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研究生:陳佳翰
研究生(外文):Chia-Han Chen
論文名稱:提昇番茄抗生物及非生物逆境之內生菌特性分析研究
論文名稱(外文):Characterization of endophytes with the ability to enhance tomato tolerance to biotic and abiotic stresses
指導教授:林乃君林乃君引用關係
口試委員:鍾嘉綾洪挺軒鄧文玲葉信宏
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物醫學碩士學位學程
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:2019
畢業學年度:108
語文別:中文
論文頁數:85
中文關鍵詞:番茄內生細菌非生物逆境生物性逆境
外文關鍵詞:Solanum lycopersicumendophytic bacteriaabiotic stressbiotic stress
DOI:10.6342/NTU201904164
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番茄 (Solanum lycopersicum) 為台灣重要蔬果之一,於台灣栽培面積大約有 4, 400 公頃,種植區域主要分布在中南部,年產量約 11 萬公噸。由於台灣地處亞熱帶地區,夏季高溫多濕,且屢有颱風或豪雨侵襲,往往造成病蟲害盛行與植株生理性障礙,包括青枯病、細菌性斑點病、高溫、乾旱、鹽害和淹水等,因此研發出新的策略以克服這些逆境為科學家重要的課題。近年來的研究顯示,利用有益微生物增加逆境下植物活性極具應用潛力,因此,本研究即以篩選出能應用於提高番茄抗逆境能力的內生菌株為目標。首先,將從番茄根系分離出的內生細菌,接種於植株後,測試其在不同逆境下的生長指標,並利用培養基測試其溶磷、螯鐵和脫氨酶 (1-aminocyclopropane-1-carboxylase, ACC) 等與可促進植物生長及抗逆境有關之活性,及其對番茄青枯病菌和細菌性斑點病菌的拮抗情形。篩選的 100 支內生細菌中,分別有 12、17、11 和 19 支菌株能增強番茄對高溫、乾旱、鹽害及淹水逆境的耐受性,且有 19 支菌株同時具有兩種以上的功能。進一步分析後發現,在潛力菌株中,Pseudomonas protegens XH1-2a 與 P. kribbensis XP1-6 有相對較佳的表現,且又以 P. kribbensis XP1-6 展現最佳的逆境耐受性。在高溫逆境下,接種 P. protegens XH1-2a 及 P. kribbensis XP1-6 後的植株,其脯胺酸含量有顯著性提昇,而Malondialdehyde (MDA) 含量及過氧化酶 (peroxidase) 活性則與對照組無差異。利用表現有 green fluorescence protein (GFP) 的菌株,可觀察此二菌株均會內生於番茄根系中。未來期望將這些內生菌導入番茄害物整合管理策略中,以減少番茄生產過程中因逆境所造成的損失。
Tomato (Solanum lycopersicum) is one of the most important fruits in Taiwan. Approximately 4, 400 ha, mainly in the middle and south parts of Taiwan, were used for tomato production, and the annual yield is about 110 thousand tons. Located in the subtropical region, hot and humid weather in summer accompanied with occasionally occurred typhoons and thunderstorm has put plants at risk of prevailing pest infestation and physiological disorder in Taiwan. Tomato plants are often threatened by different stresses, such as bacterial wilt, bacterial spot disease, heat, drought, salt and flooding. Therefore, development of strategies for farmers to counteract the outcome of such stresses becomes an important issue for scientists. Successful cases using beneficial microorganisms to increase plant vitality under stresses have been reported in recent years. In this study, endophytic bacteria isolated from tomato roots were used to investigate their in vitro plant growth-promoting (PGP) and resistance enhancing activities, including phosphate solubilization, siderophore production, and 1-aminocyclopropane-1-carboxylase (ACC) deaminase production as well as the antagonistic effects against different tomato pathogens. Afterwards, in vivo growth indices under different abiotic and biotic stresses were obtained. In total, 100 endophytic bacteria were screened for abovementioned activities, and 12, 17, 11 and 19 strains showed potentials to enhance tomato tolerance to heat, drought, salt and flooding, respectively. Moreover, 19 isolates have more than two activities. Further analysis indicates that among those candidates, XH1-2a and XP1-6, identified as Pseudomonas protegens and P. kribbensis, by 16S rDNA sequencing, exhibited better performance, and XP1-6 showed the highest tolerance under abiotic stress and biotic stress. Under heat stress, the amount of proline detected in XH1-2a or XP1-6-inoculated plants increased dominantly, but the amount of malondialdehyde and the peroxidase activity were similar to the control plants. Using green fluorescence protein-expressing derivative strains of XH1-2a and XP1-6, their colonization in the tomato roots was confirmed. In the future, these bacteria will be introduced into the integrated pest management strategies for increasing tomato adaptability to different stresses in the field.
摘要 i
Abstract ii
目錄 iv
表目錄 viii
圖目錄 ix
壹、前言 1
一、 番茄簡介 1
1. 栽培歷史 1
2. 營養與經濟價值 2
3. 栽培條件 2
二、 非生物性逆境 3
1. 高溫逆境 3
2. 乾旱逆境 3
3. 鹽害逆境 4
4. 淹水逆境 5
三、 番茄生物性逆境 6
1. 青枯病 (Bacteria wilt) 6
2. 細菌性斑點病 (Bacterial spot disease) 8
四、 促進植物生長根棲細菌與內生菌 9
1. 定義 9
2. PGPR 對作物的影響 10
3. PGPR 可增加植物抵抗生物與非生物逆境能力 10
4. PGPR或內生菌之共存性 11
貳、研究動機與目的 12
參、 材料與方法 13
一、供試植株與栽培條件 13
二、供試菌株培養與保存 13
三、內生細菌菌種鑑定 14
1. Genome DNA萃取 14
2. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 15
3. 搭配 TA cloning 進行16S rDNA定序 15
四、內生菌之特性分析 17
1. 促進生長能力測試 17
2. 拮抗病原菌能力測試 18
3. 化學藥劑感受性 18
4. 內生菌親和性試驗 19
五、測試菌株內生特性分析 20
1. 浸泡處理時間對共生效果的影響 20
2. 內生細菌於植物中分布的位置 20
六、病原菌接種試驗 20
1. 青枯病 20
2. 細菌性斑點病 21
七、非生物性逆境處理 21
1. 高溫逆境 21
2. 乾旱逆境 21
3. 鹽害逆境 22
4. 淹水逆境 22
八、相對含水量測定 22
九、生理活性分析測定 22
1. 脯胺酸 (Proline) 測定 22
2. 脂質過氧化程度測定 23
3. Peroxidase (POD) 測定 23
十、統計分析 24
肆、結果 25
一、篩選具有 ACC deaminase 活性之菌株 25
二、初步篩選具提高番茄抗逆境潛力之菌株 25
三、候選菌株對病原菌之拮抗作用 26
四、接種內生菌對番茄抗生物性逆境之影響 27
1. 青枯病菌接種試驗 27
2. 細菌性斑點病菌接種試驗 28
五、接種內生菌對番茄抗非生物性逆境之影響 28
1. 高溫逆境 28
2. 乾旱逆境 29
4. 淹水逆境 32
六、XP1-6 和 XH1-2a 之內生特性分析 34
1. 浸泡處理時間對纏據效果 (colonization) 之影響 34
2. 共生位置 34
3. 潛力內生菌的定殖效果 35
七、相關酵素之活性測定 35
伍、討論 37
陸、結論 42
柒、參考文獻 43
捌、表 50
玖、圖 68
拾、附錄 79
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