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研究生:葉孟韋
研究生(外文):Meng-Wei Ye
論文名稱:四種台灣代表性水稻種植於酸性土及鹼性土中「可培養型」內生細菌群相鑑定及其功能性研究
論文名稱(外文):Phylogenetic Study and Functional Characterization of Culture-Dependent Endophytes in Four Representative Native Rice Cultivars (Oryza sativa L.) Planted in Acidic and Alkaline Soil
指導教授:楊禮亘
指導教授(外文):Li-Sen Young
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:80
中文關鍵詞:水稻內生細菌16S rRNA基因功能性測試生物肥料生物農藥
外文關鍵詞:RiceEndophyteMicrobiome16S rRNA geneFunctional analysisBiofertilizerBiological control agents
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水稻為世界主要糧食作物之一,包含我國在內,半數以上之世界人口以稻米為主要糧食,因此水稻品質的維持及增產是非常重要的課題。台灣致力於水稻品種的改良有目共睹,在栽培耕種技術上亦是國際學習參考的榜樣,過去曾孕育出許多秈稻、稉稻等高品質新穎品種以提供消費者不同選擇同時具對抗不良環境逆境之能力。水稻產量除受各品種基因型之差異影響外(genetic effect),亦受許多土壤環境因子,包括物理、化學及生物性因子所影響(soil effect)。其中,土壤環境的物理及化學性因子包括土壤質地、水分、養分、礦物質、鹽鹼度等可透過栽培耕種技術做某種程度的補足。生物性因子除了根圈微生物(rhizobacteria)外,存在於植物體內的內生微生物(endophyte)亦扮演重要植物生長發育之調節角色。然而,我們國內對這些已被證實具有促進植物生長、提高環境耐抗性及增加抗病能力等功能之內生微生物的了解非常缺乏。為了解本土水稻基因型與土壤環境因子交互作用如何影響其內生細菌之群相,我們選擇了台灣本土具代表性的稉稻(台稉8號、台農71號)與秈稻(台中在來1號、台中秈10號)各兩個品種分別種植於酸性及鹼性土壤中。利用表面殺菌及序列稀釋塗抹培養方法可於四種水稻品種於兩種土壤中的根、莖、葉共分離出125株可培養型內生細菌,再經16S rRNA基因序列分析得到40株不同之水稻內生細菌。菌相比較分析發現相同水稻品種於不同土壤中,所分離得到之菌株菌綱有一定差異性;而相同土壤種植不同水稻品種所得到菌綱結果也有所差異性,因此可得知不同水稻品種及不同土壤會影響到水稻內生細菌的多樣性及獨特性。我們針對所分離出的40株菌株進行功能性測試及病原菌對峙試驗,發現具有可溶性澱粉、纖維素及蛋白質分解功能菌株分別佔了32.5%、22.5%、65%,而溶磷作用、鐵載體及IAA類似物質合成功能則分別佔了35%、25%、55%。此外,本實驗中所篩選出代號為LS0018及LS0023之菌株對Alternaria brassicae病原真菌具有拮抗作用,而代號LS0029菌株對Sclerotium rolfsii sacc病原真菌具有拮抗作用。另外,為瞭解這些具合成促進植物生長物質能力之內生細菌具有潛力開發成有效的生物製劑,我們進行了水稻內生細菌的回接種處理試驗(re-inoculation),結果顯示於實驗A30組,水稻種子處理編號LS0009、LS0026、LS0029、LS0077及LS0088菌株與控制組比較,其植株地上部及地下部長度均有顯著增加,而實驗B30組,於植株地上部及地下部長度有顯著增加的唯有編號LS0018菌株。這些具有農業及工業用途之多功能性內生細菌值得進一步研究其對水稻生長及產量之影響,以利於未來在面對氣候變遷和能源成本高漲時,開發成生物肥料或生物農藥,以替代化學肥料及農藥的施用及改善化學物質對於土壤及水源的環境汙染。

Rice is one of the world''s major food crops and more than half of the world''s population consumes rice as their main diet, including our country, thus maintaining and increasing rice production and quality is a very important issue. Taiwan is well known for its commitment in the breeding of novel and various high quality rice cultivars, including indica and japonica varieties, for consumer’s choice and for improved fitness against adverse environmental stresses. The cultivating technologies are also a model for the international society. Rice production not only is dependent on the genetic background, but also affected by the soil. Physicochemical factors of the soil environment such as soil texture, moisture, nutrients, minerals, salinity, can be amended by cultivation techniques. In addition to the above, rhizobacteria and/or endophytes that reside in plants are another aspect in the biological factor that act as important plant growth regulators. Although the functions of these endophytes, which include plant growth promoting, increased stress tolerance and disease resistance have been proven, yet our knowledge of these endophytes are lacking. In order to understand the interaction between genetic and soil effects and how they influence the microbiome of the endophytic bacteria in rice, we chose four native representative japonica (Taikeng 8, TNG 71) and indica (Taichung Native 1, Taichung Sen 10) rice cultivars and cultivated them in acidic and alkaline soils. Using surface sterilization and serial dilution methods, a total of 125 endophytic bacteria isolates were obtained from the root, stem and leaf sections of four rice cultivars planted in two different soils. 16S rRNA gene sequencing identified 40 different rice endophytic bacterial strains. Comparative analyses of the bacterial identities found that the same rice cultivar showed differences in the endophytic bacterial composition when cultivated in different soil. Comparatively, different rice cultivars planted on the same soil also showed differences in the endophytic bacterial composition. Therefore, it can be concluded that different rice varieties and soil environments affect the diversity and uniqueness of rice endophytic bacteria. Functional assays on the 40 rice endophytic isolates were performed. Isolates possessing amylose, cellulose and protein dissolving capabilities accounted for 32.5%, 22.5% and 65% of the total identified isolates, respectively. In addition, phosphate solubilization, siderophore and IAA production capabilities accounted for 35%, 25% and 55%, respectively. In addition, strains LS0018 and LS0023 showed antagonistic effects on pathogenic fungi Alternaria brassicae, and LS0029 showed antagonistic effect on Sclerotium rolfsii sacc. In order to understand whether those endophytes that could produce plant growth promoting substances and could be potentially developed into bioagents, we carried out a re-inoculation assay of endophytes on rice seeds. The results showed that rice seeds inoculated with strains LS0009, LS0026, LS0029, LS0077 and LS0088 exhibited significantly increased shoot and root length as compared to the control in A30 experiment, while in experimental group B30 only LS0018 strain showed significantly increase in plant shoot and root length. Indeed, these multiple functional endophytic bacteria may possess agricultural and industrial purposes that deserve further study on their effects on rice growth and yield. In the face of climate change and rising energy costs, the development of bio-fertilizers and bio-pesticides to substitute chemical usage can alternatively improve the pollutions of the soil and the water environment.

摘要......................i
ABSTRACT.................iii
誌謝......................v
圖目錄....................ix
第一章 前言.................1
1.1 功能性微生物之應用..... ..1
1.2 植物內生菌..............1
1.3 內生細菌與宿主的共處方式 ..2
1.4 內生菌鑑定方法...........3
1.4.1 非培養型內生菌鑑定方法 ...3
1.4.2 可培養型內生菌鑑定方法 ...3
1.5 植物內生細菌群落之研究.....3
1.6 內生細菌之植物生長促進功能(plant growth-promoting; PGP)..4
1.7 植物內生菌文獻研究.......4
1.8 水稻.......6
1.9 水稻種類分析.......6
1.9.1 秈稻 (Indica).......6
1.9.2 稉稻 (Japonica).......6
1.9.3 糯稻 (Javanica).......7
1.10 水稻內生菌發展與應用.......7
1.11 研究目的.......8
2.1實驗儀器設備.......9
2.2實驗材料與藥品.......10
2.3溶液配製.......12
2.4 Medium配製.......12
2.4.1 固體培養基.......12
2.4.2 液體培養基.......13
2.5 水稻前處理與初步種植.......13
2.6 水稻植株表面殺菌.......13
2.7 序列稀釋.......14
2.8菌數(colony forming unit; CFU)測量及菌株之分離篩選.....14
2.9 菌種保存.......14
2.10取菌種保存菌液.......15
2.11 Genomic DNA 萃取.......15
2.12 DNA電泳分析.......16
2.13菌株16S rRNA增量.......16
2.14菌種功能性測試.......18
2.14.1可溶性澱粉分解功能測試....... 18
2.14.2纖維素分解功能測試.......19
2.14.3蛋白質分解功能測試.......20
2.14.4溶磷作用功能測試.......21
2.14.6生產IAA類似物質的分析 .......22
2.15病原菌平盤對峙實驗.......23
2.16水稻內生菌株回接種試驗.......23
2.17 葉綠素萃取及測量.......25
第三章 結果.......26
3.1水稻內生菌之菌數測量(colony forming unit; CFU).......26
3.2建立水稻內生菌之鑑定平台.......26
3.3水稻內生菌鑑定結果及其組成分析.......26
3.3.1四種不同水稻品種種植於酸性土中各組織部位之內生菌相.......27
3.3.2四種不同水稻品種種植於鹼性土中各組織部位之內生菌相.......27
3.4水稻內生菌鑑定結果之菌屬菌綱差異性分析.......28
3.4.1酸性及鹼性兩種土壤中所篩得之內生菌株菌屬差異性.......28
3.5水稻內生菌種之功能性分析.......28
3.5.1 水稻內生菌抑制病原真菌分析.......29
3.6水稻內生菌株回接種水稻之促進生長試驗.......29
第四章 討論.......31
4.1 16S rRNA 基因序列分析策略.......31
4.2 本研究之台灣水稻品種內生菌數與其他文獻研究之差異性.......31
4.3水稻內生菌鑑定、及菌種功能探討.......32
4.3.1水稻品種及土壤條件對於內生菌種類及分佈位置之差異性.......32
4.3.2水稻內生菌種比較分析.......34
4.3.3內生菌種功能性分析.......35
4.4 水稻內生菌抑制病原真菌分析.......35
4.5 水稻內生菌株回接種水稻之促進生長試驗.......36
未來展望.......37
參考文獻.......38
英文論文大綱.......77
簡歷 .........80

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