摘要:一、輸入應實施隔離檢疫植物標準檢測流程之建立
本計畫旨在針對應實施隔離檢疫的重要植物項目擬訂應檢測之有害生物種類,逐一建立各有害生物之標準檢測流程,使各港站檢疫人員可有一致之標準可遵循,裨益於留檢期間的檢疫工作。依據近年來的檢疫業務的急迫性,將先聚焦於櫻、玫瑰與葡萄三樣作物的檢疫有害生物之檢測,各制訂一套標準作業流程(SOP),爾後再逐步擴及其他植物種類。本年度擬定的主要工作項目有:(1) 蒐集櫻、玫瑰(薔薇屬)與葡萄屬的全世界疫情與文獻新資料;(2) 盤點櫻、玫瑰與葡萄屬有害生物的國內外診斷鑑定技術及其相關參考文獻;(3) 擬訂櫻的重要檢疫有害生物(共計13種)的RT-PCR分子檢測方法與作業流程;(4) 擬訂玫瑰的重要檢疫有害生物(共計8種)的RT-PCR分子檢測方法與作業流程;(5) 擬訂葡萄屬的重要檢疫有害生物(共計10種)的RT-PCR分子檢測方法與作業流程。
二、植物菌質體檢疫分子資訊及檢測用PCR技術之建立與應用
植物菌質體 (phytoplasma)屬於Mollicutes綱之不具細胞壁 (cell wall-less) 的革蘭氏陽性 (Gram positive) 細菌,且為絕對寄生性的病原菌 (obligate parasite),侷限生長在植物韌皮部 (phloem) 中,是一種影響寄主植物生長之重要植物病原細菌,常造成簇葉 (witches’ broom)、花器綠化 (virescence) 葉化 (phyllody)、黃化 (yellowing) 及小葉 (little leaf) 等病徵,於自然界中主要由葉蟬 (leafhopper)、木蝨 (Psyllid) 等媒介昆蟲在植物間傳播。植物菌質體目前尚無法利用人工培養,因此其基因組成及生理生化特性仍無法做全盤深入之瞭解。全世界目前已發現之植物菌質體種類多達28群,可在數百種植物上造成1000多種病害。現今在植物菌質體之診斷或鑑定時,必須先進行16S rDNA解序及演化樹建構之繁瑣流程後方可確認植物菌質體之身分,所需時間也相當長,本計畫之目的在於針對檢疫上各群重要之植物菌質體,如aster yellows (16SrI group), X-disease (16SrIII group)等,利用 in silico RFLP技術建立檢疫用分子資訊。另利用質體鑑別性序列,針對分屬各群或亞群植物菌質體之地理或寄主菌系(strain)進行鑑別,開發新穎之診斷鑑定技術,完成並整合上述各階段快速與精確之診斷鑑定流程,以達到快速鑑別植物菌質體之目的,以提供進口種苗、種球、糧作與花卉等植物及其產品檢疫之有效偵檢技術,以有效防堵檢疫有害生物入侵,確保國內農業生產安全。
三、建立植物檢疫potyvirus之鑑定相關資料庫與標準鑑定流程
針對我國所公告的植物檢疫potyvirus收集相關資訊,建立植物檢疫potyvirus鑑定相關資料庫。對於欠缺檢測相關產品和文獻的植物檢疫potyvirus,則依據序列分析比對結果,設計Potyvirus屬廣效性引子對和該病毒的專一性引子對,供後續使用。必要時可從外國研究機構購買植物檢疫potyvirus乾燥材料,用來測試特定植物檢疫potyvirus的專一性引子對。建立一套植物檢疫potyvirus之標準鑑定流程,提供給植物檢疫單位參考使用。
四、具檢疫重要性之類病毒有害生物整合性診斷之研發與標準化流程之建立
本計畫擬執行:1.建構類病毒RNA基因體之cDNA質體。2.建立具高靈敏度之one step RT-PCR類病毒核酸偵測方法,以及分析其實用性、專一性、應用廣泛度。3.建立標準診斷鑑定流程。4.開發類病毒偵測之新技術以利於診斷。預期成果將達成:1.建立具檢疫重要性之共9種類病毒cDNA基因體資料庫。2.開發3種類病毒之核酸分子之快速診斷鑑定技術。3.完成建立2種類病毒類有害生物之標準檢驗鑑定流程。
Abstract: 1. Establishment of standard detection procedures for the plant post entry quarantine
This project is dedicated to devise detection methods of specified pests for the lawfully imported plants and plant materials during the isolated quarantine period. It is helpful in the routine work of quarantine by providing a standard operating procedure (SOP) for official staffs. The oriental cherry, rose (Rosa spp.) and grapevine (Vitis spp.) are considered to be more important crops based on the priority of plant species for post entry quarantine. Several items will be conducted this year: (1) collection of data and references associated with new pests for the oriental cherry, rose and grapevine; (2) inventory of diagnostic methods and references of specified pests for the oriental cherry, rose and grapevine; (3) drafting of standard detection procedures of the specified quarantine pests for the oriental cherry; (4) drafting of standard detection procedures of the specified quarantine pests for the rose; (5) drafting of standard detection procedures of the specified quarantine pests for the grapevine.
2. Establishment and Application of in silico RFLP Database and PCR-based Detection Techniques for Important Phytoplasmas
Phytoplasmas, former mycoplasma-like organisms (MLOs) can cause many yellows-type diseases. In Taiwan and other countries, there are more than 1000 different plant diseases are now known to be caused by over 800 phytoplasma species. Up to now, the phytopathogenic phytoplasmas still resist to be cultured in any available medium. It`s also very difficult to purify phytoplasmas from affected plants without the contamination of plant antigens. The biological and biochemical data of phytoplasmas are also next to nothing. In this proposed study, the in silico RFLP databases of 16Sr group I, III , XII phytoplasmas will be set up and the phytoplasma specific PCR primers based on the plasmid sequences will also be developed. The in silico RFLP analysis and PCR-based detection strategies thus developed will be useful for the differentiation of different groups, subgroups, species, or even strains or isolates of various important quarantine phytoplasmas. The detection system will also provide a useful tool for plant inspection and quarantine authorities and researchers to deal with phytoplasma diseases.
3. Development of an identification information database and the standard operation protocol for plant quarantine potyviruses
To establish an identification information database for plant quarantine potyviruses, we will collect and organize all required information. For the plant quarantine potyvirus lack of detection methods and related products, potyvirus broad-spectrum primer pairs and the virus-specific primer pairs will be designed according to the results of multiple sequence alignment. These primer pairs will be used in later detection tests. If it is necessary, the selected phytosanitary potyvirus dried material will be purchased from foreign research institutions, and then used to test the specificity of the virus-specific primer pairs. Finally, the standard operation protocol of plant quarantine potyviruses will be established and provided to the authorities of plant inspection and quarantine.
4. Development and SOP establishment of the integral diagnostic techniques for key quarantinable viroids
This specific aims of this current proposal are 1) Establishment of the cDNA bank for viroids; 2) Development of fast and accurant detection techniques for key quarantinable viroid-infected crops; 3) An integral SOP (standard of procedure) diagnosis for key quarantinable viroids. The first aim is attempting to construct and collect more quarantinable cDNA clones by recombinant DNA techniques and imported from overseas according to the literatures. Then, preparations of the viroid cDNAs will be used for the templates and postive controls during the development of detection techniques and future diagnosis. In the second aim, three different appraoches will be undertaken, including dot hybridization, RT-PCR/real-time PCR, and bioassay, to provide an multi-strategy in detection of viroids. The specificity and sensitivity of various probes corresponding to different viroids will be tested. Likewise, specific and general primers sets for various viroids will be designed for the detection the specific viroid and/or a group of viroids at the same time. Real-time PCR may also provide a quantitativeand more accurant result in detection. Specific tomotoe cultivars are emloyed to be an index of bioassay for different viroids for a minimial temporal and spatial need compared to certain parental infected crops of viroids.