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非光合成窒素固定微生物群の水田土壌における時空間分布と寄与率の解明

水稻土非光合固氮微生物时空分布及贡献率阐明

基本信息

批准号：
18K18177
负责人：
藤村玲子
金额：
$ 2.75万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Early-Career Scientists
财政年份：
2018
资助国家：
日本
起止时间：
2018-04-01 至 2020-03-31
项目状态：
已结题

项目摘要

本研究は、水田土壌生態系における新規な窒素供給プロセスの解明を目指し、鉄還元細菌を含む水田下層土中の窒素固定微生物の時空間分布の変動、及び全窒素固定量に対する寄与率をFISH-NanoSIMS法を用いて明らかにすることを目的とした。H31（R1）年度は以下の項目を実施した。（１）土壌からの細菌細胞分画法の検討：前年度の結果をふまえ分散条件および密度勾配遠心分離の条件を変更し、細菌細胞の濃縮と土壌粒子からの分画に大幅な改善が見られた。（２） FISH法の水田土壌細菌への適用に向けた検討：前年度行った検証結果を参考に、分画した土壌細菌のFISH観察条件の検討を行なった。分画した土壌細菌細胞を全細菌およびGeobacter属細菌のプローブで試験を行なった。前項の分画法の変更により、顕微鏡観察時の視界は改善された。FISH法の試験では、全細菌用プローブとDAPI染色を比較した結果、７割程度の細胞に対してハイブリダイゼーションが成功していた。Geobacterに対しては検出されず、プローブ配列やバッファー濃度の選択など、さらなる検証が必要と言える。また、未培養の土壌を使用しており、鉄還元細菌の数が少ない可能性が考えられた。（３）土壌の窒素固定活性および窒素固定細菌数の変動解析：水田土壌の窒素固定活性が高まる条件を検討した。培養後１日程度で窒素固定活性は炭素（セロビオース）添加区で大きく上昇した。同時に細菌数および窒素固定細菌数を定量PCR法により検証した結果、鉄還元細菌の窒素固定遺伝子は非添加区では変化がなく、添加区では増加が確認された。活性および窒素固定遺伝子の量はいずれも培養1日目が最も高かった。従って、FISH-NanoSIMSに資する土壌の培養は、基質添加がより高い同位体窒素の窒素取り込みを促進し、検出の精度を上げるために必要と考えられた。

In this study, the new asphyxiant in paddy soil was supplied to the asphyxiant in the paddy soil, and the asphyxiant fixed microorganism in the paddy soil was used to determine the time and space distribution of asphyxiating microorganisms, and the total asphyxiant fixed quantity of asphyxiant was determined by FISH-NanoSIMS method. The following projects will be implemented in H31 (R1) year. (1) the method of cell division of soil bacteria: the results of the previous year showed that there was a significant improvement in the distribution conditions, density matching, center separation conditions, cell separation conditions, soil particles and so on. (2) FISH method is used to determine the conditions of soil bacteria in paddy fields: in the previous year, the results were reviewed, and the conditions were examined by FISH. It is divided into four parts: local bacteria, bacteria, whole bacteria, Geobacter, bacteria, bacteria, bacteria. The sub-drawing method referred to in the preceding paragraph will improve the performance of the system and improve the boundary of the micro-observation. The results of FISH method, whole bacteria staining and DAPI staining were used to compare the results, and the cells of 7 degrees of cutting were detected. Geobacter, please tell me that you need to make sure that you have the necessary information. The possibility of reducing the number of bacteria and reducing the number of bacteria in uncultivated and uncultivated soil is important. (3) Analysis of the number of bacteria in the fixed activity of asphyxiant in paddy field: the fixed activity of asphyxiant in paddy field was high. One day after incubation, asphyxiant fixed active carbon (asphyxiant) was added to the area of asphyxiation. At the same time, the number of bacteria was fixed by asphyxiant, and the results of quantitative PCR method were used to determine the results of the results, the results of the fixed asphyxiant, the addition of asphyxiant, the addition of asphyxiant, and the addition of asphyxiant. The activity of asphyxiant was fixed and the highest concentration of asphyxiate was found in one day. It is necessary to improve the accuracy of FISH-NanoSIMS, such as the addition of high temperature, high temperature, asphyxia, asphyxia, asphyxiation, asphyxia, asphy