Genome Analysis of Anthracnose-Resistance Using Hot Pepper as the Model Host (II)

目前台灣地區之作物栽培受到炭疽病（Anthracnose）危害嚴重，炭疽病之常見病原菌有Colletotrichum acutatum、Co. gloeosporioides及Co. capsici等。而辣椒（Capsicum annuum）為國內主要香辛作物之一，炭疽病更是辣椒著果期的重要病害之一，對果實損害極劇，嚴重影響其經濟價值。至今尚無經濟、有效的方法來防除上述病害，也無抗病的商業品種可供利用，故若能培育出同時抗多種病害之辣椒品種（系），將是最有效且治本的防治措施。因此本研究計畫目的在於研究辣椒中之抗炭疽病相關基因表現情形與寄主辣椒本身抗病機制之關連性，並選殖辣椒的抗病相關基因，以便將來研究該些防禦基因之功能，以作為後續抗病機制研究、抗病育種篩選標的之參考，甚至可以利用遺傳工程技術開發出可持續表現此基因且對病原菌具有抗性之轉基因辣椒。本研究以世界蔬菜中心的抗病辣椒種原（Ca. chinense AVRDC PBC932）果實，利用同一種但具不同強弱毒力的炭疽病菌株 [同為Co. acutatum，編號各為Coll-365（弱毒力）、Coll-153（中毒力）及Coll-524（強毒力）] 進行微注射（microinjection）接種，並收取接種後一至三天之辣椒果實，以抽取總量RNA。再以「差異展示-反轉錄-聚合酵素連鎖反應（differential display-reverse transcript-polymerase chain reaction, DD-RT-PCR）」與「互補DNA-增幅片段長度多型性（cDNA-amplified fragment length polymorphism, cDNA-AFLP）」技術篩選出可能參與抗病反應之辣椒基因。本研究成果對茄科作物之抗病品種開發，或抗病機制之研究將有助益，並期能在植物防疫及農業生產方面有所貢獻。Anthracnose, caused by Colletotrichum acutatum, Co. gloeosporioides, and Co. capsici, is one of the major pepper (Capsicum annuum) fruit diseases. So far, no effective measures could be applied for disease control. Breeding for anthracnose-resistant hot pepper cultivars could be the best strategy for disease management. In this study, we try to isolate genes involved in resistance to Co. acutatum. The World Vegetable Center (the former Asian Vegetable Research and Development Center) had isolated some high virulent (i.e. Coll-524), moderate virulent (i.e. Coll-153), and low virulent isolates (i.e. Coll-365) of Co. acutatum. These fungal isolates will be used to inoculate the resistant pepper fruits (AVRDC PBC932, Ca. chinense) by microinjection to study the factors involved in pepper anthracnose resistance. The genes involved in anthracnose resistance will be screened by using differential display-reverse transcript-polymerase chain reaction (DD-RT-PCR) and cDNA-amplified fragment length polymorphism (cDNA-AFLP) techniques. These genes will be cloned and analyzed for their functions, involvement in anthracnose resistance, and be further used for breeding program as screening markers, genetic materials, or even as targets for transgenic pepper or Solanaceous crops

Developing A Molecular Identification Method of Fusarium Oxysporum F. sp. Lactucae

台灣栽植萵苣自1996年在雲林縣西螺鎮萵苣栽培田中分離到對萵苣具有專一性的萵苣萎凋病菌(Fusarium oxysporum f.sp. lactucae，簡稱 Fola)後，目前萵苣萎凋病已成為台灣夏季萵苣生產的主要限制因子之一。為能有效制止病原菌的傳播，本計畫之目標在開發能快速檢驗萵苣萎凋病菌的分子鑑定核心技術。本年度預計自台灣各地大量收集菌株，以DNA指紋法篩選出更多Fola核酸中所特有的片段，嘗試依此特異性核酸片段架構Fola之分子鑑定系統，達到快速測定病原菌之目的，並確認此檢測系統是否對特定生理小種的Fola具有專一性，同時對國外已發表之萵苣萎凋病菌分子標誌進行最佳化條件測試，增加檢測系統的專一性與靈敏度，此些PCR檢測法最佳化後，未來將應用於植物樣品檢測，以監測萵苣種子是否帶菌，及診斷植株是否遭受萵苣萎凋病菌侵染，未來並將對帶菌土壤樣本進行先期條件測試分析。這些研究成果將可作為田間健康管理之依據，祈能有效制止病原菌的傳播，使之不再成為台灣萵苣夏季生產的限制因子，以突破極具內、外銷市場之萵苣受到萎凋病影響之瓶頸。The wilt disease of lettuce was first found in Taiwan in 1996. This disease, caused by Fusarium oxysporum f.sp. lactucae (Fola), has become one of the limiting factors for lettuce production during summer season in Taiwan. The goal of this research project is to develop a rapid identification method for Fola to limit the spray of this wilt pathogens. The rapid detection technique is based on DNA fingerpring technique to screen Fola specific markers, and then to design the PCR primers for amplification of DNA fragments specific to Fola pathogen or even specific to certain race of Fola. In addition, we also will optimize the published methods for increasing sensitivity and specificity. The developed detection technique will be applied to detect Fola in lettuce seeds, lettuce plants, or even cultivation soil in the future. This will help to monitor the disease incidence of Fusarium wilt of lettuce and to control this wilt disease

Survey of field distribution of Fusarium oxysporum f. sp. cubense in Taiwan using molecular detection technique

尖鐮孢菌為造成萎凋之病原，可危害許多重要經濟作物。其中由 Fusarium oxysporum f. sp. cubense 引起之香蕉黃葉病乃世界香蕉栽培的主要限制因子之一，不僅在巴拿馬及中美洲各地造成嚴重的經濟損失，並在台灣肆虐蔓延導致台灣香蕉嚴重減產。快速且良善的檢測系統，可為作物病害管理的基石。計畫主持人已開發出一套快速檢測流程，能快速、簡便且可靠的進行香蕉黃葉病菌生理小種第四型之鑑別工作，經評估後可應用於田間病原菌之檢測，本計畫擬將此快速檢測技術應用於田間實務上，藉此自疑似帶菌的香蕉組織與田區土樣中快速檢測出是否為攜帶該病原菌之檢體 ，同時對全台香蕉黃葉病菌之生理小種分布作全盤的調查，祈能對香蕉黃葉病之防治工作有所助益。此技術平台之發展經驗，也將可作為開發其他重要經濟作物之鐮孢萎凋病菌的分子鑑定與檢測技術之重要參考依據。Fusarium oxysporum is one of the most important pathogens which cause wilt in many economically important crops. Fusarium wilt of banana, commonly known as Panama disease of banana, has become epidemic in Panama since 1890. The Panama disease of banana caused by F. oxysporum f. sp. cubense (Foc) (E. F. Smith) Snyder & Hansen is a potentially devastating disease throughout the world and the major limiting factor for banana production. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. We have develpoed an optimized PCR assay, the developed molecular method was rapid, simple, and sensitive, and could identify Foc race 4 specifically. We also demonstrated that Foc race 4 in naturally infected banana tissues could be easily tagged by using our PCR assay. In this research, the rapid identification technique will be applied for the detection of Foc pathogen in the field samples of infected plants and soils. This detection technique will also be used to analyze the distribution of the Foc races in Taiwan. This will help to control the world-wide important wilt disease of banana. The developed technology platform will be further used for molecular identification and detection of the Fusarium wilt pathogens infecting other economically important crops

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

[[alternative]]Application of the Transtheoretical Model to exercise behavior among junior college female students

[[abstract]]The main purpose of this study is to investigate the stages of exercise behavior change and their relationship among junior college female students through application the transtheoretical model (TTM). The subjects of this survey were selected from junior female students in the Hsing Wu College. By using a stratified cluster sampling method, 852 respondents（90. 2％ response rate）was selected to participate in the survey. The data were collected with a self-administered questionnaires. Chi-square test, t-test, one-way ANOVA, and one-way MANOVA were used to analyze the data. The results of this research were as follows: 1. In stages of exercise behavior change, 25.6% of the subjects are in Precontemplation, 20% in Contemplation, 45.7% in Preparation, 4.5% in Action, and 4.3% in Maintenance. 2. Grade, residence, participated in exercise team, perceived health status were significantly related to stages of exercise behavior change. 3. In perceived benefits of exercise, there are significant differences among subjects of the five stages of exercise behavior change, Precontemplation was the lowest score. 4. In perceived barriers of exercise, there are significant differences among subjects of the five stages of exercise behavior change, namely, Precontemplation > Contemplation > Preparation> Action/ Maintenance. 5. In self-efficacy for exercise, there are significant differences among subjects of the five stages of exercise behavior change, that is, Precontemplation < Contemplation < Preparation < Action / Maintenance. 6. The variables of 10 strategies of processes of change were significantly related to the stages of exercise behavior change. Those in the Precontemplation group use each process of change significantly less than those in other stages, either in experiential processes or in behavioral processes. 7. All in all, the 13 variables of TTM structure, including perceived benefits of exercise, perceived barriers of exercise, self-efficacy for exercise, and 10 strategies of processes of change, made significant differences to distinguish stage from stage. According to the finding of this study could present as references for school health practice to promotion the exercise behavior among junior female students and further research in this field.

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 (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