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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鍾嘉綾 | |
| dc.contributor.author | Shiang-Shiuan Yu | en |
| dc.contributor.author | 余祥萱 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:32:07Z | - |
| dc.date.available | 2028-08-15 | |
| dc.date.copyright | 2018-08-23 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-16 | |
| dc.identifier.citation | Ahiladevi, P., and Prakasam, V. 2013. Determination of Colletotrichum capsici toxins by gas chromatography mass spectrometry analysis. Int. J. Plant Prot. 6:323-329.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78956 | - |
| dc.description.abstract | 草莓為台灣重要的高經濟作物，炭疽病菌可潛伏感染於草莓種苗中，至本田期大規模發病，造成嚴重危害。前人研究發現，辣椒及菸草炭疽病菌分泌之毒性代謝物屬小分子化合物，接種於寄主會呈現類似病原菌感染之病徵，但目前對於炭疽病菌毒性代謝物之化學結構，及毒性代謝物在侵染寄主過程中扮演之角色均有待釐清。本研究欲探討草莓炭疽病菌之毒性代謝物種類與功能，首先建立草莓葉圓葉片生物測定法 (leaf disc bioassay)，同時以加入草莓葉汁液等方法調整液態培養條件，增加毒素產量，發現培養過草莓炭疽病菌之濾液可在離葉與圓葉片產生和病原菌相似的壞疽病徵。為瞭解毒性代謝物之相關特性，將濾液接種於草莓、菸草、木瓜、青江菜、小麥、香蕉與水稻等不同單子葉與雙子葉植物之圓葉片，確認毒性代謝物不具寄主專一性。另將濾液以高溫高壓滅菌和蛋白質酶處理後，仍無法抑制其誘導產生壞疽病徵之活性，確認活性物質具耐熱性且並非蛋白質。透過Diaion HP20 open column chromatography純化毒性代謝物，再配合高效液相色譜法 (high performance liquid chromatography)、質譜法 (mass spectrometry) 與核磁共振 (nuclear magnetic resonance) 等分析技術，鑑定活性物質為2,5-bis(hydroxymethyl)furan (BHMF)；購買商品化之BHMF接種草莓葉圓片與離葉，確實可產生類似炭疽病菌感染之病徵。本研究亦透過農桿菌轉殖法，篩選出一株毒性代謝物缺陷型突變株 (BHMF產量較野生型低~36%)，將其接種於草莓離葉後，造成的病斑比野生型顯著較小，顯示BHMF可能是草莓炭疽病菌的毒力因子。至於BHMF在草莓葉片或冠部組織之作用濃度，以及是否有其他因子協力作用等，則有待進一步釐清。 | zh_TW |
| dc.description.abstract | Strawberry is a high value fruit crop in Taiwan. Latent infected of Colletotrichum gloeosporioides in strawberry seedlings can cause severe damage to field-grown plants. In previous studies, the toxic metabolite(s) of Colletotrichum sp. were identified as low molecular weight substances; inoculation of the culture filtrates led to lesions similar to that caused by pathogen infections. However, for Colletotrichum spp., precise chemical structures of the toxic metabolite(s) and the roles of toxic metabolite(s) in pathogenesis remain to be clarified. To investigate the mechanism underlying pathogenesis of C. gloeosporioides, this research focused on the identification and functional characterization of toxic metabolite(s) produced by C. gloeosporioides. We optimized toxic metabolite(s) production by supplementing the medium with strawberry leaf extract and developed a leaf disc bioassay method for phytotoxic activity evaluation. The culture filtrate of C. gloeosporioides caused necrosis on leaf discs and induced anthracnose symptoms on detached leaves of strawberry. Assays on the leaf discs of several dicot and monocot crops, including strawberry, tobacco, papaya, bok choy, wheat, banana, and rice suggested host nonspecificity of the toxic metabolite(s). Autoclaving or pretreatment with proteinase K did not inhibit the necrosis-inducing activity of the culture filtrate, indicating that the active ingredient is a thermostable and non-protein substance. By employing Diaion HP20 open column chromatography, RP-amide high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, the toxic metabolite was isolated and identified as 2,5-bis(hydroxymethyl)furan (BHMF). Commercially available BHMF was able to induce anthracnose-like symptoms on detached leaves and leaf discs of strawberry. A toxin-deficient mutant which produced ~36% less BHMF than the wild type was generated by using Agrobacterium-mediated transformation. Inoculating the toxin-deficient mutant on detached strawberry leaves resulted in significantly smaller lesions than the wild type, suggesting that BHMF is a virulence factor of C. gloeosporioides in strawberry. Future work will focus on investigating the effective concentration of BHMF in strawberry leaf and crown tissues, as well as understanding whether there is synergistic factor(s) of BHMF. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:32:07Z (GMT). No. of bitstreams: 1 ntu-107-R05633002-1.pdf: 4485376 bytes, checksum: d998a12dd5b17358d32e9ef7f7fd0dda (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 論文口試委員審定書 I
致謝 II 中文摘要 III ABSTRACT IV CONTENTS VI LIST OF FIGURES X LIST OF TABLES XII LIST OF SUPPLEMENTARY XIII Chapter 1 Introduction 1 1.1 Research motivations 1 1.2 The strawberry cultivars in Taiwan 2 1.3 Anthracnose disease in strawberry 3 1.4 Virulence factors in the pathogenicity of Colletotrichum sp. 5 1.5 Toxic metabolite(s) of the plant pathogens 7 1.6 Phytotoxic metabolites used in screening crops for disease resistance 8 1.7 Previous studies on the toxic metabolite(s) of Colletotrichum sp. 9 Chapter 2 Materials and Methods 11 2.1 Plant materials and growth conditions 11 2.2 Fungal pathogen and culture conditions 11 2.3 Preparation of the culture filtrate containing the toxic metabolite(s) 12 2.4 Evaluation of phytotoxic activity 13 2.4.1 Leaf disc bioassay 13 2.4.2 Whole plant sprayed with culture filtrate and carborundum 13 2.4.3 Wound inoculation of the detached leaf 14 2.4.4 Detached leaf incubated in culture filtrate 14 2.5 Characterization and identification of the toxic metabolite(s) 15 2.5.1 Thermal stability test 15 2.5.2 Host-specificity test 15 2.5.3 Proteinase K Test 16 2.5.4 Isolation and purification of the toxic metabolite(s) 16 2.5.5 Identification of the toxic metabolite(s) 17 2.5.5.1 NMR analysis.........................................................................17 2.5.5.2 MS analysis............................................................................18 2.5.6 Phytotoxic activity of the candidate compound 18 2.6 Roles of the toxic metabolite(s) in the pathogenicity and virulence of C. gloeosporioides 19 2.6.1 Agrobacterium-mediated transformation (AMT) 19 2.6.2 Mycelial growth and sporulation of the wild type and toxin-deficient mutants 21 2.6.3 Quantification of the toxic metabolite(s) produced by the wild type and toxin-deficient mutants using UV-HPLC 21 2.6.4 Evaluation of the pathogenicity and virulence of the wild-type and toxin-deficient mutants 22 Chapter 3 Results 23 3.1 Preparation of the culture filtrate containing the toxic metabolite(s) 23 3.2 Inoculation and bioassay methods for the toxic metabolite(s) 23 3.3 Characterization and identification of the toxic metabolite(s) 24 3.3.1 Thermal-stability test 24 3.3.2 Host-specificity test 25 3.3.3 Proteinase K test 25 3.3.4 Identification of the toxic metabolite(s) 26 3.4 Roles of the toxic metabolite(s) in the pathogenicity and virulence of C. gloeosporioides 27 3.4.1 Agrobacterium-mediated transformation 27 3.4.2 Mycelial growth and sporulation of the wild type and toxin-deficient mutants 28 3.4.3 Quantification of the toxic metabolite(s) by UV-HPLC 28 3.4.4 Evaluation of the pathogenicity and virulence of the wild type and toxin-deficient mutants 29 Chapter 4 Discussion 31 4.1. Properties of the toxic metabolite(s) 31 4.2. Isolation and identification of the toxic metabolite(s) 34 4.3. Roles of the toxic metabolite(s) in ML133 pathogenesis 37 References 39 Tables 51 Figures 53 Supplementary Figures 69 | |
| dc.language.iso | en | |
| dc.subject | 毒性代謝物缺陷株 | zh_TW |
| dc.subject | 草莓炭疽病 | zh_TW |
| dc.subject | 毒性代謝物 | zh_TW |
| dc.subject | 高效液相色譜法 | zh_TW |
| dc.subject | 質譜法 | zh_TW |
| dc.subject | 5-bis(hydroxymethyl)furan | zh_TW |
| dc.subject | strawberry anthracnose | en |
| dc.subject | toxin-deficient mutant | en |
| dc.subject | 5-bis(hydroxymethyl)furan | en |
| dc.subject | mass spectrometry (MS) | en |
| dc.subject | high performance liquid chromatography (HPLC) | en |
| dc.subject | toxic metabolite | en |
| dc.title | 草莓炭疽病菌之毒性代謝物鑑定與功能性分析 | zh_TW |
| dc.title | Identification and functional characterization of the toxic metabolite(s) produced by Colletotrichum gloeosporioides in strawberry | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 李宗徽 | |
| dc.contributor.oralexamcommittee | 曾顯雄,陳昭瑩,林乃君 | |
| dc.subject.keyword | 草莓炭疽病,毒性代謝物,高效液相色譜法,質譜法,2,5-bis(hydroxymethyl)furan,毒性代謝物缺陷株, | zh_TW |
| dc.subject.keyword | strawberry anthracnose,toxic metabolite,high performance liquid chromatography (HPLC),mass spectrometry (MS),2,5-bis(hydroxymethyl)furan,toxin-deficient mutant, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU201803660 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-16 | |
| 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 |
| dc.date.embargo-lift | 2028-08-15 | - |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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