In temperate forest ecosystems that are not markedly affected by anthropogenic emissions, nitrogen (N) is a major limiting nutrient (Vitousek et al., 2002). The turnover of litter accumulated on the forest floor potentially represents the main source of N for plant growth (Hart & Firestone, 1991). In addition to soil microbes, ectomycorrhizal fungi (EMF), which are associated with the root tips of the dominant tree species in these ecosystems, contribute to the degradation of leaf litter and the release of nutrients through the secretion of hydrolytic and oxidative enzymes (Näsholm et al., 2009). Depending on the environmental conditions, different EMF species exude different exoenzymes to make nutrients available (Abuzinadah & Read, 1986; Courty et al., 2005; Buée et al., 2007; Koide et al., 2008; Bödeker et al., 2009). Furthermore, the morphology of external hyphae emanating from the colonized root tips differs strongly among EMF species. Based on structure, abundance and lengths of the external mycelia, EMF have been classified as contact, short-distance, medium-distance, and long-distance exploration types, which, in addition to their hydrophilic and hydrophobic properties, may contribute differentially to host nutrient acquisition (Agerer, 2001). Although the potential of EMF for mobilization of nutrients from different sources and their ability to explore different soil zones are well known (Hobbie & Horton, 2007; Pritsch & Garbaye, 2011), the analysis of distinct functions of individual EMF species within their natural community is notoriously difficult. Stable isotope signatures, which are the result of discrimination of naturally occurring 15N/14N or 13C/12C in metabolic processes, have been employed to distinguish major N and carbon (C) sources of fungi (Hobbie & Högberg, 2012). EMF-ensheathed root tips generally show higher natural 15N abundance than nonmycorrhizal root tips because of fungal 15N enrichment (Zeller et al., 2007; Högberg et al., 2008; Tedersoo et al., 2012). In a detailed analysis, Tedersoo et al. (2012) showed that the interspecific variation of the natural 15N signature of ectomycorrhizas was related to fungal lineage, but correlations with exoenzyme activities or exploration types were not found (Tedersoo et al., 2012). Hobbie & Högberg (2012) linked interspecific variation in the mean 15N signatures of EMF to preferential N uptake from the mineral horizon or the organic layer. However, the implications of different EMF species, which co-occur in assemblages, for in situ N acquisition from the same resource still remain unclear.
Beech (Fagus sylvatica), a dominant tree species forming large monospecific forests in Central Europe (Ellenberg & Strutt, 2009), produces leaf litter that is decomposed very slowly compared with that of other deciduous tree species (Jacob et al., 2010). To trace the fate of N from this recalcitrant source, 15N-labelled beech litter was applied to the forest floor (Zeller et al., 2000). The label appeared in ectomycorrhizas beneath the 15N litter cover after 6 months and reached the leaves of the mature trees after 9 months (Zeller et al., 2000). Whether the EMF species, which ultimately mediated N transfer, have different N acquisition rates within their field assemblages is currently unclear.
In the present study, we investigated the accessibility of litter-derived N for a typical EMF assemblage associated with beech roots in an old-growth beech forest. The goal was to identify interspecific differences in N acquisition from leaf litter by analysing the temporal pattern of N accumulation in root tips colonized by different EMF species in comparison with N accumulation in fine roots, soil microbial biomass, and soil. With regard to functional differences among EMF, we hypothesized that ectomycorrhizas with intermediate- and long-distance hyphae would have direct access to N in the litter bags and therefore accumulate 15N more rapidly than short-distance or contact EMF. We expected, therefore, to find interspecific differences in 15N enrichment initially in EMF species. We furthermore hypothesized that these differences would disappear over time as short-distance and contact EMF would obtain increasing access to dispersed 15N. To test these hypotheses, mesh bags were filled with 15N-labelled beech leaf litter and deposited for up to 18 months in the top soil layer (0.1 m) of an old-growth beech forest. This layer contains the majority of fine roots (Meinen et al., 2009). The mesh width of the litter bags allowed ingrowth of fungal hyphae but prevented direct access of the roots. 15N enrichment was measured in soil, microbial biomass, fine roots and root tips colonized by different EMF species during 18 months. To distinguish between N uptake from litter bags and from the soil, half of the litter bags were removed after 14 months and the 15N enrichment in microbial biomass, ectomycorrhizas and roots in the presence and absence of the litter bags was measured after further incubation for 4 months. Thereby, ectomycorrhizas foraging in the litter bags and the surroundings were distinguished.