The Impact of Climate Change on Plant Health and Quarantine

氣候變遷對植物健康與防檢疫措施之影響的相關研究不多，大多數的研究工作集中於對於植物病害與病原之影響。本計畫藉由邀請國內外學者專家舉辦” 氣候變遷對植物健康與防檢疫措施之影響研討會” 進行相關資訊之收集及專業人員交流，以協助建立國內相關專業能力，進而擬定因應策略。Research on impacts of climate change on plant health and quarantine has been limited, with most work concentrating on the effects of global climate change on the development and management of plant pathogens, pests, and diseases. This project intends to invite foreign and domestic experts to join the symposium of the impacts of climate change on plant health and quarantine

Development of the Tools and Techniques for Detecting Seed-Borne Viruses of Economically Important Vegetables

病毒藉由寄主的種子傳播是一個普遍的現象，因而造成種子在進出口時常成為檢疫的對象。本計畫將針對數種重要的蔬菜種傳病毒開發種子檢測工具和檢測技術，以利種子檢疫工作之遂行。本年工作以豆科蔬菜之種傳病毒為主要檢測對象。工作項目包括病毒外鞘蛋白基因之選殖與定序、病毒抗血清之製備與血清學試驗等預期將可產出專一性抗血清、分子探針與引子對，並發展包括ELISA, RT-PCR, IC-RT-PCR等技術，供種子檢測之用。Plant viruses can be transmitted by their hosts' seeds. This common phenomenum makes plant seed an inspection target of quranteen. This project will develop detection tools and techniques for the inspection scheme of some important seed-borne viruses of vegetables. The viruses infecting Leguminoseae crops will be focused. The viruses will be isolated and purified, the viral CP genes will be cloned and sequenced, and the specific antisera as well as molecular probes will be produced. The produced tools, i.e. antisera, probes, primers, etc, and the developed techniques will provide sensitive and reliable ways of detection for the inspection scheme of vegetable seed-borne viruses

The Construction of Transgenic Viral Resistance in Lily and Gladiolus and Their Transformation and Regeneration

百合與唐菖蒲為本國重要的經濟花卉作物。其生產，不可避免地，與其他經濟作物同樣面臨病蟲害之威脅。在百合與唐菖蒲的重要病害當中，病毒病害以其能引起重大損失及難以傳統方法防治等因素，向被列為影響其生產的主要限制因子之一，其中以Cucumoviruses和Potyviruses為主要病毒。植物病毒病害的傳統防治方法一向只能治標，而能治本的抗病育種，則由於天然的抗病基因取得不易，且耗時耗力而顯得緩不濟急。所幸近年來，結合病原衍生型抗病性(pathogen-derived resistance, PDR)概念與基因轉殖技術所建構之轉基因抗病性(transgenic resistance)，已被廣泛且有效地應用於多種植物以抵抗植物病毒之為害。本計畫擬針對感染百合與唐菖蒲的胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)與Potyviruses選殖適用的病毒基因或基因片段建構具轉基因抗性的百合與唐菖蒲以解決CMV與Potyviruses為害百合與唐菖蒲的問題。在過去十數年間，病毒的基因如鞘蛋白基因(coat protein gene, CP)，複製酵素基因(replicase gene)等，均已被實際應用於病原衍生型抗病理論之實踐，然而成效參差不一。早期的研究顯示，轉殖病毒鞘蛋白基因於植物可有效地達到抗病毒的目的；然而近來也有研究顯示病毒CP雖可在轉基因植物內大量表現卻不能提供有效的抗性者。在數種目前可行的方法中，RNA中介式轉基因抗病性(RNA-mediated transgenic resistance)因能提供幾近於免疫的超強抗病能力而被認為是達成此目標之最佳選項。RNA中介式轉基因抗病性之產生即是由轉錄後基因消寂作用(post-transcriptional gene silencing, PTGS；或稱為RNA silencing)的現象所導致。目前已知雙股形態的RNA (dsRNA)是植物體啟動PTGS的主要啟始因子。本計畫擬利用CMV和Potyviruses病毒的基因片段分別構築出一個具有inverted repeat sequences的載體，使其能在轉錄時形成雙股核醣核酸(dsRNA)錄本，進而啟動PTGS機轉使轉基因百合或唐菖蒲得以表現RNA中介式轉基因抗病性而達到抗病毒的目的。本計畫之執行，預期將能產生抗CMV或抗Potyviruses的轉基因百合或唐菖蒲，將有助於本國之花卉產業，亦將有助於提升我國花卉種苗在國際市場銷售的競爭力。Lily and gladiolus are important economic ornamental crops to our floral industry. However, the production of lily and gladiolus have been suffered from the infection by several pests and diseases, including diseases caused by viruses. Among those lily- and gladiolus-infecting viruses, Cucumoviruses and Potyviruses are the major notorious viral agents. The establishment of resistance in plants through conventional plant breeding is one of the major ways for plant disease control. However, it is not a suitable one for viruses not only for lack of natural resistance genes but also being laborious and time-consuming. Alternative approaches, based on the concept of pathogen-derived resistance (PDR), such as transgenic expression of viral coat protein (CP) or (defective) replicase in plants to confer resistance against virus infection have been reported over the years, albeit with rather differing outcomes. Transgenic virus resistance can be engineered into plant genetically by using either functional virus genes or dysfunctional viral genomic fragments to provoke resistance at protein-level (protein-mediated) or at RNA level (RNA-mediated resistance). Among the various strategies that have been applied, RNA-mediated transgenic resistance has aroused as a promising one because of the extreme resistance it conferred. Plants showing RNA-mediated resistance were either completely resistant to virus infection or recovered from the initial infection. RNA-mediated resistance can be easily obtained by introducing constructs that formed double-stranded RNA (dsRNA) during the transcription of the transgenes. It is known that dsRNA forms are the key triggers of RNA silencing mechanisms. During induction of RNA silencing mechanisms, short small interfering RNAs (siRNAs) are generated from dsRNA by digestion with an RNaseIII-like enzyme (DICER). The siRNAs are proposed to subsequently guide an endonuclease complex (RISC) to RNA that shares sequence similarity with the inducing dsRNA and ensures that this RNA is specifically degraded. However, RNA-mediated viral resistance is a highly sequence specific process in which only RNA having homology to the transgenic inducer RNA is specifically degraded. This project specifically focuses on the establishment of transgenic resistance against CMV and potyviruses in lily and gladiolus by using inverted repeat constructs derived from CMV or potyvirus genomic fragments to provoke RNA silencing and, thus, transgenic resistance

Development of Detection Techniques and Field Survey of Viroids on Ornamental Crops

類病毒(viroid)是最小的植物病原，由數百個核苷酸所組成，不具鞘蛋白。類病毒在寄主上引起類似病毒造成的病徵。早期對類病毒的診斷與檢測相當不易，近年以核酸雜合和RT-PCR建立快速可靠的診斷檢測方法，類病毒病害才大量被報導。近來，在歐盟的花卉等園藝作物上有多種類病毒被檢出。我國有關的研究少，於類病毒之相關資訊及檢測工具與技術均不甚完善。我國自歐盟進口園藝作物種類與數量均多，防檢疫上對類病毒幾乎毫無設防。本計畫擬對重要園藝作物進行類病毒之檢測與鑑定，建立類病毒基本資料，開發專一性偵檢工具與技術，俾類病毒檢防疫之需。本計畫將與荷蘭農業部植物保護中心和Wageningen University合作，進行類病毒探針之製備與檢測技術之建立，以利田間檢測之進行。Viroid is the smallest plant pathogen consisting of several hundreds of ribonucleic acids (RNA) but not encoding coat protein. The symptoms caused by viroids are similar to that caused by plant viruses. The diagnosis and detection of viroids are not easy until the development of the techniques of nucleic acid hybridization and RT-PCR which are based on genomic RNA sequences of viroids. A lot of viroids had been identified on ornamental crops in European countries in recent years. Only a few viroid infections have been reported in Taiwan. Since the viroid-related information are lack and the detection techniques for viroids are not well established yet, this project will develop specific detection tools and techniques for the detection and survey of the ornamental viroids in Taiwan. This project will search collaboration and assistance from the Plant Disease Service and the Wageningen University of the Netherlands in establishing standard detection process for viroids on ornamental crops

Identification and diagnosis of new viral diseases of lisianthus and the development of detection tools and techniques

洋桔梗為重要切花作物, 田間調查發現有疑似病毒感染之病株. 本計畫擬就該病毒進行分離純化等工作, 並對病毒之基本化學及生物性狀進行探討. 同時製備病毒之抗血清與分子檢測工具, 以建立快速偵測及診斷系統, 並有助於健康種苗驗證制度之建立.Lisianthus plants showing necrotic ringspots and chlorotic spots on leaves were found in the fields. The electron microscopic data showed virusparticles exist inside the diseased lisianthus and thought to be the possible causal agents of symptoms described. In the proposed study, diseased samples will be collected for virus isolation and purification. With purified virus preparations, the basic shemical and biological properties will be investigated. The purified virus samples will also be used to immunie rabbits for polyclonal antibody production. The produced antisera will be used to establish rapid detection techniques for viral infection. The data obtained from this study will also be useful to establish certification system for healthy germplasms of lisianthus

Development of the techniques for detecting crucifer comoviruses and vectors and for disease control

十字花科蔬菜作物發現有新興的蕪菁輪點病毒(Turnip ringspot virus, TuRSV)和蘿蔔嵌紋病毒(Radish mosaic virus, RaMV)之發生。兩病毒均屬豇豆嵌紋病毒屬(genus Comovirus)的成員，且均由黃條葉蚤(Phyllotreta striolata)及 Epitrix hirtipennis 、 Diabrotica undecimpunctata等甲蟲傳播。本計畫擬針對TuRSV與RaMV發展血清及分子檢測工具，應用於偵檢田間十字花科病株與帶毒蟲媒，除開發檢測標準流程外，亦將進行田間TuRSV與RaMV病害發生與帶毒蟲媒族群之調查及開發防疫技術之研究。The Turnip ringspot virus (TuRSV) and Radish mosaic virus (RaMV) were emerging novel viruses found in crucifer vegetables during past few years. Both viruses are Comovirus transmitted by striped flea beetle (Phyllotreta striolata) and others (Epitrix hirtipennis and Diabrotica undecimpunctata). This proposed study will develop virus-specific antesera and molecular detection methods to detect both comoviruses specifically and sensitively in crucifer crops and beetle vectors. The developed detection tools and methods will further apply to field survey and inspection of the occurrence of crucifer comoviruses in the field, not only on crucifer crops but also on viruliferous beetle vectors

Development of detection techniques and field survey on viroids of ornamental crops

類病毒(viroid)是最小的植物病原，由數百個核?酸所組成，不具鞘蛋白。類病毒在寄主上引起類似病毒造成的病徵。早期對類病毒的診斷與檢測相當不易，近年以核酸雜合和RT-PCR建立快速可靠的診斷檢測方法，類病毒病害才大量被報導。近來，在歐盟的花卉等園藝作物上有多種類病毒被檢出。我國有關的研究少，於類病毒之相關資訊及檢測工具與技術均不甚完善。我國自歐盟進口園藝作物種類與數量均多，防檢疫上對類病毒的偵檢有持續加強之必要。本計畫擬持續對重要園藝作物進行類病毒之檢測，建立類病毒基本資料，俾類病毒檢防疫之需。本計畫將與荷蘭農業部植物保護中心和Wageningen University合作，進行類病毒探針之製備與檢測技術之建立，以利田間檢測之進行。Viroid is the smallest plant pathogen consisting of several hundreds of ribonucleic acids (RNA) but not encoding coat protein. The symptoms caused by viroids are similar to that caused by plant viruses. The diagnosis and detection of viroids are not easy until the development of the techniques of nucleic acid hybridization and RT-PCR which are based on genomic RNA sequences of viroids. A lot of viroids had been identified on ornamental crops in European countries in recent years. Only a few viroid infections have been reported in Taiwan. This project will keep developing specific detection tools and techniques for viroids and keep detecting and surveying the occurrence of local and invading viroids on ornamental and solanaceous crops in Taiwan. This project will collaborate with the Plant Disease Service of the Netherlands in establishing standard detection process for viroids

Virus Diseases of Lisianthus in Taiwan

洋桔梗(lisianthus, Eustoma russellianum) 病毒病害於過去20 年間陸續在台灣地區發生，目前已有9 種病毒病害被發現，包括菜豆黃化嵌紋病毒( Bean yellow mosaic virus , BYMV)、洋桔梗嵌紋病毒( Lisianthus necrosis virus ,LNV)、胡瓜嵌紋病毒( Cucumber mosaic virus , CMV)、蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)、蠶豆萎凋病毒2 ( Broad bean wilt virus 2, BBWV-2)、番茄嵌紋病毒 ( Tomato mosaic virus , ToMV)、藿香薊黃脈病毒(Ageratum yellow vein virus, AYVV)、康乃馨斑駁病毒(Carnation mottle virus, CarMV)和番椒葉脈斑駁病毒 (Pepper veinal mottle virus, PVMV)。新興的洋桔梗病毒仍陸續被發現，其中包括黃金葛潛隱病毒(Pothos latent virus, PoLV)和咸豐草斑駁病毒(Bidens mottle virus , BiMoV)，將於本文中略述之，並及於部分洋桔梗病毒之研究情形。 Lisianthus (Eustoma russellianum) with virus infections were found field during the past decades in Taiwan. Nine different viruses have been identified in the past decade. Bean yellow mosaic virus (BYMV) was the first being detected in lisianthus, followed by Lisianthus necrosis virus (LNV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV), Tomato spotted wilt virus (TSWV), Broad bean wilt virus (BBWV), Tomato mosaic virus (ToMV), Tomato yellow leaf curl virus (TYLCV) , and Pepper veinal mottle virus (PVMV). Researchers have characterized most of these viruses in Taiwan serologically and molecularly. However, new viral diseases keep emerging in the field still. Some of the new lisianthus diseases caused by viruses, such as Carnation mottle virus (CarMV), Bidens mottle virus (BiMoV), and Pothos latent virus (PoLV), will be discussed in this seminar