Effects Of Elevated Carbon Dioxide And Nitrogen Fertilizer On The Abundance And Community Structure Of Root-associated Nitrogen-fixing Bacteria In Rice Paddy

Since the industrial revolution,the concentration of CO₂ in the atmosphere has reached 394μmol·mol^-1 by the year 2012,and is rising still.And CO₂ is the primary greenhouse gas and contributes to 60%of the global warming effect.Rice is the staple food for Asians,a large number of microorganisms exist in its root zone,which is an important place for the global carbon and nitrogen cycle.Although root associated microbial communities and abundance were often influenced by nitrogen and elevated atmospheric CO₂ concentration（FACE）in rice paddy,However,so far,the effect of atmospheric CO₂ concentrations（Ambient and FACE）and nitrogen fertilizer（low nitrogen fertilizer,LN;high nitrogen fertilizer,HN）on root associated nitrogen fixing-bacteria has not been reported.In this study to examine the combination effect of elevated atmospheric CO₂and nitrogen fertilizer on the root association nitrogen-fixing bacterial community structure,rice plant（WuYun Jing 23）were sampled in July（tillering stage）and September（heading stage）of 2014 and performed by quantitative real-time PCR,Next Generation Sequencing and cloning sequencing.The nifH gene was used as target functional gene.The results of the study are as follows:（1）Qualitative PCR analysis of nif H gene showed that the abundances of nitrogen-fixing bacterial community in the heading stage of root（6.8×10⁸²×10⁹copies/g）of rice plant on September were not only significant higher than that of rhizosphere Soil（8.9×10⁷³.6×10⁸ copies/g）but also higher than both root（2.2×10⁸⁴.6×10⁸ copies/g）and rhizosphere soil（1.95×10⁸².5×10⁸ copies/g）on July tillering stage.In addition,under low nitrogen conditions,FACE treatment reduced the abundance of nitrogen-fixing bacteria in the root and rhizosphere on September.（2）To examine the effect of FACE treatment and nitrogen fertilizer on root zone nitrogen-fixing bacteria communities,the clone library analysis（nifH）were conducted.Principal Component Analysis（PCA）component analysis indicated that the influence of nitrogen treatment on nitrogen-fixing bacteria community structure is higher than the FACE treatment.However,the nitrogen fixing bacteria community structure was changed by FACE treatment under low nitrogen condition.（3）According to the results,in the heading stage（September）rice roots to Methylosinus（17.1%）of Methylocystaceae TypeII methane oxidizing bacteria is advantage of nitrogen-fixing bacteria.Clone library sequencing results are consistent with the result of high flux.（4）Diazotrophic Methylosinus of Methylocystaceae predominant in rice roots of Heading stage and its relative abundances was decreased significantly from 14.8-7.3%with FACE treatment under low nitrogen conditions.However,relative abundance of Rhizobum sp.R2-708 was significantly increased from 1.1-4.3%by FACE treatment under high nitrogen condition.（5）Under low nitrogen conditions,relative abundance of Methylosinus and Rhizobum sp.R2-708 in heading stage were higher than these of tillering stages.This study revealed that the root-associated nitrogen-fixing bacteria responded to climate change and nitrogen fertilizer concentration in rice paddy.It will provide the basic data of the nitrogen cycle for global response to climate change and guidance for rational fertilization in rice fields.