Molecular identification of Fusarium oxysporum f. sp. cubense and its detection in infected banana seedlings

香蕉黃葉病 (Fusarial wilt) 又稱巴拿馬病 (Panama disease)，是由Fusarium oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen (Foc) 引起的系統性病害；近20年來，由於香蕉黃葉病肆虐，造成台灣香蕉嚴重減產；而於無病土中種植不帶病的健康香蕉苗為防治此病害的最佳策略之ㄧ，因此開發一套方便快速的檢測方法實為當務之急。本研究先將17個由罹患香蕉黃葉病之香蕉植株所分離的F. oxysporum菌株接種北蕉，待產生香蕉黃葉病病徵，確定為具有病原性之Foc race 4後，再利用「隨機增幅核酸多型性分析法 (random amplified polymorphic DNA, RAPD)」發展出專一性引子對，配合「聚合酵素連鎖反應 (polymerase chain reaction, PCR)」法來偵測香蕉黃葉病菌，期能有效杜絕此類病原菌的傳播。結果顯示，前人使用之OPA-02引子確實可在所有具病原性之香蕉黃葉病菌株 (race 4) DNA中，增幅出一特定片段OPA02400，且回收此片段作為探針進行「南方雜合 (Southern hybridization)」分析，證實其對香蕉黃葉病菌具專一性。取此序列設計三對引子，其中以OPA02400A1/OPA02400S2引子對進行PCR，可於香蕉黃葉病菌 (race 4) 的DNA中增幅出242 bp的專一性片段，其靈敏度可達10 pg的Foc DNA；在反應物中含有1,000 ng香蕉葉片DNA時，其PCR靈敏度降為100 pg的Foc DNA (降低10倍)。但若以OPA02400序列所設計之另外二對引子進行PCR增幅，則對Foc不具專一性。其中以OPA02400F1/OPA02400R1為引子對進行PCR，在香蕉黃葉病菌、百合萎凋病菌、茼蒿萎凋病菌、番茄萎凋病菌、絲瓜萎凋病菌、西瓜蔓割病菌、金線連基腐病菌和水稻徒長病菌的DNA中都可以增幅出特定的302 bp片段，顯示其對Foc不具專一性；而以OPA02400F2/OPA02400R2引子對進行PCR，則可於所有供試之Fusarium屬菌株的DNA中增幅出特定的148 bp片段。目前將接種Foc並已罹病的香蕉苗之DNA進行PCR檢測，已在同一植株之有病徵及無病徵部位分別偵測到Foc DNA。另外，將Kim等人 (1999) 所發表之微波爐萃取孢子DNA法加以修改，利用微波爐法抽取菌絲DNA，所得之DNA再進行PCR檢測，前後所需時間總共只要4小時，可提高病原菌分子鑑定之效率。此外，本研究比對所有供試之Fusarium菌株與基因庫中已發表之核糖體核酸 (nrDNA) 的內轉錄區間 (internal transcribed spacer, ITS) 序列，結果顯示序列相似度高達92-97%；但是所有供試Foc race 4的nrDNA在ITS2區間之特定位置皆有一個核苷酸為C，而在Foc race 1、其它不同生理分化型的F. oxysporum與Fusarium屬等供試菌株中則為T的差異。Fusarial wilt of banana, commonly known as Panama disease, is caused by Fusarium oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen (Foc). In recent twenty years, this disease has severely destroyed banana production in Taiwan. One of the best way to control Panama disease is to grow healthy banana seedling in virgin soil. It is thus necessary to use a conventient and fast method to detect the Foc pathogen in order to control this disease. In this study, 17 isolates of F. oxysporum obtained from the banana plants showing Fusarial wilt symptoms were used to inoculate banana seedling (cultivar ‘Giant Cavendish'), and their pathogenicity indicated that these isolates were all Foc race 4. Random amplified polymorphic DNA (RAPD) analysis was used to develop a specific and fast detection method for Foc. A 400 bp fragment (OPA02400) amplified only in Foc race 4 isolates was identified by polymerase chain reaction (PCR) using OPA-02 random primer. This OPA02400 fragment was cloned and used as a probe for Southern hybridization. The results suggested that the OPA02400 was specific to Foc. Based on the nucleotide sequence of the OPA02400, three primer pairs were designed for PCR amplification of the Foc DNA. A 242 bp DNA fragment specific to Foc was amplified by PCR using primer pair OPA02400A1 (A1)/ OPA02400S2 (S2), and the detection sensitivity was 10 pg of fungal genomic DNA. In the presence of 1,000 ng banana leaf genomic DNA, the sensitivity of PCR using A1/S2 primer pair decreased 10-fold to 100 pg of fungal genomic DNA. The other two primer pairs, OPA02400F1 (F1)/ OPA02400R1 (R1) and OPA02400F2 (F2)/ OPA02400R2 (R2) designed were not specific to Foc. Using F1/R1 primer pair for PCR, a 302 bp DNA fragment was ampfied from the DNA of Foc, F. oxysporum f. sp. lilii, F. oxysporum f. sp. chrysanthemi, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. luffae, F. oxysporum f. sp. niveum, F. oxysporum (infecting anoectochilus) , and F. moniliforme. Whereas, a 148 bp DNA fragment was ampfied from the DNA of all tesed Fusarium isolates using F2/R2 primer pair. The developed PCR technique using of A1/S2 primers also successfully detected Foc DNA in infected banana stem or leaf tissues whether showing symptom or not. In addition, a microwave method for fungal spore DNA extraction, published by Kim et al. (1999), was modified for DNA extraction from fungal myclia. By combining the modified microwave method for DNA extraction with the PCR assay, molecular identification of fungal pathogens could be achieved within 4 hours. Besides, we also sequenced the internal transcribed spacer (ITS) regions of the nrDNA cluster from all tested Fusarium isolates. The DNA sequences of ITS1 and ITS2 obtained showed 92-97% identity. In all Foc race 4 isolates tested, however, there is one nucleotide difference within the ITS2 region comparing to the Foc race 1 isolates and all the other Fusarium isolates tested (cytosine instead of thymidine).一、中文摘要…………………………………………………………………… 1 二、英文摘要......................................................................................................... 3 三、緒言………………………………………………………………………… 6 四、材料與方法………………………………………………………………… 12 (一)香蕉黃葉病菌之病原性測試………………………………………….. 12 (二)供試菌株之培養……………………………………………………….. 13 (三)抽取總量DNA…………………………………………………………. 13 (1)微波爐法…………………………………………………………….. 13 (2)傳統抽取法………………………………………………………….. 14 (四)應用RAPD技術分析香蕉黃葉病菌………………………………….. 15 (五)病原菌之標的DNA的選殖及定序…………………………………… 15 (六)定序結果分析………………………………………………………….. 16 (七)南方雜合分析………………………………………………………….. 16 (八)專一性引子之設計與PCR測試………………………………………. 17 (1)不同黏合溫度測試………………………………………………….. 18 (2)不同Mg2+濃度測試…………………………………………………. 18 (3)專一性PCR反應……………………………………………………. 18 (4)靈敏度測試………………………………………………………….. 19 (5)在不同濃度香蕉DNA中偵測定量香蕉黃葉病菌DNA之測試….. 19 (6)在定量香蕉DNA中偵測不同濃度香蕉黃葉病菌DNA之測試….. 19 (7)香蕉黃葉病菌race 1菌株之測試…………………………………... 20 (九)應用PCR技術增幅nrDNA的ITS以分析Fusarium spp…………… 21 五、結果………………………………………………………………………… 22 六、討論………………………………………………………………………… 29 七、引用文獻…………………………………………………………………… 36 八、圖表………………………………………………………………………… 46 九、附錄………………………………………………………………………… 6

縣(市)風景區旅客行為之研究-以高雄縣級風景區為例

出處：教育

PRIMER, METHOD AND KIT FOR DETECTING FUSARIUM WLIT PATHOGEN(FUSARIUM OXYSPORUM F. SP. CUBENSE) OF BANANA

本發明為提供一種用於偵測香蕉黃葉病菌生理小種第四型(Fusarium oxysporum f. sp. cubense race 4)之寡核苷酸序列，及利用其用於診斷鑑定香蕉黃葉病菌之方法與套組

PRIMER, METHOD AND KIT FOR DETECTING FUSARIUM WILT PATHOGEN (FUSARIUM OXYSPORUM F. SP. NIVEUM) OF WATERMELON

本發明為提供一種用於偵測西瓜蔓割病菌(Fusarium oxysporum f. sp. niveum)之寡核苷酸序列，及利用其用於診斷鑑定西瓜蔓割病菌之方法與套組