臺灣博碩士論文加值系統

土壤中存有大量的固定性磷化合物，經溶磷微生物作用，可轉換成植物可吸收利用的有效性磷源。目前針對溶磷細菌進行溶磷作用機制之研究，大部份是以有機酸的分泌，而達到溶解難溶性無機磷化合物，為了了解更多在台灣亞熱帶地區溶磷細菌與有機酸之間的關係及其多樣性，本研究共分離出36株菌株，利用16S rDNA基因定序法，鑑別不同菌株在系統分類所在位置，及RAPD (Random Amplified Polymorphic DNA) 分子標示法分析菌株與菌株之間基因的差異性，並進一步測定其溶磷能力，分析有機酸物質。結果顯示36株菌株經16S rDNA定序，共鑑別出7屬34株 (Arhtrobacter nicotinovorans 2株，Arhtrobacter ureafaciens 5株，Chryseobacterium indoltheticum 1株，Gordonia sp. 1株，Nocardioides simplex 1株，Phyllobacterium rubiacearum 1株，Rhodococcus erythropolis 8株及Serratia sp. 5株)，二株未命名菌株，其中一株 (bc13，unnamed organism) 在基因序列比對上與Cucurbit yellow vine disease bacterium接近，另一株 (bc21，unnamed organism) 在RDP的資料庫中則無相近的菌種序列，有可能為台灣本土的特有菌株。此外Arthrobacter nicotinovorans，Arthrobacter ureafaciens，Nocardioides simplex，Phyllobacterium rubiacearum及Rhodococcus erythropolis是以往所提出常見的溶磷微生物中所沒有的，可見具溶磷能力之微生物，其分佈範圍甚廣。RAPD試驗以UPGMA程序所建構的遺傳相關樹狀圖，顯示菌株與菌株間的差異很明顯，產生4個群叢，具相當高的歧異度，有效的區分出同一菌種中菌株之差異性，但所產生的群叢與溶磷量並無顯著相關。測定36株菌株之溶磷能力，顯示培養基pH值及活菌數對溶磷量呈顯著相關 (P<0.05)，但菌株Chryseobacterium indoltheticum (bc05) 其pH值為6.0，溶磷量卻相當高 (289.8 mg L-1)，利用高效能液體層析儀分析菌體分泌液也發現此菌株另有unknown3物質的分泌，是否此物質是利用鉗合作用或其他因子的作用而造成溶磷現象，而非環境pH值降低所致，則需進一步的再探討。此外視菌種產生不同的有機酸及生成量，而達到溶磷能力的效果，並且大部份的菌株所產生的有機酸物質不只一種，隨著溶磷量增高，有機酸含量也有相對增加之趨勢，以菌株Arthrobacter nicotinovorans (bc03，bc34) 之有機酸差異最為明顯，bc03產生大量的磷酸根及有機酸unknown1物質，相對於bc34則是低磷酸根及低有機酸，其他菌株也有產生如葡萄醣酸、檸檬酸、乳酸、琥珀酸、丙酸及未知的有機酸unknown2等。本研究所分析的36株菌株，顯示其有機酸在溶解難溶性磷酸鹽扮演了很重要的角色，在台灣亞熱帶地區溶磷微生物具有其多樣性的特徵。

In soil, a lot of soluble inorganic phosphate is fixed and rapidly immobilized to forms which are unavailable to plants. The phosphate-solubilizing microorganisms (PSM) are capable of solubilizing insoluble inorganic phosphate into forms that can be utilized by plants. It is generally accepted that the major mechanism of mineral phosphate solubilization is the action of organic acids secreted by microorganisms. Our objective was to understand the diversity of phosphate-solubilizing microorganisms in the subtropical zone, Taiwan. Thirteen six strains were isolated and identified by sequencing partial 16S rDNA. Isolates were analysed by RAPD to examine in detail the divergence among genomes and the secreted organic acids were analyzed by HPLC. The results of 34 identified strains are Arthrobacter nicotinovorans, 1 strain, Arthrobacter ureafaciens, 5 strains, Bacillus sp., 10 strains, Chryseobacterium indoltheticum, 1 strain, Gordonia sp., 1 strain, Nocardioides simplex, 1 strain, Phyllobacterium rubiacearum, 1 strain, Rhodococcus erythropolis, 8 strains and Serratia sp., 5 strains. One unnamed organism (bc13) is close to cucurbit yellow vine disease bacterium in the alignment of RDP database. Another unnamed organism (bc21) shows no close relationship to any bacteria specie in RDP database. The unnamed organism (bc21) may be a new local strain in Taiwan. Among the identified strains, Arthrobacter nicotinovorans, Arthrobacter ureafaciens, Nocardioides simplex, Phyllobacterium rubiacearum and Rhodococcus erythropolis were not ever reported in their phosphate solubilizing capability. It is thus clear that phosphate-solubilizing microorganisms distribute widely. The result of RAPD analysis can be divided into 4 clusters. The correlation coefficient of isolates among populations, pH values and P-concentrations showed significance (P<0.05). Chryseobacterium indoltheticum (bc05) had a lot of P-concentrations (289.8 mgL-1) with middle pH value at 6.0. We found there was unknown3 substance in HPLC analysis. Whether unknown3 substance effected capability of solubilizing phosphate or other factors existed, that needs to research further. The more organic acids secreted and the more P-concentrations existed in the media which was clearly demonstrated in Arthrobacter nicotinovorans (bc03, bc34). Other isolates also produced more than one organic acid. Some organic acids secreted such as gluconic acid, citric acid, lactic acid, succinic acid, unknown1 and unknown2 were found among other strains. In this study, organic acids truly play an important role in solubilizing phosphate and these strains which we isolated in the subtropical zone, Taiwan, are divergent in genome and metabolism of organic acids.

目錄
中文摘要……………………………………………………………. I
英文摘要……………………………………………………………. III
目錄…………………………………………………………………. V
表次………………………………………………………………….. VII
圖次………………………………………………………………….. IX
壹、前言………………………………………………………….. 1
貳、前人研究…………………………………………………….. 3
一、土壤中磷的型態………………………………………… 3
二、溶磷微生物種類………………………………………… 4
三、溶磷微生物溶磷機制之探討…………………………… 6
四、利用16S rDNA做為細菌分類之特徵………………….. 8
五、溶磷微生物之分子遺傳指紋…………………………… 9
六、接種溶磷微生物對植物的效應………………………… 10
參、材料與方法
一、菌株來源及鑑別………………………………………… 12
二、逢機增幅多形性DNA分析……………………………. 17
三、pH值、菌落數及水溶性磷濃度分析………………….. 19
四、菌體培養液之有機酸分析……………………………… 20
五、資料庫之建立…………………………………………… 21
肆、結果
一、菌株鑑別………………………………………………… 22
二、逢機增幅多形性DNA分析……………………………. 23
三、pH值、菌落數及水溶性磷濃度分析………………….. 24
四、菌體培養液之有機酸分析……………………………… 26
五、資料庫之建立…………………………………………… 28
伍、討論…………………………………………………………... 29
陸、結論…………………………………………………………... 36
柒、參考文獻……………………………………………………… 38
附錄………………………………………………………………….. 86
表次
表一、不同微生物在無機磷酸鹽類培養基中之磷含量…………. 5
表二、不同菌株之鑑定…………………………………………… 46
表三、RAPD分析中使用的引子序列…………………………… 47
表四、RAPD放大分析結果之二元數據資料…………………… 49
表五、不同菌株於液體鈣磷培養基培養三天後活菌數、pH及
水溶性磷濃度……………………………………………… 56
表六、標準有機酸之滯留時間…………………………………… 58
表七、不同菌株在鈣磷培養基中，以HPLC分析胞外分泌物中
的有機酸種類……………………………………………… 59
表八、不同菌株在鈣磷培養基中，以HPLC分析胞外分泌物中
的unknown物質…………………………………………… 60
表九、以HPLC分析不同菌株胞外分泌物中unknown物質之滯
留時間………………………………………………………. 61
表十、菌株在鈣磷培養基中unknown1物質與水溶性磷含量
之相關………………………………………………………. 62
表十一、Bacillus sp.在鈣磷培養基中有機酸與水溶性磷含量之相關
…………………………………………………………….. 63
表十二、Serratia sp.及unnamed organism bc13在鈣磷培養基中有
機酸與水溶性磷含量之相關……………………………. 64
表十三、Rhodococcus erythropolis在鈣磷培養基中有機酸與水溶性磷含量之相關……………………………………………….. 65
圖次
圖一、根據RAPD相似度所得6株溶磷菌株之親緣關係圖……. 9
圖二、菌株經菌合脢鏈反應後之產物……………………………. 66
圖三、利用UPGMA方法分析三十六株菌株及RDP資料庫中
相近菌株的16S rDNA 500個鹼基對之關係圖…………… 67
圖四、不同菌株逢機增幅多形性DNA分析之產物……………… 68
圖五、根據RAPD相似度得到的三十六株菌株之親緣關係圖….. 69
圖六、不同菌株pH值與溶磷量之關係圖………………………… 70
圖七、不同菌株活菌數與溶磷量之關係圖………………………... 71
圖八、不同菌株分泌液中有機酸之HPLC分析結果……………… 72
圖九、溶磷微生物資料庫系統之架構圖…………………………... 82
圖十、溶磷微生物資料庫之首頁…………………………………… 83
圖十一、溶磷微生物資料庫之簡述………………………………... 83
圖十二、溶磷微生物基本資料查詢………………………………… 84
圖十三、溶磷微生物生理特徵查詢………………………………... 84
圖十四、溶磷微生物生理特徵查詢………………………………… 85

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