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|標題:||Development of Management Technology for the Control of New Plant Diseases and Other Pests
|作者:||林益昇, 楊恩誠,陳家鐘,陳美娥,林長平,齊心,張誠,路光暉||關鍵字:||應用研究;Root Rot/Vine Decline of Muskmelon;植物保護類;洋香瓜黑點根腐病;胡瓜萎凋病;東方果實蠅;瓜實蠅;克蠅;雙性基因;卵黃原蛋白基因啟動子;釋放攜帶致死基因昆蟲作用機制;生命表;化學訊息物;嫁接防治;梨衰弱病;植物菌質體;中國梨木蝨;黔梨木蝨;蛋白質水解物;微膠囊;反向核酸聚合連鎖反應;轉殖蠅;四環素調控系統;致死基因;整合性防治;氣相層析-觸角電圖;無病原性尖鐮胞菌;Fusarium Wilt of Cucumber;Oriental Fruit Fly (Bactrocera dorsalis Hendel);Bactrocera cucurbitae;Cuelure;Doublesex Gene;Vitellogenin Gene Promoter;Release of Insects Carrying a Dominant Lethal (RIDL);Life Table;Semiochemical;Grafting Control;Pear Decline;Phytoplasma;Cacopsylla chinensis;Cacopsylla qianli;Protein Hydrolysates;Microcapsule;Inverse PCR;Transgenic Fly;Tetracycline Regulation System;Lethal Gene;Integrated Control;GC-EAD;nonpathogenic Fusarium oxysporum||摘要:||
1.瓜類重要作物病害之防治：(a)洋香瓜黑點根腐病之生態與嫁接防治：The past experimental results showed squash rootstock could be used to reduce the incidence of the root rot/vine decline of muskmelon in field. However, the squash was also the host of Monosporascus cannonballus in field, so the mechanism of tolerance of rootstock to M. cannonballus will be studied further.(b)胡瓜萎凋病之生物防治：The GUS or GFP reporter genes are being used to observe the infection, colonization and distribution of both Fusarium oxysporum f. sp. cucumerinum and nonpathogenic F. oxysporum in plant tissues. Otherwise, the strains of nonpathogenic F. oxysporum which had been tested their control ability against Fusarium wilt at greenhouse are going to be retested in field.2.台灣梨樹梨衰弱病之病原與其媒介昆蟲之探討：In 1994, pears with decline symptoms similar to the slow decline form of pear decline (pear decline-Taiwan, PDTW) were observed in orchards of Asian pear (Pyrus pyrifolia (Burm. f.) Nakai) in Dungshr and Heping. PCR that can specifically amplify the conserved 16S rDNA sequence of PDTW phytoplasma was conducted to detect the presence of PDTW phytoplasma in plants and vectors. According to the analyses of the 16S rDNA and 16S-23S rDNA spacer sequence, the PD-like disease was confirmed to be caused by phytoplasma closely related to those of the apple proliferation group. In this work, PCR was also applied in the study of vectorship. On the basis of rDNA sequence of PDTW phytoplasma, two specific primer pairs APf2/ L1n and APf3/ rPD1s were designed for the detection of the agent in insect vector. Two species of pear psyllas Cacopsylls qianli and C. chinensis were discovered in 1994 and 2002, respectively. PCR-based detection was applied in the study of vectorship and the result implied C. qianli is a candidate of the vector of PDTW. Pear decline phytoplasma was observed in pear leaf vein and two insect vectors by transmission electron microscopy. Based on the sequence analyses, phytoplasmas of two different 16S rDNA groups, 16SrII and 16SrX, were revealed to be associated with PDTW-affected pear trees and carried by C. chinensis. Specific primers IIPf1/ IIPr1 along with IIPf2/ IIPr1 for PDTWII phytoplasma, and primers fPD2/ rPDs1 along with APf3/ rPDs1 for PDTW phytoplasma had been applied in semi-nested PCR. The 16S rDNA sequences of PDTW and PDTWII phytoplasmas were amplified by PCR from genomic DNA of Cacopsylla chinensis in Jianshih, Hsinchu county. The pear trees in Jianshih began to show the symptom of redden leaves in 2006. 16S rDNA sequences of PDTW and PDTWII phytoplasmas were amplified with PCR from genomic DNA of diseased pear tree in Jianshih and were identical to those sequences obtained from the phytoplasmas carried by C. chinensis. 3.瓜果實蠅誘引劑之新劑型開發II：The major goal of this study is to improve the performance of protein hydrolysate and Cue-Lure so that these two substances may increase their efficacy to attract female fruit fly and melon fly . The methods may include the adjustment of pH value of the protein hydrolysate at 8-9. The preservative will be used to decrease the degradation speed of protein hydrolysate. The softener will also be used to keep protein moisted to maintain its effect. The reports of chemical decomposition mechanism of Cue-Lure will be reviewed. Possible stabilizers for Cue-Lure will be added to slow down the decomposition rate. The improved protein hydrolysate and Cue-Lure will be tested on their bio-efficacy in the field as the forms of aqueous paste and oily paste. According to the test results, the formulations will be modified for efficacy improvement.4.評估雌性雙性基因干擾現象在轉殖東方果實蠅個体及族群中之穩定性：The goal of this project is to develop a useful method to control oriental fruit fly (Bactrocera dorsalis Hendel), a pest leads to a huge economic loss every year in Taiwan. Recent years, many new strategies for pest management have proposed by researchers around the world. Since both male and female doublesex proteins (DSXs) are transcriptional factors, they regulate many sex-specific gene expressions, for example yolk protein gene, and control the development of fly. In this project, we determined the gene structure of Bactrocera dorsalis doublesex (dsx); found out the physiological function of dsx through RNA interference (RNAi); and created a transgenic oriental fruit fly line that expresses dsxf dsRNA in last year. Our preliminary data point out the internal expressed dsxf dsRNA can inhibit the normal expression level of yolk protein gene, and results in the significant reduce of offspring number of transgenic oriental fruit flies. This result indicates the introduced dsxf dsRNA molecules can effectively disrupt the normal development of female flies. In this coming year, we will focus on the determination of doublesex RNAi stability in individual and fly population. We plan to use invert PCR technique to determine the insertion position on chromosome. Subsequently, we will develop a rapid PCR assay to study the stability of introduced gene fragment in individual flies and populations.5.應用基因轉殖防治東方果實蠅之技術開發：The aim of this study is to develop a strategy using transgenic technique to control the oriental fruit fly Bactrocera dorsalis (Hendel). In this continuous study, we will keep work on the following objects: (1) finish the activity analysis of the yolk protein promoter: A yolk protein promoter-driven EGFP (an enhanced green florescent protein, as an indicator of gene expression) was transformed onto B. dorsalis, and the EGFP expression pattern in the fly will be analysed using RT-PCR or western blot; (2) start to construct the tetracycline regulatory system: Nased on the results of the first item, we will start to construct the regulatory system that is able to conditionally control the expression of a lethal gene in females; and (3) evaluate the efficiency of this transgenic population on controlling wild type oriental fruit fly, including stability of the transgene inherity, mating competition and survivability of the transgene fly.6.以生命表為基礎之瓜實蠅整合性防治：Because Bactrocera cucurbitae attacks a variety of fruits of cucurbits and non-cucurbit hosts in Taiwan, the effect of host species on the development and reproduction needs to be investigated. I propose to study the life table of B. cucurbiate on spongy gourd under the field conditions. To collect the population dynamics for many years in field, I plan to continue the sampling of field population. In this year, I will continue the experiment on the age-specific response of male melon fly to the cue lure. Life table is the major scientific basis of ecological pest management. Based on the life tables collected in the laboratory and in the field, we can then recognize the potential of population growth of B. cucurbitae on different hosts. This knowledge is also useful for the understanding of the variation in life expectancy. Only then, an economical and effective control strategy can be planed based on pest ecology and computer simulation. However, because life table varies with host species and environmental conditions, it is necessary to carry out this project for many years in order to collect life tables for application.7.雌性東方果實蠅誘引物質之開發研究：The oriental fruit fly is the most singnificant fruit pest in the tropical area. The host range is more than 150 species over the world, and more than 50 species in Taiwan. The main method of oriental fruit fly managemet is . the earlist male attractant is made by methyl eugenol. And protein bait is developed as a food attractant. In addition to these two attractants, female attractant is the method with the most direct and study potential. It is species specific to attract and capture females, but avoiding killing non-target insects. This project researches the volatile of the host plant-guava and the non-host plant-Garcinia dulcis by combining chemical analyzing and neurophysiology techniques. In order to find out the chemical that female locates host plants, safer and more effective attractant is developed for reduceing the economic loss caused by the oriental fruit fly.This study tests the attractive chemicals by four steps: first, collecting the plant votalite; second, identified by GC-MS ; third, testing the neuron reaction by GC-EAD; last, behavioral test.Controling the oriental fruit fly by attractants is not only reducing the usage of pesticides and avoiding the problem of pesticide residue but also decreasing the ecological impact of local environment.Comparing to the methyl eugenol, the female attractant is more efficient in controlling. Both of methyl eugenol and the female attractant could be exercised in the population detection simultaneously.
1. 瓜類重要作物病害之防治： (1) 洋香瓜黑點根腐病之生態與嫁接防治：本研究先前發現嫁接南瓜根砧可於田間防治洋香瓜黑點根腐病，但供試根砧植株亦為本病原菌的田間寄主。因此本年度將進一步探討供試根砧的耐病機制。 (2) 胡瓜萎凋病之生物防治：本研究利用Gus/GFP gene fusion system來觀察胡瓜萎凋病菌之侵染過程及無病原性尖鐮胞菌的可能防治機制與兩者在植物中之分佈位置。此外，並嘗試進行田間防治試驗，確認所篩選之無病原性鐮胞菌對胡瓜萎凋病的防治效果。 2. 台灣梨樹梨衰弱病之病原與其媒介昆蟲之探討：探討目前嚴重發生之台灣梨樹衰弱病之病因，釐清植物菌質體與此病害病徵發展之關係，並探討病原菌族群消長與季節性之關係。完成田間媒介昆蟲之PCR偵測，以利進行發病生態之研究，並進一步提供相關防治方法之參考。監測媒介昆蟲攜菌情形、蟲相擴散、田間梨樹植株PCR偵測及發病情形之相關性。完成罹病梨樹組織及媒介昆蟲之TEM鏡檢，獲得台灣梨衰弱病原菌質體之電顯切片證據，並檢視病原菌質體之形態構造與所含相對濃度及其增殖情形。協助昆蟲專家於其進行傳菌試驗時梨株及蟲體之PCR檢測，確定媒介昆蟲傳菌能力與發病條件。對台灣地區梨樹及進口之梨接穗與害蟲提供快速、簡便、精確之診斷及鑑定工具，並進行防疫及檢疫工作之偵檢策略之制定。完成當年田間罹病株採樣與媒介昆蟲DNA抽取及病原菌質體PCR檢測，並完成當年植株發病生態之調查。完成媒介昆蟲與植物組織之超薄切片製作、以穿透式電子顯微鏡(TEM)鏡檢組織內病原菌質體之存在情形。完成台灣梨衰弱病PCR專一性引子對靈敏度之測定，並擬定植株與媒介昆蟲病原菌質體檢測技術如multiplex PCR、nested PCR、RFLP-PCR及booster PCR等之檢測流程。配合real-time PCR檢測技術，監測複合感染之病株及媒介昆蟲體內第十群(PDTW)與第二群(PDTWII)梨衰弱病菌質體之相對濃度。持續監測單一及複合感染之罹病株在病徵表現上之差異。完成台灣梨樹梨衰弱病植株與其媒介昆蟲之檢測流程及防治策略之制定。針對台灣梨衰弱病病害之病原與媒介昆蟲作深入之研究探討，配合分子檢測技術如real-time PCR、multiplex PCR、nested PCR、RFLP-PCR及booster PCR等，發展快速靈敏之檢測方式，並針對各個重要梨產區之單一及複合感染的梨株採樣進行檢測，同時也有利於進口接穗與媒介昆蟲之防疫檢疫篩檢。監控台灣梨衰弱病高風險地區之媒介昆蟲傳播梨衰弱病與梨株發病之情形，將研究成果應用於田間媒介昆蟲防治策略之擬定。持續進行台灣梨樹衰弱病發病生態之研究，以利台灣梨樹衰弱病之檢測流程及防治策略之擬定。 3. 瓜果實蠅誘引劑之新劑型開發II：本計畫目標以改善目前市售之賜諾殺與及蛋白質水解物於田間的有效範圍及延長其藥效，以改善防治瓜果實蠅的效果。其中添加緩衝溶液調整酸鹼值在8-9之間，再加入防腐劑以減緩其腐敗之速度，加入軟化劑以延長藥效及耐陽光曝曬。另ㄧ方面，將克蠅易水解的缺點列入研究重點之ㄧ，將了解克蠅分解的化學途徑，尋找適合克蠅的保護劑，使克蠅之化學安定性達到改善，減緩水解速率。將上述改善後之成品進行生物性試驗及化學物理安定性測試。並將持續開發可誘引雄、雌蟲之水性、油性膏劑的有效配方，由於此部份需要更多的資訊，在本年度計畫中將持續蒐集資料及藥品，進行生物性試驗，篩選最佳之劑型配方。 4. 評估雌性雙性基因干擾現象在轉殖東方果實蠅個體及族群中之穩定性：發展出有效的抑雌方法，控制東方果實蠅(Bactrocera dorsalis Hendel)的危害是本計畫研究之主要目標。由於近年來對昆蟲性別控制機制之研究獲得許多進展，基於這些昆蟲生理資料之累積，國際上也逐漸探討發展新的蟲害防治策略之可行性。雌性和雄性雙性蛋白都是轉錄控制因子，能夠決定與性別相關蛋白之表現與否。本研究計畫用雙性doublesex (dsx)基因為實驗切入點，先決定東方果實蠅雙性基因之結構，接著以核醣核酸干擾(RNA interference)確証了dsx對果實蠅雌性發育之影晌，其實際參與了東方果實蠅生殖發育之調控。由於本實驗研究之主要目標在於探尋控制雌蠅發育之方法，因而配合研發中之果實蠅轉殖技術，計畫針對東方果實蠅的『性別專一性』防治策略。在去年計畫中特別針對轉殖東方果實蠅體內dsx dsRNA之表現及其對雌蟲生育能力的干擾能力加以評估。實驗結果指出dsxf RNAi轉殖東方果實蠅G1子代數目都較正常東方果實蠅為少，此一子代減少共現象顯示東方果實蠅生育的確受到外源doublesex dsRNA之影晌。此外，以東方果實蠅dsxf RNAi轉殖品系，進行原卵黃蛋白(yolk protein, yp)之表現分析，再度証實內生性雙性基因dsRNA能干擾東方果實蠅雙性蛋白之正常功能，進而造成生育機制之混亂。此法能藉由導入外源dsx dsRNA之作用造成子代數目減少，若能再進一步確認植入dsx基因之穩定性及雄性轉殖蠅的正常生理功能，將可發展出一個具有創新性和對東方果實蠅有專一性的抑雌防治方法。今年計畫中將主要針對轉殖東方果實蠅個體體內及族群中dsx dsRNA之表現及其對雌蟲生育能力的干擾能力穩定性加以評估。先用反向核酸聚合反應(Inverse PCR)決定dsxf RNAi基因片段在轉殖東方果實蠅染色體上的扦入位置；再經由特定PCR實驗確認植入dsxf干擾基因之穩定性或非轉移性。 5. 應用基因轉殖防治東方果實蠅之技術開發：本期計畫係延續前期計畫，繼續執行以下各項內容： (1) 完成啟動子(promoter)轉殖與活性專一性測試。完成以卵黃原蛋白基因啟動子在蟲體上之表現特性分析； (2) 依據第一項之分析結果構築致死基因轉殖載體並建立基因轉殖蟲群。以scorpin基因為致死基因，將之構築於Tet-off gene expression system並同時建立分別具有此兩系統的個體。 (3) 蟲體轉殖效果之測試與評估。將所建立成功之果實蠅品系，於實驗室或溫室中進行誘發試驗，評估致死基因表現情形與其殺蟲效果。 6. 以生命表為基礎之瓜實蠅整合性防治：由於瓜實蠅能危害本省多種瓜類與非瓜類之寄主植物，必須研究各種寄主植物種類對瓜實蠅之發育與繁殖之影響。本年度擬繼續於實驗室與田間研究瓜實蠅在絲瓜上之田間生命表。為收集瓜實蠅族群之多年田間動態，擬繼續調查田間之族群動態。本年度亦擬繼續試驗不同年齡成蟲對誘引劑的反應。生命表研究為生態蟲害防治之科學基礎。依據瓜實蠅之室內生命表與田間生命表可了解瓜實蠅之族群在不同寄主上之增長潛能，亦可明瞭田間期望壽命之變異性，唯有如此方可能以害蟲生態為基礎配合電腦模
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