Presence of nitrogen-fixing microorganisms in seagrass, Posidonia oceanica, roots and nitrogen isotopic signature of seagrass tissues

Triplicate samples of Posidonia oceanica were randomly collected at 26 locations along the Balearic Islands (Mediterranean Sea) during the summers of 2005 and 2006. Roots were subjected to a surface-sterilization protocol prior to nucleic acid extraction. The nitrogenase was amplified by PCR using degenerate primers for nifH gene sequences. The PCR products were checked by electrophoresis on 1.5% agarose gels. The youngest leaf (free of epiphytes) of three shoots and three young rhizome fragments were dried at 60oC for 48 h and ground to a fine powder. All isotopic analyses were measured using standard elemental analyzer isotope ratio mass spectrometer (EA-IRMS) procedures. Isotopic ratios (R) are reported in the standard delta notation (‰), deltasample=1000((Rsample/Rstandard)-1), where R = 15N/14N. These results are presented with respect to the International standard of atmospheric nitrogen (AIR, N2). Analytical reproducibility of the reported delta values, based on sample replicates, was better than ±0.2‰.