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(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

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 (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