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標題: Characteristics of Azotobacter species isolated from rice rhizosphere soils of organic and conventional farmings in Taiwan
作者: Meng-Ke Tsai
關鍵字: Azotobater;rice;free-living nitrogen fixation bacterium;固氮菌屬;水稻;有益菌;有機與慣行水稻田
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Azotobacter, a free-living nitrogen fixation bacterium, has been proved in promoting rice growth through releasing some beneficial compounds. However, the function of Azotobacter spp. are highly specific, and rarely studies focus on the comparisons of their status in Taiwan organic and conventional paddy rice fields. The purposes of this study were: 1. to screen and classify with 16S rDNA-based of the Azotobacter isolates from 8 organic and 8 conventional paddy rice fields distribution around different Taiwan area. 2. to determine and compare the characters of the isolates between organic and conventional fields. 3. to select the highly potential isolates and evaluate their apparent beneficial function on the growth of rice seedlings. Results showed that there were 40 Azotobacter isolates identified, based on 16S rDNA sequence analysis.There are five Azotobacter spp. been found, including A. armeniacus, A. beijerinckii, A. chroococcum, A. tropicalis and A. vinelandii. There were 12.5% isolates from conventional farms, and 87.5% from organic farms. In acid soils A. beijerinckii (53.3%, 43.8%) is the most popular and A chroococcum (40.8%, 43.8%) is the second at soil pH 4.5 - 5.5 and pH 5.5 - 6.5, respectively. As the soil pH increased, the percentage of A. Beijerinckii isolates decreased. In soil pH ranged 6.5 - 7.5 the most popular species are A. chroococcum (33.3%), A. vinelandii (33.3%) and followed by A. beijerinckii (22.2%). The ability of Pyoverdine production, tricalcium phosphate dissolution and gelatin hydrolysis of Azotobacter isolates which screened from organic farms is more than from conventional farms. In this study, a total of 14 Azotobacter isolates produce Pyoverdine after subculture, 16 isolates can use the carboxymethyl cellulose as carcon source, the colonies of 17 isolates will turn into brown after 3 days, and 5 isolates (CHB202, CHB214, CHB251, CHB257 and CHB278) have the ability to use lignin. In addition, CHB194, CHB213, CHB216 and CHB251 have been identified as the nif H genes owner, but their ethylene reduction ability is too low to be considered as nitrogen fixer. All isolates could grow at tricalcium phosphate medium, but there are more than half of the isolates (22/40) have no transparent ring around the colonies. There were 5 potential Azotobacter isolates, CHB196, CHB198, CHB188, CHB197, CHB260 have been evaluated as potential beneficial microorganisms by thier performances on nitrogen fixation, IAA production and tricalcium phosphate solubilization. In bioassay of these five isolates on the growth of rice seedlings, results showed that all of the inoculants increased the growth of rice seedling shoot and root. Furthermore, the promotion effect of CHB188, CHB196, CHB197 isolates reached significant level compared to the control. The present work provided the information that the Azotobacter species screened from Taiwan paddy rice fileds have potential in increasing the production of rice.

接種游離固氮菌屬Azotobacter被證實可增加土壤中氮含量及促進水稻生長。然而Azotobacter spp.的功能依據地區而有高度特異性且台灣水稻田之栽培農法與土壤性質對分離之 Azotobacter 菌種的影響研究較少。本研究的目的可分為1.分離與以16S rDNA 序列鑑定自台灣本土水稻田中分離之Azotobacter spp.。2.探討台灣本土有機與慣行農法水稻根圈土壤中Azotobacter 分離菌株種類及其生理、生化特性與土壤性質的相關性。3.評估Azotobacter 分離株對於水稻苗生長之影響。
本研究從8處有機耕種及8處傳統耕種水田土壤樣品中分離共40株Azotobacter菌株,依據分離株之16S rDNA 序列分析結果,所分離菌株屬A. armeniacus (亞美尼亞短桿固氮細菌)、A. beijerinckii (倍傑林客短桿固氮細菌)、A. chroococcum (色球短桿固氮細菌)、A. tropicalis (熱帶短桿固氮細菌) 和A. vinelandii (葡萄園短桿固氮細菌)等5種。分離之菌株12.5% 來自慣行田區、87.5% 來自有機田區。土壤pH 4.5 - 5.5及pH 5.5 - 6.5兩範圍中,Azotobacter分離株菌種組成以A. beijerinckii為主(53.3%及43.8%),A. chroococcum(40.8%及43.8%)次之。隨著土壤pH值上升,A. beijerinckii之比例下降。在土壤樣品pH 6.5 - 7.5主要菌種組成以A. chroococcum (33.3%)及A. vinelandii (33.3%),其次才是A. beijerinckii (22.2%)。另慣行農法田區之Azotobacter分離株生成載鐵物質- Pyoverdine的比例(56%) 高於有機農法田區 (32%),在載鐵物質、磷酸三鈣溶解能力及水解明膠的能力測試也有同樣的趨勢。
本研究所篩選之 Azotobacter 有14菌株在繼代培養後仍能生成Pyoverdine並於235nm (UVC)波長下產生生物性螢光。有17株菌在經過繼代培養三天後會轉成褐色。CHB202、CHB214、CHB251、CHB257及CHB278等5分離株有分解木質素之透明環。有16分離株具分解羧甲基纖維素後形成之透明圈。另外,CHB194、CHB213、CHB216 及CHB251等4菌株具有nif H固氮基因,在繼代培養後所測試固氮活性數值太低無法視為具有游離固氮活性。所有分離株均能在磷酸三鈣培養基上生長良好,然而卻有半數以上分離株 (22/40) 無法在菌落周圍形成透明環。所篩選Azotobacter分離株所合成IAA濃度範圍0.49~15.59 μg/ml。綜合評估游離固氮活性、溶解磷酸三鈣能力及IAA生成量等有益特性,有明確表現者由高到低為CHB196、CHB198、CHB188、CHB197、CHB260。這5菌株可提高稻穀發芽率及水稻苗地上部及根部生長且顯著高於對照組。從本研究之結果證明可以由本土土壤篩選有利水稻栽培的Azotobacter菌株。
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