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研究生:廖仁宏
研究生(外文):Jen-Hung Liao
論文名稱:液化澱粉芽孢桿菌Ba-BPD1及其抗菌脂胜肽防治作物病害之研究
論文名稱(外文):Biocontrol of crop disease with Bacillus amyloliquefaciens BPD1 and its major antifungal lipopeptide
指導教授:劉永銓
口試委員:謝奉家李國欽吳建一黃振文
口試日期:2017-05-18
學位類別:博士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:78
中文關鍵詞:液化澱粉芽孢桿菌Ba-BPD1伊枯草菌素豐原素抗菌脂胜肽生物質譜組織成像分析液相層析串聯質譜分析
外文關鍵詞:Bacillus amyloliquefaciensBa-BPD1IturinFengycinAntagonistic lipopeptideMALDI-TOF IMSLC/MS
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近年來,廣泛地開發農用微生物製劑來進行田間作物栽培的生物綜合防治,已經成為友善農業的一種趨勢。搭配農用微生物製劑進行生物防治,可以達到化學農藥減量使用與合理化施肥的目標。液化澱粉芽孢桿菌是經美國FDA審核認可的安全性菌種。本研究便是以液化澱粉芽孢桿菌(Bacillus amyloliquefaciens BPD1)開發成同時兼具藥效與肥效的農用微生物製劑。
本論文主要包含三個部分,第一部分利用gyrA基因鑑定由農業藥物毒物試驗所謝奉家實驗室在梨山土壤篩到的潛力菌株,確認為液化澱粉芽孢桿菌(Bacillus amyloliquefaciens),命名為BPD1。本土潛力菌株Ba-BPD1,在病害防治方面,具有廣譜的抑菌效果,可拮抗21種植物病原真菌與12種植物病原細菌。另外,Ba-BPD1也會產生螯鐵蛋白與病原菌競爭三價鐵離子,由於鐵質是一般細菌啟動生理反應的催化劑,阻斷病原菌對鐵質的吸收,進而達到抑制病原菌的目的。在促進作物生長方面,Ba-BPD1具有溶磷效果,可溶解土壤中化合態的磷礦,使其釋放出可溶性的有效磷,提供作物吸收利用,達到促進作物生長的目的。
第二部分研究胺基酸的添加對Iturin A產量的影響。氨基酸添加的構想是來自於iturin A結構式中原有的氨基酸組成,嘗試在複合培養基中添加氨基酸來促進iturin A的合成,初始添加量為0.8%,結果顯示serine是最佳添加胺基酸,對iturin A產量的提升最有幫助,可使產量達883 mg/L,約為對照組157 mg/L的5.63倍。進而探討添加量,發現以添加0.8%與1.2%的serine對iturin A的增產最有幫助,iturin A產量分別為899 mg/L與914 mg/L。最後探討添加含有不同氨基酸的複合氮源,嘗試提高iturin A產量,降低成本,因純氨基酸太貴,不符成本,故尋求serine含量較高的複合氮源來取代。複合氮源的添加則以soy peptone E110的添加對Iturin A產量提升最有幫助,iturin A產量可達500 mg/L,是對照組產量178 mg/L的2.81倍。
第三部分研究主要在釐清液化澱粉芽孢桿菌(Bacillus amyloliquefaciens, BPD1)所產生之脂胜肽對稻熱病菌(Pyricularia oryzae Cavara, PO)的拮抗效用。為了找出拮抗稻熱病菌的主要抗真菌脂胜肽,利用脂胜肽的標準品來進行平板拮抗試驗,結果顯示fengycin是拮抗稻熱病菌主要關鍵抗菌脂胜肽。另外,分別以單獨培養與共培養的平板來測試,並透過MALDI-TOF-IMS即時監測來觀察抗菌脂胜肽在菌落處與拮抗區域的空間分佈與消長情形。同時以掃描式電子顯微鏡(SEM)來觀察經脂胜肽處理的病原真菌菌絲型態變化。經fengycin處理過的稻熱病菌菌絲與孢子明顯的變形與腫大。此研究使我們對Ba-BPD1拮抗稻熱病菌的作用機制有了更深入的瞭解,也促進了生物農藥應用的發展。
In recent years, extensive development of agricultural microbial agents for biocontrol of crop diseases has become a trend of friendly agriculture. With the agricultural microbial agents for biological control, the decrease in chemical insecticides application and the rational use of fertilizers can be achieved. Bacillus amyloliquefaciens is a safety strain approved by the US FDA. This study is based on a newly isolated Bacillus amyloliquefaciens BPD1 and its development into the biopesticide and biofertilizer.
A stain screened and isolated from the soil in Lishan was identified via the gyrA gene as Bacillus amyloliquefaciens BPD1 (Ba-BPD1). This strain has a broad spectrum of antibacterial effect, capable of antagonizing 21 kinds of plant pathogenic fungi and 12 kinds of plant pathogenic bacteria. In addition, Ba-BPD1 can also produce siderophore competing ferric ions with pathogen. Because the iron is the general catalyst for bacteria to start the physiological reaction, iron stripping can achieve the purpose of inhibiting pathogens. In the promotion of crop growth, Ba-BPD1 has the ability to dissolve phosphorus from the phosphate around the root of corps in soil, the soluble phosphorus was easily absorbed and utilized by the crop.
Effect of the addition of amino acids on Iturin A production was also studied. The idea of amino acid addition is derived from the amino acid composition of the iturin A structure. Various amino acids were applied as the precursor to promote the synthesis of iturin A. The results show that serine is the best addition of amino acids, the production of iturin A is about 883 mg/L, a 5.63-fold increase to that of the control. To test the concentration of serine, it was found that 1.2% of serine was the most helpful on the increase of iturin A, where iturin A production could be enhanced to 914 mg/L. To try to reduce the production cost, the complex nitrogen sources containing various amino acids were used to replace pure amino acids addition. Soy peptone E110 was found to be the best complex nitrogen source for the increase of Iturin A production. The production of iturin A was enhanced to 500 mg/L, a 2.81 times increase to that of the control.
The antagonistic effect of the lipopeptides secreted by Bacillus amyloliquefaciens strain BPD1 (Ba-BPD1) against Pyricularia oryzae Cavara (PO) was studied. To determine the major antifungal lipopeptides effective against PO, single and dual cultures were carried out in solid-state media. The matrix-assisted laser desorption/ionization–time of flight imaging mass spectrometry (MALDI-TOF IMS) was used to identify the most effective lipopeptide in situ. Meanwhile, the morphology of pathogen fungi treated with lipopeptides was observed via the SEM. Among the three lipopeptide families, i. e., surfactin, iturin, and fengycin, the last one was found to be the most effective for inhibiting mycelial growth and conidial germination of PO. The conidia and hyphae of fengycin-treated PO were shown to become deformed and tumorous under exposure. This study provides deeper understanding about the antagonistic effect of Ba-BPD1 against fungal phytopathogens, which might be helpful in the development of reagents for biological control applications.
中文摘要 i
Abstract iii
第一章 緒論 1
1.1前言 1
1.2研究目的 4
第二章 文獻回顧 6
2.1液化澱粉芽孢桿菌簡介 6
2.2國內外微生物製劑的發展與挑戰 12
2.3抗菌脂胜肽質荷比的分析與鑑定 12
2.4抗菌脂胜肽在空間分佈與消長的分析與鑑定 13
2.4.1微生物IMS分析簡介 14
2.4.2微生物IMS的挑戰 16
2.4.3微生物IMS應用實例 17
第三章 本土液化澱粉芽孢桿菌Ba-BPD1的分離鑑定與特性 20
3.1前言 20
3.2材料與方法 21
3.2.1菌株與試驗培養基組成 21
3.2.2 Ba-BPD1之Iturin A, fengycin與surfactin基因確認 21
3.2.3 MALDI-TOF-MS 分析 21
3.2.4 Ba-BPD1之抗真菌與抗細菌之平板拮抗試驗 22
3.2.5螯鐵蛋白(siderophore)篩選試驗 22
3.2.6溶磷(Phosphate solubilization)篩選試驗 23
3.3結果與討論 23
3.3.1. Ba-BPD1的菌種鑑定 23
3.3.2 ituD, fenD and sfp 基因分析 24
3.3.3 Ba-BPD1產生之抗菌脂胜肽之MALDI-TOF MS鑑定 25
3.3.4 Ba-BPD1與植物病原真菌及植物病原細菌的拮抗試驗 26
3.3.5 Ba-BPD1之溶磷效果與螯鐵蛋白產生之平板快速篩選 31
3.4結論 32
第四章 氨基酸與複合氮源添加對Iturin A 產量提升之研究 33
4.1前言 33
4.2材料與方法 34
4.2.1試驗培養基組成 34
4.2.2菌株與其培養條件 35
4.2.3 Iturin A的萃取分析方法 36
4.3結果與討論 36
4.3.1複合培養基添加棕梠酸、棕梠油、大豆油對Iturin A產量影響 36
4.3.2複合培養基添加氨基酸對Iturin A產量影響 37
4.3.3合成培養基添加氨基酸對Iturin A產量影響 38
4.3.4不同比例之Serine添加對Iturin A產量影響 39
4.3.5含有多種氨基酸的複合氮源添加對Iturin A產量影響 40
4.4結論 41
第五章 液化澱粉芽孢桿菌Ba-BPD1防治稻熱病機制之研究 43
5.1前言 43
5.2材料與方法 44
5.2.1試驗培養基組成 44
5.2.2菌株與其培養條件 44
5.2.3水稻病原真菌的平板拮抗試驗 45
5.2.4 LC/MS與LC-MS/MS分析 45
5.2.5 LC各fraction之拮抗試驗與相對應的MS分析 46
5.2.6 MALDI-TOF IMS分析 46
5.2.7抗真菌生物檢定分析與菌絲形態觀察 47
5.2.8稻熱病菌孢子形態的觀察 47
5.2.9稻熱病防治田間試驗 47
5.3結果與討論 48
5.3.1水稻病原真菌的平板拮抗試驗結果 48
5.3.2 Ba-BPD1產生之脂胜肽鑑定 50
5.3.3 HPLC之各fraction與病原菌的拮抗試驗及MS分析 51
5.3.4 MALDI-TOF IMS分析 57
5.3.5提升豐原素產量的培養基測試 59
5.3.6生物檢定平板拮抗試驗與SEM觀察 60
5.3.7 Ba-BPD1發酵液對稻熱病菌孢子發芽的影響 61
5.3.8田間試驗結果 62
5.4結論 63
第六章 結論與未來展望 64
參考文獻 65
附錄 72
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