Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89347
DC FieldValueLanguage
dc.contributor李敏惠zh_TW
dc.contributor.authorKuan-Ting Liuen_US
dc.contributor.author劉冠霆zh_TW
dc.contributor.other植物病理學系所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-07T07:25:34Z-
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Antimicrobial effects of volatiles produced by two antagonistic Bacillus strains on the anthracnose pathogen in postharvest mangos. Biological Control 65: 200-6.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/89347-
dc.description.abstract芒果為台灣重要的外銷出口作物,位居果品出口總產值第一,而炭疽病菌Colletotrichum gloeosporioides Penz.為芒果栽培之最重要病原菌,可以感染芒果樹的葉、花穗、枝條及採收前與採收後果實,並造成產量及品質嚴重受損。目前以套袋及化學藥劑噴灑為主要防治方式,然而後者所衍生的病原菌抗藥性及藥劑殘留過量等問題亦相應而生,本研究透過微生物源與植物源資材的篩選與運用,提供防治芒果炭疽病的參考。由於芒果果實一年一穫,因此以芒果切離葉片作為分析之材料。為建立切離葉接種平台的穩定性,進行不同葉齡葉片接種,結果顯示8天內之葉齡發病穩定。葉表與葉背在接種後24小時,孢子發芽率與附著器生成率並無顯著差異。利用乙醇萃取17種植物材料,發現其中4種萃取液對C. gloeosporioides TYC-2菌絲生長抑制率將近50%。植物A乙醇萃取液於濃度0.08 mg/ml下可完全抑制孢子發芽。將植物A乙醇萃取液與TYC-2孢子同時接種至果實上,分別於0.08 mg/ml下可完全抑制病害發生。自土壤、芒果葉片及芒果花中分離出45株酵母菌及83株細菌具有抑制TYC-2菌絲生長能力。酵母菌分離株中,3H-4菌株於PDA培養基上與TYC-2菌株共培養後,具有優異之競爭能力;於細菌分離株中,菌株A與菌株B對於15株不同寄主來源之炭疽病菌亦具有抑制效果,經由16S rDNA以及Biolog鑑定後,皆為Bacillus amyloliquefaciens。經由切離葉與果實接種試驗發現菌株A與菌株B於培養液 (SSM) 4天之發酵液經稀釋100倍後,可抑制病斑之產生,並且於切離葉接種試驗中發現菌株B於稀釋600倍後仍具有80%之病斑面積抑制效果。將菌株B之SSM發酵液靜置於室溫下20週,其菌量可由1.48 × 109cfu/ml上升至2.6 × 109cfu/ml;經由100℃處理菌株B之SSM發酵濾液20分鐘,其抑菌能力並無降低的現象。為了促進菌株B發酵液之抑病能力,因而添加不同植物油於SSM培養液中,在SSM中添加0.5%植物油A稀釋200倍後,可提升菌株B發酵液之病斑面積抑制率由43%至100%。經由切離葉接種分析,發現菌株B SSM發酵液之有效成分為發酵濾液,將發酵濾液與TYC-2孢子於玻片上共培養後,發現其具有抑制發芽並造成發芽管不正常膨脹的能力。經由掃描式電子顯微鏡觀察,芒果葉片上菌株B SSM發酵液可使TYC-2發芽管形態異常。在果實上則顯示菌株B SSM發酵液處理32小時後,菌絲產生許多空泡結構,並於接種後72小時仍無法觀察到果表壞疽病斑的產生,而對照組於接種48小時後即產生顯微壞疽病斑。利用薄層色層分析法分析菌株B於SSM與SSM添加0.5%植物油A之酸沉降萃取液,皆可發現Rf值0.045與Rf值0.38具有抗生活性,將此兩區間之TLC活性回收液與酸沉降萃取液進一步使用高效液相層析法分析,皆可發現於Rf值0.38之TLC活性回收液具有與iturin A標準品相同滯留時間的波峰,此外,兩發酵液之酸沉降萃取液皆可發現與iturin A標準品及surfactin標準品相似滯留時間的波峰。綜合上述結果,植物A乙醇萃取液與B. amyloliquefaciens 菌株B具有進一步發展為田間應用的生物防治藥劑潛力。zh_TW
dc.description.abstractMango is one of the most important fruit crops and is the highest value of exported fresh fruits in Taiwan. Colletotrichum gloeosporioides causes mango anthracnose and is one of the major pathogens in mango. It can infect mango leaves, flowers, branches and fruit, and causes considerable fruit disqualified during postharvest period. Currently, the disease management strategies majorly rely on fruit bagging and chemical fungicides application. However, fungicide application can result in fungicide resistance and excessive chemical residues. The purpose of this study is to screen and apply microbe- and plant-derived materials to control mango anthracnose. Since mango fruit is seasonally available, a stable inoculation platform for using mango leaf as inoculation material was established in the beginning in my thesis study. Mango (cv. Irwin) leaf with age within 8-days is highly susceptible to the infection of C. gloeosporioides TYC-2 and the infection is stable and repeatable. In addition, anthracnose lesions appeared earlier on the abaxial than the adaxial surface of a leaf, which is not related to the ability of germination and appressorial formation of the pathogen on both surfaces. Among 17 tested plants, ethanol extracts from 4 different plants showed nearly 50% of inhibition on mycelial growth of TYC-2. Plant A ethanol-extracts showed complete inhibition on TYC-2 spore germination at 0.08 mg/ml in vitro. The anthracnose lesion was not appeared when mango leaf and fruit were co-inoculated with TYC-2 and plant A ethanol-extracts (0.08 mg/ml). Total 45 yeast isolates and 83 bacterial isolates were isolated from the soil, leaves or flowers of mango and showed inhibitory ability to the mycelial growth of TYC-2. Among all yeast isolates, isolate 3H-4 showed strong competition against the growth of C. gloeosporioides in PDA medium. Among all bacterial isolates, strain A and B has significant inhibitory effect to 15 Colletotrichum isolates which cause the anthracnose of mango, Chinese cabbage and chili pepper. Strain A and B were identified as Bacillus amyloliquefaciens based on the comparison of 16S rDNA sequence and Biolog analysis. The 100-fold dilution of 4-day-old culture liquid of strain A or B cultured in culture medium (strain A-SSM or strain B-SSM) could completely inhibit anthracnose lesion production on detached leaves and fruit. Moreover, it showed 80% lesion area reduction when treated with 600-fold dilution of strain B-SSM on detached leaf. The bacteria population of strain B -SSM could increase slightly from 1.48 × 109cfu/ml to 2.6 × 109cfu/ml after stored for 20 weeks under room temperature. In addition, the antagonistic activity of strain B -SSM was resistant to heat treatment, 100℃ for 20 minutes. It indicates that strain B -SSM had great stability during short-time storage assay. To improve the antagonistic activity of strain B, various plant oils were added into SSM to culture this bacterial strain. The results showed that 0.5% (v/v) plant oil A amended SSM could increase the antagonistic activity of strain B by increasing the lesion area reduction from 43% to 100% when 200-fold diluted bacterial culture was applied. The active ingredient of strain B -SSM remained in the culture filtrate but not the culture pellets after the bioactivity assay on the detached leaf. Culture filtrate of strain B -SSM could inhibit spore germination and cause abnormal swelling of germ tubes of TYC-2 in vitro and in planta under the examination of light microscopy as well as scanning electron microscopy. There were many vacuole-like structures formed in hyphal 32 h after treated with strain B -SSM, and no necrosis lesion was observed on fruit 72 h after treatment. Thin layer chromatographic (TLC) analysis revealed that two regions (Rf 0.045 and 0.38) with antifungal activity were identified in strain B -SSM and strain B -SSM with 0.5% plant oil A. The bioactive components were recovered from the TLC plate and analyzed by high-performance liquid chromatography (HPLC). The data revealed that the bioactive components contain iturin A. Based on the results presented in this study, the ethanol-extracts of plant A and bacterial strain B has great potential for further development of biological control agents in field applications to control mango anthracnose.en_US
dc.description.tableofcontents摘要 i Abstract iii 目次 vi 前言 1 材料與方法 6 一、 儀器設備及藥品 6 二、 供試菌株來源、培養、接種源製備及保存 6 (一) 菌株來源 6 (二) 培養、接種源製備及保存 6 三、 芒果切離葉接種平台建立 7 (一) 不同葉齡之芒果葉對芒果炭疽病菌菌株TYC-2之感受性 7 (二) TYC-2於葉片上之發芽、附著器形成及侵染試驗 8 (三) 角質層染色觀察 8 (四) 不同品種芒果成熟葉片葉背接種 8 四、 具拮抗效力之植物萃取液篩選 9 (一) 植物萃取液製備 9 (二) 對菌絲生長之抑制效果篩選 9 (三) 迷迭香品系A萃取液對TYC-2孢子發芽之抑制效果 9 (四) 迷迭香品系A乙醇萃取液於植物體上之防治效果 10 五、 拮抗微生物之篩選 10 (一) 拮抗微生物之分離與保存 10 (二) 對峙培養試驗 11 (三) 拮抗細菌分離株4A-1、4A-24與4nC-7菌株與其他炭疽病菌分離株之對峙效果 11 (四) 酵母菌分離株3H-4、3H-8及3L-16菌株之競爭性對峙測試 12 (五) 拮抗細菌之鑑定 12 六、 拮抗細菌4A-1與4A-24之發酵基質配製 14 (一) 培養液之製備 14 (二) 拮抗菌發酵液之製備 15 七、 拮抗細菌4A-1與4A-24菌株發酵基質篩選試驗 15 (一) 於固態發酵用培養液上之對峙效果 15 (二) 切離葉生物分析法 15 (三) 不同培養基之拮抗菌發酵液對於防病效果評估 16 (四) 病害調查 16 八、 拮抗細菌4A-24菌株SSM發酵液特性分析 16 (一) 發酵液之逐日取樣分析 16 (二) 儲架壽命試驗 17 (三) 溫度耐受性試驗 17 九、 各類油劑對拮抗細菌4A-24菌株SSM發酵液之抑菌及抑病評估 17 (一) 添加不同植物油之發酵液的抑病效果評估 17 (二) 添加不同濃度葵花油之發酵液的抑病效果評估 18 十、 果實採收後之防病測試 18 (一) 採收後果實生物分析法 18 (二) 拮抗細菌4A-1與4A-24菌株芒果果實病害防治試驗 18 十一、 拮抗細菌拮抗機制探討 18 (一) 拮抗細菌4A-1與4A-24菌株發酵液有效性成分分析 18 (二) 拮抗細菌4A-24菌株發酵濾液對TYC-2孢子發芽之影響 19 (三) 以掃描式電子顯微鏡觀察4A-24菌株發酵液於芒果葉片上對TYC-2菌株之影響 19 (四) 拮抗細菌4A-24菌株發酵液於芒果果實對TYC-2菌株之影響 20 (五) 拮抗細菌4A-24菌株發酵濾液之拮抗物質分離及生物活性檢測 20 (六) 高效液相層析 (High-performance liquid chromatography, HPLC) 分析 22 十二、 統計分析 22 結果 23 一、 芒果切離葉接種平台之建立 23 (一) 不同葉齡之芒果葉對芒果炭疽病菌菌株TYC-2之感受性 23 (二) TYC-2於葉片上之發芽、附著器形成及侵染試驗 23 (三) 角質層染色觀察 24 (四) 不同品種芒果成熟葉片葉背接種 24 二、 具拮抗效力之植物萃取液篩選 24 (一) 對菌絲生長之抑制效果篩選 24 (二) 迷迭香品系A萃取液對TYC-2孢子發芽之抑制效果 25 (三) 迷迭香品系A乙醇萃取液於植物體上之防治效果 25 三、 拮抗微生物之篩選 26 (一) 拮抗微生物之分離 26 (二) 對峙培養試驗 26 (三) 酵母菌分離株3H-4、3H-8及3L-16菌株之競爭性對峙測試 27 (四) 拮抗細菌分離株4A-1、4A-24及4nC-7菌株與其他炭疽病菌分離株之對峙效果 27 (五) 拮抗細菌之鑑定 27 四、 拮抗細菌4A-1與4A-24菌株發酵基質篩選試驗 28 (一) 於固態發酵用培養液上之對峙效果 28 (二) 不同液態培養基之拮抗菌發酵液對於防病效果評估 28 五、 拮抗細菌4A-24菌株SSM發酵液特性分析 29 (一) 發酵液之逐日取樣分析 29 (二) 儲架壽命試驗 29 (三) 溫度耐受性試驗 30 六、 各類油劑對4A-24菌株SSM發酵液之抑菌及抑病評估 30 (一) 添加不同植物油之發酵液的抑病效果評估 30 (二) 添加不同濃度葵花油之發酵液的抑病效果評估 30 七、 果實採收後之防病測試 31 (一) 拮抗細菌4A-1與4A-24菌株芒果果實病害防治試驗 31 八、 拮抗菌拮抗機制探討 31 (一) 拮抗細菌4A-1與4A-24菌株發酵液有效性成分分析 31 (二) 拮抗細菌4A-24菌株發酵濾液對TYC-2孢子之發芽影響現象 31 (三) 以掃描式電子顯微鏡觀察4A-24菌株發酵液於芒果葉片上對TYC-2菌株之影響 32 (四) 拮抗細菌4A-24菌株發酵液於芒果果實對TYC-2菌株之影響 32 (五) 拮抗細菌4A-24菌株發酵濾液之拮抗物質分離及生物活性檢測 33 (六) 高效液相層析 (HPLC) 分析發酵濾液之拮抗物質 34 討論 35 參考文獻 43 附錄一、供試植物病原真菌菌株來源 83 附錄二、17種供試植物來源列表 84 附錄三、具有抑制Colletotrichum spp.生長能力植物列表 85 附錄四、芒果防治田間試驗 86 附錄五、拮抗細菌4A-24菌株發酵液於白菜炭疽病害之防治 91zh_TW
dc.language.isozh_TWzh_TW
dc.rights不同意授權瀏覽/列印電子全文服務zh_TW
dc.subject芒果炭疽病zh_TW
dc.subjectColletotrichum gloeosporioideszh_TW
dc.subject生物防治zh_TW
dc.subject植物萃取液zh_TW
dc.subjectMango anthracnoseen_US
dc.subjectColletotrichum gloeosporioidesen_US
dc.subjectbiological controlen_US
dc.subjectplant extractsen_US
dc.title篩選及應用拮抗細菌與植物萃取液防治芒果炭疽病zh_TW
dc.titleScreening and application of antagonistic bacteria and plant extracts to control anthracnose disease on mangoen_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-08-07zh_TW
dc.date.openaccess10000-01-01-
Appears in Collections:植物病理學系
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