Symbiotic N2-fixation by Trifolium repens using 15N natural abundance method (δ15Ν)

15N natural abundance method (δ15Ν) is used with success for over two decades for N2-fixation determination in natural, semi-natural and cultivated ecosystems. The application of this method is based on the differences that exist in 15N natural abundance (δ15Ν) in the plant tissues between a N2-fixing species (δ15Ν≤0‰) and non-fixers which are used as a reference. During the 1998 growing season, four leaf samplings were conducted in an upland grassland of Northern Greece and δ15Ν values were determined in Trifolium repens and 6 co-existing species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale, Plantago lanceolata, Achillea millefolium) which served as references. Festuca valida, Taraxacum officinale and Achillea millefolium were found to be appropriate reference species, since they had significantly different δ15Ν values compared to Trifolium repens and the N2-fixation determinations calculated using these values were acceptable (positive). As well as, the average of δ15Ν values of all non-legume species gave acceptable N2-fixation percentages. The percentages of N2-fixation determined for the four reference species (Festuca valida, Taraxacum officinale, Achillea millefolium and the average δ15Ν values) did not differ significantly. Significant differences were found for N2-fixation percentages between sampling dates. The reliability of the method of 15Ν natural abundance is high when the percentages of N2-fixation determined are high (>70%). In our study, for all reference species (Festuca valida, Taraxacum officinale, Achillea millefolium and the average of δ15Ν values) and samplings, N2-fixation was higher than 75%, which reveals that the use of 15N natural abundance method was pertained.