臺灣博碩士論文加值系統

現代農業由於大量使用化學農藥來防治植物病害，隨長時間的使用和濫用，造成作物農藥殘留過多或抗藥性菌株的出現，因此開發低毒性、低用量、高藥效、高選擇性的抗真菌劑是刻不容緩的；篩選新型抗真菌劑，需要一個大量的篩選樣本及有效的篩選系統，本研究室於近幾年以動物糞便為樣本，設計不同樣本前處理法及篩選培養基已經分離出上千種放線菌，且以Candida albican做為主要之檢定菌，分離出104株具生產抗真菌劑之菌株，保存於食品工業研究所菌種保存中心，本論文之研究目標有二：(1)以10種動物糞便樣本探討所使用之樣品前處理法及分離培養基之菌株分離效果；(2)利用Pythium splendens、Rhizoctonia solani、Phytophthora capsici三種國內重要之植物病原真菌做為檢定菌，自上述本研究室分離之104株具生產抗真菌劑之菌株中挑選48株進行抗真菌活性檢測，選擇抗菌活性較特別之菌株，分離其抗真菌物質。
在糞生放線菌分離效果探討方面，探討乾熱法、濕熱法、鹼處理法、SDS-yeast extract處理法、phenol處理法五種樣本前處理法及M3、AY、HV、GA、AI五種分離培養基之分離效果，挑選十種動物糞便樣本，利用上述樣品前處理法及分離培養基，共篩選出459株放線菌；每種動物糞便樣本所篩得之放線菌種類多寡不一，且在不同前處理法及篩選培養基亦影響可篩得之放線菌種類。其中以SDS-yeast extract前處理法所篩得之放線菌種類最多，共249株；分離培養基以M3、AI及HV所篩得種類較多，而GA篩得種類最少。
在篩選抗真菌劑生產株方面，挑選出48株已知具生產抗真菌劑之放線菌當做篩選樣本，利用對峙培養及paper disc agar diffusion assay兩種抗真菌活性檢測法，以Pythium splendens、Rhizoctonia solani、及Phytophthora capsici三株植物病原真菌做為檢定真菌，篩選出編號84A22放線菌，其對P. splendens和P. capsici有抑菌活性，但對R. solani無抑菌活性，做抗真菌物質的分離純化。以醱酵培養的方式取得大量活性物質，經由乙酸乙酯萃取、silica gel液相管柱層析法、高效能液相管柱層析法（HPLC）純化出一抗真菌活性物質，其分子量約1100，進行進一步的結構鑑定。

Fungicides are often used to control fungal diseases for plant protection in agriculture. Because of the increasing arise of drug resistant fungi and most of the fungicides are not biodegradable that causes residual toxicity to other organism. The development of new antifungal agents that possess low toxicity, high efficacy, and high selectivity is necessary. In this study, methods for the isolation of bacterial strains from animal feces is evaluated.
From a collection of 10 different animal feces, a total of 459 Actonimycetes strains were isolated. We found that pretreatment of animal feces with SDS-yeast extract and use of M3, A1, or HV media gave better recovery of Actinomycetes strains. In addition, using phytopathogenic fungi Pythium splendens, Rhizocotonia solani, and Phytophthora capsici as the tested strains for antifungal agent-screening model, the antifungal activity of 48 laboratory antifungal strains were analyzed. These 48 strains were previously collected for their inhibitory activity in Candida albican growth. The culture medium prepared from strain 84A22, a Streptomyces alboviridis, inhibited the growth of both P. splendens and P. capsici. The active component existing in the culture medium of 84A22 was purified by ethyl acetate extraction, silica gel column chromatography, and HPLC. Mass spectrometric analysis indicated that the molecular weight of this active component is ~1100. Determination of the structure of this active component is currently in progress.

壹、 緒論
貳、實驗材料與方法
一、 實驗材料
1. 培養基及培養液
2. 菌株
3. 試藥與耗材
4. 管柱及其材質
二、 實驗方法
1. 放線菌分離條件探討
2. 篩選生產菌株
3. 菌種鑑定
4. 醱酵培養
5. 活性物質之萃取與純化
6. 結構鑑定
參、結果
1. 放線菌分離條件探討
2. 篩選生產菌株
3. 菌種鑑定
4. 醱酵培養
5. 活性物質之萃取與純化
6. 結構鑑定
肆、討論
1. 放線菌分離條件探討
2. 篩選生產菌株
3. 醱酵培養
4. 活性物質之萃取與純化
5. 結構鑑定
6. 未來之工作
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