Increased anthropogenic nitrogen (N) deposition is a major contributor to alteration of soil nutrient cycles particularly in nutrient poor ecosystems, such as the Mediterranean basin, where co-limitation of N and phosphorus (P) occurs and N addition might thus lead to an exacerbation of P limitation. Here, we measured the effect of medium term (6 years) N fertilization in different forms and doses (40 kg N ha−1 yr−1 as 1:1 NH4Cl and (NH4)2SO4; 40 and 80 kg N ha−1 yr−1 as NH4NO3) on nutrient stoichiometry, potential turnover rates and abundance of roots, ectomycorrhizal (ECM) root tips and adjacent soil in Cistus ladanifer L. In order to assess the impact of N addition at its most extreme point, we sampled roots and topsoil (10 cm) with and without plant influence in the summer months. We analysed N and P concentrations in soil and roots and determined the abundance of the most dominant mycorrhizal root tip morphotypes. We also assessed nutrient turnover in soil, roots and mycorrhizal root tips by measuring their N and C related enzyme activities (EAs) as well as acid phosphatase (AP) activity.

Results

showed decreased soil Pinorg and increased soil N:Pinorg in the treatment plots. Also, a decline in Cenococcum geophilum in N addition plots was found and a general reduction in ECM colonization in the treatment receiving ammonium without nitrate. We also detected a decrease of C. geophilum absolute EA and AP, as well as N related EA in the whole soil compartment. Furthermore, we observed lower root AP activity and found a loss of correlation between N related EA and AP activity in all treatments, while a high correlation between N related EA and C related EA persisted in all plots. EA was also generally negatively related with root P/soil P, which we used as a measure for plant P status.

The negative effect of ammonium on the ECM community of C. ladanifer and a putative loss of short distance exploration morphotypes, such as C. geophilum, together with decreased AP activity in the plant roots, might be connected with low Pinorgavailability in soil with plant influence, thus being in line with the hypothesis of P depletion due to N addition. Furthermore, the decrease of N related EA in the soil compartment, as well as the decoupling of N and P cycles, might be signs of altered soil microbial communities. This decoupling, together with the strong dependence of EA on plant P status, could point to a shift from N and P scavenging ECM communities to more copiotrophic saprophytic fungi that rely on C and N acquisition from soil organic matter rather than plant C inputs. We posit that a decline in root colonization by ECM fungi and changes in N:P cycling could be detrimental to ecosystem development, as C. ladanifer is a common ECM species in early successional stages, providing a host for ECM fungi that also colonize late-successional plants.