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研究生:陳和緯
研究生(外文):Her-Wei Chen
論文名稱:Bacillusmycoides、水楊酸類似物及harpin蛋白對萵苣幼苗生長及抗病反應的效果
論文名稱(外文):Effects of Bacillus mycoides, SA analogue, and harpin on the growth and disease-resistant response of lettuce seedlings
指導教授:張碧芳張碧芳引用關係
指導教授(外文):P. F. L. Chang
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
畢業學年度:96
語文別:中文
論文頁數:65
中文關鍵詞:植物根圈促生細菌蕈狀芽孢桿菌BTH萵苣萎凋病健旺harpin
外文關鍵詞:Plant growth-promoting rhizobacteriaBacillus mycoidesBTHFusarium wilt of lettuceMessenger STSharpin
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植物根圈促生細菌 (Plant growth-promoting rhizobacteria, PGPR) 可分泌植物生長的調控因子以活化根部的代謝作用,如植物荷爾蒙,或是藉由分泌抗生物質、嵌鐵物質、競爭生態位與養份及誘導植株抗性來降低植物病原菌的侵害等方式來促進植物生長。本研究目的為施用 BM CHT2402 於不同作物上觀察其促進作物生長的能力,且進一步針對萵苣萎凋病進行防治試驗並找出可能的防禦反應。本研究中發現 BM CHT2402具有產生吲哚乙酸 (Indole-3-Acetic Acid, IAA) 與氨 (NH3) 之特性,將 BM CHT2402的細菌懸浮液混拌入栽培介質後種植番茄、西瓜及萵苣植株三週後,具有促進植物生長的趨勢,其中以萵苣明豐三號品種與番茄農友 301 品種有顯著差異。進一步嘗試以澆灌法施用 BM CHT2402 細菌懸浮液於不同作物,結果並無促進植物生長的趨勢。由萵苣萎凋病菌 (Fusarium oxysporum f. sp. lactucae) 所引起的萵苣萎凋病 (Fusarium wilt of lettuce) 為台灣葉萵苣夏季生產之主要限制因素,本研究中利用BM CHT2402 混拌栽培介質處理、或以水楊酸類似物,植物活化劑苯基 (1,2,3) 噻二唑-7-硫代羧酸硫甲酯 [benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, BTH] 及主成分為 harpin 蛋白的健旺肥料澆灌處理萵苣幼苗後,分析其對防治萵苣萎凋病的效果,結果顯示 BTH 澆灌處理與BM CHT2402 混拌栽培介質處理都有降低萵苣萎凋病罹病度的能力,而健旺澆灌處理則是各重複試驗的差異很大,以統計分析後,其效果並不顯著。BM CHT2402本身對於 F. oxysporum f. sp. lactucae FO-18 菌株沒有直接的拮抗效果,以 BM CHT2402 混拌栽培介質處理後,萵苣植株的苯丙氨酸氨裂解酶 (phenyl alanine ammonia lyase, PAL) 的基因表現量有降低的現象,而可溶性酚化物 (soluble phenolic compound) 的含量與 PAL 的酵素活性皆沒有提升,且會降低 guaiacol peroxidase (GPX) 的酵素活性,目前推測 BM CHT2402 施用後可搶先佔據生態位以降低萵苣萎凋病的罹病度或誘發植物體內其他的防禦反應。BTH 澆灌處理萵苣植株後,萵苣地上部與根部的 PAL 基因表現明顯增強,而萵苣根部中 PAL 與 GPX 的酵素活性都高於對照組,此現象可能與 BTH 澆灌處理可以降低萵苣萎凋病的罹病度相關。不同重複的健旺處理對降低罹病度的差異很大,應不適合用來防治萵苣萎凋病。
Plant growth-promoting rhizobacteria could enhance plant health by providing plant growth regulators such as phytohormones to activate the metabolic activities of plant roots, secreting antibiotics or siderphores, competing for the ecological niches or nutrition, and inducing plant defense responses, so as to protect plants from attack by phytopathogens or reduce the disease severity. In this study, application BM CHT2402 observed the ability of promotion different crops growth, controlling Fusarium wilt of lettuce and found possible plant defense-related response. In this study, the abilities of BM CHT2402 to produce indole-3-acetic acid (IAA) and ammonia (NH3) were observed. Soil mixed with cell suspension of BM CHT2402 had the potential to promote the growth of tomato, watermelon, and lettuce plants, Ming-feng NO. 3 cultivar of lettuce and KNOWN-YOU Farmers 301 cultivar of tomato had significant different. However, soil irrigated with cell suspension of BM CHT2402 had no obvious growth-promoting effects on these plants. Fusarium wilt caused by Fusarium oxysporum f. sp. lactucae (Fola) has become one of the most severe diseases of lettuce in Taiwan during summer. In this study, applications of BM CHT2402, SA analogue, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH), and Messenger STS (MSTS) on lettuce plants were tested on controlling this wilt disease. We found that both BTH irrigation and soil amendment with BM CHT2402 could significantly reduce the disease severity of Fusarium wilt of lettuce, but the MSTS treatment was unable to control this disease. According to the results of duel culture, BM CHT2402 itself did not have antagonistic activity against Fola FO-18. For soil amendment with BM CHT2402, the gene expression of phenyl alanine ammonia lyase (PAL) in lettuce was reduced, the content of soluble phenolic compounds and PAL enzyme activity in lettuce plants did not increase, and the enzyme activity of guaiacol peroxidase (GPX) was reduced. The mechanism of BM CHT2402 to reduce disease severity of Fusarium wilt of lettuce was still not known, but it may relate to the ability of BM CHT2402 to colonize on lettuce roots, or to induce other defense response in lettuce. BTH treatment could induce gene expression of PAL, and increase the enzyme activity of PAL and GPX. This may be the reason why BTH treatment could reduce the disease severity of Fusarium wilt of lettuce. The potential of MSTS to reduce the disease severity caused by Fola was not significant, so MSTS treatment may not be suitable for controlling Fusarium wilt of lettuce.
中文摘要…………………………………………………………i
英文摘要…………………………………………………………iii
前言………………………………………………………………1
材料與方法………………………………………………………9
供試菌株來源與培養……………………………………………9
供試植株…………………………………………………………10
BM CHT2402的 PGPR 特性分析…………………………………10
(一) 吲哚乙酸 (indole-3-acetic acid, IAA) 產量分析…10
(二) 產氨 (NH3) 能力分析……………………………………11
(三) 產氰化物 (HCN) 能力分析………………………………12
(四) 幾丁質分解酵素 (chitinase) 活性分析………………12
(五) 溶磷 (Phosphate solubilization) 能力分析……… 12
BM CHT2402 對植株生長之影響……………………………… 13
(一) 混拌栽培介質施用……………………………………… 13
(二) 澆灌施用………………………………………………… 13
萵苣萎凋病防治試驗……………………………………………14
(一) 病原菌製備……………………………………………… 14
(二) 罹病等級與罹病度……………………………………… 14
(三) 萵苣植株接種…………………………………………… 15
對峙培養測試……………………………………………………15
北方雜合法分析萵苣植株苯丙氨酸氨裂解酶 (phenyl alanine ammonia lyase,PAL) 之表現………………………………… 16
(一) 傷口處理萵苣葉片……………………………………… 16
(二) 植物總量 RNA 之萃取……………………………………16
(三) RNA變性膠體電泳之分析…………………………………17
(四) 反轉錄-聚合酵素連鎖反應 (reverse transcription -polymerase chain reaction, RT-PCR)………………………18
(五) PAL 基因之選殖及定序………………………………… 19
(六) 北方雜合法分析 PAL 基因之表現………………………20
可溶性酚化物萃取………………………………………………22
酵素活性測定……………………………………………………23
(一) PAL 活性測定…………………………………………… 23
(二) 萵苣植株之 Guaiacol peroxidase (GPX) 活性測定…24
結果………………………………………………………………26
BM CHT2402菌株的 PGPR 特性分析……………………………26
BM CHT2402菌株對植株生長之影響……………………………26
(一) 混拌栽培介質施用……………………………………… 26
(二) 澆灌施用………………………………………………… 26
萵苣萎凋病防治試驗……………………………………………27
對峙培養測試……………………………………………………27
萵苣植株的 PAL 基因表現…………………………………… 28
可溶性酚化物萃取分析……………………………………… 28
萵苣植株的 PAL 酵素活性測定……………………………… 29
萵苣植株之 GPX 活性測定…………………………………… 29
討論………………………………………………………………31
參考文獻…………………………………………………………36
圖表………………………………………………………………44
附錄………………………………………………………………62

圖表目次
表一、B. mycoides CHT2402 菌株的 PGPR 特性分析………44

圖一、以 B. mycoides CHT2402 懸浮液混拌栽培介質處理種植三週後對不同作物鮮重的影響。……………………………………45

圖二、以 B. mycoides CHT2402 懸浮液澆灌處理後對不同作物鮮重的影響……………………………………………………………47

圖三、利用 B. mycoides CHT2402、BTH 及健旺處理以防治萵苣萎凋病之效果…………………………………………………………49

圖四、B. mycoides CHT2402 與 Fusarium oxysporum f. sp. lactucae FO-18 菌株在不同培養基上的對峙情形………… 52

圖五、以北方雜合法分析 B. mycoides CHT2402、BTH 及健旺處理對萵苣地上部及根部中的苯丙氨酸氨裂解酶基因表現之影響…54

圖六、利用 B. mycoides CHT2402、BTH 及健旺處理對萵苣植株可溶性酚化物含量之影響……………………………………………56

圖七、利用 B. mycoides CHT2402、BTH 及健旺處理對萵苣的苯丙氨酸氨裂解酶活性之影響…………………………………………58

圖八、利用 B. mycoides CHT2402、BTH 及健旺處理對萵苣的 guaiacol peroxidase 活性之影響……………………………60
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