃度為1.9ng。利用菌落雜配分析法,從罹病甘藍葉部及白菜、芥藍藍種子所分離的菌落中,以pXcc70-8-1探針,可正確的將黑腐病菌菌落鑑定出,另外,從白菜及芥藍種子抽出液直接以點漬配法亦可偵測到黑腐病菌。

A genomic library of strain Xcc 70 of Xanthomonas campestris pv. campestris was constructed in the phagmid pBluescript ⅡKS- and transformed into E. coli strain DH 10B .The recombinant plasmind DNAs from 10 clones selected randomly were labeled by the nontadioactive system with Digoxigenin -11-dUTP using the random method . In Dot-blot hybridization and Southern-blot hybridization analysis, all probes hybridized tensively with genomic DNA of Xcc70. One of the clones, pXcc70-8, after digestion with EcoR I, yield fragment of 2.7, 1.6 and 0.6 kilobases (kb).When a subclone, pXcc70-8-1, containing the 0.6 kb fragment was used as a probe ,it hybridized with all 51 strains of Xcc , but not to the other 55 bacterial strains tested , which inclued X. campestris pv. begoniae (Xcb), X. campestris pv. citri (Xcci), X. campestris pv diffenbachiae (Xcd), X campestris pv. glycines (Xcg), X. campestris pv.mangiferaecindicae(Xcm). X. campestris pv.oryzae(Xco), X. campestris pv. phaseoli (Xcp), X. campestris pv . pruni (Xcpr), X campestris pv. uppalii (Xcu),X campestris pv. vesicatoria (Xcv), X. albilineans (Xa), X fragariae (Xf), Pseudomonas cichorii(Pci), P. glumae (Pg), P. solanacearum(Ps), P. syringae pv. syringae (Pssi), Erwinia carotovora subsp. carolovora (Ecc), E. chrysanthemi (Ech), Escherichia coli (E. coli ), P. fluorescens (Pf), P. putida (Pp), and saprophytic strains isolated from cabbage leaves.Two other subclones pXcc70-30-1 (contained 1.5 kb EcoR I fragment) and pXcc 70-25-2 (contained 1.1kb BamHⅠfragment ) and were obtained pXcc 70-30 and pXcc 70-25, respectively and were also tested for their specificity for detection of Xcc. The probe pXcc 70-30-1 hybridized with all Xcc strains, but also with Xcb, Xcci, Xcg, Xco, Xcpr and Xcu. The probe pXcc70-25-2 hybridized with all Xcc strains, but also with most pathovars of X. campestris, Pci, Pf and Pp. The specific probe pXcc70-8-1detected the total DNA of Xcc70 to 0.19 ng in dot-blot hybridization, whereas probes pXcc 70-30-1 and pXcc70-25-2, were only one-tenth as sensitive . Using colony hybridization method, the pXcc70-8-1probe specifically detected colonies of Xcc plated directly from infected leaves of cabbage and seeds of Pai-Tsai and Chinese kale. Xcc in seed extracts of Pai-Tsai and Chinese kale could be also detected by the probe in the dot blot assay.
Appears in Collections:植物病理學系

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