Development of Quarantine Techniques for Seeds, Seedlings, Mushroom and the Material of Agricultural Production

本計畫擬針對進出口及國際上種子、種苗、菇類和栽培資材之重要檢疫病原菌之檢疫技術開發，運用以PCR為基礎的檢測技術可以更靈敏與迅速診斷鑑定比傳統的生物檢測來得精準，在本研究中運用以RAPD與ITS序列之結果設計檢測種子與其栽培資材之專一性引子，並研發更精確之種苗及使用資材帶菌之分離純化及其核酸萃取技術。運用以PCR為基礎的診斷鑑定技術檢測來自不同的種子、種苗與栽培資材並且建立相關的重要檢疫病原菌之資料庫。In this study, we plan to develop quarantine techniques for seed, seedling, mushroom and the material of agricultural production . Polymerase chain reaction (PCR) techniques offer advantages over traditional methods of detection and diagnosis because fungi do not need to be cultured prior to detection by PCR, and the technique is rapid and sensitive. In this plan, PCR primers derived from RAPD results and ITS sequences were developed for the specific detection of seed and the material of agricultural production. Besides,we will study and develop DNA extraction of vegetable seeds and the material of agricultural production. We use PCR-based methods to diagnose different fungi from seed, seedling, and the material of agricultural production and set up the database of the important fungi of quarantine

Molecular Strategies for the Analysis of Pepper Plants and Phytophthora capsici Interactions

計畫目標：在亞洲蔬菜研究發展中心使用之疫病菌菌株與抗感性之番椒、番茄品種，進行病原性測試與生理小種區分，選拔出抗、感病之番茄與番椒品種以及對應之毒力與非毒力菌系。進一步建立番茄、番椒抗感病品系與疫病菌分子圖譜，進而篩選分子標記。並以非毒力菌株接種抗病品系、毒力菌株接種感病品系分析其不親合反應及親合反應在不同時期基因表現之差異；同時構築番椒、蕃茄親合、不親合反應與之cDNA基因庫，進一步分析基因庫之分子特性。架構(重要工作項目)：在亞洲蔬菜發展中心便用之疫病菌菌株與不同品種之茄科植物，進行病原性測試與生理小種區分，選拔出抗疫病菌、感病之番茄與番椒品種以及對應之毒力與非毒力菌系。建立茄科作物疫病菌分子 標記圖譜，篩選與非毒力基因連鎖之分子標記，建立各菌株之RAPD等圖譜，應用混合分離分析(bulked segregant analysis)進行篩選與非毒力基因連鎖分子標記。並探討不親合反應中的基因表現差異，構築cDNA基因庫並加以分析其意義。預期效益：a.毒力、非毒力菌株遺傳差異圖譜之建立。b.篩選非毒力基因連鎖之分子標記。c.抗感病番椒、番茄品系之基因型分析。d.比較不親合反應中的基因表現差異。Aims of this project: According to pathogenicity and virulence assays with various tomato and pepper cultivars inoculated with P. capsici isolated from AVRDC, we can obtain resistant, susceptible plants and virulent, avirulent races. Then we set up the molecular markers and map the avirulence genes-linked to these DNA markers. Additionally, we use biotechnology with a number of compatible and incompatible interactions to determine that different expressed genes between susceptible and resistant plants. Meanwhile, we want to construct the partial cDNA libraries of peppers and tomatos in the compatible and incompatible reaction for plant disease resistance genes isolation. Frame: we select various cultivars from AVRDC and inoculate with Phytophthora infestans and P. capsici isolates to obtain resistant, susceptible plants and virulent, avirulent races. With map-based cloning strategy, we map avirulence genes with DNA markers and pre-select markers using bulked segregant analysis. We also construct the the partial cDNA libraries of pepper and tomato in the compatible and incompatible reaction and use cDNA analysis to establish gene expression profiles. Prediction and prospect: 1. Establishment of molecular markers of virulent and avirulent race. 2. Selecting molecular markers linked to avirulence genes. 3. Analysis of resistant and susceptible tomato and pepper cultivars' genotype. 4. Gene expression profiles with some biotechnological methods

Improvement of Cultural Techniques and Supplemental Materials for the Lentinula edodes and Pleurotus spp.

本計畫研究主旨在於收集台灣不同香菇品系與鮑魚菇品系，並且運用基礎科學與生化分生技術來加以區別台灣品系與國外品系間的問題，並選育優質的栽培品種，以增加產業的競爭能力;此外對農業廢棄機質回收在利用，運用鮑魚菇強力分解酵素，對台灣廢棄農業機質再度良善運用，建立良好的國內菇類生產體系與改進生產技術培育具有競爭力且優質的菇類產品。The purpose of the project is to investigate the characteristics of the isolates of the (Lentinus edodes) and the (Pleurotus) mushroom in Taiwan, and using basic and biotechnological studies to distinguish the question among the strains of Taiwan and foreign country, and screen and repair the quality healthy fungous strain, to increase the competitive power of the industry with it. In addition retrieve and utilize agriculture's discarding machine quality, using (Pleurotus) mushrooms brute force to resolve ferments, discard the agriculture machine quality to use to Taiwan kindheartedly once again and set up good domestic mushroom production system and healthy and high-quality mushrooms

Diversity and the development of integrated controls for the rice sheath blight pathogen in Taiwan

計畫目標為持續收集國內各地區水稻紋枯病菌株，利用分子分析其遺傳差異性，了解毒力與環境、水稻品種之間的關聯，配合國內各地區農藥使用情形，調查抗藥性問題，搭配本實驗室所發現可能與稻紋枯病菌致弱毒力相關的影響因子及耐紋枯病的水稻品系，並探討現今減農藥及有機農業下水稻紋枯病之管理技術及策略，提供一個全面低農藥、優質、有效的綜合防治管理。Rice sheath blight disease is one of the endemic rice diseases in Tawian. The general control recently by using chemical fungicide could affects the environment and causes the generation of resistance of pathogen to the fungicide. The purposes of this project is to collect the rice sheath blight pathogen in Taiwan and by analyzing the diversity, the relationship of virulence, environment and host cultivar could be clarified. With the condition of the use of chemical fungicides, the isolates which might be resistant to fungicide were surveyed. Besides, the hypovirulent isolates were screened from the field and the factors including dsRNA and mycovirus were discussed. As to the prevalence of organic cultivation, the management and strategy under different cultivation system were compared and discussed for the effective, low fungicide, and efficient integrated management

Establishment and Application of Diagnostic and Detection Techniques for Botrytis spp.

收集台灣各地的灰黴病菌株，進行專一性核酸探針的選殖與專一性的評估，以開發偵測與診斷用試劑，並建立灰黴病指紋圖譜資料庫以供日後鑑定及應用發展之所需。研究架構為完成此目標首先必須收集灰黴病菌菌株與進出口種苗種球，進行菌株之分離及鑑定，而後進行專一性核酸片段之選殖與進行專一性核酸探針之設計，並利用逆點漬雜合技術以確定專一性核酸探針之專一性。同時發展應用於植物體上之檢測技術與檢測流程的建立。並以隨機增幅多型性分析 (RAPD) 及核酸限制酵素剪切片段多型性分析 (RFLP) 等技術建立灰黴病菌之核酸指紋圖譜，以作為日後鑑定灰黴病菌之依據。For the purpose of develop detection and diagnosis kits of Botrytis spp. First we collect the Botrytis isolates from Taiwan, and clone the specific DNA fragment for design the specific primer for detection of Botrytis spp. Use reverse dot blot hybridization to sure the specificity of the primer, and establish the process for detection and diagnosis. At same time we construct the fingerprint database of Botrytis spp. for the bases of identification and estimate for the occurrence of new strain of Botrytis spp. After we finished these work. We can know the time to control the disease caused by Botrytis spp. by understanding the physical and the ecology of Botrytis spp., and decrease the economical lose caused by Botrytis spp. By the development of the detection and diagnosis kits we can detect and diagnose Botrytis spp. faster. For the construction the database of Botrytis fingerprint we can use it for the identification risk assesment of the occurrence of new strain of Botrytis spp

Development of Molecular Diagnosistic and Detection Kits for Botrytis spp.

計畫目標: 收集台灣各地的灰黴病菌株, 進行專一性核酸探針的選殖與專一性的評估, 以開發偵測與診斷用試劑, 並建立灰黴病指紋圖譜資料庫以供日後鑑定及應用發展之所需.架構: 為完成此目標首先必須收集灰黴病菌菌株與賴俊枝種苗種球, 進行菌株之分離及鑑定, 而後進行專一性核酸片段之選殖與進行專一性核酸探針之設計, 並利用逆點漬雜合技術以確定專一性核酸探針之專一性.同時發展應用於植物體上之檢測技術與檢測流程的建立.並以隨機增幅多型性分析( RAPD )及核酸限制酵素剪切片段多型性分析( RFLP )等技術建立灰黴病菌之核酸指紋圖譜, 以作為日後鑑定灰黴病菌之依據.預期效益: 完成以上的工作預計將可藉由對灰黴病菌生理生態的瞭解而掌控防治的時機以減少經濟損失, 並藉由專一性核酸探針的開發與標準檢測流程的建立將可加速對灰黴病菌的診斷與偵測速度.並藉由指紋圖譜的建立可作為鑑定灰黴病菌之依據與新菌系產生之評估指標.For the purpose of develop detection and diagnosis kits of Botrytis spp.First we collect the Botrytis isolates from Taiwan, and clone the specific DNA fragment for design the specific primer for detection of Botrytis spp.Use reverse dot blot hybridization to sure the specificity of the primer, and establish the process for detection and diagnosis.At same time we construct the fingerprint database of Botrytis spp.for the bases of identification and estimate for the occurrence of new strain of Botrytis spp.After we finished these work.We can know the time to control the disease caused by Botrytis spp.by understanding the physical and the ecology of Botrytis spp., and decrease the economical lose caused by Botrytis spp.By the development of the detection and diagnosis kits we can detect and diagnose Botrytis spp.faster.For the construction the database of Botrytis fingerprint we can use it for identify and estimate for the occurrence of new strain of Botrytis spp