摘要:(I) 具外銷潛力的柑橘種苗病害診斷與健康管理套裝技術之研發
柑橘類果樹是台灣栽培面積最大的果樹,為無性繁殖的作物,此類作物容易受到多種系統性病原(systemic pathogens)的感染危害。台灣重要柑橘系統性病原包括黃龍病細菌、萎縮病毒、破葉病毒與鱗砧次病毒等,這些病原經由帶病種苗普遍傳播而危害嚴重,防治上通常須採用健康管理(Health management)之策略,包括無病種苗之生產與種植,撲滅傳染源,媒介昆蟲之防除及合理化肥培管理等。多年來,台灣防治工作獲有成效,尤其苗期病害防治得宜,至今仍可維持各重要柑橘品種的生產,具備優勢與潛力於未來進行種苗外銷或海外布局。有基於此,本計畫擬先盤點與補強我國柑橘種苗的病害診斷與健康管理各項技術,再將所有研發的相關技術予以整合,構築成套裝技術鏈;並篩選外銷潛力品種,以\"客製化\"的思考方向,應用這研發的套裝技術鏈促成繁苗效率與品質之最佳化,以達成外銷柑橘種苗之目標。本計畫擬進行三年的研究試驗,本年度(第一年)的具體工作目標包括: (1)盤點我國目前所有柑橘種苗病害診斷與健康管理技術;(2)完成國家柑橘原種圃內重要原種母樹的健康認證;(3)「原種圃」、「採穗圃」與「健康苗圃(繁殖圃)」病害監測技術之強化;(4)柑橘育苗期病害診斷技術之補強與新技術研發;(5)柑橘健康種苗「去毒」與繁苗技術之改良與效率提升;(6)柑橘育苗期其他重要疫病蟲害的整合防治技術之研發。
(II) 建立具外銷潛力之無病毒紅龍果種苗生產體系
為了建立具外銷潛力之無病毒紅龍果種苗生產體系,我們將針對紅龍果植株中普遍存在的仙人掌X病毒(Cactus virus X, CVX)、蟹爪蘭X病毒(Zygocactus virus X,ZyVX)和紅龍果X病毒(Pitaya virus X, PiVX),研發multiplex RT-PCR檢測方法,協助其他農業研究單位篩選無病毒材料,或開發去病毒技術,以確保健康紅龍果種苗之優良品質。因此103年度將先進行multiplex RT-PCR之研發,首先收集CVX和ZyVX等Potexvirus屬病毒的序列資料,根據序列比對結果分別設計多組CVX和ZyVX專一性引子對,對本實驗室新發現的PiVX亦根據基因選殖所獲得的PiVX序列,設計多組PiVX專一性引子對。以病毒選殖株配合PCR,測試CVX、ZyVX和PiVX病毒引子對之專一性,之後再以RT-PCR篩選效果最佳的引子對組合。接著先進行multiplex PCR反應,同時調整引子對相對濃度、PCR溫度控制及時間長短等參數;最後再利用罹病組織之總量RNA進行multiplex RT-PCR檢測方法的最佳化。此multiplex RT-PCR檢測方法將利用在無病毒材料之篩選,以及應用於開發去病毒技術。此外,亦可應用於紅龍果病毒之田間調查,提供全省紅龍果栽種地區CVX、ZyVX和PiVX發生率的重要資訊。收集CVX和ZyVX等Potexvirus屬病毒的序列資料,根據序列比對結果分別設計多組CVX和ZyVX專一性引子對,對本實驗室新發現的PiVX亦根據所獲得的PiVX序列,設計多組PiVX專一性引子對。以病毒選殖株配合PCR,測試CVX、ZyVX和PiVX病毒引子對之專一性,之後再以RT-PCR篩選效果最佳的引子對組合。接著先進行multiplex PCR反應,同時調整引子對相對濃度、PCR溫度控制及時間長短等參數;最後再利用罹病組織之總量RNA進行multiplex RT-PCR檢測方法的最佳化。研發multiplex RT-PCR檢測方法,以診斷鑑定紅龍果植株中普遍存在的病毒CVX、ZyVX和PiVX。此multiplex RT-PCR可協助其他農業研究單位篩選無病毒材料,藉以生產具競爭力之健康紅龍果種苗。此外,利用我們所研發multiplex RT-PCR,對國內的紅龍果栽種地區進行調查,可提供CVX、ZyVX和PiVX發生率的重要資訊。
(III) 香蕉優質健康種苗繁苗體系之建立及外銷輸出規劃
建立外銷主力華蕉品種及少量外銷之芭蕉品種之種苗無病原檢測工作及生產體系。藉由台蕉健康種苗認證體系及標準繁苗流程之創新規劃,進而推廣民間種苗業者穩定生產健康蕉苗,避免當今全球繁苗產業常見之蕉苗高變異率及品質不一的缺失,並配合國外品種權授權業務,開拓台蕉蕉苗海外新市場。
Abstract: (I) Development of integrated techniques of disease-diagnosis and health management for citrus seedlings with export potential
Citrus is an important crop with the widest cultivated areas among fruit trees in Candidatus Liberibacter asiaticus (the bacteria causing huanglongbing), Citrus tristeza virus, Citrus tatter leaf virus and Citrus exocortis viroid. These pathogens causing serious diseases bring tremendous losses to the citrus industry. The good control strategies against these diseases are based on the principles of “health management” including the production and cultivation of pathogen-free seedlings, elimination of inoculum sources, control of vector insects, and appropriate fertilizer application. These systemic diseases have impacted the citrus industry for many years in Taiwan, and we do have the strength and potential to plan the export and oversea arrangement of citrus seedlings in the near future. Therefore, this project is dedicated to inventory and strengthen various techniques associated with disease-diagnosis and health management, then integrate them into a systemic technology chain. We will be able to select special citrus varieties and apply this technology chain to efficiently produce healthy citrus seedlings for export. The whole project will be conducted for 3 years. Several research items will be executed this year (the first year): (1) inventory of all existing techniques associated with disease-diagnosis and health management; (2) certification of pathogen-free mother trees in the national nursery foundation; (3) strengthening of disease-monitoring techniques in foundation-tree, scion-tree and multiplication nurseries; (4) improvement of original diagnostic methods and development of new diagnostic methods for different diseases in the nursery stage; (5) improvement of the shoot-tip micrografting technique and elevation of the efficiency of seedling productions; (6) development of the integrated control techniques for the other important pests in the nursery stage.
(II) Development of production system for virus-free pitaya seedlings with export potential
In order to establish the production system for virus-free pitaya seedlings with export potential, we plan to develop a multiplex RT-PCR for Cactus virus X (CVX), Zygocactus virus X (ZyVX) and Pitaya virus X (PiVX). This detection method can help us to screen virus-free plant material or to remove viruses from infected plants. To achieve the research goal of 2014 year¸ we will collect the sequence information of CVX and ZyVX, and then design several pairs of specific primers for each virus. PiVX specific primers will also be signed according to the sequences we cloned and obtained. The primer pair of each virus with best specificity will be selected according to the results of PCR as well as RT-PCR tests. After that, we will utilize multiplex PCR to optimize the primer concentration, PCR temperature and time, etc. Finally, total RNA purified from infected plants will be used to test the performance of multiplex RT-PCR. This detection method can not only be used in eliminating pitaya viruses and screening virus-free plants, but also in investigating the infection frequency of CVX, ZyVX and PiVX in pitaya orchards in Taiwan.
(III) Establishment of standardized micro-propagation system and development of export capacity of high-quality banana seedling in Taiwan
The objective of this study is to establish systems for efficient banana seedling production and for pathogen-free detection process at various stages of seedling production for the major Cavendish bananas and the minor cultivars for export purpose. By means of establishments of the certification system for healthy banana seedling and the standard procedure for seedling propagation, the private seedling producers would be able to produce stable and healthy banana seedlings to lower the offtype rate and the level of inconsistent quality. In addition, Taiwan banana seedlings may have the opportunity to open its overseas market through the overseas authorization.