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標題: 十字花科蔬菜種媒黑斑病菌之分子檢測
Molecular detections for seed-borne Altelnaria brassicicola on crucifer seeds
作者: 張裕民
關鍵字: crucifer seeds;十字花科蔬菜種子;Alternaria brassicicola;seed health test;quantitive;molecular diagnosis;probe;RAPD;PCR;黑斑病;種子檢查;定量;分子診斷;探針;專一性引子對;真菌
出版社: 植物病理學系
本研究應用PCR以及PCR-ELISA來檢測十字花科蔬菜的種媒真菌。首先根據國際種子檢查協會推薦的培養皿檢查法,以吸濕紙法檢查不同品種之芥菜、甘藍、蘿蔔、白菜、花椰菜與青花菜六種十字花科蔬菜種子,一週後以Alternaria spp. 檢測出現的頻率最高,分別為青花菜14.75%、芥菜9.25%、甘藍二個樣本分別為3.75%與5.75%、花椰菜二個樣本分別為5%與2.75%、蘿蔔二個樣本分別為8%與10.75%、白菜三個樣本分別為9%、5%與5.5%帶有Alternaria spp.,其中多為腐生之A. alternata與黑斑病菌A. brassicicola,亦可分離到Aspergillus spp.、Cladosporium spp.、Curvularia spp.、Fusarium spp.、Penicillium spp.、Trichurus spp.、 Rhizopus spp.,並進行菌株收集培養及保存的工作。因此本研究利用RAPD隨機引子OPA-09篩選的A. brassicicola 830 bp的專一性片段;另經南方轉漬分析此專一性片段均存在所有A. brassicicola菌株的總量基因體核酸中。經pCRâII-TOPOâ vector選殖後,再經DNASTARâ軟體分析設計出專一性引子對Ab536r (5´-ATATAAAGGCGGGTAACG-3´)及Ab536f (5´-AGCGCCTTATACTCCTT CT-3´),可針對有病原性之A. brassicicola增幅出536 bp的專一性條帶,而對於其他種子分離到的真菌及健康種子的基因體核酸皆無法增幅出此片段。此專一性引子對靈敏度可達50 pg μl-1,可以檢測到接種100個黑斑病菌孢子的種子之基因體核酸。我們進一步利用篩選出的830 bp專一性片段,設計一個生物素標定的捕捉探針(5´biotin- TAAGGTATAAATAGTAA AGTAAC-3´),並將專一性引子標定DIG物質進行PCR反應,攜有DIG的單股PCR產物會生物素標誌捕捉探針(biotinylated capture probe)雜合,而此探針附著在微力價盤(microtiter plate)上的鏈黴卵白素結合,經由anti-DIG-POD與DIG聯結後,再以特定基質呈色於波長450 nm下觀察吸光值,發展出PCR-ELISA技術,我們研究顯示,當鏈黴卵白素濃度為50 μg ml-1,以及探針濃度為1 pmol時,以DIG標定於primer進行之PCR-ELISA有較佳的讀值表現,比使用DIG標定於dNTP進行之PCR-ELISA較為經濟,此技術有著高靈敏度、無EtBr與放射線標定污染之優點,並可像ELISA一般進行大量樣本和研究罹病組織內病原菌之定量偵測,可作為病害感染程度或病害綜合管理發病門檻的參考。

Two techniques for simultaneous detection for seed-borne fungi on crucifer seeds, Polymerase chain reaction (PCR) and PCR-based enzyme-linked immunosorbant assay (PCR-ELISA), have been developed in this study. Thirty isolates of Alternaria brassicicola, 25 isolates of Alternaria, 2 isolates of Aspergillus, Curvularia, Fusarium, Penicillium, Rhizopus, 1 isolate of Cladosporium and Trichurus were isolated respectively from 50 crucifers. The blotter method was used to survey 6 different crucifer cultivars seeds Alternaria spp. in total 11 samples, have the highest frequency. The percentages were: 14.75% in broccoli; 9.25% in mustard; 3.75% and 5.75% in 2 cabbage samples; 5% and 2.75% in 2 cauliflower samples; 8% and 10.75% in 2 radish samples; 9%, 5% and 5.5% in 3 pai-tsai samples. Though this method is simple to manipulate, it need the long culture periods and does not have species-specificity. PCR technology has the potential for widespread application in the field of plant pathogen detection and indexing programs. eleven random primers were used for random amplified polymorphic DNA (RAPD) analysis to find specific DNA markers of A. brassicicola. The primer OPA-09 (5´-GGGTAACGCC-3´) amplified a distinct fragment, subsequently cloned in pCRÒII- TOPOÒvector, and designed the A. brassicicola-specific primer pairs Ab536R (5´-ATATAAAGGCGGGTAAACG-3´) and Ab536F (5´-AGCGCCTTATACTCCTTCT-3´) according to the specific sequence of 830-bp DNA fragment. The primers could amplify a specific 536-bp fragment from genomic DNA of A. brassicicola by PCR. The sensitivity of the primer pairs could detect 50 pg of genomic DNA of A. brassicicola, and could detect seeds with at least 100 A. brassicicola spores. Another parallel diagnosis system, PCR-ELISA; enable immunoenzymatics determination of PCR products in the liquid phase and does not need for electrophoresis, thereby simplifying the analysis of the results with an ELISA reader. Amplified PCR products generated using a digoxigenin-labeled primer or dNTP, heat-denatured and hybridize with a biotin-labelled capture probe (5´biotin- TAAGGTATAAATAGTAAAGTAAC -3´) immobilized onto streptavidin coated microtiterplates, was used to capture the digoxigenin-labeled fragments that were detected with a peroxidase antidigoxigenin conjugate. Subsequent enzymic conversion of substrate was measured at 450 nm with an ELISA reader. Our results showed that when utilizing DIG-labeled primer, 1 pmol probe and 50 μg ml-1 streptavidin, will near the results of utilizing DIG-labeled dNTP, 0.5 pmol probe and 10μg ml-1 streptavidin, but is more economic. Applying the species-specific primer pairs by PCR technology for seed healthy test could shorten the test periods. Furthermore, the high sensitivity PCR-ELISA system could detect PCR products, transverse electrophoresis results to quantity data. In further research we shall match the disease database for seed health test or other disease predictive models.
Appears in Collections:植物病理學系

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