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

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

Development of Molecular Tools for Differentiating Races in Fusarium Oxysporum F. Sp. Cubense, the Causing Agent of Banana Wilt Disease (II)

尖鐮孢菌為造成許多重要經濟作物萎凋之病原。其中由Fusarium oxysporum f. sp. cubense引起之香蕉黃葉病為全世界香蕉栽培的主要限制因子，不僅在台灣肆虐蔓延，導致台灣香蕉嚴重減產，也在巴拿馬及中美洲各地造成嚴重的經濟損失。由於尖鐮孢菌可形成厚膜孢子殘存於土壤或植物殘株，病原菌一旦經由土壤、種苗、農機具與車輛、灌溉水或人員傳入處女地則無法根除，防治不易。因此開發快速檢測技術，有效制止病原菌的傳播，實為當務之急。本年度將自台灣主要香蕉產區蒐集香蕉黃葉病菌菌株，並進行病原性測試；同時也將蒐集感染其他作物之鐮孢菌屬菌株；另一方面藉美國農部分子植物病理學專家—翁溥博士之協助購買香蕉黃葉病菌各生理小種之標準菌株，並以純培養方式維持其生長以供後續DNA萃取及PCR分析用，並將利用修正過的微波爐法以快速萃取少量菌絲之DNA，並將此技術應用於香蕉黃葉病菌之檢測上。此外，也將利用「增幅片段長度多型性」（AFLP）及「隨機增幅多型性核酸」（RAPD）等分析來篩選具生理小種專一性之核酸指紋標記（DNA marker），並由美國農部翁溥博士協助DNA指紋圖譜（fingerprinting）之分析。本計畫之最終成果為開發一套PCR檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌不同生理小種之鑑別工作，未來更可進一步應用於偵測土壤及植物組織中的病原菌，期望對於香蕉黃葉病之防治工作有所助益。以此發展技術為平台，也可應用於開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定及檢測技術。Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarial wilt of banana, commonly known as Panama disease of banana, became epidemic in Panama as early as 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. The goal of this research is to develop a rapid, simple, and reliable identification method for the fungal pathogen Fusarium oxysporum f. sp. cubense (FOC). The developed PCR methods could be applied to other Races of FOC in addition to Foc Race 4 of Taiwan origin or all FOC races. FOC isolates and other formae speciales of F. oxysporum (affecting crops other than bananas) and other Fusarium spp. will be collected from different geographic locations in Taiwan. One of the principal investigators (PI), Dr. C. P. Chao, will provide FOC isolates in his collections and do pathogenicity test with banana plantlets. The other PI, Dr. P.-F. L. Chang, in addition to collect FOC isolates and do pathogenicity test with banana plantlets, has developed a microwave method for rapid genomic DNA extraction from fungal mycelia and establish a specific polymerase chain reaction (PCR) method for rapid FOC identification. This technique will be used for detecting pathogen in plant diseased tissues and soils in the future. In addition, the PIs will collaborate with Dr. Peter P. Ueng of USDA-ARS, Beltsville, Maryland, USA to obtain the available type cultures of all FOC races (Races 1, 2, and 4) in US. In the future, the rapid identification technique will be further developed to apply for the detection of F. oxysporum f. sp. cubense pathogen in soils and plant tissues. This will help to control the world-wide important wilt disease of banana. The developed platform technology will be further used for molecular identification and detection of the Fusarium wilt pathogens of other economically important crops

開發香蕉黃葉病菌不同生理小種之分子鑑定技術(II)

Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarial wilt of banana, commonly known as Panama disease of banana, became epidemic in Panama as early as 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. The goal of this research is to develop a rapid, simple, and reliable identification method for the fungal pathogen Fusarium oxysporum f. sp. cubense (FOC). The developed PCR methods could be applied to other Races of FOC in addition to Foc Race 4 of Taiwan origin or all FOC races. FOC isolates and other formae speciales of F. oxysporum (affecting crops other than bananas) and other Fusarium spp. will be collected from different geographic locations in Taiwan. One of the principal investigators (PI), Dr. C. P. Chao, will provide FOC isolates in his collections and do pathogenicity test with banana plantlets. The other PI, Dr. P.-F. L. Chang, in addition to collect FOC isolates and do pathogenicity test with banana plantlets, has developed a microwave method for rapid genomic DNA extraction from fungal mycelia and establish a specific polymerase chain reaction (PCR) method for rapid FOC identification. This technique will be used for detecting pathogen in plant diseased tissues and soils in the future. In addition, the PIs will collaborate with Dr. Peter P. Ueng of USDA-ARS, Beltsville, Maryland, USA to obtain the available type cultures of all FOC races (Races 1, 2, and 4) in US. In the future, the rapid identification technique will be further developed to apply for the detection of F. oxysporum f. sp. cubense pathogen in soils and plant tissues. This will help to control the world-wide important wilt disease of banana. The developed platform technology will be further used for molecular identification and detection of the Fusarium wilt pathogens of other economically important crops.尖鐮孢菌為造成許多重要經濟作物萎凋之病原。其中由Fusarium oxysporum f. sp. cubense引起之香蕉黃葉病為全世界香蕉栽培的主要限制因子，不僅在台灣肆虐蔓延，導致台灣香蕉嚴重減產，也在巴拿馬及中美洲各地造成嚴重的經濟損失。由於尖鐮孢菌可形成厚膜孢子殘存於土壤或植物殘株，病原菌一旦經由土壤、種苗、農機具與車輛、灌溉水或人員傳入處女地則無法根除，防治不易。因此開發快速檢測技術，有效制止病原菌的傳播，實為當務之急。本年度將自台灣主要香蕉產區蒐集香蕉黃葉病菌菌株，並進行病原性測試；同時也將蒐集感染其他作物之鐮孢菌屬菌株；另一方面藉美國農部分子植物病理學專家—翁溥博士之協助購買香蕉黃葉病菌各生理小種之標準菌株，並以純培養方式維持其生長以供後續DNA萃取及PCR分析用，並將利用修正過的微波爐法以快速萃取少量菌絲之DNA，並將此技術應用於香蕉黃葉病菌之檢測上。此外，也將利用「增幅片段長度多型性」（AFLP）及「隨機增幅多型性核酸」（RAPD）等分析來篩選具生理小種專一性之核酸指紋標記（DNA marker），並由美國農部翁溥博士協助DNA指紋圖譜（fingerprinting）之分析。本計畫之最終成果為開發一套PCR檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌不同生理小種之鑑別工作，未來更可進一步應用於偵測土壤及植物組織中的病原菌，期望對於香蕉黃葉病之防治工作有所助益。以此發展技術為平台，也可應用於開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定及檢測技術

Development of Molecular Tools for Differentiating Races in Fusarium oxysporum F.sp. cubense, the Causing Agent of Banana Wilt Disease

尖鐮孢菌為造成許多重要經濟作物萎凋之病原。其中由Fusarium oxysporum f. sp. cubense引起之香蕉黃葉病為全世界香蕉栽培的主要限制因子，不僅在台灣肆虐蔓延，導致台灣香蕉嚴重減產，也在巴拿馬及中美洲各地造成嚴重的經濟損失。由於尖鐮孢菌可形成厚膜孢子殘存於土壤或植物殘株，病原菌一旦經由土壤、種苗、農機具與車輛、灌溉水或人員傳入處女地則無法根除，防治不易。因此開發快速檢測技術，有效制止病原菌的傳播，實為當務之急。本年度將自台灣主要香蕉產區蒐集香蕉黃葉病菌菌株，並進行病原性測試；同時也將蒐集感染其他作物之鐮孢菌屬菌株；另一方面藉美國農部分子植物病理學專家—翁溥博士之協助購買香蕉黃葉病菌各生理小種之標準菌株，並以純培養方式維持其生長以供後續DNA萃取及PCR分析用，擬將利用修正過的微波爐法快速萃取少量菌絲之DNA。接下來測試所開發之PCR偵測技術是否可適用於香蕉黃葉病菌各生理小種，或僅只適用於台灣所分離之Race 4；此外，也將利用「增幅片段長度多型性」(AFLP)及「隨機增幅多型性核酸」(RAPD)等分析來篩選具生理小種專一性之核酸指紋標記(DNA marker)，並由美國農部翁溥博士協助DNA指紋圖譜(fingerprinting)之分析。本計畫之最終成果為開發一套PCR檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌不同生理小種之鑑別工作，未來更可進一步應用於偵測土壤及植物組織中的病原菌，期望對於香蕉黃葉病之防治工作有所助益。以此發展技術為平台，也可應用於開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定及檢測技術。Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarial wilt of banana, commonly known as Panama disease of banana, became epidemic in Panama as early as 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. The goal of this research is to develop a rapid, simple, and reliable identification method for the fungal pathogen Fusarium oxysporum f. sp. cubense (FOC). We would determine if the developed PCR method could be applied to either only FOC Race 4 of Taiwan origin or all FOC races. FOC isolates and other formae speciales of F. oxysporum (affecting crops other than bananas) and other Fusarium spp. will be collected from different geographic locations in Taiwan. One of the principal investigators (PI), Dr. C. P. Chao, will provide FOC isolates in his collections and do pathogenicity test with banana plantlets. The other PI, Dr. P.-F. L. Chang, in addition to collect FOC isolates and do pathogenicity test with banana plantlets, will develop a microwave method for rapid genomic DNA extraction from fungal mycelia and establish a specific polymerase chain reaction (PCR) method for rapid FOC identification. This technique will be used for detecting pathogen in plant diseased tissues and soils in the future. In addition, the PIs will collaborate with Dr. Peter P. Ueng of USDA-ARS, Beltsville, Maryland, USA to obtain the available type cultures of all FOC races (Races 1, 2, and 4) in US. In the future, the rapid identification technique will be further developed to apply for the detection of F. oxysporum f. sp. cubense pathogen in soils and plant tissues. This will help to control the world-wide important wilt disease of banana. The developed platform technology will be further used for molecular identification and detection of the Fusarium wilt pathogens of other economically important crops

Analysis of Fusarium oxysporum f. sp. cubense Using Random

中 文 摘 要 香蕉黃葉病 (Fusarium wlit of banana) 係由 Fusarium oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen 所引起，因於1890年在巴拿馬大發生，又稱巴拿馬病 (Panama disease) 。此病害分布極廣，而且目前除了栽培抗病品種外，並無其他有效防治方法，對世界各地的香蕉產量造成嚴重的威脅，台灣自1967年屏東縣出現疑似罹患巴拿馬病的病株之後，此病即嚴重摧毀台灣的香蕉產業。由於香蕉乃利用無性繁殖之種苗栽培，使用不帶病原菌之健康種苗為防治此土壤傳播性病害方法之一，因此建立快速篩選無病原菌香蕉苗之偵測法，為香蕉健康種苗驗證所必須。本研究利用「隨機增幅核酸多型性分析」 (random amplified polymorphic DNA, RAPD) 開發香蕉黃葉病菌之快速偵測技術，並設計專一性引子對供聚合酵素連鎖反應 (polymerase chain reaction, PCR) 檢測之用，以達到快速並準確偵測病原菌之目的。目前已自全省各地收集不同寄主的 F. oxysporum 菌株計十種15個分離株及香蕉黃葉病菌株15個分離株，經大量培養後抽取其菌絲 DNA ，並以健康香蕉組織 DNA、一種其他 Fusarium sp. 及三種非 Fusarium 屬之病原菌 DNA作為對照。利用 OPA、OPAT 及 OPAW 引子組 (Operon Technologies, Inc., Alameda, CA) 和文獻中分析香蕉黃葉病菌所使用之44個引子，針對上述各種 DNA 進行 RAPD 分析。結果顯示以 OPA-02 (5’-TGCCGAGCTG-3’) 引子進行 RAPD 增幅測試，可在香蕉黃葉病菌株 DNA 中增幅出約 400 bp 之專一性片段，經純化回收此片段作為探針，並將上述 RAPD 結果進行南方雜合分析，可知在香蕉菌株之增幅樣品中，能辨識 400 bp 專一性的條帶，此 400 bp 片段經解序分析已知和目前 GenBank 中已發表的核酸序列並無相似性，利用此序列設計二組正向及反向之專一性引子對 OPA02400A1 (5’-CAGGGGATGTATGAGGAGGCT-3’)、OPA02400A2 (5’-CGGTACTTGCTGTGCGGGGA-3’)、OPA02400S1 (5’-CAGCTATGACAAGAACACCAGA-3’) 與 OPA02400S2 (5’-GTGACAGCGTCGTCTAGTTCC -3’)，利用 OPA02400A1/OPA02400S2 及 OPA02400A2/OPA02400S2 二種組合於 PCR 反應中，在合適的PCR條件下皆可分別增幅出特定之專一性片段，並可由南方雜合分析確定其專一性。英 文 摘 要 ABSTRACT Fusarium wilt of banana, the so- called Panama disease, caused by Fusarium oxysporum Schlechtend.:Fr. f. sp. cubense (E.F. Smith) W.C. Snyder & H.N. Hansen (Foc). In 1890, the outbreak of this disease in Panama almost completely destroyed the export trade of banana. Foc is distribute worldwide and is difficult to control in agricultural practice except planting wilt-resistant banana hybrids. Panama disease is a serious threat to banana production in the whole world. In 1967, the suspected Fusarium-wilted of banana was discovered in Pingtung, Taiwam, and this disease seriously destroyed the banana export industry there after in Taiwan. Since banana is propagated by asexual plantlets, in order to control the soilborne Panama disease, it is necessary to use health banana plantlets for production. Hence, the development of a fast detect Foc in banana plantlet is essential for rapid screening of health banana plantlets. In this study, random amplified polymorphic DNA (RAPD) analysis was first used to develop a rapid detection method for Foc. Next, specific primers for polymerase chain reaction (PCR) was designed for quick and accurate detection of Foc pathogen. The individually purified DNA from mycelia of fifteen isolates of Foc, fifteen F. oxysporum (Fo) isolates from ten species of different hosts and one other Fusarium sp. was used for RAPD and PCR analyses. The DNA sample from healthy banana tissue and three kinds of non-Fusarium pathogens pathogens were also used as control. Forty-four 8 to 15 nt of short random primers were used for analysis of the aboved DNA samples. A specific 400-bp fragment was amplified from DNA of 14 Foc and another Fo (Fo-L1) isolated from anoectochilus using OPA-02 (5’-TGCCGAGCTG-3’) primer. This 400-bp fragment (OPA02400) obtained from Foc samples was cloned and used as probe for Southern blot analysis. The OPA02400 probe could identify the specific 400-bp fragment amplified from the DNA samples of forteen Foc and Fo-L1. The sequence of OPA02400 showed no similarity to any sequence deposited in GenBank. The OPA02400 sequence was used to design two forward and reverse specific primers: OPA02400A1 (5’-CAGGGGATGTATGAGGAGGCT-3’)、OPA02400A2 (5’-CGGTACTTGCTGTGCGGGGA-3’)、OPA02400S1 (5’-CAGCTATGACAAGAACACCAGA-3’) and OPA02400S2 (5’-GTGACAGCGTCGTCTAGTTCC -3’). The results suggested that the OPA02400A1/OPA02400S2 and OPA02400A2/OPA02400S2 primer pairs could amplify specific fragments only in the 15 Foc and Fo-L1 DNA samples under proper PCR conditions, and the specificity was further confirmed by Southern blot analysis.目 錄 壹、緒論 2 貳、材料與方法 8 供試菌株的來源與培養 8 各菌株在不同培養基上的菌落型態比較 8 菌絲總量DNA之萃取 9 RAPD 分析 10 核酸探針之製備 10 設計專一性引子及其 PCR 分析 11 南方雜合分析 11 專一性 PCR 最佳條件之測試 11 參、結果 15 香蕉黃葉病菌在不同培養基上的菌落型態比較 15 RAPD 分析 16 RAPD 之南方雜合分析 18 專一性片段 OPA02400 的序列及專一性引子對的設計 18 專一性 PCR 之結果 19 PCR 之最佳條件 19 肆、討論 21 伍、參考文獻 26 陸、中文摘要 31 柒、英文摘要 33 捌、圖表說明 35 玖、附錄 7

開發香蕉黃葉病菌不同生理小種之分子鑑定技術(III)

尖鐮孢菌為造成許多重要經濟作物萎凋之病原。其中由Fusarium oxysporum f. sp. cubense引起之香蕉黃葉病為全世界香蕉栽培的主要限制因子，不僅在台灣肆虐蔓延，導致台灣香蕉嚴重減產，也在巴拿馬及中美洲各地造成嚴重的經濟損失。由於尖鐮孢菌可形成厚膜孢子殘存於土壤或植物殘株，病原菌一旦經由土壤、種苗、農機具與車輛、灌溉水或人員傳入處女地則無法根除，防治不易。因此開發快速檢測技術，有效制止病原菌的傳播，實為當務之急。本年度將自台灣主要香蕉產區蒐集香蕉黃葉病菌菌株，並進行病原性測試；同時也將蒐集感染其他作物之鐮孢菌屬菌株；另一方面藉美國農部分子植物病理學專家—翁溥博士之協助購買香蕉黃葉病菌各生理小種之標準菌株，並以純培養方式維持其生長以供後續DNA萃取及PCR分析用。本計畫之成果為開發一套PCR檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌不同生理小種之鑑別工作，並可應用於田間帶菌植株或種苗的檢測，未來更可進一步應用於偵測土壤中的病原菌，期望對於香蕉黃葉病之防治工作有所助益。以此發展技術為平台，也可應用於開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定及檢測技。Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarial wilt of banana, commonly known as Panama disease of banana, became epidemic in Panama as early as 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. The goal of this research is to develop a rapid, simple, and reliable identification method for the fungal pathogen Fusarium oxysporum f. sp. cubense (FOC). The developed PCR methods could be applied to other races of FOC in addition to Foc race 4 of Taiwan origin or all FOC races. FOC isolates and other formae speciales of F. oxysporum (affecting crops other than bananas) and other Fusarium spp. will be collected from different geographic locations in Taiwan. One of the principal investigators (PI), Dr. C. P. Chao, will provide FOC isolates in his collections and do pathogenicity test with banana plantlets. The other PI, Dr. P.-F. L. Chang, in addition to collect FOC isolates, will do pathogenicity test with banana plantlets, and establish a specific polymerase chain reaction (PCR) method for rapid FOC identification. In this project, the technique developed for detecting pathogen in plant diseased tissues, will be applied for tagging infected soils in the future. In addition, the PIs will collaborate with Dr. Peter P. Ueng of USDA-ARS, Beltsville, Maryland, USA to obtain the available type cultures of all FOC races (races 1, 2, and 4) in US. In the future, the rapid identification technique will be further developed to apply for the detection of F. oxysporum f. sp. cubense pathogen in soils. This will help to control the world-wide important wilt disease of banana. The developed platform technology will be further used for molecular identification and detection of the Fusarium wilt pathogens of other economically important crops

Development of Molecular Tools for Differentiating Races in Fusarium oxysporum f. sp. cubense, the Causing Agent of Banana Wilt Disease (III)

尖鐮孢菌為造成許多重要經濟作物萎凋之病原。其中由Fusarium oxysporum f. sp. cubense引起之香蕉黃葉病為全世界香蕉栽培的主要限制因子，不僅在台灣肆虐蔓延，導致台灣香蕉嚴重減產，也在巴拿馬及中美洲各地造成嚴重的經濟損失。由於尖鐮孢菌可形成厚膜孢子殘存於土壤或植物殘株，病原菌一旦經由土壤、種苗、農機具與車輛、灌溉水或人員傳入處女地則無法根除，防治不易。因此開發快速檢測技術，有效制止病原菌的傳播，實為當務之急。本年度將自台灣主要香蕉產區蒐集香蕉黃葉病菌菌株，並進行病原性測試；同時也將蒐集感染其他作物之鐮孢菌屬菌株；另一方面藉美國農部分子植物病理學專家—翁溥博士之協助購買香蕉黃葉病菌各生理小種之標準菌株，並以純培養方式維持其生長以供後續DNA萃取及PCR分析用。本計畫之成果為開發一套PCR檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌不同生理小種之鑑別工作，並可應用於田間帶菌植株或種苗的檢測，未來更可進一步應用於偵測土壤中的病原菌，期望對於香蕉黃葉病之防治工作有所助益。以此發展技術為平台，也可應用於開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定及檢測技。Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarial wilt of banana, commonly known as Panama disease of banana, became epidemic in Panama as early as 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. The goal of this research is to develop a rapid, simple, and reliable identification method for the fungal pathogen Fusarium oxysporum f. sp. cubense (FOC). The developed PCR methods could be applied to other races of FOC in addition to Foc race 4 of Taiwan origin or all FOC races. FOC isolates and other formae speciales of F. oxysporum (affecting crops other than bananas) and other Fusarium spp. will be collected from different geographic locations in Taiwan. One of the principal investigators (PI), Dr. C. P. Chao, will provide FOC isolates in his collections and do pathogenicity test with banana plantlets. The other PI, Dr. P.-F. L. Chang, in addition to collect FOC isolates, will do pathogenicity test with banana plantlets, and establish a specific polymerase chain reaction (PCR) method for rapid FOC identification. In this project, the technique developed for detecting pathogen in plant diseased tissues, will be applied for tagging infected soils in the future. In addition, the PIs will collaborate with Dr. Peter P. Ueng of USDA-ARS, Beltsville, Maryland, USA to obtain the available type cultures of all FOC races (races 1, 2, and 4) in US. In the future, the rapid identification technique will be further developed to apply for the detection of F. oxysporum f. sp. cubense pathogen in soils. This will help to control the world-wide important wilt disease of banana. The developed platform technology will be further used for molecular identification and detection of the Fusarium wilt pathogens of other economically important crops