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研究生:蔡效耕
研究生(外文):Hsiao-Keng Tsai
論文名稱:利用溶磷特性及分泌物探討誘發性溶磷菌之溶磷現象
論文名稱(外文):Characteristic of Induced Phosphate-solubilizing Activities and Exudates of Bacteria
指導教授:楊秋忠楊秋忠引用關係
指導教授(外文):Chiu-Chung Young
學位類別:碩士
校院名稱:國立中興大學
系所名稱:土壤環境科學系
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:109
中文關鍵詞:溶磷菌大腸桿菌誘發溶磷2-酮基葡萄糖酸PQQ
外文關鍵詞:phosphate-solubilizing bacteriaEscherichia coliinduced phosphate-solubilizing2-keto-D-gluconic acidpyrroloquinoline quinone
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受到他種微生物代謝產物而誘發溶磷能力的誘發性溶磷菌可能普遍存在於土壤中,但尚無人加以探討。因此本實驗探討誘發溶磷菌的溶磷現象與其分泌物加以推測可能的溶磷誘導物質。實驗以Pseudomonas cepacia Al74 - Escherichia coli JM109作為標準溶磷誘導系統,在不同誘導距離下觀察在平面培養基中菌株之溶磷圈與菌落大小,同時觀察實驗室中已知菌種15株溶磷菌與24株根瘤菌與大腸桿菌之平面誘導溶磷現象。並將經篩選具有平面誘導溶磷現象之溶磷菌Al74,bc29,bc30與根瘤菌A1,A9與大腸桿菌混合培養於液態鈣磷培養基中,於180小時內以鉬藍法及HPLC測量有效性磷含量與有機酸。此外設計之胞膜葡萄醣去氫酶相關引子經聚合酶鏈反應圖譜用於探討15株溶磷菌株之基因差異。結果顯示,在本實驗溶磷微生物的溶磷誘導特性與菌種有關,具有誘導溶磷現象的根瘤菌株皆為溶磷菌。以 P. cepacia Al74-E. coli JM109作為標準系統,其平面培養基之溶磷環溶磷圈比值與誘導距離相關,可以作為平面誘導特性數值計量。而以液態鈣磷培養基混合培養顯示在初期,混合大腸桿菌會降低有效性磷水準,而在後期增加有效性磷水準,但在鈣磷液體不若平面培養基觀測顯著;此外在有機酸分泌物上,溶磷菌Al74,bc29,bc30與根瘤菌A1分泌與葡萄醣產酸假說的有機產物2-酮基葡萄糖酸 (2-keto-D-gluconic acid),但分泌大量多醣體之根瘤菌A9則無法偵測到此一物質。引子圖譜顯示大部份的菌株為多片段的,PqC與GDH兩引子在預測亮帶的有無與溶磷誘導作用有0.73以上的相關,而PqA與平面培養基的溶磷圈有0.74相關,葡萄醣氧化酶輔因子PQQ可能是造成溶磷誘導作用的原因之一。

The non-phosphate-solubilizing bacteria may be induced by metabolites of ether phosphate-solubilizing bacteria. We try to investigate the characteristic of induced phosphate-solubilizing (IPS) activities and exudates of bacteria. The Pseudomonas cepacia Al74 and Escherichia coli JM109 were applied as standard IPS system for measuring the diameter between clear zone and colony. Besides, we analysised of organic acids and water-soluble phosphorus in tricalcium phosphate medium during 180 hr of 5 strain including phosphate- solubilizing bacteria (PSB) strain (Al74, bc29, and bc30), and Rhizobium sp. (A1, and A9) by HPLC and spectrometer after observation the phenomenon of 15 PSB and 24 Rhizobium strain on the tricalcium phosphate solid medium. In addition to activities and exudates study, glucose dehydrogenase (GDH)-related primers were used to study the genotype of 15 PSB strain. The result of IPS on the solid medium is related to bacteria species, and Rhizobium both having having IPS activity and phosphate-solubilizing ability. In standard IPS system, the ratio, which was used in quantilification of the effects of IPS, of phosphate-solubilizing ring to circle was related to IPS distance. In liquid system, co-incubation with E. coli JM109 were reduced the amount of water-soluble phosphorus at the initial stage, and increased later. Except polysaccharid-richness Rhizobium sp. A1, the secretions of Al74, bc29, bc30, and Rhizobium sp. A1 all have secreted 2-keto-D-gluconic acid, which was cope with phosphate-solubilizing hypothesis. The pattern of most PSB PCR products of dehydrogenase-related primers was variety. The primer PqC and GDH used in prediction IPS have correlation value over 0.73, and correlation between PqA and phosphate-solubilizing clear zone is 0.74. Therefore the pyrroloquinoline quinone, cofactor of GDH is one factor in the IPS system.

壹、目錄2
貳、圖表目錄4
參、摘要6
肆、前言9
伍、前人研究11
一、土壤中無機磷的型態及其重要性11
二、溶磷微生物之研究發展11
三、溶磷菌溶磷機制之探討12
(一)有機酸之分泌13
(二)微生物於銨同化作用或呼吸作用時釋出質子之效應17
(三)無機酸之產生17
(四)CO2之作用17
(五)H2S之作用18
(六)腐質酸及黃酸之作用18
四、溶磷菌在酵素與基因學的研究18
五、溶磷菌接種至土壤中之效應23
六、接種溶磷菌對植物的效應23
(一)接種溶磷微生物對植物生長的效應23
(二)聯合接種溶磷菌與囊叢枝菌根菌對植物之效應24
(三)聯合接種溶磷菌與固氮根瘤菌對植物之效應25
七、以PCR為基礎的DNA指紋(FINGERPRINTER)25
陸、材料與方法26
一、平面培養基誘導性實驗26
(一)標準系統平面培養基溶磷誘導現象之觀察26
(二)不同菌株在平面培養基溶磷誘導現象之觀察27
二、菌株胞外分泌物之探討27
(一)初步篩選與保存27
(二)菌株生長與誘導特性分析28
三、專一性引子及聚合酶鏈索反應32
柒、結果與討論37
一、平面培養基誘導性實驗37
(一)標準系統平面培養基溶磷誘導現象之觀察38
(二)不同菌株在平面培養基溶磷誘導現象之觀察47
二、菌株胞外分泌物之探討51
(一)初步篩選與保存51
(二)菌株生長與誘導特性分析54
三、PQQ與GDH基因引子75
四、誘發性溶磷模式探討81
(一)固體培養基誘發性溶磷模式81
(二)液體培養基誘發性溶磷模式82
(三)固液體培養基誘發性溶磷模式差異性87
(四)利用特殊培養基修正土壤溶磷菌溶磷潛能87
捌、結論88
玖、參考文獻89
壹拾、附錄103
一、複序列分析序列比對資料103

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