BG Biogeosciences BG Biogeosciences 1726-4189 Copernicus Publications Göttingen, Germany 10.5194/bg-10-8269-2013 A reduced fraction of plant N derived from atmospheric N (%Ndfa) and reduced rhizobial nifH gene numbers indicate a lower capacity for nitrogen fixation in nodules of white clover exposed to long-term CO<sub>2</sub> enrichment Watanabe T. 1 Bowatte S. 2 Newton P. C. D. 2 Laboratory of Soil Biology and Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan AgResearch Grasslands, Private Bag 11008, Palmerston North 4442, New Zealand 16 12 2013 10 12 8269 8281 Copyright: © 2013 T. Watanabe et al. 2013 This work is licensed under the Creative Commons Attribution 3.0 Unported License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/3.0/ This article is available from https://bg.copernicus.org/articles/10/8269/2013/bg-10-8269-2013.html The full text article is available as a PDF file from https://bg.copernicus.org/articles/10/8269/2013/bg-10-8269-2013.pdf

Using the δ<sup>15</sup>N natural abundance method, we found that the fraction of nitrogen derived from atmospheric N (%Ndfa) in field-grown white clover (<i>Trifolium repens</i> L.) plants was significantly lower (72.0% vs. 89.8%, <i>p</i> = 0.047 in a grassland exposed to elevated CO<sub>2</sub> for 13 yr using free air carbon dioxide enrichment (FACE). Twelve months later we conducted an experiment to investigate the reasons behind the reduced N fixation. We took cuttings from white clover plants growing in the FACE and established individual plants in a glasshouse using soil from the appropriate ambient or elevated CO<sub>2</sub> treatments. The established plants were then transplanted back into their "rings of origin" and sampled over a 6-week period. We used molecular ecological analyses targeting <i>nifH</i> genes and transcripts of rhizobia in symbiosis with white clover (<i>Trifolium repens</i> L.) to understand the potential mechanisms. Shoot biomass was significantly lower in eCO<sub>2</sub>, but there was no difference in nodule number or mass per plant. The numbers of <i>nifH</i> genes and gene transcripts per nodule were significantly reduced under eCO<sub>2</sub>, but the ratio of gene to transcript number and the strains of rhizobia present were the same in both treatments. We conclude that the capacity for biological nitrogen fixation was reduced by eCO<sub>2</sub> in white clover and was related to the reduced rhizobia numbers in nodules. We discuss the finding of reduced gene number in relation to factors controlling bacteroid DNA amount, which may imply an influence of nitrogen as well as phosphorus.

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