檢測及檢疫技術之研發與應用

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計畫名稱: 檢測及檢疫技術之研發與應用
計畫主持人: 蔡佳欣
共同計畫主持人: 劉豐瑞;林鳳琪;黃巧雯;吳意真;謝廷芳;許如君;陳宗祺;鄭櫻慧;蘇俊峰;路光暉;周明儀;邱一中;陳珮臻;陳思翰;蔣國司;蕭旭峰;陳延禎;邱燕欣;王智立;阮明淑;江昭皚;林志鴻;陳美娥;葉文斌;洪挺軒;陳淑佩;李奇峰;劉弘一;林美賢;楊爵因;顏聖紘;柯俊成;陳文華;林慧玲;何琦琛;王文裕;陳啟予;許洞慶;李後峰;郭美華;張雅君;朱家慶;林宗岐;顏志恒;李永安
計畫編號: 109農科-8.4.3-子-C1
計畫主管機構: 行政院農業委員會
計畫執行機構: 行政院農業委員會農業試驗所植物病理組
全程計畫年: 2020
關鍵字: 檢疫整合型系統;疫情分析;檢疫害物;檢疫處理;檢測;檢疫;Quarantine integrated system ;epidemic situation analysis;Quarantine pest;Qarantine treatment;detection;quarantine
摘要: 一、植物及其產品輸出入風險分析、檢疫技術與程序之研發、改進與應用(一)邊境查驗檢出檢疫有害生物之整合鑑定及分析系統本專家系統主要在邀集國內鑑定專家進行樣品鑑定分析,強化我國進、出口農產品之病、蟲、蟎等有害生物,以為國內植物檢疫處理之依據。本(109)年度工作重點及預期效益包括,1.強化進出口農產品檢疫鑑定作業分層負責系統(如附表),可深化國內有害生物鑑定系統之功能。2. 開辦重要檢疫害物真菌及線蟲鑑定技術訓練,可提升基層檢疫人員鑑定能力,加強檢疫樣品處理能力急件定樣品之正確性。3. 制度化緊急檢疫樣品鑑定規劃與處理,可完整化整體檢疫鑑定系統之功能提升檢疫鑑定之效率。此系統將有利於國內檢疫鑑定系統完整化,並與國際系統調和一致化。本計畫檢疫對象包括植物病害之真菌、細菌、病毒及線蟲;植物之半翅目(蚜蟲、木蝨及葉蟬)、膜翅目、鱗翅目等害蟲及蟎類。 (二)建立檢疫重要性果實蠅診斷鑑定技術、疫情資訊及教育訓練系統本計畫擬建立的果實蠅物種診斷鑑定參考資訊,內容包含:1)臺灣地區檢防疫果實蠅名錄之修訂,2)國際上重要果實蠅害蟲疫情資訊及3)強化果實蠅診斷鑑定能力之教育訓練及所需素材。農業貿易諮商談判,常涉及國內外果實蠅物種疫區確認與否的問題,為避免談判中不必要的困擾,必須優先釐清國內外果實蠅害蟲分布現況。建立果實蠅之形態鑑定圖譜,可以直接提供診斷鑑定人員使用,以爭取時效上之先機。並規劃相關資料庫,提供學術、教學、農業及商業上之所需的數位化資訊。 (三)我國重要檢疫病毒類有害生物之國際疫情分析及檢測技術開發為維持我國有害生物清單中病毒類資料之準確性,我們將繼續檢視並修正病毒清單中100筆資料的學名與分類,同時增列重要植物檢疫病毒至有害生物清單。此外,我們也將蒐集感染番茄與番椒之番茄褐斑皺果病毒(<em>Tomato brown rugose fruit virus</em>, ToBRFV)相關資料,收集其病毒序列資料,設計ToBRFV專一性引子對,以兩階段的RT-PCR測試方法的準確性。必要時可從外國研究機構購買ToBRFV乾燥材料或核酸,用來測試ToBRFV專一性引子對的效果。將修正的病毒類有害生物清單,以及所研發之ToBRFV檢測方法,提供給植物檢疫單位參考使用。(四)具跨大陸入侵臺灣疑慮之鱗翅目害蟲之風險評估與鑑識秋行軍蟲(Spodoptera frugigperda,英名縮寫為FAW)為近年全球雜糧作物之重大害蟲。由於這種昆蟲快速地在2019年6月入侵我國,為了即早建立防治體系所需要之基礎知識,我們將利用生態棲位模擬(ecological niche modeling)與預測物種分布(predicted species distribution model)分析技術,推測此種蛾類在臺灣本島未來的擴張範圍。釐清秋行軍蟲與同屬物種與不同屬但形態上易混淆物種,以及所有在臺灣的作物上可出現的夜蛾總科昆蟲之鑑識方法,並藉由比較博物館標本典藏瞭解各地秋行軍蟲之表型差異。採集因應分子親緣關係與族群遺傳學所需之樣本,以利在未來持續監測臺灣島內族群遺傳結構之動態、抗藥性相關遺傳特質的變動,以及入侵來源。我們希望這個計畫的成果除了能夠協助防治之外,還能夠培訓新興有害生物之鑑識與診斷人才,發表相關科學成果,並提供政府、農民與試驗單位鑑識服務。 (五)無花果苗木之病毒及類病毒風險評估及其檢測技術研發與應用近年來,我國種植無花果的風氣相當盛行,栽培品種多自國外引進,業者持續向防檢局提出自未有核准輸入紀錄國家輸入無花果植株之申請,爰需對可感染無花果之病毒及類病毒等有害生物進行相關風險評估,以掌握其疫情及輸入風險,做為輸入風險評估之參考。另國內盛行的無花果栽培品種多來自美國及日本等FMD的發生地區,故引進FMV及其他病毒與類病毒的可能性極高。為了防止上述病毒及類病毒隨著無花果苗木引入台灣而影響產業,亟需針對FMD的相關病毒及類病毒研發診斷及檢測方法,用以篩檢進口無花果苗木,以及監測FMV在國內田間之發生情形。 (六)核准輸入植物清單與檢疫條件查詢系統維護與資料優化於109年計畫彙整現行核准輸入植物清單之植物學名之分類階層(門、科及屬)對應清單,並規劃將已核准輸入之植物之學名及其所有分類階層比對現行檢疫規定,使能夠精確檢索已核准輸入植物之檢疫條件,並優化檢疫條件之檢索功能,利於農產品進口業務管理與風險管控。 (七)我國重要檢疫真菌類及類病毒有害生物之國際疫情分析及檢測技術開發本計畫擬重新檢視「中華民國輸入植物或植物產品檢疫規定」之「乙、有條件輸入植物或植物產品之檢疫條件」的第十項盤點「有害生物清單」的所有類病毒名單,依發生地區、寄主範圍與疫情發生狀況進行風險分析,按其重要性加以分類並蒐集完整的國際疫情,開發快速檢疫方法,以提供港站或支援單位的檢疫人員有一致之標準可遵循,裨益檢疫工作。,另外針對「有害生物清單」真菌類中的42種病原真菌(依屬名英文字母排列順序,A~L),並根據目前的最新文獻報導及國內疫情,提供有害生物清單修正意見。  二、輸出入植物檢疫有害生物之診斷鑑定技術研發(一)小果番茄早年自國外引進國內種植,經業者與農民在生產與品種改良等技術持續努力下,使得國產小果番茄鮮果質優量豐,除供國內消費外,亦可輸銷國外。臺灣因屬東方果實蠅、瓜實蠅及南瓜實蠅等疫區,故欲外銷至非疫區國家須施行檢疫處理。本試驗係針對此三種果實蠅為殺蟲標的進行低溫冷藏處理試驗,以期研發檢疫處理方式突破檢疫障礙,尋求適合之外銷檢疫方法。(二)因應進口栽培介質及植株附帶栽培介質中檢出之重要植物檢疫線蟲有比例逐漸增加之趨勢,且為求有效預防高風險性外來種之入侵與散佈,本計劃針對葉芽線蟲屬線蟲之生物學與分子生物學相關資訊,以及國際間既存之快速分子檢測技術進行完整評估,開發符合我國檢疫及防疫需求之實用檢測流程與方法。本計畫開發葉芽線蟲屬內種間之PCR快速分子檢測技術,建立屬內重要檢疫線蟲之分子檢驗作業流程,以供未來檢疫人員快速鑑定相關檢疫線蟲使用。(三)本年度計畫將 持續由邊境攔截驗本中收集莖線蟲屬線蟲,並建立可以穩定培養食菌性莖線蟲的平台,利用培養出的族群增加邊境攔截以及本土莖線蟲屬的型態與分生資料庫,在這些基礎上嘗試開發可以區別不同食性莖線蟲種類的鑑別方法。因台灣地屬熱帶亞熱帶交界區,又因為高海拔地區也有許多蔬菜果樹作物,故溫帶地區輸入有害生物,在台建立族群的風險也不小。邊境檢疫是我們進口農產品的第一道防線,建立省時並且精確的檢測方式,可以節省許多人力以及倉儲成本,也有助加速進出口業務處理速度。食菌性莖線蟲因曾於邊境檢疫時,以及國內栽培介質中被檢出,也在過去的台灣田間調查中被報導存在,此類檢疫風險較小之線蟲因型態難與幾個重要檢疫種類區分,增加邊境檢疫的工作難度。108年度進行計畫過程已收集至少4個族群的莖線蟲屬族群,不只訓練學生的鑑定線蟲能力。收集與培養莖線蟲族群的過程,也讓我們看見這屬重要的經濟線蟲其種類許多,但非有嚴重危害的這些種類,相關資料非常少,建立型態與分生資料的過程,應有助於開發未來不僅限於這屬線蟲一個資料檢索與搜尋的系統。此資料庫建立之後隨之地搜尋比對系統建立,相信不只是對於防檢疫相關工作,對於學術界也是一個重要的工作。 三、植物檢疫疫病害蟲偵測鑑定資訊系統精進計畫為更新現有系統技 術架構以符合資安及使用者需求,並解決各分局登打資料承辦人登打資料不統一之情形。擬依據最新資安規範及使用者需求,辦理植物疫病害蟲偵測鑑定資訊系統精進計畫,以強化邊境有害生物鑑定及風險管理。 四、輻射檢疫處理技術在出口番石榴鮮果實之開發與應用     本計畫擬以番石榴為材料,測試東方果實蠅(<em>Bactrocera dorsalis</em>)在其內對輻射照射處理之耐受性,藉以測試得適用於番石榴輻射照射檢疫處理之劑量與流程,提供防檢局做為輸美番石榴檢疫處理之參考。若本計畫執行時程許可,擬再加入測試瓜實蠅(<em>B. cucurbitae</em>)及南瓜實蠅(<em>B. tau</em>)兩種果實蠅。 五、重要作物病蟲害診斷、檢測鑑定技術之研發及應用 (一)番茄系統性病害之鑑定與防治技術開發番茄為台灣重要果菜類蔬菜,病害種類眾多,目前雙生病毒感染引起的番茄捲葉病與黃化捲葉病主要以抗(耐)品種及媒介昆蟲粉蝨為主要防治對策,但調查發現雙生病毒在番茄上常見複合感染或基因重組,使得病毒變異大,引進抗病基因的番茄品種抗病效果逐年遞減。因粉蝨已適應台灣自然環境且雜食性強,要以藥劑控制粉蝨達到防治此病害的效果必須密集施藥,抗耐病品種培育仍是最有效且環保的方法,目前抗耐病品種篩選仍以置於溫網室中利用粉蝨媒介感染,篩選結果因粉蝨族群及傳毒效率而異,使實驗變數增大。而在細菌病害上,台灣田間可造成番茄萎凋的病原菌主要有青枯病菌(<em>Ralstonia solanacearum</em>)及莖腐病(<em>Pectobacterium carotovorum </em>subsp. <em>carotovorum</em>)。近年田間零星出現新興髓壞疽病(病菌<em>Pseudomonas viridiflava</em>、<em>P. mediterranea</em>)。萎凋型病害常造成番茄產業的大量損失,且無防治藥劑可推薦,因此本試驗擬調查番茄捲葉病病因之變化,了解抗病品種抗病能力下降的原因,並構築其農桿菌媒介接種系統,定量穩定篩選抗病品種,期能加速抗病品種育成,降低目前因防制粉蝨大量使用農藥所付出的經濟成本,並對番茄萎凋病原細菌建立田間防治技術,分別針對各病菌開發適合的生物及藥劑資材以供農友實際防治細菌性病害。 (二)斑翅果蠅在台灣分布探究及其與相近種間分子鑑定研究 本計畫擬調查及採集國內草莓及葡萄園中果蠅種類,並與國外研究單位取得斑翅果 蠅樣本,進行快速鑑定技術開發,使鑑定工作能達到簡易、快速的目標,並釐清台 灣是否有斑翅果蠅的分布危害。計畫目標著重在 1. 調查國內草莓及葡萄園分布的 果蠅種類。2. 建立重要檢防疫害蟲斑翅果蠅之快速鑑定技術及標準流程。3. 利用 生物技術建立快速、簡易的台灣常見果蠅標本鑑定技術 ,使能實際應用於害蟲檢疫。重要工作項目如下:1. 主要草莓及葡萄產地之果蠅類蟲相調查及採集;2. 快速 診斷鑑定技術標準流程之建立。預期計畫完成後,可提供下列具體貢獻,有助於我 國檢疫體系之建立:1. 協助國內檢防疫單位進行農產品之斑翅果蠅害蟲樣本鑑定 ;2. 建立分子快速診斷鑑定技術標準流程,可作為海關檢疫之用;3. 建立重要檢 防疫重要斑翅果蠅及其相近果蠅種類標本之快速鑑定資料庫,提供海關檢疫人員進 行檢疫業務。 (三)開發農業害蟲之診斷鑑定技術—毛螢金花蟲屬 (3/3)之形態診斷鑑定及危害影響評估金花蟲科(Chrysomelidae)為為鞘翅目(Coleoptera)中種類最多樣的一個科,台灣已記錄的種類超過六百種;往昔的研究都只著重於新種及新紀錄的發表,且採集地點相當零散。本研究為現代化分類學研究的開端,針對毛螢金花蟲屬Pyrrhalta 作分類修訂,以全面性及定期性的方式採集標本,探討屬級的分類地位,詳細研究每個種類的診斷特徵、成蟲出現時期、寄主植物及地理分布,並重新檢查已知種的模式標本,以建立一個扎實且有系統性的診斷鑑定研究。 六、運用智能化排程分析高通量小分子RNA比對重要番茄種子病原技術建立本計畫擬針對進出口茄科種子,以小分子RNA作為檢定標定,釐清病原相,初步以LINUX系統架構CLC NGS系統,建庫5種茄科寄主小分子RNA資料庫,並建構小分子RNA解序試驗流程。建立高通量分析運算計算機計算流程(analysis work flow) (並搭載CLC NGS分析平台),建立番茄及甜椒重要茄科種子的小分子RNA檢定病原技術,批次收集進出口種子,找出潛隱地種傳病原。 七、重要外銷蘭花病蟲害整合性防治技術開發臺灣外銷蘭花主要有蝴蝶蘭、蕙蘭與嘉德麗雅蘭等,在面對國際化的出口競爭壓力中,如何提升臺灣蘭花出口的育成率,是增加競爭優勢的可行辦法之一。然而蘭花在栽培過程中,病蟲害的發生是造成育成率下降的主要原因之一。過去常以單一病蟲害的發生,研擬可行的病蟲害管理策略,雖然可以對症下藥,但也常導致栽培業者在面對不同病蟲害發生時,因誤診或不瞭解病蟲害的發生生態,非但無法對症下藥,甚至延誤預防病蟲害發生的最佳時機。另外在出口檢疫時,病蟲害的發生,特別是害蟲的存在,往往導致出口障礙與出貨品質不佳。本研究擬針對蝴蝶蘭、蕙蘭與嘉德麗雅蘭常見病蟲害,從清潔種苗制度、園區病蟲害管理作為與病蟲害發生時的防治作法,建立病蟲害的整合式防治技術,期能於田間實際推廣使用。1. The development, improvement and application of risk analysis, quarantine technology and procedures for import and export plants and plant products(1)Integrated system for identification and analysis of quarantine pests intercepted by border inspectionThe goal of the integrated system mainly invites and integrates domestic pest identification experts to provide scientific support for the identification of plant pests, including plant diseases, insects, mites, and other pests on imported and exported agricultural products. The main research topics and expected benefits of this year include 1. to strengthen the expert identification system to deepen the function of domestic quarantine identification of pests of plant pathogens, insects, and mites; 2. to set up the identification training course of fungi and nematodes for the quarantine officers in each branch bureau to enhance the ability of quarantine identification of pests and assure the safety of domestic agricultural products; 3. to systemize the standard procedure for an emergency event of quarantine plant pest identification to enhance the function of the efficiency of quarantine pest identification. This system will facilitate the completion of the domestic quarantine identification system and reconcile consistency with the international system. The quarantine objects include fungi, bacteria, viruses, and nematodes of plant diseases; plant pests of Hemipteran (aphids, thrips, scale insects, psyllids, and leafhopper, etc.), Coleopteran, Lepidopteran, and Hymenopteran insects and mites.(2)Establishment of diagnostic technique, epidemic information and education training systems of fruit flies (<em>Diptera: Tephritidae</em>) with quarantine importance.This project aims to establish a more concise and accurate fruit fly information and references database, including 1) Revising fruit fly species checklist in Taiwan; 2) Collecting international fruit fly epidemic data; 3) Strengthening fruit fly diagnosis ability and providing pest species information. Confirming fruit fly species of quarantine importance is usually an important issue in agricultural trade negotiations. To prevent unnecessary difficulty during quarantine inspection and trade negotiations, setting up the species diagnostic atlas and species, distribution and host information will be useful, not only for the quarantine and trade negotiation purpose, but also for educational or agricultural perspectives. (3)International outbreak analysis and development of detection methods for important quarantine virusesIn order to maintain the accuracy of our quarantine pest list, we will continue investigating and correcting the classification and scientific name of 100 listed viruses and also add new important quarantine viruses to the pest list. In addition, we will collect the information of seed-transmitted <em>Tomato brown rugose fruit virus</em> (ToBRFV) as targets. ToBRFV-specific primers will be designed based on viral sequence data. The detection accuracy will be tested by two step RT-PCR. If it is necessary, ToBRFV dried material will be purchased from foreign research institutions, and then used to test the specificity of our primer pairs. Finally, the revised quarantine virus list and the detection method for ToBRFV will be provided to the authorities of plant inspection and quarantine.(4)Assessments and identifications of the lepidopterous pests with cross-continental invasion risk for TaiwanFall armyworm (<em>Spodoptera frugiperda</em>, FAW) is known as one of the most severe and rapidly increasing risks for more than 370 crops in the world. Due to the fact that FAW suddenly invaded Taiwan in June, 2019, establishment of the following knowledge bases gain priority for immediate and future planning for management and control of this pest. We will first simulate the potential geographical expansion and spread of FAW in Taiwan by employing ecological niche modeling (ENM) and predicted species distribution model (PSD). We then will clarify the identification and diagnosis of FAW and other species associated with the crops in Taiwan by using morphological characters, including those of adult, larval and pupal stages. We will also collect samples of FAW from the surrounding countries being invaded by this insect in order for better comparison in morphology as well as for future uses in other studies, such as population genetics, genetic basis of resistance, and the geographical source(s) of the invading population. We wish the results of this project can provide information for future IPM, train people for fast identification, publish scientific reports and offer identification services for government agency and farmers. (5)Pest risk assessment and development detection techniques for viruses and viroids for fig seedlings (<em>Ficus carica</em>)In recent years, the cultivation of fig trees is very popular in Taiwan. Many fig cultivars are imported from abroad. Traders frequently submit applications to the Bureau of Animal and Plant Health Inspection and Quarantine for importing fig plants from countries that have not been approved. Risk assessments for the FMD-associated viruses and viroids should be carried out to grasp their epidemics and imported risks as a reference for input risk assessment. Besides, most of the domestically cultivated fig cultivars come from the prevalent areas of FMD, such as the United States and Japan, thus the possibility of introducing FMV and other viruses and viroids is extremely high. Development of diagnosis and detection techniques for inspecting FMD-causing viruses and viroids is urgently needed to prevent the epidemics of FMD in Taiwan. The methods will be used to inspect FMD-associated viruses and viroids in imported seedling shoots and monitor the occurrence of FMV in field fig trees. (6)The Maintenance and Data Optimization of Search System of Approved Plant List for Importation and its Quarantine RequirementsIt is planned to consolidate the corresponding hierarchical classification list of botanical names of the currently approved import plant list in 2020, and plan to compare it with the current quarantine regulations. This makes it possible to accurately search the quarantine conditions of approved imported plants, and optimizes the query system function of quarantine conditions, which is beneficial to the management and risk control of agricultural product import. (7)International epidemic situation analysis and development of diagnostic technologies for important quarantine plant pests in fungi and viroidsThis project is dedicated to review the quarantine pests in fungi and viroids based on their current epidemic situations and propose a new and update list for quarantine pests. For the newly emerged fungal and viroid pathogens in the world, the data associated with their biological and pathological information will be collected and provided for the risk assessment in quarantine. Besides, the detection methods of new fungi and viroids will be also inventoried and gathered to develop and establish a standard quarantine procedure and avoid the invading of important pests.   2. Development of the identification techniques for pests on the imported and exported plants(1)The small-size tomato has been introduced many years ago.  In the cooperation between domestic seed producers and the cultured farmers, the small-size tomato fruit are produced with good quality. In addition to domestic consumption, it can also be exported. However, Taiwan is an epidemic area of fruit flies such as oriental fruit flies, melon flies and pumpkin fruit flies. Therefore, exporting the tomato fruit to non-epidemic areas, it is necessary to have a quarantine treatment to disinfest the possibility fruit flies. The study herein is aimed at the low temperature treatment to disinfest the three species of fruit flies possibly infected with tomato fruit. And then establish the quarantine treatment for a optimal method to export the tomato fruits.(2)Due to the increasing detection of important plant parasitic nematodes in the inspected importing planting materials and in the planting materials attached to the importing plants, the importance of applying effective and efficient inspection and identification methods at quarantine have been elevated. To prevent the entrance of high risk quarantine nematode pests, the goal of this project is to evaluate existing detections assays and develop suitable protocol for the detection of the important nematodes in the <em>Aphelenchoides </em>genus. This project first evaluates the biological aspects, the molecular information and the existing DNA based molecular detection assays targeting the quarantine nematodes, and then develop effective and efficient molecular assays and optimize the best available assay to meet our quarantine inspection needs. Also, this project develops molecular detection techniques, providing quarantine inspection tool for rapid identification of the important quarantine nematodes.(3)This project will help to identify quarantine samples sent by the BAPHIQ, in the samples, all <em>Ditylenchus</em> spp. would be collected, the morphometric data and the basic 18S rDNA data will also be collected as well to help to identify them to species. In the lab, several fungal strain will be used to cultural the intercepted nematodes, any samples containing more than one nematodes that have the potential to be <em>D. dipsaci </em>will also be challenged to alfalfa. The morphometric, molecular sequences data, as well as the potential feeding hosts, will be compared and contrasted in between the Ditylenchus populations, in hope of establishing a database that would be helpful to distinguish the hypha-feeding <em>Ditylenchus</em> spp. group from the important quarantine species. Taiwan is situated on tropical and subtropical area, the high altitude regions were planted with vegetable and fruit tree crops, agricultural products imported from the temperate region still have potential threats to bring in harmful pests that could establish their population in Taiwan. Precise and efficient technology methods should be developed to help lower the input of quarantine works. Hypha-feeding <em>Ditylenchus</em> spp. were found in the intercepted samples as well as the routine survey of Taiwan local crops. These <em>Ditylenchus</em> spp. had low quarantine risks but are very hard to tell apart from the quarantine species based on their morphology, and usually have very little differences in their conservative nuclear regions. Morphometric and molecular data of four hypha-feeding <em>Ditylenchus</em> sp. populations were collected during the 108 project, and in the process, we found very few literatures could be found for these group of nematodes. Establishing a database for the <em>Ditylenchus</em> spp. both from intercepted and local origin will help to clarify criteria for the database. In the future, a quick indexing system could be established based on this information. The results are very helpful not only for quarantine works but the scientific research groups.  3. Improvement of Plant Quarantine Disease and Pest Detection Information SystemFor updating the technical structure of the existing system to meet the needs of information security and users, and to resolve the inconsistencies in the registration data of the log-in data organizers of the branches. According to the latest information security regulations and user needs, an advanced plan for the Plant Disease and Pest Detection Quarantine Information System will be processed to strengthen the identification and risk management of pests at the border. 4. Development and application of irradiation quarantine treatment technique on exporting guava fruitThis project aims to use guava as a material to test the tolerance of the oriental fruit fly (<em>Bactrocera dorsalis</em>) to radiation treatment to obtain the optimal dose and treatment procedures that is suitable for guava phytosanitary treatment. These results achieved will be provided to the Bureau of Animal and Plant Health Inspection and Quarantine as a reference for the quarantine treatment of guava exported to the United States. If there is still more time, it is planned to add two other fruit flies, i.e., <em>B. cucurbitae</em> and <em>B. tau</em>, for the testing. 5. Development and application of diagnosis and identification of crop diseases and pests (1)Development of identification and control technology of tomato systemic disease<strong>   </strong> Tomato crop is one of the important vegetable in Taiwan. Many diseases of tomato could be found in the field. At present, tomato leaf curl and yellow leaf curl caused by geminivirues infection are mainly controlled by resistant (tolerant) varieties and vector insect whitefly. However, the investigation found that the commonly mixed infection or genetic recombination of geminiviruses in tomato, which makes the virus with high mutation rate and the disease resistance of tomato varieties with disease resistance gene is decreasing year by year. Because whitefly has adapted to Taiwan's natural environment and is omnivorous, it is difficult to control the disease by using pesticides. Currently, screening for resistant varieties is still placed plant in a net house and infected with whitefly. The screening results varied with the population of the whitefly and the efficiency of the transmission, which increased the experimental variables.     In addition, many bacterial diseases of tomato could be found in the field. The bacterial pathogen included <em>Ralstonia solanacearum</em>, <em>Pectobacterium carotovorum </em>subsp. <em>carotovorum</em>, <em>Pseudomonas viridiflava</em>, and<em> P. mediterranea </em>could result in wilt symptom on tomato and caused large losses and few control agents are recommended.    This study is to perform quantitatively and stably screen resistant varieties, which can accelerate the breeding of resistant varieties and then reduce the use of pesticides for whitefly management. Moreover, for control pathogen causing bacterial wilt symptoms, the bio-control agent and agrochemicals will be will be established. (2)The development of rapid diagnosis techniques and distribution survey of spotted wing drosophila (<em>Drosophila suzukii</em>) in TaiwanThis project will to survey and collect fruit fly species in strawberry orchards and vineyards, obtain specimens of <em>Drosophila suzukii</em> with foreign research institutes. Use these specimens for rapid identification of technical developments to make identification easier and faster. It also clarified whether there is a distribution of <em>Drosophila suzukii</em> in Taiwan. The project aims to focus on the following items: 1. Investigation of <em>Drosophila</em> species distributed in strawberry orchards and vineyards. 2. Establish a rapid identification technique and standard operation procedure (SOP) for important pests (<em>Drosophila suzukii</em> , spotted wing drosophila). 3. Establish a rapid and simple identification technique for common <em>Drosophila</em> specimens in Taiwan to enable practical application to pest quarantine. Important work items are as follows: 1. Investigation and collection of <em>Drosophila</em> species in strawberry and grape orchards; 2. Establishment of rapid diagnostic and identification technical standard operation procedure. After the completion of the project, we can provide the following specific contributions to help establish the quarantine system in Taiwan: 1. Assist domestic inspection and quarantine organization in the identification of <em>Drosophila suzukii </em>on agricultural products; 2. Establish a standard operation procedure for the rapid diagnosis and identification technique. It can be used as a quarantine for customs; 3. Establish a rapid identification database for important specimens of <em>Drosophila suzukii</em> and its similar species, and provide customs quarantine operations. (3)Identification of the insect pests of crops: the genus <em>Pyrrhalta</em> Joannia (3/3)Chrysomelidae is one of the most diverse families of Coleoptera. More than six hundred species have been described or recorded from Taiwan. However, most of taxonomic studies on chrysomelids were restricted to short descriptions of new species or announcement of new records. The collecting sites of the studied specimens were always scattered and the number of the studied specimens were fewer as expected. This research is dedicated to the modern taxonomic studies on Chrysomelidae of Taiwan. The species belonging to the genus Pyrrhalta are revised comprehensively. Specimens will be collected intensively and extensively.  Diagnostic characters will be searched throughout all members of the genus. Adult appearance, host plants, and the geographic distribution of each species will be major components of this research. The type specimen of the known species will be reexamined. A sound classification of Chrysomelidae in Taiwan is expected to be established. 6. The establishment of high through-put sequencing workflow to detect pathogens of tomato seedsThis plan intends to use small molecule RNA(sRNAs) as a detect target to clarify the consortia and identities of pathogens in import and export of tomato seeds. Specifically, a CLC NGS analysis system based on LINUX will be built up. Small RNAs from 5 tomato samples will be extracted and used for sequencing. The raw data of these sequences will be processed using a serial of bioinformatics tools and programs. Then a analysis workflow for deconstructing sequencing data into plant pathogen identities and a sRNA database of tomato host plant will be set up. Along with hardware updating with high-performance computing processors, this workflow will help us to establish sRNA detection procedures for tomato pathogens. Moreover, batched of import and export tomato seeds will be collected and tested. And hopefully, it could help to detect hidden seed-borne pathogens in practice. 7. Development of integrated control techniques of important diseases and pests for exported orchidsTaiwan's mainly export orchids are <em>Phalaenopsis</em>, <em>Cymbidium</em> and <em>Cattleya</em> and so on. Those orchids have export pressures from international competition, and enhancement of yield and quality is one of the feasible ways to increase competitive advantage. However, during the cultivation of orchids, the occurrence of pests and diseases is one of the main reasons for the decline of yield and quality. In the past, the development of a viable pest management strategy was following specific single disease or pest. But, practitioners may misunderstand or don't know about the occurrence of pest and disease ecology when they faced with different diseases and pests. This not only failed to remedy the problem but also delayed the best time to prevent pests. In addition, the occurrence of pests and diseases, especially the presence of pests, often leads to export barriers and poor quality of delivery at the time of export quarantine. Focusing on the perspective of clean seedling system and pest management in the greenhouse, this study will aim at development an integrated pest management in common pests and diseases of <em>Phalaenopsis</em>, <em>Cymbidium</em> and <em>Cattleya</em>. 
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