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Assimilation and isotopic fractionation of nitrogen by mycorrhizal fungi

2001; Wiley; Volume: 151; Issue: 2 Linguagem: Inglês

10.1046/j.1469-8137.2001.00178.x

1469-8137

K. S. Emmerton, Terry V. Callaghan, Helen E. Jones, Jonathan R. Leake, Anders Michelsen, D. J. Read,

Geology and Paleoclimatology Research

New PhytologistVolume 151, Issue 2 p. 503-511 Free Access Assimilation and isotopic fractionation of nitrogen by mycorrhizal fungi K. S. Emmerton, Corresponding Author K. S. Emmerton Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Author for correspondence: K. S. Emmerton Fax: +44 (0)2392 872 022 Email:[email protected]Search for more papers by this authorT. V. Callaghan, T. V. Callaghan Abisko Scientific Research Station, Royal Swedish Academy of Sciences, S-981 07, Abisko, Sweden; Sheffield Centre for Arctic Ecology, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this authorH. E. Jones, H. E. Jones Institute of Terrestrial Ecology, Merlewood Research Station, Grange-over-Sands, Cumbria LA11 6JU, UK;Search for more papers by this authorJ. R. Leake, J. R. Leake Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this authorA. Michelsen, A. Michelsen Department of Plant Ecology, University of Copenhagen, Øster Farimagsgade 2 D, DK-1353 Copenhagen K, DenmarkSearch for more papers by this authorD. J. Read, D. J. Read Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this author K. S. Emmerton, Corresponding Author K. S. Emmerton Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Author for correspondence: K. S. Emmerton Fax: +44 (0)2392 872 022 Email:[email protected]Search for more papers by this authorT. V. Callaghan, T. V. Callaghan Abisko Scientific Research Station, Royal Swedish Academy of Sciences, S-981 07, Abisko, Sweden; Sheffield Centre for Arctic Ecology, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this authorH. E. Jones, H. E. Jones Institute of Terrestrial Ecology, Merlewood Research Station, Grange-over-Sands, Cumbria LA11 6JU, UK;Search for more papers by this authorJ. R. Leake, J. R. Leake Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this authorA. Michelsen, A. Michelsen Department of Plant Ecology, University of Copenhagen, Øster Farimagsgade 2 D, DK-1353 Copenhagen K, DenmarkSearch for more papers by this authorD. J. Read, D. J. Read Department of Animal and Plant Sciences, The University of Sheffield, Sheffield S10 2TN, UK;Search for more papers by this author First published: 21 December 2001 https://doi.org/10.1046/j.1469-8137.2001.00178.xCitations: 62AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Summary • The patterns of nitrogen (N) utilization and of N isotope fractionation were determined when two ecto-(ECM) and an ericoid (ERM) mycorrhizal fungus were grown with inorganic (ammonium or nitrate) or organic (glutamic acid or glycine) N sources of predetermined N isotope composition. • All N sources were readily utilized by each of the fungi but substantial differences in the pattern of N isotope fractionation were observed both between the fungi and the N sources. • Whereas several of the ECM-N source combinations exhibited significant net fractionation in favour of 15N, no such effect was seen in the ERM fungus, where, on ammonium, there was preferential assimilation of 14N. • It is concluded that isotopic fractionation during N uptake and metabolism can cause significant shifts in the 15N abundance of mycorrhizal fungi and that, as a result, any attempt to use the tissue 15N abundance as a means of identifying the substrates being exploited by mycorrhizal fungi, or their plant partners, in nature, are likely to be unrealistic. Introduction It has been suggested that the enrichment in the heavy isotope of N (15N) seen in fruit bodies of some ectomycorrhizal fungi is a reflection of their ability to use organic fractions of soil organic matter, which have been assumed to be enriched with 15N (Gebauer & Dietrich, 1993). The relatively high values of 15N natural abundance in the fungal tissues contrasts with the low levels of 15N abundance seen in the tissues of coexisting vascular plants (Handley et al., 1996; Taylor et al., 1997; Michelsen et al., 1998; Gebauer & Taylor, 1999). The distinctive patterns of enrichment were highlighted by the observation of Högberg et al. (1996), that the fungal mantles of the ECM roots of Fagus sylvatica may be enriched in 15N by as much as 6.4‰ relative to the tissues of the root axes which they ensheath. Such relative enrichment of the fungal tissues may be attributable to a number of factors including: preferential transfer of 14N relative to 15N from the fungus to the plant; selective uptake by the fungi of 15N-enriched sources; and net fractionation of N isotopes during assimilation or exudation of individual N sources in favour of 15N. With regard to preferential transfer of 14N relative to 15N from the fungus to the plant, it is widely acknowledged that the products of enzymic reactions involved in the metabolism of N are characteristically depleted in 15N relative to the source compounds (Shearer & Kohl, 1986; Högberg, 1997). This would clearly help to explain the depletion in the tissues of ECM plants like Fagus, since a large proportion their N supplies are received through the fungal symbiont. While it is acknowledged that the transfer of N between fungal and plant partners via the glutamine-glutamate shuttle can be bi-directional (Martin & Botton, 1993) the net flux will normally be in the direction of the plant, which would, as a consequence, be expected to become progressively enriched in 14N relative to its fungal partner. The number of amino-transfer reactions and the extent to which the products transferred to the plant consist of end of chain compounds like serine, which are known to be relatively 15N depleted (Macko et al., 1987; Bol et al., 1998; Ostle et al., 1999; Schmidt & Stewart, 1999), will all influence the extent of disparity in 15N abundance between the mycorrhizal fungus and the host plant. However, since both ECM and saprotrophic fungi in nature are highly enriched in 15N relative to levels seen in atmospheric N2 (Gebauer & Taylor, 1999), it is apparent that factors other than transfer enrichment are involved. The possibility that selective uptake of 15N-enriched sources and/or net fractionation in favour of 15N during assimilation or exudation by the heterotrophs contributes to the observed disparities, cannot be excluded. It is, however, extremely difficult to gain evidence of isotopic selectivity in the soil environment, which is characterized by an enormous heterogeneity of substrate types and transformation pathways. For this reason, as part of a wider investigation of the extent and nature of nitrogen isotope fractionation in ECM and ERM systems, we carried out a study under axenic laboratory conditions in which individual fungal species were grown on pure inorganic or organic N sources of known initial δ15N content. The N fractionation patterns were followed sequentially by mass spectrometric determination of the natural abundance of 15N in the fungal tissues and in their culture media. Materials and Methods Origin of mycorrhizal fungi The ECM fungus Paxillus involutus (Fr.) Fr (code 80871) was obtained from the laboratory culture collection of D. J. Read, University of Sheffield, UK. This strain originates from Scotland, where it was isolated from a sporocarp associated with Pinus sylvestris L. The ECM fungus Leccinum scabrum Kallio (code LS97–1) was isolated from a sporocarp located close to Betula nana L. and Betula pubescens Ehrh. ssp. tortuosa Led. Nyman at a dry heath near Abisko, Sweden. The ericoid mycorrhizal (ERM) fungus (morphologically similar to Hymenoscyphus ericae (Read) Korf & Kernan) (code CAS-1) was obtained from a culture collection of the Department of Plant Ecology, University of Copenhagen, Denmark. This isolate originates from Abisko, Sweden, where it was isolated from roots of the ericaceous dwarf shrub Cassiope tetragona L. The mycorrhizal ability of each of these fungal strains was confirmed during mycorrhizal synthesis with aseptically grown hosts (K. S. Emmerton, unpublished; Emmerton et al., 2001). Nitrogen sources and nutrient solutions The ECM fungus P. involutus, L. scabrum and the ERM fungus were grown individually in liquid culture with ammonium, nitrate, glutamic acid or glycine as sole sources of N. Nitrogen sources (Table 1) were added at 20 mg N l−1 (which represents an intermediate concentration of 0.5 M K2SO4 extractable soil ammonium-N in organic horizons in Scandinavian heath tundra – see Michelsen et al., 1996, 1998) to a basal medium of N-free 1/5th Rorison's nutrient solution (Hewitt, 1966) with calcium supplied at 29 mg l−1. Supplementary glucose (to provide 400 mg carbon l−1) was added to nutrient solutions to provide sufficient quantities of C to sustain fungal growth. Nutrient solutions were adjusted to pH 5 (which represents an intermediate pH of tundra bulk soil in Abisko, Sweden – see Michelsen et al., 1998) and dispensed in 400 ml aliquots, to give a total of 8 mg N in each 500 ml treatment bottle. Treatment bottles were sterilized by autoclaving (Bajwa & Read, 1985) at 121°C for 15 min. Table 1. Specification of N sources used N sources δ15N (‰) Ammonium sulphate −0.7 or 2.4a Calcium nitrate −0.1 or 0.3a L-Glutamic acid −4.8 Glycine 7.7 a Depending on the experiment. Growth of mycorrhizal fungi on inorganic and organic nitrogen sources Paxillus involutus and Leccinum scabrum Inoculum cores, cut from the growing margins of cultures of P. involutus and L. scabrum on nutrient agar were transferred aseptically to Petri dishes containing water agar. These dishes were incubated upside down, in the dark in a constant temperature room at 20°C for 10 d. The inoculum cores, 'fluffed-up' with new growth, were then transferred aseptically to the experimental treatments. A subsample of these inoculum cores was collected and analysed for initial d. wt, N content and 15N natural abundance as described below. Ericoid mycorrhizal fungus Inoculum cores, cut from the growing margins of cultures of the ERM fungus on nutrient agar, were transferred aseptically to a sterile macerator vial containing c. 10 ml sterile distilled water and macerated. Treatment bottles were then inoculated aseptically with 100 µl aliquots of the ERM fungi macerate. Growth conditions Mycorrhizal fungi pure cultures were incubated in a static condition in the dark at 20°C and harvests were taken at 15, 20 and 40 d. There were four or five replicate flasks for each species, harvest and treatment combination. Fungal mycelium was separated from the culture solutions by filtration (Whatman No. 1), filter rinsed with 100 ml of 1 mM CaCl2, oven dried (80°C for 48 h) and weighed. Elemental and isotopic analyses of fungal samples and nitrogen sources Fungal samples and N sources were analysed for their percentage N and natural abundance of 15N using a Dumas-type Roboprep continuous-flow CHN analyser, coupled to a Tracermass isotope ratio mass spectrometer (both instruments from Europa Scientific Ltd., Crewe, UK). Results expressed in δ15N units denote deviations in parts per thousand (‰) from the 15N : 14N ratio of the atmospheric N2 standard of 0.0036765 and corresponds to 0.3663 atom percentage 15N. Therefore, (Eqn 1) (R, the 15N : 14N ratio.) The Rreference is calibrated against air, the δ15N of which is arbitrarily set to zero. The standard deviation of δ15N for replicated reference material was < 0.3‰. Units The δ15N of fungal samples are presented throughout as assimilated (A) source-relative (S-R)δ15N values (δ15NAS-R). The δ15NAS-R-values take account of: the N present in the organism at the time of initiation of the experiment (T=0) (Högberg et al., 1994); and the difference between the δ15N of the harvested sample and that of the initial δ15N of the N source supplied (δ15NS). Fungal δ15NAS-R-values were calculated using an isotope dilution method based on mass-balance techniques. This technique works on the basis that the 15N abundance of the N-bearing compound (in this case the fungal sample) is the product of the total N and 15N abundance of its constituent parts: (Eqn 2) (c, the fungal sample; and a and b, constituent parts.) Therefore, based on the isotope dilution method of Eqn 2, by substituting the organism parameters at the time of the initiation of the experiment (T=0) for a, and the end of the experiment (sample) for c, we found that: (Eqn 3) (NA, the N assimilated during the course of the experiment (i.e. b in Eqn 2).) Whilst: (Eqn 4) Eqn 3 takes into account the dilution effect that the initial N would have upon the δ15N at each harvest. The application of Eqn 4 similarly enables more meaningful comparisons to be made between samples of different treatments; since treatments that have different δ15NS can subsequently be compared. At each harvest, the δ15NA of ERM samples have been assumed to be analogous to the δ15Nsample. This is justifiable since the 100 µl aliquots of inoculum would only have introduced c. 0.084 µg N, with a δ15N of c. 2.5‰ (as calculated from previous analyses), which equates to between 7000- and 50 000-fold fewer 15N atoms than were assimilated by the fungus from any of the treatments during the course of the experiments. It is also substantially less than the analytical error. The δ15N of mycorrhizal fungi culture filtrates (δ15Ncf) have been calculated by mass-balance from Eqn 2. By substituting the harvested fungal sample (sample) for a and cf for b into Eqn 2 we found that: (Eqn 5) (S, the N source supplied (the arbitrary N compound (c) in Eqn 2); and Ncf, NS − Nsample.) Statistical analyses All statistical analyses were performed with SPSS (version 8.0, SPSS Science Software, Birmingham, UK) or Sigma Plot (version 4.0, SPSS Science Software) (regression analysis). The Levene test was employed to assess homogeneity of variances. Where appropriate, data were transformed prior to statistical analysis. Significant differences were determined by regression or ANOVA followed by the Tukey multiple comparison test. Results Growth and nitrogen content of mycorrhizal fungi At the 15 d and 20 d harvest, growth and N uptake by P. involutus was higher on ammonium than on nitrate, glutamic acid or glycine (1, 2). Biomass and N uptake by P. involutus was lowest at 20 d and 40 d when glycine was supplied as the sole source of N. At 40 d yield and N uptake by P. involutus was similar on ammonium, nitrate and glutamic acid. Figure 1Open in figure viewerPowerPoint Mycelial dry weight of (a) Paxillus involutus; (b) Leccinum scabrum; and (c) an ericoid mycorrhizal fungus, when grown with either ammonium (open columns), nitrate (closed columns), glutamic acid (light grey columns) or glycine (dark grey columns) for 15 d, 20 d or 40 d. Means (+1 SE) with the same letters are not significantly different (Tukey multiple comparison test for unequal replication; α, 0.05; n, 4 or 5). Figure 2Open in figure viewerPowerPoint Total N assimilated by (a) Paxillus involutus; (b) Leccinum scabrum; and (c) an ericoid mycorrhizal fungus, when grown with either ammonium (open columns), nitrate (closed columns), glutamic acid (light grey columns) or glycine (dark grey columns) for 15 d, 20 d or 40 d. Means (+1 SE) or medians with the same letters are not significantly different (Tukey multiple comparison test for unequal replication; α, 0.05; n, 4 or 5). No difference in biomass or N uptake was exhibited by L. scabrum, at 15 d or 20 d, when supplied with either ammonium, nitrate, glutamic acid or glycine as the sole source of N (1, 2). However, at 40 d its yield and N uptake were significantly lower on ammonium than nitrate (P < 0.05). The high standard error for biomass of L. scabrum and its N uptake (compared with P. involutus and the ERM fungus) reflected the high variability in growth of L. scabrum observed in stock cultures maintained on plates of MMN agar medium. At 15 d, growth of the ERM fungus was highest when nitrate or glutamic acid were supplied as the sole sources of N. However, at the later harvests biomass was equally high when grown with ammonium, nitrate, glutamic acid or glycine (Fig. 1c). N uptake was most rapid to 20 d when the ERM fungus was grown with ammonium or glutamic acid as the sole source of N (Fig. 2c). Uptake of N was significantly lower on glycine than on ammonium, nitrate or glutamic acid until 40 d, when the median N uptake did not vary significantly between N treatments (Fig. 2c). Growth medium pH changes The pH of the growth medium exhibited a marked shift in most fungal species-N source combinations as biomass and N uptake increased (Fig. 3). When N was added in the cationic form as ammonium, the growth medium pH of all fungi showed a pronounced decrease with increasing yield and N uptake. When N was supplied in the neutral amino acid form as glycine, the ERM fungus growth medium pH decreased, whereas that of the ECM fungi remained at c. pH 5. When N was added in the anionic form as nitrate or in the acidic form as glutamic acid, the pH of growth medium either remained relatively stable or increased, with increasing yield and N uptake. Figure 3Open in figure viewerPowerPoint Relationship between the amount of N taken up by Paxilus involutus (open circles), Leccinum scabrum (open squares) or an ericoid mycorrhizal fungus (closed triangles) and the pH of the growth media when mycorrhizal fungi are supplied with 8 mg N as (a) ammonium (b) nitrate (c) glutamic acid, or (d) glycine. Best-fit relationships are shown as solid lines. Fractionation of N isotopes in ammonium Fractionation of ammonium N isotopes by P. involutus, L. scabrum and the ERM fungus was highly significant. The 15N abundance of N assimilated by each species was correlated positively with the amount of N assimilated (Fig. 4, Table 2). Compared with the initial δ15N of the ammonium supplied, the δ15N of N assimilated by P. involutus, L. scabrum and the ERM fungus ranged from −1‰ to 4‰, −3‰ to 3‰ and −11‰ to −15‰, respectively. However, fungal fractionation of N isotopes was substantially greater than this suggests. This is because fungal fractionation of N isotopes led to significant changes in the δ15N of residual N (the N that was not assimilated that remained in the culture media). Whilst the best-fit relationships between the δ15N of N assimilated by fungi and the amount of N assimilated were linear regressions (Table 2), the relationship between the δ15N of residual N in the culture medium and the amount of N assimilated was a power two-parameter regression for P. involutus and a polynomial-cubic regression for L. scabrum and the ERM fungus (Table 3). These contrasting regression expressions, for each fungal species, demonstrate that fractionation against 14N and 15N by the ECM fungi and the ERM fungus, respectively, intensified highly significantly, particularly when N became more depleted from the nutrient medium. Figure 4Open in figure viewerPowerPoint Relationship between the 15N abundance of N taken up by mycorrhizal fungi (closed circles), or 15N abundance of N remaining in culture nutrient solution (open circles) and the amount of N taken up by mycorrhizal fungi when supplied with 8 mg N as ammonium, nitrate, glutamic acid or glycine. The initial 15N abundance of the N source supplied is represented as a dotted line at zero on the y-axis. Linear regressions of the relationship between 15N abundance of the N taken up and the amount of N taken up by mycorrhizal fungi are shown as solid lines. Significance levels for regression coefficients are shown in Table 2 and Table 3. Note the different y-axis scale for plots with ammonium. Table 2. Significance levels for linear regression coefficients (elevation and slope), and r2 values of the relationship between the δ15N of N assimilated by mycorrhizal fungi and the amount of N assimilated when supplied with ammonium, nitrate, glutamic acid or glycine N source Coefficient Paxillus involutus Leccinum scabrum Ericoid fungus Ammonium Elevation *** *** *** Slope *** (+) *** (+) * (+) r 2 0.93 0.95 0.37 Nitrate Elevation *** *** ns Slope ns *** (+) ns r 2 0.10 0.79 0.08 Glutamic acid Elevation ns ** ns Slope ns ns ns r 2 0.06 0.24 0.09 Glycine Elevation *** ns *** Slope *** (−) ns ns r 2 0.52 0.00 0.09 * P < 0.05; ** P < 0.01; and *** P < 0.001. Slope regression coefficients are proceeded by (+) or (−) indicating a positive or negative relationship, respectively (see Fig. 4 for plots of data and fitted lines). ns, not significant. Table 3. Significance levels for linear (and nonlinear) regression coefficients1 (elevation and slope), and r2 values of the relationship between the δ15N of N remaining in the culture nutrient solution and the amount of N assimilated by mycorrhizal fungi N source Coefficient Paxillus involutus Leccinum scabrum Ericoid fungus Ammonium Elevation ns2 ns3 ns3 Slope *** (−) *** (−) *** (+) r 2 0.98 0.95 0.99 Nitrate Elevation ns ns ns Slope *** (+) ns ns r 2 0.92 0.24 0.02 Glutamic acid Elevation ns ns ns Slope *** (+) * (+) ns r 2 0.61 0.37 0.04 Glycine Elevation *** ns ns Slope ns ns *** (+) r 2 0.09 0.21 0.92 * P < 0.05, ** P < 0.01, and *** P < 0.001. Slope regression coefficients are proceeded by (+) or (−) indicating a positive or negative relationship, respectively (see Fig. 4 for plots of data). 1Statistics relate to linear regression unless otherwise indicated. 2Statistics relate to a power-two parameter regression. 3Statistics relate to a polynomial-cubic regression. ns, not significant. High N uptake and fungal fractionation of N isotopes by P. involutus and L. scabrum caused the ammonium source δ15N to decrease significantly (P < 0.001) to −18‰ and −10‰, respectively (Table 3, Fig. 4). Consequently, δ15N in mycelium of P. involutus and L. scabrum became 22‰ and 13‰ more enriched in 15N, respectively, relative to the residual N in culture filtrates. By contrast, the ERM fungus showed highly significant preferential assimilation of 14N-ammonium so that the δ15N of the residual ammonium increased significantly (P < 0.001) to 14‰ (Table 3, Fig. 4). Consequently, the ERM fungus became 21‰ more depleted in 15N relative to the residual N in the culture solution as N uptake increased. Fractionation of N isotopes in nitrate When the ERM fungus was grown with nitrate no source-relative N isotope fractionation was formed (Fig. 4, Table 2). By contrast, the δ15N of N assimilated by both ECM fungi was significantly lower than the initial δ15N of nitrate. The mean 15N abundance of N assimilated by P. involutus remained at −1‰ irrespective of the amount of N assimilated (Fig. 4, Table 2). Although L. scabrum also fractionated strongly against 15N-nitrate (P < 0.001), the 15N abundance of N assimilated was correlated positively to the amount of N assimilated (Table 3, Fig. 4) so that by the final harvest at 40 d the mean difference between the initial δ15N of nitrate and that of L. scabrum was only 1‰. Fractionation of N isotopes in glutamic acid When P. involutus or the ERM fungus were grown with glutamic acid no N isotope fractionation was detected, relative to the source N (Table 2). By contrast, the mean 15N abundance of N assimilated by L. scabrum remained depleted relative to the initial δ15N of glutamic acid, at −1‰, irrespective of the amount of N assimilated (Table 2, Fig. 4). Fractionation of N isotopes in glycine Fractionation in favour of 15N-glycine by P. involutus was highly significant (Fig. 4, Table 2). However, the 15N abundance of N assimilated by P. involutus was correlated negatively to the amount of N assimilated (Fig. 4, Table 2), so that by 40 d mycelial 15N abundance was no different from that of initial glycine supplied. When L. scabrum was grown with glycine no N isotope fractionation was detected, relative to the source N (Fig. 4, Table 2). By contrast to both ECM fungi, the ERM fungus fractionated against 15N-glycine (P < 0.001) and the mean 15N abundance of the ERM fungus remained at −1‰ compared with the initial δ15N of glycine supplied, irrespective of the amount of N assimilated (Fig. 4, Table 2). The effect of fungal fractionation against 15N, in conjunction with the amount of N assimilated by the ERM fungus, was that the δ15N of the glycine source increased significantly (P < 0.001) by 1‰ (Table 3, Fig. 4). Discussion Gebauer & Dietrich (1993) hypothesized that the 15N enrichment that they observed in tissues of ECM and saprotrophic fungi was attributable to the assimilation of soil organic residues that were similarly enriched. While the ability of both ECM and ERM fungi to utilize organic sources of nitrogen has been demonstrated previously (Abuzinadah & Read, 1986, 1988; Bajwa & Read, 1985) and confirmed in the present study, it is also shown here that in the course of assimilation of both inorganic and organic forms of N considerable fractionation of N isotopes takes place. If, as seems likely, such fractionation occurs in the course of assimilation and transformation of naturally occurring N sources, the 15N abundance of fungal tissues are unlikely to directly reflect those of the original soil N sources. The situation is further complicated by the fact that the observed pattern of fractionation is both fungus and N source specific. Clear differences emerge between the ECM and ERM fungi, particularly in relation to assimilation of ammonium where, at high levels of uptake, the ECM fungi become 15N enriched while the ERM fungus show strong depletion in its 15N abundance. Once accumulation or exclusion of the heavier isotope begins the fractionation becomes progressively enhanced over time because the proportional abundance of 15N decreases or increases in the pool from which the N is being assimilated. On theoretical grounds the intensification of fractionation should be particularly enhanced as 15N becomes enriched in the tissue of the organism rather than depleted (Emmerton, 2000). Intense net fractionation against 15N of the kind seen in the ERM fungus has been reported previously in cultures of Anabaena (Macko et al., 1987) but in that case it was observed when the cyanobacterium was grown with N2, and nitrate as well as with ammonium as N sources. In the same study, Macko et al. (1987) found fractionation in favour of 15N in the heterotrophic bacterium Vibrio harveyi, of the kind we now report in the ECM fungi. At present the mechanisms responsible for the overall differences in N isotope fractionation patterns, seen both between fungal species and N sources, remain unclear. However, it is widely recognized that N assimilation by mycorrhizal fungi is achieved by different pathways (Smith & Read, 1997) and each, according to the extent of its involvement in processing assimilated N atoms, may produce a distinctive fractionation pattern. In the case, for example, of ammonium, the ion may be assimilated by the glutamine synthetase-glutamate synthase (GS-GOGAT) or the glutamate-dehydrogenase glutamine snythetase (GDH-GS) pathways. Because the two pathways have different affinities for ammonium (Marschner, 1986) it is probable that each will yield a particular fractionation pattern. Since mycorrhizal fungal species (Genetet et al., 1984; Chalot et al., 1991; Brun et al., 1992) and partners in a given mycorrhizal association (Martin et al., 1986; Dell et al., 1989) can each use one or the other, or a combination of both of these pathways, it is clear that the outcome may be a distinctive set of fractionation patterns in each case. Further, the activities of the enzymes involved in each of the pathways has been shown to be influenced by the concentration of N supplied. Thus St. John et al. (1985) showed that when the ERM fungus H. ericae was grown with a low external concentration of ammonium GS-GOGAT activity was maximal, whereas at high concentrations of this ion GDH activity exceeded that of the GS-GOGAT system by six times. The reverse effect has been reported in the saprotrophic fungus Neurospora crassa (Lomnitz et al., 1987). It becomes very evident from considerations of these kinds that if, as is most likely, metabolic processes exert a strong fractionating effect upon the nitrogen isotope composition of organisms, the differences in 15N abundance between them may be at least partly explained by the time integrated relative importance of each pathway. The results of the present work should be interpreted with caution because the experimental conditions are very different from those prevailing in soil. In addition to the fact that the natural environment will contain a mixture of N sources, there will be differential fractionation of each of these by a diverse microbial population. Such effects will further complicate any attempt to interpret the 15N abundance of a single mycorrhizal fungus or plant–fungus partnership in terms of N source utilization. The relationships between the patterns of fractionation observed here under axenic conditions and those prevailing when the fungi are grown on the same substrates but in association with their autotrophic partners are examined in the following paper. Acknowledgements We thank Darren Sleep and Chris Quarmby of I.T.E. Merlewood for technical advice and assistance, the N.E.R.C. and the Swedish Academy of Science for financial support, and the Abisko Scientific Research Station for support during fieldwork. References Abuzinadah RA & Read DJ. 1986. The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. 3. Protein utilization by Betula, Picea and Pinus in mycorrhizal association with Hebeloma crustuliniforme. New Phytologist 103: 507– 514. Abuzinadah RA & Read DJ. 1988. Amino-acids as nitrogen-sources for ectomycorrhizal fungi – utilization of individual amino-acids. Transactions of the British Mycological Society 91: 473– 479. Bajwa R & Read DJ. 1985. 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