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研究生:蕭鈺霏
研究生(外文):Yu-Fei Hsiao
論文名稱:C1L細菌處理與葉枯病菌感染改變玉米根圈之細菌族群
論文名稱(外文):Bacterial community in maize rhizosphere affected by C1L bacterization and Cochliobolus heterostrophus infection
指導教授:陳昭瑩陳昭瑩引用關係
口試委員:張雅君李佳音郭志鴻黃祥恩
口試日期:2014-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
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:2014
畢業學年度:102
語文別:中文
論文頁數:64
中文關鍵詞:根圈細菌族群臘狀芽孢桿菌玉米葉枯病菌假單胞菌屬
外文關鍵詞:Rhizobacterial communityBacillus cereusCochliobolus heterostrophusPseudomonasBacillus
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植物根圈存在大量的微生物,整體根圈的微生物相組成與植物健康有高度的關聯性。臘狀芽孢桿菌C1L為臺灣百合根圈篩選出的生物防治潛力菌株,目前已被研究證實具有誘導玉米系統性抗葉枯病的能力。本研究中探討施用C1L菌株於玉米根圈,對根圈細菌族群之影響,並分析葉枯病菌感染玉米葉片後,所引發的根圈細菌族群變化,及從中找出新的具生物防治潛力菌株。研究結果顯示玉米根圈在施用C1L菌株後3天,Proteobacteria門假單胞菌屬根圈細菌之豐度大幅增加;而玉米根圈在施用C1L菌株後6天,假單胞菌屬根圈細菌之豐度則回復至原始狀態。當玉米根圈在施用C1L菌株後3天,接種葉枯病菌3天後的假單胞菌屬根圈細菌之豐度比未施用C1L菌株但接種葉枯病菌者高。這些結果指出,假單胞菌屬根圈細菌族群的增加與植物抗病狀態有相當的關聯性。此外接種葉枯病菌後3天,腸桿菌屬、假單胞菌屬、類芽孢桿菌屬與芽孢桿菌屬根圈細菌的豐度,相對於對照組有增加的情形。另一方面,從施用C1L菌株後3天,再接種葉枯病菌後3天的玉米根圈中,分離到對葉枯病可降低至少30%罹病度的假單胞菌屬細菌12株以及芽孢桿菌屬細菌14株,推測這些分離株之施用能加強玉米植株之抗病性,有進一步應用發展的潛力。

A large number and wide variety of microorganisms are associated with plant roots and the genetic elements of rhizosphere microbiome determine the plant health. Bacillus cereus C1L screened from the rhizosphere of Lilium formosanum could reduce severe symptom development of southern corn leaf blight (SCLB) caused by Cochliobolus heterostrophus. In this research, the changes of maize rhizobacterial community post C1L application and C. heterostrophus infection were analyzed by metagenomic mining. The abundance of Pseudomonas rhizobaceria remarkably increased at 3 days post C1L application, then decreased to the original state at 6 days after C1L application. Moreover, the abundance of Pseudomonas rhizobacteria was higher at 3 days post inoculation with C. heterostrophus on maize plants 3 days post C1L application as compared with that without C1L pretreatment. These results indicated a high relatedness between Pseudomonas and disease resistance state of maize. Besides, the relative abundance of Enterobacter, Pseudomonas, Paenibacillus and Bacillus rhizobacteria increased in maize rhizosphere 3 days post inoculation with C. heterostrophus as compared with that in the control. On the other hand, 12 isolates of Pseudomonas and 14 isolates of Bacillus, reducing at least 30% disease severity of leaf blight in corn seedlings, were screened from maize rhizobacterial community. These bacterial isolates capable of enhancing plant disease resistance were presumed and have potential for further development of application.

目錄
壹、 中文摘要 1
貳、 英文摘要 2
參、 前言 3
肆、 前人研究 5
一、玉米與玉米葉枯病 5
二、臘狀芽孢桿菌 C1L 6
三、植物根圈細菌族群 6
四、植物對根圈微生物族群之影響 8
五、植物促生根圈細菌 9
六、細菌鞭毛與植物抗性誘導 11
伍、 材料與方法 12
一、供試菌株之培養與保存 12
1. 供試菌株之培養 12
2. 供試菌株之保存 12
二、C1L菌株施用與葉枯病菌接種對玉米根圈細菌族群之影響 13
1. 溫室試驗之植株栽培 13
2. C1L菌株對玉米葉枯病之防治效果確認 13
3. 玉米根圈細菌族群分析 13
4. 根圈微生物相總體DNA之萃取 14
5. 根圈細菌之16S rDNA片段增幅 14
6. 16S rDNA PCR產物之純化與序列確定 15
7. 454焦磷酸定序(454 pyrosequencing) 15
8. 序列資訊分析 15
三、玉米根圈ISR菌株之分離篩選 16
1. 植株栽培及處理 16
2. 根圈細菌之分離純化 16
3. 根圈細菌之ISR能力測試篩選 17
4. 根圈細菌ISR菌株之溶磷及固氮能力測試 17
四、根圈細菌菌株鞭毛蛋白之ISR作用測試 18
1. 溫室試驗之植株栽培 18
2. 細菌鞭毛萃取 19
3. SDS-聚丙烯醯胺膠體電泳分析 19
4. 細菌鞭毛之蛋白質含量測定 20
5. 人工合成短片段胜&;#32957;flg22C1L與flg22Pss 20
6. 細菌鞭毛蛋白與flg22之ISR作用測試 20
7. 癒傷葡聚醣(callose)累積測試 21
陸、 結果 22
一、C1L菌株對玉米葉枯病之防治效果 22
二、施用C1L菌株與接種葉枯病菌對玉米根圈細菌族群之影響 22
1.玉米根圈細菌族群之454焦磷酸定序結果 22
2.原始玉米根圈細菌族群分析 23
3.施用C1L菌株對玉米根圈細菌族群之影響 23
4. 施用C1L菌株防治葉枯病對玉米根圈細菌族群之影響 24
5. 接種葉枯病菌對玉米根圈細菌族群之影響 24
三、玉米根圈ISR菌株之分離篩選 25
1. 根圈細菌之分離純化 25
2. 根圈細菌之ISR菌株篩選 25
3. 根圈細菌ISR菌株之溶磷及固氮能力測試 25
4.根圈ISR菌株鑑定 26
四、根圈ISR菌株鞭毛蛋白之ISR作用測試 26
1. 細菌鞭毛蛋白與flg22之ISR作用測試 26
2.細菌鞭毛蛋白與flg22誘導阿拉伯芥癒傷葡聚醣累積測試 26
柒、 討論 28
捌、 參考文獻 34
玖、 圖表集 46
圖一、玉米根圈細菌族群分析之試驗設計 47
圖二、C1L細菌處理可降低玉米葉枯病的罹病度 48
圖三、處理組A~F各組重複樣品之玉米根圈細菌族群主座標分析 49
圖四、處理組A~F各組重複樣品之玉米根圈細菌族群親緣距離階層式群集分析 50
圖五、玉米根圈細菌族群組成豐度比較圖 51
圖六、處理組A ~F之玉米根圈細菌族群組成豐度比較圖 52
圖七、不同處理組之玉米根圈細菌族群組成豐度比較圖 53
圖八、處理組D~F之玉米根圈細菌族群中Proteobacteria門四個主要綱之相對豐度比較圖 54
圖九、處理組D~F之玉米根圈細菌族群γ-Proteobacteria綱中數個主要屬之相對豐度比較圖 55
圖十、處理組C之玉米根圈細菌族群Proteobacteria門中四個主要綱之相對豐度比較圖 56
圖十一、處理組C之玉米根圈細菌族群γ-Proteobacteria綱數個主要屬之相對豐度比較圖 57
圖十二、處理組C之玉米根圈細菌族群Firmicutes綱之相對豐度比較圖 58
圖十三、玉米根圈假單孢菌屬分離株之誘導系統性抗病能力測試 59
圖十四、玉米根圈革蘭氏陽性菌分離株之誘導系統性抗病能力測試 60
圖十五、flg22C1L與flg22Pss之胺基酸序列比對 61
圖十六、芽孢桿菌鞭毛蛋白及flg22C1L誘導系統性抗病作用測試 62
圖十七、C1L菌株鞭毛蛋白與flg22C1L誘導阿拉伯芥葉片之癒傷葡聚醣累積情形 63
圖十八、芽孢桿菌鞭毛蛋白誘導阿拉伯芥葉片之癒傷葡聚醣累積情形 64



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