重要植物有害生物診斷鑑定技術研發及綜合管理

1. 茶樹神澤氏葉蟎生物防治天敵效能評估：台灣的包種茶及烏龍茶品質極佳，茶葉除內銷外，外銷方面以日本、美國、香港及中國大陸為主，外銷市場潛力豐厚。拓展外銷市場，亦有助於國內市場價格的提高及穩定。 出口茶葉推展的重要議題，為農藥殘留問題，尤其近年屢次發生新殺蟎殘留量過高，遭退貨的案例。因此發展安全、無毒的生產技術益發重要。本生物防治栽培管理方法將能生產無農藥、高品質及產量的茶葉，此為世界各國目前作物生產的潮流。建立田間觀測用族群密度調查法，省時、省力又可較準確估計葉蟎族群密度的取樣方法，並監測葉蟎族群終年發生頻度，可有效擬訂施用藥劑與生物防治的適當時機。大量飼育溫氏捕植蟎以提供釋放，充分供應試驗茶園捕植蟎之需求，以防治葉蟎危害。捕植蟎施放比率測試，有助於提供今後生物防治茶樹神澤氏葉蟎技術參考，往昔用藥與產量品質品質調查、成本分析與效果評估，可幫助落實非農藥防治及生物技術之利用。釋放溫氏捕植蟎，配合整體茶葉栽培管理技術，提升品質與產量，增進農民經濟收益，促進產品外銷及提昇我國農產品競爭力，祈開創茶葉市場之新藍海。 2. 葉牡丹黑腐病菌之鑑定與致病性：本計畫之目標為自葉牡丹的黑腐病株中分離並鑑定出黑腐菌株，亦將嘗試自葉牡丹種子中分離黑腐菌株，並將這些分離株與本省分離自甘藍的黑腐菌株相比較。我們將以接種試驗比較這些菌株對二種十字花科蔬菜(甘藍與小白菜)，三種十字花科花卉(葉牡丹、香雪球及紫羅蘭)，及一種十字花科模式植物-阿拉伯芥的致病性；亦將以脈衝式電泳比較這些菌株的基因型。另外，我們先前曾自一株分離自甘藍的黑腐菌株Xc17中，分離到orfF'致病基因及OrfF'致病蛋白，並構築出orfF'缺失的Xc17無致病株，稱為Xc17#34。我們將以PCR及西方雜配檢查分離自葉牡丹與甘藍的黑腐菌株是否皆具有orfF'致病基因及OrfF'致病蛋白，並將測試Xc17與Xc17#34對二種十字花科蔬菜(甘藍與小白菜)，三種十字花科花卉(葉牡丹、香雪球及紫羅蘭)，及一種十字花科模式植物-阿拉伯芥的致病性。本計畫之預期效益為自葉牡丹分離並鑑定出黑腐菌株；得知分離自葉牡丹與甘藍的黑腐菌株的基因型、orfF'致病基因、及OrfF'致病蛋白是否有差異；及獲得這些菌株、Xc17及Xc17#34菌株對二種十字花科蔬菜、三種十字花科花卉、一種十字花科雜草模式植物(阿拉伯芥)的致病性。 3. 十字花科露菌病菌菌相調查及綜合防治管理之探討：十字花科露菌病嚴重影響台灣全省各地十字花科蔬菜產量與品質，然卻極少有關此病原菌台灣本土性菌株菌相及藥劑特性之相關研究，也尚無推薦之生物製劑以供選擇利用。本研究擬進行台灣本土性菌株收集及rDNA ITS區間序列分析，以了解台灣菌株與國外菌株在遺傳特性之相似或相異度。另針對推薦使用，卻具抗藥性風險之苯醯胺類，如：滅達樂與史托比系列藥劑，如：亞托敏，及非專一性多點作用之藥劑，如：鋅錳乃浦及錳乃浦等藥劑，評估台灣菌株對此些藥劑之抗感藥性。研究中亦擬測試對卵菌綱病原菌Phytophothora spp.具防治潛力之鏈黴菌生物製劑，在防治十字花科露菌病菌之應用性。此些資訊之建立，將提供擬定十字花科露菌綜合防治策略之依據。 4. 農藥管理法子法規相關資料之蒐集研析：本計畫主旨在以團隊力量蒐集國際間與農藥應用有關之法規資訊，以協助甫完成公告「農藥管理法」相關系列子法規之修訂與增訂。本計畫中主要工作將邀集國內農藥應用管理學有專精之專家學者組織諮詢團隊，提供防檢局同仁修法工作進行中必要資訊與協助。另一工作重點則將致力於彙集與生物農藥應用推動攸關的合理化管理系統之建立所需資訊，期有助於生物農藥產業化應用之落實。 5. 應用水楊酸及甲殼素提高蝴蝶蘭種苗對抗蕙蘭屬崁紋病毒(CYMMV)之抗性：本計畫之年度目標在於了解可以保護蝴蝶蘭及朵麗蝶蘭出瓶苗免於感染CymMV感染之SA及CHT之處理濃度，以及噴施的頻率。 6. 櫻屬外來種簇葉病之調查與評估：櫻花簇葉病其主要病因為來自真菌中的子囊菌(Taphrina wiesneri)引起，此病原是透過孢子和雨水傳播，目前台灣並無此疫情發生，也並無此病原菌發生的紀錄。本計畫擬調查防檢局檢疫站及隔離栽培區的櫻花，抽樣檢驗是否已有簇葉病的發生，若有疑似發生狀況時，除先通知防檢局相關單位外，並攜回實驗室進行分離檢驗，已確定是否為T. wiesneri所造成的簇葉病。最後並評估若有此情況發生時，對台灣農業影響的情況評估。 7. 蘋果蠹蛾PCR檢測試劑套組之開發與改良：本研究主要目的在開發可供快速鑑定蘋果蠹蛾(Cydia pomonella)之PCR反應試劑套組。此套組係根據本研究室近五年來使用於檢測蘋果蠹蛾的PCR方法加以測試與改良。除了DNA模版與taq聚合酶之外，本試劑套組擬將其他所有PCR反應所需試劑(包括專一性引子)預先混合，使用者僅需依所提供的操作手冊逐步加入適量的檢體DNA與taq聚合酶，之後再進行PCR反應即可得到檢測結果。我們預期將開發出具有高敏感度與準確性且方便使用之檢測試劑套組供如防檢疫人員等需要鑑定蘋果蠹蛾者使用。同時，我們亦擬將此試劑套組申請專利與技術轉移給適當廠商。 8. 利用恆溫式圈環形核酸增幅法(LAMP)快速鑑定地中海果實蠅(Ceratitis capitata)：地中海果實蠅(Ceratitis capitata (Wiedemann))是世界上危害最嚴重的果實蠅種類之一，亦被列為台灣重要的檢疫害蟲。除成蟲外，其它齡期皆不易由形態加以鑑定，目前可用PCR、PCR-RFLP、RAPD、生物晶片等方法來鑑定，此類方法皆基於PCR，故需不同的溫度周期反應，花費時間較長，仍有發展更快速更方便檢測方法的空間。LAMP (loop-mediated isothermal amplification method)是利用特殊設計的引子對，在單一的溫度(65℃)下以特別的聚合酶反應，快速增幅DNA。本研究擬尋找適合LAMP反應的快速抽取果實蠅DNA方法及適合鑑定地中海果實蠅的LAMP引子、條件，快速及精確檢測地中海果實蠅。 9. 外銷農產品檢疫技術開發： (1) 1969年日本首次報導蘭花斑點病毒(Orchid fleck virus, OFV)之鑑定，隨後德國、澳洲、巴西、南非及哥斯大黎加也陸續發現此病毒之存在。OFV可感染虎頭蘭、文心蘭、石斛蘭、蝴蝶蘭及托鞋蘭等蘭花於葉片造成壞疽、黃色斑點或輪斑。目前國際命名委員會(ICTV)將其暫時視為未歸類病毒群(The unassigned viruses)。OFV病毒顆粒為槍彈型，大小約為40 x 150 nm。此病毒可經由機械接種感染部分草本植1.An evaluation of biocontrol agent against Tetranychus kanzawai on tea.High quality and preference of Taiwan Wu-Loong and Pao-Chun tea were produced for domestic and international market in past decades, exported majorly to Japan, USA, Hong-Kong, and Mainland China. The incremental sales of Taiwan tea in recent years revealed a high demand of the international market. A diversity of sale market ensured the wholesale price and Taiwan tea industry. Pesticide residue of tea has been a major issue and makes tea production and exportation business so vulnerable. In past years, a few Taiwan tea consignments were rejected due to the residue of chemical pesticide. A safe and non-chemical control of tea pests became essential for the tea production. Biological control of tea spider mites is one of the major issue, we integrate other control measures against tea pests to produce non-chemical residue produce. Developing a sampling technique on tea mites, we periodically sample the tea leaves to establish a precision level of mite population assessment. The results are used to construct a monitoring system with frequencies of mite occurrences in the field that are used to assess a better timing and an optimal numbers of predatory mite releases, and chemical control application as well. We mass rear and release Amblyseius womersleyi Schicha, a predatory mite to control tea spider mites. The amount of predatory mites released will depend on the ratio between predators and kanzawa spider mites. The quality of tea and the cost of biological control using the predators will be assessed to make comparison with chemical control measures. We also integrated the biological control with non-chemical control measures and/or cultural practices to ensure the biological control agents. With integrated control of spider mites and other tea pests using the latest information and techniques, we shall be able to promote the quality and sale market of Taiwan tea besides a high benefit return for the farmers.2.Identification and pathogenicity of black-rot causiong bacteria recovered from ornamental kale. The purpose of this project is to isolate and identify the black-rot causing agent from ornamental kale, and to compare these isolates with those that were isolated from cabbage. The pathogenicities to two cruciferous vegetables (cabbage and Chinese cabbage), three cruciferous flowers (gillyflower, alyssum, and ornamental kale) and one cruciferous weed (Arabidopsis) of these black-rot isolates will be compared by inoculation to plant leaves. The genome of these isolates will be compared by performing pulse field gel electrophoresis of the chromosome. Previously, we isolated the orfF' virulence gene and the OrfF' vitulence protein from a cabbage black-rot isolate Xc17, and constructed its avirulent orfF' knock-out mutant, Xc17#34. For these black-rot isolates recovered either from cabbage or ornamental kale, the presence of orfF' virulence gene and the OrfF' vitulence protein will be examined by PCR and western analysis,respectively. The pathogenicities of Xc17 and Xc17#34 to the above-mentioned two cruciferous vegetables, three cruciferous flowers and one cruciferous weed will be examined. It is expected that, by executing this project, black-rot isolates will be obtained from ornamental kales, and their genomes can be compared with those isolates recovered from cabbage. The pathogenicities of all these black-rot isolates including Xc17 and Xc17# 34 to two cruciferous vegetables, three cruciferous flowers, and one cruciferous weed will be obtained and compared. 3.Population diversity and integrated pest management of downy mildew pathogen on crucifers. Downy mildew is an important plant disease on crucifers in Taiwan and worldwide. This disease causes severe reduction in vegetable yield. However, little is known about the population diversity of and resistance or susceptibilities to fungicides by downy mildew pathogens isolated in Taiwan. In this project, the phylogenetic relationships based on rDNA sequences of isolates from Taiwan and worldwide will be compared. The susceptibilities/ resistance to fungicides eg. azoxystrobin, metalaxyl, mancozeb and maneb will be assessed. In addition, the potential application of Stereptomyces sp. as a biocontrol agent for downy mildew on crucifers will be tested. Information gained from this study will provide strategies in integrated pest management of downy mildew pathogen on crucifers.4.Collection and integration of international information for supporting revision and establishment of by-laws, administrative guidelines in accordance with the new, promulgated Agricultural Pesticides Act. The main objectives of this investigation is to collect by cohorts effort the available information among the international realm for supporting the revision of our by- laws and guidelines in accordance with the newly promulgated, Agricultural Pesticide Act. Authorities with expertise in all aspects of pesticide application will be invited to form an advisory board to provide needed hands and resources for BAPHIQ staffs while carrying out the attempted revision works. Special effort will be also devoted to pool together useful information to facilitate the establishment of our national regulation system for the application of biopesticide.5.Enhance the disease resistance of Phalaenopsis spp. and Doritaenopsis spp. to Cymbidium mosaic virus by salicylic acid and chitosan. To test the available concentrations and application frequency of salicylic acid (SA) and chitosan (CHT) for increasing resistance of Phalaenopsis spp. and Doritaenopsis spp. seedlings to Cymbidium mosaic virus (CymMV) in greenhouses.6.Botanized and assessed bioinvasion pathogen caused witchs' broom on Pruns. Its main cause of disease of leaf disease, in order to come from the ascus fungus in the fungi that the oriental cherry forms a cluster (Taphrina wiesneri) Caused, this disease travelled through spore and rainwater, there is not this epidemic situation to take place in Taiwan at present, record that there is no this pathogen to take place. This plan plans to investigate that defends the oriental cherry that examined the Quarantine Office of the office and isolated the cultivating area, is it is it is it form a cluster emergence, leaf of disease to have already to examine to sample, when there is state of suspected to be taking placing, besides notifying and defending and examining the relevant units of the office first, bring back to separate for examining the laboratory, have already confirmed whether it is T or not.Assess it when this situation takes place finally, assess the situation that the agriculture of Taiwan influences.7.Development and improvement of a molecular detection kit for codling moth identification. The purpose of this research is mainly to develop a PCR detection kid for rapid identification of the codling moth, Cydia pomonella. The components of the kit will be tested and modified based on the protocol that we have applied to identify the codling moth in passed 5 years. The format of the kit will be premixed which is that all the PCR reagents are mixed excluding DNA template (i.e. sample DNA) and taq polymerase. To obtain identification result, users only need to follow the provided instruction manual to add appropriate sample DNA and taq polymerase, and then carry out the PCR reaction. We expect to develop an accurate, sensitive, and user-friendly PCR reaction kit for the people who need to rapidly identify codling moth such as quarantine inspectors. Furthermore, this kit will be patented and technical transfer to a cooperative company.8.Rapid identification of Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) by loop-mediated isothermal amplification. The Mediterranean fruit fly Ceratitis capitata (Wiedemann)(Diptera: Tephritidae) is one of the world's most widespread and damaging pests in agriculture and also an important quarantine pest in Taiwan. While adult medfly can be identified by well developed morphological keys, it is difficult to differentiate different species of genus Ceratitis at pupa, larva and egg stages. In addition to morphological characters, molecular tools have been widely used in the diagnosis of quarantine insects for those stages. For example, Polymerase Chain Reaction (PCR) based methods, such as PCR-restriction fragment length polymorphism (PCR-RFLP), Randomly Amplified Polymorphic DNAs (RAPD), and oligonucleotide arrays, have been applied for medfly identification. Although PCR based methods can produce robust results for species identification, it requires a precise and costly thermal cycler which is only applicable in well-controlled laboratory condition. For this reason, the development of a rapid and robust method for species identification at early stage of medfly is necessary. A new technique with isothermal conditions, the loop-mediated isothermal amplification method (LAMP), would be a valuable alternative for rapid and robust diagnostic tool as this method demands only a thermostat instrument. In this study, we developed a simple and rapid method which combines an easy DNA extraction procedure with LAMP to detect the existence of medfly DNA in the sample.9.Development of detection techniques of Orchid fleck virus. (1).Orchid fleck virus (OFV) was first identified and reported in Japan by Doi et al. in 1969. Since then the same virus was also found in Germany, Australia, Costa Rica, South Africa, and Brazil. OFV could infect many species of orchids including Phalaenopsis, Oncidium, Canlanthe, Cymbidium and Paphilpedium, mainly inducing yellow or necrotic spots or ringspots. Currently, ICTV assigned this virus in the group of “The unassigned viruses” for its unique properties of being transmitted persistently by mite (Brevipalpus californicus). In the past, OFV has never been found to occur in Taiwan, however, in 2004 a cymbidium plant with yellow spotting foliar symptom was found in Lu-Ku and subsequently identified to be infected by OFV. Since this is the first case of OFV found in Taiwan, we decided to eradicate all the infected orchids to prevent OFV from further dissemination. After eradication, a 3-year field surveillance program was conducted and OFV was not found again. Currently, OFV can be detected only by RT-PCR, however, the procedure is tedious and expensive comparing with serological methods. This project attempts to clone and express the NP gene of OFV in bacteria culture and use it as immunogen for antiserum preparation. Hopefully, the antiserum can be applied in high throughput sample detection system, for example, in the field surveillance program leading to a more efficient and cost effective survey of OFV in Taiwan.(2).Relationships between tomato ripeness and fruit fly infestation and monitoring of the fruit flies occurred in tomato field under screenhouse cultivation. The oriental fruit fly is present in Taiwan and tomato is one of the hosts of this pest. Therefore, a quarantine treatment is required in order to disinfest the eggs and larvae of the fruit fly that may exist in the fruit before the tomato is allowed to export to the pest-free areas or countries. Although the use of the quarantine treatment meets the requirement from the importing countries, it may affects the ripeness stage of the tomato to be harvested, reduces the quality of the fruit during postharvest ripening, increases production costs, and reduces the competitiveness of exportation. The cultivation of tomato in well-designed screenhouse has been adopted by some growers in Taiwan, and the use of screenhouse effectively protects the tomato from infestation by the fruit flies and greatly reduces the risk that the exporting tomatoes may carry quarantine pests. However, frequently getting in and out of the screenhouse or not taking good care of the screenhouse may provide the chance for the fruit flies entering into the fields. In general, the fruit flies lay eggs in the host fruits only when they reach preferred degree of ripeness. If we can make sure that the tomato is harvested at a safe ripening stage and can avoid the infestation by the fruit flies under the protection of the screenhouse during the production period, the combination of these two applications may serve as an alternative to the quarantine treatments that required by the importing countries. The major working items of this project are: (1) maintaining a fruit fly colony for use, (2) determining the rates of fruit fly infestation for the tomatoes with different degrees of ripeness, (3) monitoring the occurrence of fruit flies in screenhouse-cultivated tomato fields. This study will document the stage of ripeness for tomato that harvested without the risk of infestation by the fruit flies, confirm the effectiveness of screenhouse in the protection of tomato fruits, and find the way to improve the protection structure of screenhouse. According to the research results, hopefully an alternative to the quarantine treatment can be proposed and used to reduce the production costs of the tomatoes and increase the competitiveness in the exportation

重要植物有害生物診斷鑑定技術研發及綜合管理

1.An evaluation of biocontrol agent against Tetranychus kanzawai on tea.High quality and preference of Taiwan Wu-Loong and Pao-Chun tea were produced for domestic and international market in past decades, exported majorly to Japan, USA, Hong-Kong, and Mainland China. The incremental sales of Taiwan tea in recent years revealed a high demand of the international market. A diversity of sale market ensured the wholesale price and Taiwan tea industry. Pesticide residue of tea has been a major issue and makes tea production and exportation business so vulnerable. In past years, a few Taiwan tea consignments were rejected due to the residue of chemical pesticide. A safe and non-chemical control of tea pests became essential for the tea production. Biological control of tea spider mites is one of the major issue, we integrate other control measures against tea pests to produce non-chemical residue produce. Developing a sampling technique on tea mites, we periodically sample the tea leaves to establish a precision level of mite population assessment. The results are used to construct a monitoring system with frequencies of mite occurrences in the field that are used to assess a better timing and an optimal numbers of predatory mite releases, and chemical control application as well. We mass rear and release Amblyseius womersleyi Schicha, a predatory mite to control tea spider mites. The amount of predatory mites released will depend on the ratio between predators and kanzawa spider mites. The quality of tea and the cost of biological control using the predators will be assessed to make comparison with chemical control measures. We also integrated the biological control with non-chemical control measures and/or cultural practices to ensure the biological control agents. With integrated control of spider mites and other tea pests using the latest information and techniques, we shall be able to promote the quality and sale market of Taiwan tea besides a high benefit return for the farmers.2.Identification and pathogenicity of black-rot causiong bacteria recovered from ornamental kale. The purpose of this project is to isolate and identify the black-rot causing agent from ornamental kale, and to compare these isolates with those that were isolated from cabbage. The pathogenicities to two cruciferous vegetables (cabbage and Chinese cabbage), three cruciferous flowers (gillyflower, alyssum, and ornamental kale) and one cruciferous weed (Arabidopsis) of these black-rot isolates will be compared by inoculation to plant leaves. The genome of these isolates will be compared by performing pulse field gel electrophoresis of the chromosome. Previously, we isolated the orfF' virulence gene and the OrfF' vitulence protein from a cabbage black-rot isolate Xc17, and constructed its avirulent orfF' knock-out mutant, Xc17#34. For these black-rot isolates recovered either from cabbage or ornamental kale, the presence of orfF' virulence gene and the OrfF' vitulence protein will be examined by PCR and western analysis,respectively. The pathogenicities of Xc17 and Xc17#34 to the above-mentioned two cruciferous vegetables, three cruciferous flowers and one cruciferous weed will be examined. It is expected that, by executing this project, black-rot isolates will be obtained from ornamental kales, and their genomes can be compared with those isolates recovered from cabbage. The pathogenicities of all these black-rot isolates including Xc17 and Xc17# 34 to two cruciferous vegetables, three cruciferous flowers, and one cruciferous weed will be obtained and compared. 3.Population diversity and integrated pest management of downy mildew pathogen on crucifers. Downy mildew is an important plant disease on crucifers in Taiwan and worldwide. This disease causes severe reduction in vegetable yield. However, little is known about the population diversity of and resistance or susceptibilities to fungicides by downy mildew pathogens isolated in Taiwan. In this project, the phylogenetic relationships based on rDNA sequences of isolates from Taiwan and worldwide will be compared. The susceptibilities/ resistance to fungicides eg. azoxystrobin, metalaxyl, mancozeb and maneb will be assessed. In addition, the potential application of Stereptomyces sp. as a biocontrol agent for downy mildew on crucifers will be tested. Information gained from this study will provide strategies in integrated pest management of downy mildew pathogen on crucifers.4.Collection and integration of international information for supporting revision and establishment of by-laws, administrative guidelines in accordance with the new, promulgated Agricultural Pesticides Act. The main objectives of this investigation is to collect by cohorts effort the available information among the international realm for supporting the revision of our by- laws and guidelines in accordance with the newly promulgated, Agricultural Pesticide Act. Authorities with expertise in all aspects of pesticide application will be invited to form an advisory board to provide needed hands and resources for BAPHIQ staffs while carrying out the attempted revision works. Special effort will be also devoted to pool together useful information to facilitate the establishment of our national regulation system for the application of biopesticide.5.Enhance the disease resistance of Phalaenopsis spp. and Doritaenopsis spp. to Cymbidium mosaic virus by salicylic acid and chitosan. To test the available concentrations and application frequency of salicylic acid (SA) and chitosan (CHT) for increasing resistance of Phalaenopsis spp. and Doritaenopsis spp. seedlings to Cymbidium mosaic virus (CymMV) in greenhouses.6.Botanized and assessed bioinvasion pathogen caused witchs' broom on Pruns. Its main cause of disease of leaf disease, in order to come from the ascus fungus in the fungi that the oriental cherry forms a cluster (Taphrina wiesneri) Caused, this disease travelled through spore and rainwater, there is not this epidemic situation to take place in Taiwan at present, record that there is no this pathogen to take place. This plan plans to investigate that defends the oriental cherry that examined the Quarantine Office of the office and isolated the cultivating area, is it is it is it form a cluster emergence, leaf of disease to have already to examine to sample, when there is state of suspected to be taking placing, besides notifying and defending and examining the relevant units of the office first, bring back to separate for examining the laboratory, have already confirmed whether it is T or not.Assess it when this situation takes place finally, assess the situation that the agriculture of Taiwan influences.7.Development and improvement of a molecular detection kit for codling moth identification. The purpose of this research is mainly to develop a PCR detection kid for rapid identification of the codling moth, Cydia pomonella. The components of the kit will be tested and modified based on the protocol that we have applied to identify the codling moth in passed 5 years. The format of the kit will be premixed which is that all the PCR reagents are mixed excluding DNA template (i.e. sample DNA) and taq polymerase. To obtain identification result, users only need to follow the provided instruction manual to add appropriate sample DNA and taq polymerase, and then carry out the PCR reaction. We expect to develop an accurate, sensitive, and user-friendly PCR reaction kit for the people who need to rapidly identify codling moth such as quarantine inspectors. Furthermore, this kit will be patented and technical transfer to a cooperative company.8.Rapid identification of Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) by loop-mediated isothermal amplification. The Mediterranean fruit fly Ceratitis capitata (Wiedemann)(Diptera: Tephritidae) is one of the world's most widespread and damaging pests in agriculture and also an important quarantine pest in Taiwan. While adult medfly can be identified by well developed morphological keys, it is difficult to differentiate different species of genus Ceratitis at pupa, larva and egg stages. In addition to morphological characters, molecular tools have been widely used in the diagnosis of quarantine insects for those stages. For example, Polymerase Chain Reaction (PCR) based methods, such as PCR-restriction fragment length polymorphism (PCR-RFLP), Randomly Amplified Polymorphic DNAs (RAPD), and oligonucleotide arrays, have been applied for medfly identification. Although PCR based methods can produce robust results for species identification, it requires a precise and costly thermal cycler which is only applicable in well-controlled laboratory condition. For this reason, the development of a rapid and robust method for species identification at early stage of medfly is necessary. A new technique with isothermal conditions, the loop-mediated isothermal amplification method (LAMP), would be a valuable alternative for rapid and robust diagnostic tool as this method demands only a thermostat instrument. In this study, we developed a simple and rapid method which combines an easy DNA extraction procedure with LAMP to detect the existence of medfly DNA in the sample.9.Development of detection techniques of Orchid fleck virus. (1).Orchid fleck virus (OFV) was first identified and reported in Japan by Doi et al. in 1969. Since then the same virus was also found in Germany, Australia, Costa Rica, South Africa, and Brazil. OFV could infect many species of orchids including Phalaenopsis, Oncidium, Canlanthe, Cymbidium and Paphilpedium, mainly inducing yellow or necrotic spots or ringspots. Currently, ICTV assigned this virus in the group of “The unassigned viruses” for its unique properties of being transmitted persistently by mite (Brevipalpus californicus). In the past, OFV has never been found to occur in Taiwan, however, in 2004 a cymbidium plant with yellow spotting foliar symptom was found in Lu-Ku and subsequently identified to be infected by OFV. Since this is the first case of OFV found in Taiwan, we decided to eradicate all the infected orchids to prevent OFV from further dissemination. After eradication, a 3-year field surveillance program was conducted and OFV was not found again. Currently, OFV can be detected only by RT-PCR, however, the procedure is tedious and expensive comparing with serological methods. This project attempts to clone and express the NP gene of OFV in bacteria culture and use it as immunogen for antiserum preparation. Hopefully, the antiserum can be applied in high throughput sample detection system, for example, in the field surveillance program leading to a more efficient and cost effective survey of OFV in Taiwan.(2).Relationships between tomato ripeness and fruit fly infestation and monitoring of the fruit flies occurred in tomato field under screenhouse cultivation. The oriental fruit fly is present in Taiwan and tomato is one of the hosts of this pest. Therefore, a quarantine treatment is required in order to disinfest the eggs and larvae of the fruit fly that may exist in the fruit before the tomato is allowed to export to the pest-free areas or countries. Although the use of the quarantine treatment meets the requirement from the importing countries, it may affects the ripeness stage of the tomato to be harvested, reduces the quality of the fruit during postharvest ripening, increases production costs, and reduces the competitiveness of exportation. The cultivation of tomato in well-designed screenhouse has been adopted by some growers in Taiwan, and the use of screenhouse effectively protects the tomato from infestation by the fruit flies and greatly reduces the risk that the exporting tomatoes may carry quarantine pests. However, frequently getting in and out of the screenhouse or not taking good care of the screenhouse may provide the chance for the fruit flies entering into the fields. In general, the fruit flies lay eggs in the host fruits only when they reach preferred degree of ripeness. If we can make sure that the tomato is harvested at a safe ripening stage and can avoid the infestation by the fruit flies under the protection of the screenhouse during the production period, the combination of these two applications may serve as an alternative to the quarantine treatments that required by the importing countries. The major working items of this project are: (1) maintaining a fruit fly colony for use, (2) determining the rates of fruit fly infestation for the tomatoes with different degrees of ripeness, (3) monitoring the occurrence of fruit flies in screenhouse-cultivated tomato fields. This study will document the stage of ripeness for tomato that harvested without the risk of infestation by the fruit flies, confirm the effectiveness of screenhouse in the protection of tomato fruits, and find the way to improve the protection structure of screenhouse. According to the research results, hopefully an alternative to the quarantine treatment can be proposed and used to reduce the production costs of the tomatoes and increase the competitiveness in the exportation.1. 茶樹神澤氏葉蟎生物防治天敵效能評估：台灣的包種茶及烏龍茶品質極佳，茶葉除內銷外，外銷方面以日本、美國、香港及中國大陸為主，外銷市場潛力豐厚。拓展外銷市場，亦有助於國內市場價格的提高及穩定。 出口茶葉推展的重要議題，為農藥殘留問題，尤其近年屢次發生新殺蟎殘留量過高，遭退貨的案例。因此發展安全、無毒的生產技術益發重要。本生物防治栽培管理方法將能生產無農藥、高品質及產量的茶葉，此為世界各國目前作物生產的潮流。建立田間觀測用族群密度調查法，省時、省力又可較準確估計葉蟎族群密度的取樣方法，並監測葉蟎族群終年發生頻度，可有效擬訂施用藥劑與生物防治的適當時機。大量飼育溫氏捕植蟎以提供釋放，充分供應試驗茶園捕植蟎之需求，以防治葉蟎危害。捕植蟎施放比率測試，有助於提供今後生物防治茶樹神澤氏葉蟎技術參考，往昔用藥與產量品質品質調查、成本分析與效果評估，可幫助落實非農藥防治及生物技術之利用。釋放溫氏捕植蟎，配合整體茶葉栽培管理技術，提升品質與產量，增進農民經濟收益，促進產品外銷及提昇我國農產品競爭力，祈開創茶葉市場之新藍海。 2. 葉牡丹黑腐病菌之鑑定與致病性：本計畫之目標為自葉牡丹的黑腐病株中分離並鑑定出黑腐菌株，亦將嘗試自葉牡丹種子中分離黑腐菌株，並將這些分離株與本省分離自甘藍的黑腐菌株相比較。我們將以接種試驗比較這些菌株對二種十字花科蔬菜(甘藍與小白菜)，三種十字花科花卉(葉牡丹、香雪球及紫羅蘭)，及一種十字花科模式植物-阿拉伯芥的致病性；亦將以脈衝式電泳比較這些菌株的基因型。另外，我們先前曾自一株分離自甘藍的黑腐菌株Xc17中，分離到orfF'致病基因及OrfF'致病蛋白，並構築出orfF'缺失的Xc17無致病株，稱為Xc17#34。我們將以PCR及西方雜配檢查分離自葉牡丹與甘藍的黑腐菌株是否皆具有orfF'致病基因及OrfF'致病蛋白，並將測試Xc17與Xc17#34對二種十字花科蔬菜(甘藍與小白菜)，三種十字花科花卉(葉牡丹、香雪球及紫羅蘭)，及一種十字花科模式植物-阿拉伯芥的致病性。本計畫之預期效益為自葉牡丹分離並鑑定出黑腐菌株；得知分離自葉牡丹與甘藍的黑腐菌株的基因型、orfF'致病基因、及OrfF'致病蛋白是否有差異；及獲得這些菌株、Xc17及Xc17#34菌株對二種十字花科蔬菜、三種十字花科花卉、一種十字花科雜草模式植物(阿拉伯芥)的致病性。 3. 十字花科露菌病菌菌相調查及綜合防治管理之探討：十字花科露菌病嚴重影響台灣全省各地十字花科蔬菜產量與品質，然卻極少有關此病原菌台灣本土性菌株菌相及藥劑特性之相關研究，也尚無推薦之生物製劑以供選擇利用。本研究擬進行台灣本土性菌株收集及rDNA ITS區間序列分析，以了解台灣菌株與國外菌株在遺傳特性之相似或相異度。另針對推薦使用，卻具抗藥性風險之苯醯胺類，如：滅達樂與史托比系列藥劑，如：亞托敏，及非專一性多點作用之藥劑，如：鋅錳乃浦及錳乃浦等藥劑，評估台灣菌株對此些藥劑之抗感藥性。研究中亦擬測試對卵菌綱病原菌Phytophothora spp.具防治潛力之鏈黴菌生物製劑，在防治十字花科露菌病菌之應用性。此些資訊之建立，將提供擬定十字花科露菌綜合防治策略之依據。 4. 農藥管理法子法規相關資料之蒐集研析：本計畫主旨在以團隊力量蒐集國際間與農藥應用有關之法規資訊，以協助甫完成公告「農藥管理法」相關系列子法規之修訂與增訂。本計畫中主要工作將邀集國內農藥應用管理學有專精之專家學者組織諮詢團隊，提供防檢局同仁修法工作進行中必要資訊與協助。另一工作重點則將致力於彙集與生物農藥應用推動攸關的合理化管理系統之建立所需資訊，期有助於生物農藥產業化應用之落實。 5. 應用水楊酸及甲殼素提高蝴蝶蘭種苗對抗蕙蘭屬崁紋病毒(CYMMV)之抗性：本計畫之年度目標在於了解可以保護蝴蝶蘭及朵麗蝶蘭出瓶苗免於感染CymMV感染之SA及CHT之處理濃度，以及噴施的頻率。 6. 櫻屬外來種簇葉病之調查與評估：櫻花簇葉病其主要病因為來自真菌中的子囊菌(Taphrina wiesneri)引起，此病原是透過孢子和雨水傳播，目前台灣並無此疫情發生，也並無此病原菌發生的紀錄。本計畫擬調查防檢局檢疫站及隔離栽培區的櫻花，抽樣檢驗是否已有簇葉病的發生，若有疑似發生狀況時，除先通知防檢局相關單位外，並攜回實驗室進行分離檢驗，已確定是否為T. wiesneri所造成的簇葉病。最後並評估若有此情況發生時，對台灣農業影響的情況評估。 7. 蘋果蠹蛾PCR檢測試劑套組之開發與改良：本研究主要目的在開發可供快速鑑定蘋果蠹蛾(Cydia pomonella)之PCR反應試劑套組。此套組係根據本研究室近五年來使用於檢測蘋果蠹蛾的PCR方法加以測試與改良。除了DNA模版與taq聚合酶之外，本試劑套組擬將其他所有PCR反應所需試劑(包括專一性引子)預先混合，使用者僅需依所提供的操作手冊逐步加入適量的檢體DNA與taq聚合酶，之後再進行PCR反應即可得到檢測結果。我們預期將開發出具有高敏感度與準確性且方便使用之檢測試劑套組供如防檢疫人員等需要鑑定蘋果蠹蛾者使用。同時，我們亦擬將此試劑套組申請專利與技術轉移給適當廠商。 8. 利用恆溫式圈環形核酸增幅法(LAMP)快速鑑定地中海果實蠅(Ceratitis capitata)：地中海果實蠅(Ceratitis capitata (Wiedemann))是世界上危害最嚴重的果實蠅種類之一，亦被列為台灣重要的檢疫害蟲。除成蟲外，其它齡期皆不易由形態加以鑑定，目前可用PCR、PCR-RFLP、RAPD、生物晶片等方法來鑑定，此類方法皆基於PCR，故需不同的溫度周期反應，花費時間較長，仍有發展更快速更方便檢測方法的空間。LAMP (loop-mediated isothermal amplification method)是利用特殊設計的引子對，在單一的溫度(65℃)下以特別的聚合酶反應，快速增幅DNA。本研究擬尋找適合LAMP反應的快速抽取果實蠅DNA方法及適合鑑定地中海果實蠅的LAMP引子、條件，快速及精確檢測地中海果實蠅。 9. 外銷農產品檢疫技術開發： (1) 1969年日本首次報導蘭花斑點病毒(Orchid fleck virus, OFV)之鑑定，隨後德國、澳洲、巴西、南非及哥斯大黎加也陸續發現此病毒之存在。OFV可感染虎頭蘭、文心蘭、石斛蘭、蝴蝶蘭及托鞋蘭等蘭花於葉片造成壞疽、黃色斑點或輪斑。目前國際命名委員會(ICTV)將其暫時視為未歸類病毒群(The unassigned viruses)。OFV病毒顆粒為槍彈型，大小約為40 x 150 nm。此病毒可經由機械接種感染部分草本

Surveillance on Turkey, Ostrich and Waterfowl Avian Influenza

對水禽類動物鴨、鵝.雞及火雞共計265場，6000支肛門拭子或血清分別以應用反轉錄聚合鋂鏈進行檢測病毒核酸，而血清則以免疫酵素分析法及AGP等方法進行禽流感抗體之監測，如為陽性反應之檢體病進行亞型之鑑定，陽性率過高之場別，擬進行現場採集檢體進行病毒分離及以應用反轉錄聚合鋂鏈進行檢測，以早期發現未曾在台灣發生者，或者在台灣已成為地方性疾病，但有爆發成流行之趨勢者。透過台灣區人工飼養鴕鳥協會及專業獸醫師，進行鴕鳥之死亡及飼養失敗病例之收集。並進行病理之解剖、病因之探討及病原分離與鑑定。並就環境、病理學、病原所得之資料進行分析，提出解決之建議方案。Sera samples will be collected seasonally from all over the country. Avian influenza antibody of ELISA, agar gel precipitation (AGP), and hemagglutination inhibition (HI) test. Fecal samples will be collected from the antibody positive flocks, and then subjected to RT-PCR for AIV detection and subserotype determination. Domestic turkeys will detects for serological antiboies to avian influenza virus in Taiwan, sampling 20 from each group or farm to testing with enzyme-linked immunosorbent assay (ELISA). The antibody prevalence will be analyzing as a data for prevention of AI. The turkey farms showed positive reaction after serum detection will be test to viral isolation and sub-serotyping, this farm will going to sera monitoring. In this project, we will cooperate with Taiwan Domestic Ostrich Association and employ a professional veterinarian to propagate the important of monitoring disease. We will collect clinical case ofostrichs and analysis the aetiology to establish the disease database of ostrich in Taiwan

Surveillance on Waterfowl Avian Influenza

本計劃依各縣市水禽飼養比率及不同季節採集統計上足量之水禽血清樣品，分別以ELISA、AGP及HI tests測定禽流感抗體抗體並做亞型鑑定，抗體陽性場追蹤採糞材行反轉錄鋂禽流感之病毒檢測及亞型鑑定。Sera samples will be collected seasonally from all over the country. Avian influenza antibody of ELISA, agar gel precipitation (AGP), and hemagglutination inhibition (HI) test. Fecal samples will be collected from the antibody positive flocks, and then subjected to RT-PCR for AIV detection and subserotype determination

水禽及火雞類禽流感監控

Sera samples will be collected seasonally from all over the country. Avian influenza antibody of ELISA, agar gel precipitation (AGP), and hemagglutination inhibition (HI) test. Fecal samples will be collected from the antibody positive flocks, and then subjected to RT-PCR for AIV detection and subserotype determination. Domestic turkeys will detects for serological antiboies to avian influenza virus in Taiwan, sampling 20 from each group or farm to testing with enzyme-linked immunosorbent assay (ELISA). The antibody prevalence will be analyzing as a data for prevention of AI. The turkey farms showed positive reaction after serum detection will be test to viral isolation and sub-serotyping, this farm will going to sera monitoring.對水禽類動物鴨、鵝.雞及火雞共計265場，6000支肛門拭子或血清分別以應用反轉錄聚合