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標題: 檬果炭疽病的生物防治菌鑑定與其防病之雛形醱酵營養配方設計
Identification for biocontrol agents of mango anthracnose and design of their pilot-scale fermenting nutrient recipes for the disease control
作者: 江嘉容
Chiang, Chia-Jung
關鍵字: biocontrol
Bacillus licheniformis
mango anthracnose
Bacillus licheniformis
出版社: 植物病理學系所
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摘要: Colletotrichum gloeosporioides引起的檬果炭疽病,是一個重要的貯藏病害,也是造成檬果在貯藏期損失的主要原因之一。目前防治檬果炭疽病之方法中,最常用的方法是以施用化學藥劑為主,但由於抗藥性菌株的出現及環保意識的抬頭,亟須找尋替代的防治方法。本研究主要目的在於篩選有效防治檬果炭疽病的拮抗細菌,並調製培養拮抗細菌的培養基配方,祈能研製微生物植物保護製劑,用於防治檬果炭疽病。自雲林及南投分離細菌後,經由切離葉生物分析、溶血測試及 16S rDNA 分子鑑定,結果發現 Paenibacillus macerans EC-21-02、Bacillus licheniformis EC-31-02 和 EC-34-01 以及 Brevibacillus aqri NA-27-01 和 NN-13-01 等菌株可將檬果炭疽病之罹病度由 100% 降至 0%,且它們均不具有溶血反應與不屬於人體之伺機性病原。設計 25 種不同組成配方對於拮抗菌抑菌的影響,試驗結果發現將 EC-34-01、NA-27-01 及NN-13-01 分別培養於 SY+raffinose pentahydrate、SY+molasses 及 Yeast+NaCl 三種不同之培養基配方中,其醱酵液對於 C. gloeosporioides MG-2 之菌絲抑制率最佳;進一步將 EC-34-01、NA-27-01 及NN-13-01 培養於 Surimi-Molasses (SM) 及 Soybean meal-Surimi-Molasses (SSM) 培養基中,其獲得的醱酵液以切離葉評估防病之功效,結果發現 EC-34-01 培養於 SSM 培養基之 100 稀釋醱酵液可使罹病度由 100% 降至 14% 左右,其中又以培養在酸鹼值 8.0 之 SSM 醱酵液的效果最為優異。利用生理生化測試、Biolog 鑑定系統及脂肪酸鑑定系統鑑定,進一步佐証 EC-34-01 菌株的身分外,隨後利用 27 種不同的專一性引子對偵測 B. licheniformis EC-34-01 可能產生的代謝產物,結果推測其擁有可產生 4’-phosphopantetheinyl transferase、Acyl-homoserine lactonasem、Bacillomycin A、Bacillomycin D、Iturin C、Iturin A、Bacilysin、Bacillaene、Difficidin、Bacillibactin、Fengycin、Macrolactin、Surfactin 及 Pleiotropic regulator 等 14 種物質之基因序列。為提昇 B. licheniformis EC-34-01 在 SSM 培養基之醱酵液的防病效果,發現醱酵液中加入 5% 酒精之 500 倍稀釋液可使罹病度由 100% 降至 10% 左右,具有良好防治病害的效果。將 B. licheniformis EC-34-01 醱酵液之粗萃取液進行薄層生物分析,只有在酸鹼值 2 的環境下所萃取之粗萃取液可對 C. gloeosporioides MG-2 產生抑制圈,且具有抑制炭疽病菌菌絲生長與孢子發芽之效果。EC-34-01 之粗萃取液中的主要抑菌成分之 Rf 值約 0.78,歸屬於低極性分子且為耐熱之胜肽類。
Mango anthracnose, caused by Colletotrichum gloeosporioides, is not only an important postharvest disease but also one of the reasons causing crop losses. Up to now, the use of fungicides is the main approach to control mango anthracnose, however, due to the emergence of fungicide-resistant pathogens and the rise of environmental awareness, the search for alternative methods is important. The main purpose of this study is to search and formulate a medium for antagonistic bacteria to control mango anthracnose, and in turn develop it into a biopesticide to control the disease. The antagonistic bacteria obtained from Nantou and Yunlin were identified and evaluated by detached leaf bioassay, hemolysis test and molecular identification – 16S rDNA. Results indicated that five strains, including Paenibacillus macerans EC-21-02, Bacillus licheniformis EC-31-02 and EC-34-01, Brevibacillus aqri NA-27-01 and NN-13-01 were able to reduce the severity of mango anthracnose from 100% to 0%, did not produce hemolysis reactions and were not opportunistic pathogens for humans. Twenty-five cultural media containing different components were designed. The fermented broths of EC-34-01 cultured in SY+raffinose pentahydrate, NA-27-01 in SY+molasses and NN-13-01 in Yeast+NaCl did show to be the most effective in inhibiting mycelial growth of the pathogen. Then fermented broths of EC-34-01, NA-27-01 and NN-13-01 cultured in Surimi-Molasses (SM) and Soybean meal-Surimi-Molasses (SSM) media were analyzed by bioassay method of detached mango leaf. One hundredfold dilution of fermented broth of EC-34-01 cultured in SSM medium could reduce 86% anthracnose severity compared to the control, and found SSM at pH 8.0 was the most optimal for culturing EC-34-01 in controlling mango anthracnose. B. licheniformis EC-34-01 was reconfirmed via physiological and biochemical characteristics assays, Biolog MicrologTM computer software and the analysis of fatty acid methyl esters microbial identification system. In addition, twenty-seven specific primers were used to detect gene sequences in Bacillus licheniformis EC-34-01 with fourteen kinds of ability for producing 4’-phosphopantetheinyl transferase、Acyl-homoserine lactonasem、Bacillomycin A、Bacillomycin D、Iturin C、Iturin A、Bacilysin、Bacillaene、Difficidin、Bacillibactin、Fengycin、Macrolactin、Surfactin and Pleiotropic regulator. After fermentation, five hundredfold dilution of EC-34-01 fermented broth with 5% ethanol was effective in reducing disease severity level from 100% to 10%. Analysis of the crude extracts of EC-34-01 fermented broths by inhibitory activity assay showed that the crude extract obtained from the condition at pH 2 did not only show inhibitory zone for C. gloeosporioides MG-2 but also inhibited its mycelial growth and conidial germination. The Rf value of bioactive compounds of crude extract of EC-34-01 fermented broth was approximately 0.78, indicating the compounds belong to the low polarity and heat-tolerance of peptides.
其他識別: U0005-2507201116494700
Appears in Collections:植物病理學系



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