Influence of mycorrhizal associations on foliar δ15N values of legume and non-legume shrubs and trees in the fynbos of South Africa:: Implications for estimating N2 fixation using the 15N natural abundance method

In this study, we examined the use of the N-15 natural abundance method to quantify the percentage N derived from fixation of atmospheric N-2 in honeybush (Cyclopia spp.) shrubs and trees in the fynbos, South Africa. Non-fixing shrubs and trees of similar phenology to the Cyclopia species were chosen as reference plants. These reference plants were selected to cover a range of mycorrhizal associations (ericoid mycorrhizal, arbuscular mycorrhizal and non-mycorrhizal). Isotopic analysis revealed a wide range of foliar delta(15)N values for the reference plants, including many very negative values. The marked differences in delta(15)N values were defined by the mycorrhizal status of the reference plant species, with the ericoid and arbuscular mycorrhizal plants showing lower foliar delta(15)N values relative to their non-mycorrhizal counterparts. In contrast, the delta(15)N values of the N-2-fixing Cyclopia species were uniformly clustered around zero, from -0.11parts per thousand to -1.43parts per thousand. These findings are consistent with the observation that mycorrhizal fungi discriminate against the heavier N-15 isotope during transfer of N from the fungus to the host plant, leaving the latter depleted in N-15 (i.e. with a more negative delta(15)N value). However, a major assumption of the N-15 natural abundance method for estimating N-2 fixation is that both legume and reference plant should have the same level of fractionation associated with N uptake. But, because mycorrhizal associations may strongly affect the level of fractionation during N uptake and transfer, the test legume should belong to the same mycorrhizal group as the chosen reference plant species. As shown in this study, if the mycorrhizal status of the legume and the reference plant differs, or cannot be assessed, then the N-15 natural abundance technique cannot be used to quantitatively estimate N-2 fixation.