臺灣博碩士論文加值系統

物質大小介於 1-100 nm 之間會有新的特性，此尺寸大小的物質被稱為奈米材料。一些奈米材料被報導具有抗微生物的活性，具有潛力應用於農業上。然而奈米材料對於生物體會產生何種影響，目前我們所知甚少。本研究使用奈米矽片銀 (AgNP/NSP) 與奈米矽片 (NSP) 測試對西瓜蔓割病菌 Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen 的影響，希望藉由瞭解奈米矽片銀與奈米矽片對西瓜蔓割病菌的影響及其作用機制，有助於將奈米材料應用於植物病害管理上。本研究顯示奈米矽片銀對西瓜蔓割病菌的抑制效果較奈米矽片強，奈米矽片銀可有效抑制西瓜蔓割病菌之菌絲生長、菌絲細胞活性及孢子活性。半胱氨酸為銀離子的螯合劑，添加半胱氨酸於奈米矽片銀、奈米矽片或硝酸銀中，僅會降低奈米矽片與硝酸銀對西瓜蔓割病菌的抑制效果，但不影響奈米矽片銀的抑菌效果。以掃描式電子顯微鏡觀察可知奈米矽片銀與奈米矽片會附著於西瓜蔓割病菌的表面，並造成細胞皺縮，而硝酸銀則無此現象，顯示奈米矽片銀的抑菌機制與硝酸銀不同。在酵素活性測試部分，奈米矽片銀與奈米矽片可與西瓜蔓割病菌的外泌蛋白質結合，並降低纖維素分解酵素的活性。奈米矽片銀可誘導西瓜蔓割病菌細胞壁合成相關基因 gas1 與 fks1的表現。本研究結果顯示奈米矽片銀與奈米矽片對於抑制西瓜蔓割病菌具有多層面的影響。本研究也建立奈米物質對植物生長的安全評估試驗，測試上述奈米材料對於萵苣、番茄及西瓜的影響，結果可知 100 ppm 奈米矽片銀對於萵苣、番茄與西瓜的種子發芽與幼苗生長具有抑制效果，尤其明顯抑制此三種植物幼苗根部的生長，顯示奈米矽片銀應用於作物病害管理上需要審慎思考其使用方式。奈米矽片銀與奈米矽片對於植物生長的影響仍有待後續進一步的測試。

Materials will have the new characteristics when their sizes are ranged in 1-100 nm. Materials in this size are defined as nanomaterials. Some nanomaterials were reported to pose antimicrobial activity. Nanomaterials have potential to be applied in agriculture. So far, we don’t know much about the effects of nanomaterials on organisms. In this study, we used silver nanoparticles on nano-scale silica platelets (AgNP/NSP) and nano-scale silica platelets (NSP) to test their effects on Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen (Fon). Understanding the effects of nanomaterials on Fon and their possible mechanisms would be helpful to apply nanomaterials in plant disease management. The results showed that AgNP/NSP had stronger inhibitory effect on Fon than NSP. AgNP/NSP could inhibit hyphal growth, and reduce cell activity and spore bioactivity of Fon. After adding cysteine, which could bind silver ions, NSP and silver nitrate, but not AgNP/NSP, reduced their inhibitory effect of Fon. Results of scanning electron microscopy revealed that both AgNP/NSP and NSP, but not silver nitrate, could attach on the cell surface of Fon and cause cell shrinkage. It indicates that AgNP/NSP has different inhibitory mechanism from its bulk materials, silver nitrate, on Fon. In enzyme activity assay, both AgNP/NSP and NSP could absorb secreted proteins of Fon and reduced cellulase activity was observed in the secreted proteins of Fon. AgNP/NSP could induce gas1 and fks1 gene expression of F. oxysporum, both genes are involved in fungal cell wall synthesis. The results suggested that AgNP/NSP and NSP may have multiple inhibitory mechanisms on Fon. In this study, to establish the safety test about the impact of nanomaterials on plant growth, the effects of AgNP/NSP and NSP were tested on lettuce, tomato, and watermelon. AgNP/NSP showed negative effects on seed germination and seedling growth of lettuce, tomato, and watermelon at 100 ppm. The root growth of the tested seedlings was significantly inhibited. It indicates that AgNP/NSP should be used carefully in plant disease management. Effects of AgNP/NSP and NSP on plant growth need to be further tested in the future.

中文摘要 I
英文摘要 II
圖表目次 VI
前言 1
材料與方法 8
一、供試材料與來源 8
二、奈米矽片銀與奈米矽片對西瓜蔓割病菌之抑制效果 8
(一) 奈米物質對真菌菌絲生長之影響 8
(二) 奈米物質對真菌菌絲細胞活性之影響 9
(三) 奈米物質對真菌孢子活性之影響 9
三、半胱氨酸 (cysteine) 對奈米矽片銀與奈米矽片抑制西瓜蔓割病菌效果
之影響 10
四、以電子顯微鏡觀察奈米矽片銀與奈米矽片對西瓜蔓割病菌之影響 10
(一) 掃描式電子顯微鏡法 (scanning electron microscopy) 10
(二) 穿透式電子顯微鏡法 (transmission electron microscopy) 10
五、奈米矽片銀與奈米矽片對西瓜蔓割病菌菌絲細胞膜通透性之影響 11
(一) 以電解值滲漏度評估細胞膜之通透性 11
(二) 以碘化丙啶 (propidium iodide) 染色法評估細胞膜之通透性 11
六、奈米矽片銀與奈米矽片對西瓜蔓割病菌養分利用之影響 12
(一) 真菌之培養方式 12
(二) 還原醣含量之測量 12
(三) 蛋白質含量之測量 12
七、奈米矽片銀與奈米矽片對西瓜蔓割病菌外泌蛋白質活性之影響 13
(一) 真菌外泌蛋白質的收集 13
(二) 奈米物質吸附真菌外泌蛋白質之測試 13
(三) 纖維素分解酵素之活性測定 13
八、奈米矽片銀與奈米矽片對西瓜蔓割病菌合成細胞壁基因表現之影響 14
(一) 材料準備與 RNA 萃取 14
(二) RNA 品質確認與反轉錄作用 (reverse transcription) 14
(三) 以即時聚合酶連鎖反應 (real-time PCR) 分析基因表現 15
九、奈米矽片銀與奈米矽片對植物之影響 15
(一) 奈米物質對種子發芽之影響 15
(二) 奈米物質對植物幼苗生長之影響 16
十、統計分析 16
結果 17
一、奈米矽片銀與奈米矽片對西瓜蔓割病菌之抑制效果 17
(一) 奈米物質對真菌菌絲生長之影響 17
(二) 奈米物質對真菌菌絲細胞活性之影響 17
(三) 奈米物質對真菌孢子活性之影響 17
二、半胱氨酸對奈米矽片銀與奈米矽片抑制西瓜蔓割病菌效果之影響 18
三、以電子顯微鏡觀察奈米矽片銀與奈米矽片對西瓜蔓割病菌之影響 18
(一) 以掃描式電子顯微鏡觀察之結果 18
(二) 以穿透式電子顯微鏡觀察之結果 18
四、奈米矽片銀與奈米矽片對西瓜蔓割病菌菌絲細胞膜通透性之影響 18
五、奈米矽片銀與奈米矽片對西瓜蔓割病菌養分利用之影響 19
六、奈米矽片銀與奈米矽片對西瓜蔓割病菌外泌蛋白質活性之影響 20
七、奈米矽片銀與奈米矽片對西瓜蔓割病菌合成細胞壁基因表現之影響 20
八、奈米矽片銀與奈米矽片對植物之影響 21
(一) 奈米物質對種子發芽之影響 21
(二) 奈米植物對植物幼苗生長之影響 22
討論 24
參考文獻 31
圖表 44

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