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研究生:謝菘合
研究生(外文):Song-He Hsieh
論文名稱:三枯草桿菌群菌株之生物膜形成能力在番茄青枯病防治效力與生長促進能力
論文名稱(外文):Revealing the biofilm formation, efficacy in biocontrol of tomato bacterial wilt and growth promotion by three Bacillus subtilis group strains
指導教授:黃姿碧
指導教授(外文):Tzu-Pi Huang
口試委員:林宜賢段淑人
口試委員(外文):Yi-Hsien LinShu-Jen Tuan
口試日期:2018-01-18
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:50
中文關鍵詞:枯草桿菌群青枯病菌生物防治根部纏據SWEET醣類轉運蛋白
外文關鍵詞:Bacillus subtilis groupRalstonia solanacearumbiocontrolroot colonizationSWEET (sugars will be eventually effluxed transporter)
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由Ralstonia solanacearum 所引起之青枯病是番茄病害中最具危害性之細菌性病害,常導致番茄產量嚴重的損失。植物促進生長根圈菌PGPR (Plant growth promoting rhizobacteria) 例如桿菌屬 (Bacillus species) 菌株能夠產生脂肽類物質並具抑制植物病原真菌及細菌包括青枯病原之生長作用,因此具病害防治潛力。由台灣本土所分離之枯草桿菌群菌株Bacillus subtilis 151B1、GAPB2及 Bacillus amyloliquefaciens PMB05對於多種不同的病原真菌及細菌具拮抗活性,具有潛力作為生物製劑防治不同之植物病害,本研究之主要目的為比較三菌株對青枯病原之拮抗活性及防治青枯病之能力;分析三菌株之生物膜形成及根部纏據能力與病害防治效果之相關性;解析阿拉伯芥中之SWEETs醣類轉運蛋白是否參與枯草桿菌群菌株之生物膜形成及纏據能力;評估桿菌屬菌株促進植物生長之能力與相關機制。本研究結果顯示三株菌株皆能抑制番茄青枯病菌R. solanacearum RS7之生長,並且於番茄植株澆灌B. subtilis GAPB2及B. amyloliquefaciens PMB05菌株培養液,可明顯降低青枯病害之發生,其發病率較施用SYB培養液之對照組可降低約46.4及43% 青枯病發病率。利用24孔盤之生物膜形成能力測試法測試三菌株於MSgg培養基之生物膜形成能力,結果顯示GAPB2及PMB05菌株形成生物膜之能力較151B1菌株佳。另為瞭解不同碳素源對於枯草桿菌群菌株生物膜形成能力之影響,研究中將MSgg培養基中的碳素源glycerin以sucrose、fructose或glucose取代,試驗結果發現三菌株於sucrose為碳素源之培養基中生物膜形成皆較於其他條件下少。此外GAPB2菌株在阿拉伯芥SWEET2基因及SWEET16與SWEET17雙基因刪除突變植株根部纏據的能力明顯低於野生型及SWEET2基因過表現植株,此結果顯示SWEETs醣類轉運蛋白在枯草桿菌群菌株於植物根部纏據能力扮演一定角色。且施用三菌株之培養液顯著促進番茄根部之生長,並產生植物生長促進相關物質,如indole-3-acetic acid、2,3-butanediol及其溶磷能力,其中又以151B1菌株具最佳生長促進效用,且所產生之生長促進相關物質及溶磷能力亦是最佳。綜合上述結果,B. subtilis GAPB2與B. amyloliquefaciens PMB05菌株具有明顯之番茄青枯病防治效果,而B. subtilis 151B1菌株則具最優異的促進植物生長能力。
 Tomato bacterial wilt caused by Ralstonia solanacearum is one of the devastating diseases in tomatoes. Plant growth promoting rhizobacteria such as Bacillus species have been shown to produce lipopeptides and antagonize against various pathogenic fungi and bacteria including R. solanacearum, thus are potential biocontrol agents for disease suppression. Two Bacillus subtilis strains 151B1, GAPB2 and one Bacillus amyloliquefaciens strain PMB05 native in Taiwan were shown to exhibit differential activity against various bacterial and fungal pathogens and were potential biocontrol agents for various plant diseases. The main objectives of my research were to compare the antagonistic activity of the three Bacillus strains against R. solanacearum; to assess the biofilm formation and root colonization by the three Bacillus strains and to determine whether the above phenotypes are associated with their efficacy in controlling tomato bacterial wilt; to determine if sugar transporters in Arabidopsis plants may involve in biofilm formation and colonization by Bacillus species; and to evaluate the activity of plant growth promotion by the Bacillus strains and the putative mechanisms. Our results indicated that the B. subtilis 151B1, GAPB2 and B. amyloliquefaciens PMB05 all showed antagonistic activity against R. solanacearum RS7 which was isolated from a diseased tomato plant. Drenching applications of B. subtilis GAPB2 and B. amyloliquefaciens PMB05 on tomatos plants were found to suppress tomato bacterial wilt. The disease incidence of tomato bacterial wilt with the treatments of strains GAPB2 and PMB05 was reduced up to 46.4% and 43%, respectively, compared to the medium control. To determine if the types of sugars may affect biofilm formation by the Bacillus strains, the carbon source glycerin in MSgg (Minimal Salts Glutamate Glycerin) medium were replaced with sucrose, fructose or glucose, and biofilm formations by three Bacillus strains were assessed in 24 wells microplates. The biofilm formations by strains PMB05 and GAPB2 were significantly higher than strain 151B1 in the MSgg medium. All three strains formed less biofilms in the sucrose containing media than in other sugars. The root colonization of the strain GAPB2 was less on Arabidopsis SWEET2 and SWEET16/SWEET17 knock out mutants than the wild type and SWEET2 overexpression lines, suggesting a possible role of SWEETs in root colonization by Bacillus species. In addition, application of three Bacillus strains all showed significant promotion in roots growth of tomato plants and expression in plant growth promotion traits including production of indole-3-acetic acid, 2,3-butanediol and phosphate solubilization. Among three Bacillus strains, the strain 151B1 exhibited the best efficacy in plant growth promotion and its related traits. To summarize, B. subtilis GAPB2 and B. amyloliquefaciens PMB05 posessed efficacy in suppression of tomato bacterial wilt. While strain 151B1 exhibited the best ability in tomato growth promotion.
中文摘要................................................ i
英文摘要................................................ iii
目次.......................................................v
表目次...................................................vii
圖目次................................................. viii
前言.....................................................1
材料方法................................................ 10
一、供試藥品來源.........................................10
二、供試菌種來源與培養條件................................10
三、供試番茄及阿拉伯芥之種植..............................12
四、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株對番茄青枯病菌 之拮抗性測試...............................12
五、B. subtilis GAPB2及B. amyloliquefaciens PMB05菌株對番茄青枯病的防治效果測試........................................13
六、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株之pellicle 及生物膜形成能力測試............................14
七、阿拉伯芥幼苗根部纏據能力測試...................................15
八、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株培養液對番茄生 長之影響....................................16
九、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株 2,3-butanediol 之產生能力檢測............................16
十、 B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株 indole-3-acetic acid之產生能力檢測......................17
十一、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株溶磷能力測試.............................................17
結果......................................................19
一、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05菌株對番茄青枯病菌具有拮抗性...................................19
二、B. subtilis GAPB2及B. amyloliquefaciens PMB05菌株培養液具降低青枯病發生之效果.......................................19
三、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05之生物膜形成能力及於不同醣類對於其生物膜形成能力之影響...........20
四、阿拉伯芥SWEET基因突變影響B. subtilis GAPB2於根部之纏據..21
五、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05培養液之施用具促進番茄植株生長之作用............................22
六、B. subtilis 151B1、GAPB2及B. amyloliquefaciens PMB05具與促進生長相關性狀...........................................23
討論......................................................24
參考文獻..................................................28
圖表說明..................................................38
附錄......................................................48
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