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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳昭瑩(Chao-Ying Chen) | |
dc.contributor.author | Hsiao-Chin Lu | en |
dc.contributor.author | 陸曉親 | zh_TW |
dc.date.accessioned | 2021-06-08T03:29:36Z | - |
dc.date.copyright | 2021-02-22 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-03 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21254 | - |
dc.description.abstract | Colletotrichum spp.引起炭疽病使草莓產量受嚴重衝擊,過度依賴化學藥劑防治所衍生出的抗藥性及農藥殘留問題,又是草莓產業一大隱憂,開發新穎的天然源防治資材即為當前重要課題。抗菌胜肽被認為是可做為對抗微生物感染的新興防治資材。LsGRP1 (Lilium ‘Star Gazer’ glycine rich protein 1)是葵百合上的防禦蛋白,已知LsGRP1 C端區域 (LsGRP1C),是一富含半胱胺酸的抗菌區段,能有效抑制多種植物病原真菌孢子發芽且對多種細菌有致死作用。本研究以化學合成LsGRP1C對草莓炭疽病菌進行生體外的抑菌機制探討,以及於草莓植株上進行抑病機制研究。生體外抑菌試驗結果顯示,以無抑菌效力濃度的金屬螯合劑乙二胺四乙酸(ethylenediaminetetraacetic acid disodium salt dihydrate, EDTA)共同使用,可增強LsGRP1C對不同種Colletotrichum spp.的抑制作用。組織化學染色則證實在加入EDTA後,LsGRP1C能誘使Colletotrichum sp. SWHL-1產生類細胞凋亡與細胞膜滲漏的現象。值得注意的是,植體上原位保護試驗顯示,於草莓的葉、莖或果實單獨處理LsGRP1C,即可有效減少甚至完全抑制壞疽病斑的產生。進一步以Colletotrichum siamense ML133進行草莓葉片上LsGRP1C的抑病機制研究,顯示LsGRP1C在不直接與病原菌接觸下,即可使C. siamense ML133引起之病徵嚴重程度降低;LsGRP1C的處理葉及系統葉經flg22刺激均可增加癒傷葡聚醣累積。因此,推測LsGRP1C除了直接抑制病原菌生長,尚能透過引發植物誘導抗病反應的作用模式,以抑制草莓炭疽病的發生。 | zh_TW |
dc.description.abstract | Anthracnose caused by Colletotrichum spp. seriously affects strawberry production. However, heavy dependence of pesticide in strawberry industry often leads to the resistant pathogens and chemical residue problem, so the development of novel control agents from natural resources becomes an important issue. Antimicrobial peptides (AMPs) are the emerging disease management materials to combat microbial infection. LsGRP1 (Lilium ‘Star Gazer’ glycine rich protein 1) is a defense protein of Lilium ‘Star Gazer’. The cysteine-rich C-terminal region, LsGRP1C, exhibits antimicrobial activity to inhibit spore germination of various plant pathogenic fungi and confer lethal effect on a variety of bacteria. In this study, the synthetic LsGRP1C was used in vitro to dissect its antifungal activities on Colletotrichum spp., and in planta to investigate the disease suppression function on strawberry anthracnose. In vitro antifungal assays revealed that the co-treatment of ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), a chelating agent, at a concentration without antifungal activity, enhanced the inhibitory effect of LsGRP1C on different species of Colletotrichum spp. Histochemical staining demonstrated that the mixture of LsGRP1C and EDTA caused apoptosis-like programmed cell death and membrane permeabilization of Colletotrichum sp. SWHL-1. Worth to notice, in situ protection assays showed that treatment with LsGRP1C alone significantly inhibited necrotic symptom development in leaves, stem and fruits of strawberry. Furthermore, LsGRP1C could systemically enhance the leaf resistance to Colletotrichum siamense ML133. LsGRP1C treated leaves and system leaves could increase the accumulation of callose by flg22 stimulation. Callose deposition also could be triggered by flg22 in the systemic leaves. Herein, LsGRP1C is strongly speculated to combat strawberry anthracnose not only via inhibiting pathogen growth but also has a action mode of inducing plant defense. | en |
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dc.description.tableofcontents | 中文摘要 I 英文摘要 II 壹、前言 1 貳、前人研究 4 一、草莓栽培現況 4 二、草莓炭疽病菌 5 三、臺灣草莓炭疽病防治現況 6 四、葵百合富含甘胺酸之防禦相關蛋白LsGRP1 6 五、絲狀真菌細胞死亡模式 8 六、植物防禦反應 8 七、抗菌胜肽(Antimicrobial peptides, AMPs) 10 八、生物性防治資材在植物保護上之應用 11 參、材料與方法 13 一、供試植物 13 1. 桃園1號 13 2. 香水草莓 13 二、供試菌株 13 1. 菌株來源及培養 13 2. 菌株親緣性分析及鑑定 14 3. 菌株病原性測試 15 三、合成LsGRP1 C端胜肽 15 四、合成細菌之病原分子模式 15 五、生體外抑制真菌試驗 16 六、以組織化學染色法檢測真菌細胞的生理變化 16 1. 細胞膜完整性 16 2. 胞內活性氧物質累積 17 3. 染色質凝集 18 4. 菌絲細胞核DNA斷裂 18 七、原位處理LsGRP1C與EDTA對草莓不同部位炭疽病發病之影響 19 1. 草莓接種 19 2. 原位施用LsGRP1C與EDTA混合液 19 八、原位不同濃度LsGRP1C處理對草莓葉部炭疽病發病之影響 20 九、錐蟲藍染色觀察葉片組織死亡及真菌之侵入情形 20 十、伊凡思藍觀察真菌侵入構造 20 十一、桃園1號草莓主葉處理LsGRP1C對副葉炭疽病發病之影響 21 十二、草莓葉片癒傷葡聚醣之組織染色法 22 十三、數據統計及分析 22 肆、結果… 24 一、菌株鑑定 24 二、LsGRP1C對SWHL-1孢子發芽與發芽管延長之影響 24 三、添加乙二胺四乙酸輔助LsGRP1C抑制SWHL-1孢子發芽 25 四、LsGRP1C與EDTA的共處理使SWHL-1菌絲細胞死亡 26 五、LsGRP1C與EDTA共處理能夠影響SWHL-1細胞膜通透性 26 六、LsGRP1C與EDTA共處理能夠誘使SWHL-1累積細胞內活性氧物質 26 七、LsGRP1C與EDTA共處理能夠促使SWHL-1細胞染色質凝集 27 八、LsGRP1C與EDTA共處理能夠引發SWHL-1菌絲細胞核DNA斷裂 27 九、添加EDTA降低LsGRP1C生體外抑制Colletotrichum spp.分離株孢子發芽之半抑制濃度 28 十、LsGRP1C可原位抑制SWHL-1對果實的危害 28 十一、LsGRP1C可原位抑制SWHL-1對走莖的危害 29 十二、LsGRP1C可原位抑制ML133對草莓走莖的危害 29 十三、LsGRP1C可原位抑制ML133對草莓葉的危害 30 十四、LsGRP1C原位抑制草莓炭疽病的效果具有劑量依賴性 30 十五、LsGRP1C造成植體上炭疽病菌的侵入構造死亡 31 十六、LsGRP1C處理誘發草莓對炭疽病的系統抗性 31 十七、LsGRP1C能夠強化flg22所誘發的防禦相關癒傷葡聚醣累積 31 十八、LsGRP1C處理能夠活化原位防禦反應與誘發系統抗性潛勢化 32 伍、討論 33 陸、參考文獻 38 柒、圖表集 48 表一、親緣關係分析使用之引子 49 表二、LsGRP1C、LsGRP1C與EDTA混合液抑制Colletotrichum spp.分離株之IC50值 50 圖一、Colletotrichum sp. SWHL-1, SWNT-1, SWNT-2, SWNT-3, SWNT-4, SWNT-6與C. acutatum species complex菌株之親緣關係樹 52 圖二、Colletotrichum sp. SWHL-2, SWHL-3, SWHL-4, HLSL-2 及C. gloeosporioides species complex菌株之親緣關係樹 53 圖三、Colletotrichum siamense MLSF-1及C. gloeosporioides species complex菌株之親緣關係樹 55 圖四、LsGRP1C對Colletotrichum sp. SWHL-1孢子發芽與發芽管延長之影 56 圖五、LsGRP1C混合EDTA對Colletotrichum sp. SWHL-1孢子發芽之影響 57 圖六、EDTA輔助LsGRP1C抑制Colletotrichum sp. SWHL-1孢子發芽 58 圖八、以SYTOX Green染色觀察LsGRP1C與EDTA對Colletotrichum sp. SWHL-1細胞膜通透性的影響 60 圖九、以H2DCFDA染色觀察LsGRP1C與EDTA對Colletotrichum sp. SWHL-1細胞內活性氧物質累積的影響 62 圖十、以DAPI染色觀察LsGRP1C與EDTA造成Colletotrichum sp. SWHL-1細胞染色質凝集 63 圖十一、以TUNEL與DAPI染色觀察LsGRP1C與EDTA處理造成Colletotrichum sp. SWHL-1之細胞核DNA斷裂 64 圖十二、LsGRP1C可原位抑制Colletotrichum sp. SWHL-1對草莓果實的危 67 圖十三、LsGRP1C可原位抑制Colletotrichum sp. SWHL-1對草莓走莖的危害 69 圖十四、LsGRP1C可原位抑制Colletotrichum siamense ML133對草莓走莖的危害 70 圖十五、LsGRP1C可原位抑制Colletotrichum siamense ML133對草莓葉的危害 72 圖十六、以錐蟲藍染色觀察LsGRP1C原位處理干擾Colletotrichum siamense ML133侵入草莓葉片 73 圖十七、LsGRP1C原位抑制草莓炭疽病的效果具有劑量依賴性 74 圖十八、以伊凡思藍染色觀察LsGRP1C造成植體上草莓炭疽病菌的侵入構造死亡 76 圖十九、LsGRP1C處理可誘導草莓產生對抗Colletotrichum siamense ML133的系統抗性 77 圖二十、LsGRP1C強化flg22所誘發的草莓防禦相關癒傷葡聚醣累積 78 圖二十一、LsGRP1C增加草莓系統葉癒傷葡聚醣累積 80 捌、附錄 81 附錄一、多基因親緣關係分析之菌株資料 82 | |
dc.language.iso | zh-TW | |
dc.title | 外源性抗菌胜肽LsGRP1C抗草莓炭疽病之機制研究 | zh_TW |
dc.title | The mechanisms of exogenous antimicrobial peptide LsGRP1C against strawberry anthracnose | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 沈偉強(Wei-Chiang Shen),鍾嘉綾(Chia-Lin Chung),林乃君(Nai-Chun Lin),黃健瑞(Chien-Jui Huang) | |
dc.subject.keyword | 草莓炭疽病,Colltotrichum spp.,LsGRP1C,抗菌胜肽,乙二胺四乙酸,誘導抗病, | zh_TW |
dc.subject.keyword | Strawberry anthracnose,Colltotrichum spp.,LsGRP1C,antimicrobial peptide,ethylenediaminetetraacetic acid disodium salt dihydrate,induced resistance, | en |
dc.relation.page | 84 | |
dc.identifier.doi | 10.6342/NTU202100217 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-02-04 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
顯示於系所單位: | 植物病理與微生物學系 |
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