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研究生:黃培真
研究生(外文):Pei-Chen Huang
論文名稱:內生細菌應用於番茄青枯病與熱逆境管理之效果評估
論文名稱(外文):Effectiveness evaluation of the endophytic bacteria on management of bacterial wilt and heat stress in tomato
指導教授:林乃君林乃君引用關係
指導教授(外文):Nai-Chun Lin
口試委員:洪挺軒鄧文玲鍾嘉綾
口試日期:2020-07-27
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:2020
畢業學年度:108
語文別:中文
論文頁數:67
中文關鍵詞:番茄內生細菌高溫茄科青枯病
外文關鍵詞:Solanum lycopersicumEnophytic bacteriaHeat stressRalstonia solanacearum
DOI:10.6342/NTU202004038
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番茄 (Solanum lycopersicum) 為臺灣重要蔬菜作物之一,種植區主要在臺灣中南部。由於全球暖化及臺灣夏季氣候高溫多雨,番茄在栽培期間遭遇生物性及非生物逆境威脅的機率也越來越高;其中,茄科青枯病 (bacterial wilt) 和高溫逆境是目前番茄栽培管理中需重視的問題。為了尋求解決方案,過去的研究篩選出可增加番茄品種農友 301 對茄科青枯病抗性及對高溫耐受性之內生細菌 Pseudomonas kribbensis XP1-6。本研究以將 P. kribbensis XP1-6 發展成可應用於田間為目標,探討其在不同番茄品種上的效果及纏據率、不同施用方法的效果及種子包覆技術應用的可行性。結果顯示,以 P. kribbensis XP1-6 處理不同番茄品種的幼苗後,可發現 P. kribbensis XP1-6 能成功纏據在農友 301、全福 993、玉女、美女、金瑩及橙蜜香等商業栽培品種的根系;此外,P. kribbensis XP1-6 亦可以纏據於部分嫁接苗所使用的根砧品種,包括:茄子品種 Eg190、Eg195、Eg219 和 TS03 及番茄品種 H7996 的根系中。不論是以種子處理或種苗處理,P. kribbensis XP1-6 在降低青枯病病害嚴重度上皆有好的效果,能使病害嚴重度下降 15% 以上 (對照組罹病度 66.67%)。種子包覆技術發展的初步結果發現,將種子浸泡於 P. kribbensis XP1-6 中 24 小時以內,P. kribbensis XP1-6 便可成功附在種子表面並於種子發芽後進入番茄根系。耐熱試驗初步結果顯示,於幼苗期接種 P. kribbensis XP1-6 可以減少玉女番茄嫁接苗 (根砧:茄子品種勇嫁) 夏季栽培於溫室時的落花現象。為了未來在不同栽培條件與環境下均有菌株可供使用,本研究持續針對茄科青枯病篩選具有提升番茄抗性的潛力菌株。首先自之前研究的潛力菌株中另外挑選出 XK1-15、XK2-11、XK2-14、XR2-2、XS2-3及 MP4-1 六個菌株;接著針對這六個菌株,檢視其是否具有溶磷、indole acetic acid (IAA) 合成及螯鐵等促進植物生長的活性,也進行對青枯病菌之拮抗試驗及利用盆栽試驗了解其是否能提高番茄抗青枯病的能力。其中,菌株 XK1-15 和 MP4-1可以明顯降低番茄於接種青枯病菌十四天後的病害嚴重度至47.5% 和 60% 左右 (此時未接種內生菌株之對照組植株,病害嚴重度已達 90% 左右),未來將對此二菌株進行更深的探討與應用。綜合以上結果顯示,之前研究所篩選出的 P. kribbensis XP1-6 除了可用於番茄品種農友 301 之外,也可應用在其他商業品種上,亦有潛力可用於嫁接苗。而確認以種子包覆來應用 P. kribbensis XP1-6 之可行性後,未來可以利用不同的包覆技術或資材以提高包覆於種子上菌株的存活率與活性。此外,篩選出更多的菌株不僅可以適地適用或針對不同時期進行施用,也可透過混用不同菌株間產生協同效力,使提高抗病性與耐熱的效果升級,創造更多潛力內生菌株的應用價值。
Tomato (Solanum lycopersicum) is one of the important vegetables in Taiwan, mainly produced in the middle and southern parts of Taiwan. Due to global warming and the hot and humid summer in Taiwan, tomato may encounter a variety of biotic and abiotic stresses, especially bacterial wilt and high temperature, during cultivation. To seek for solutions, the endophytic bacterium Pseudomonas kribbensis XP1-6, capable of enhancing resistance to bacterial wilt and heat tolerance in tomato cultivar Knowyou 301, was identified in a previous study. In this study, whether this endophytic bacterium can be applied successfully in the tomato field in Taiwan was investigated through exploring the effect and colonization rate of P. kribbensis XP1-6 in different tomato cultivars, and feasibility of using different inoculation methods and the seed coating technique. The results showed that P. kribbensis XP1-6 has good colonization efficiency in the roots of commercialized cultivars Knowyou 301, Chuanfu 993, Yu-Nu, Beauty, Jing-Ying and Cheng-Mi-Hsiang; however, P. kribbensis XP1-6 can only colonize in some rootstock cultivars, including Eg190, Eg195, Eg219, TS03 and H7996. No matter seeds or germinated seeds (seedlings) were used for inoculation, P. kribbensis XP1-6 could reduce the disease incidence of bacterial wilt by more than 15%. In a preliminary seed coating experiment, P. kribbensis XP1-6 could successfully attach and colonize on seeds and enter seedlings after germination. Preliminary heat tolerance test indicated that inoculation of P. kribbensis XP1-6 in grafted seedlings of tomato cultivar Yu-Nu (rootstock: eggplant cultivar Yung-Chia) reduced bud drop in a greenhouse experiment in summer. In order to maintain an inventory of beneficial endophytic bacteria for different cultivation means and under different environmental conditions, I continue to screen potential endophytic bacteria which can enhance resistance to bacterial wilt. Another seven potential strains, including XK1-15, XK2-11, XK2-14, XR2-2, XS2-3, MP4-1, and XH1-2a, were selected using bacterial collection from a previous study, and their plant growth-promoting (PGP) activities and antagonistic effects against Ralstonia solanacearum were investigated. Finally, we found strains XK1-15 and MP4-1 could reduce disease incidence of bacterial wilt significantly to 47.5% and 60% (that of the control plants was about 90%). In the future, these two strains can be further explored for application in the field. Taking together, the data showed that P. kribbensis XP1-6 can be used not only on tomato cultivar Knowyou 301, but also many other commercialized cultivars and potentially on grafted seedlings. The fact that seed coating can successfully inoculate tomato with P. kribbensis XP1-6 implies a further study to increase its viability and activity on seeds using appropriate coating materials is required. In addition, possessing more potential beneficial bacteria can increase the possibility to consider such tomato management strategy in different locations and growing seasons and uses of combined strains for synergistic effect on enhancement of tomato disease resistance or heat tolerance, thus creating more application value to the potential endophytes.
誌謝 I
中文摘要 II
ABSTRACT IV
CONTENTS VI
圖目錄 X
壹、 前言 1
一、 番茄簡介 1
二、 番茄品種特性 1
三、 非生物性逆境 3
四、 生物逆境 4
五、 植物生長促進細菌 (Plant Growth-Promoting Bacteria; PGPB) 6
貳、 研究動機與目的 8
參、 材料與方法 9
一、 供試植株與栽培條件 9
二、 供試菌株培養及保存 9
三、 內生菌菌種鑑定 9
1. Genome DNA 萃取與聚合酶連鎖反應 (polymerase chain reaction, PCR) 9
四、 內生菌接種方式 11
1. 種子處理 11
2. 種苗處理 11
3. 澆灌處理 11
五、 內生菌特性分析 11
1. 纏據狀況 11
2. 生長促進 12
3. 青枯病菌拮抗試驗 12
4. 抗生素能力 13
5. 藥劑感受性測試 13
6. 生理生化測試 13
六、 耐熱試驗 16
七、 青枯病菌接種試驗 16
八、 以內生菌包覆番茄種子可行性評估 16
1. 風乾時間之影響評估 16
2. 浸泡時間之影響評估 17
3. 儲架壽命調查 17
九、土壤混拌內生菌儲架壽命 17
十、 促進生長試驗 17
十一、 統計分析 18
肆、 結果 19
一、 P. kribbensis XP1-6 應用性評估 19
1. P. kribbensis XP1-6 在不同品種番茄及根砧品種根系的纏據效果 19
2. 透過不同處理方式接種 P. kribbensis XP1-6 之番茄植株對青枯病罹病度之差異評估 19
3. 種子包覆內生菌之效果評估 20
4. 風乾種子儲架壽命 21
5. 土壤混拌內生菌之儲架壽命 21
6. 接種 P. kribbensis XP1-6 對不同品種番茄於高溫下的耐受性影響 21
6. 高溫下 P. kribbensis XP1-6 對番茄生殖生長的影響 22
二、促進番茄青枯病抗性潛力菌株之再篩選 22
1. 提高番茄抗青枯病能力潛力菌株之再篩選 22
2. 潛力內生菌株特性分析 23
3. 潛力內生菌株對青枯病菌之拮抗能力 23
4. 內生菌 XK1-15 和 MP4-1 鑑定 23
5.抗生素敏感性測試 (antibiotic sensitivity test) 24
6. 藥劑感受性測試 24
6. 生理生化測試 25
7. 促進生長試驗 25
伍、 討論 26
陸、 結論 30
柒、 參考文獻 31
捌、 表 38
玖、 圖 43
拾、 附錄 58
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