Nutrient uptake by boreal forest trees is dependent upon the symbiotic ectomycorrhizal (ECM) fungi that colonize the majority (> 95%) of the fine root tips of the trees (Taylor et al., 2000). In return for soil-derived nutrients, the fungi receive photosynthate from the host plant (Smith & Read, 1997). Nitrogen (N) is the most important macronutrient determining plant growth in these ecosystems (Barbour et al., 1987), with the majority of soil N sequestered in organic compounds (Tamm, 1991). The ECM fungi in boreal forests are adapted to the conditions of low mineral N availability, with many, if not most, capable of extracting N from organic sources (Leake & Read, 1997; Nygren et al., 2007).
In the absence of anthropogenic influences, N inputs from atmospheric deposition into boreal systems are low, c. 1–3 kg N ha−1 yr−1 (Binkley et al., 2000; Persson et al., 2000; Brenner et al., 2005), mainly in the form of nitrate () originating from lightning discharges (Aneja et al., 2001). Despite this input, nitrate concentrations in boreal soils are usually below detection limits (Andersen & Gundersen, 2000; Persson et al., 2000), although the low values may be a consequence of rapid microbial assimilation of any nitrate produced from nitrification (Stark & Hart, 1997). However, ECM fungi are likely to be exposed to higher nitrate concentrations during spring snow melts and dry/wetting cycles. By contrast to our knowledge concerning the uptake and metabolism of ammonium in ECM fungi (Chalot et al., 1990, 1991; Smith & Read, 1997), we currently know very little regarding their use of nitrate. Such knowledge is becoming increasingly important as anthropogenic inputs of nitrate into forest ecosystems continue to increase.
Mineral N inputs into boreal forest systems can increase 50–200 times as a result of forest fertilization with ammonium nitrate (c. 150–200 kg ha−1). This amount of application is a common forest practice in the boreal region carried out to increase timber yield (Pettersson, 1994). The general response of ECM fungi to N fertilization is negative (Wallenda & Kottke, 1998), with reduced species richness commonly being found in fertilized forest plots (Peter et al., 2001). Similar effects have been found on chronic N deposition gradients (Lilleskov et al., 2001). However, not all ECM fungi respond similarly to elevated inputs of mineral N. Certain ECM genera (e.g. Cortinarius, Piloderma and Suillus) are particularly negatively affected (Wästerlund, 1982; Brandrud, 1995; Lilleskov et al., 2002), while others (e.g. Laccaria, Lactarius, Paxillus and Russula) have been found to increase fruit body production with augmented concentrations of soil N (Shubin, 1988; Lilleskov et al., 2001; Avis et al., 2003).
The mechanisms involved in the response of ECM fungi to elevated soil N are unclear but a reduction in carbon allocation below ground by the host plants has been proposed as the major factor responsible (Wallenda & Kottke, 1998). However, assuming that a reduction in C availability does not stimulate fruiting in a select few ECM fungi, a general reduction in C availability below ground might be expected to manifest a similar response in all ECM fungi. One potential explanation for this differential response of ECM could be related to their relative ability to metabolize nitrate, with those species that proliferate after N additions being able to utilize nitrate more efficiently than those taxa that are negatively influenced.
In those ECM fungi that have been successfully cultured, the ability to use ammonium () is a universal trait (Smith & Read, 1997). The use of nitrate as an N source has so far only been examined in a small number of ECM fungi and the results suggest that utilization is very variable, both between and within species (France & Reid, 1984; Ho & Trappe, 1987; Anderson et al., 1999). A few ECM species (e.g. some Pisolithus isolates) seem to prefer to grow on rather than on (Scheromm et al., 1990; Aouadj et al., 2000; Sangtiean & Schmidt, 2002), while others show limited (Sawyer et al., 2003) or no growth (Norkrans, 1949) on nitrate. The different nitrate uptake capacities are also reflected in the nitrate-reducing capabilities which might vary strongly between species (Sarjala, 1990).
When nitrate is assimilated into fungi, it is transported across the plasma membrane by a high-affinity nitrate transporter (Jennings, 1995; Jargeat et al., 2003). Once inside the cell, nitrate is reduced to nitrite by the enzyme nitrate reductase (NR, EC 220.127.116.11) and then further to ammonium by nitrite reductase, before being incorporated into amino acids, amino sugars, nucleic acids and other biomolecules (Takaya, 2002). To date, the genes encoding for nitrate reductase (nar genes) have been characterized from only two ECM fungal species, one from the basidiomycete Hebeloma cylindrosporum (Jargeat et al., 2000) and one from the ascomycete Tuber borchii (Guescini et al., 2003). One single copy of the gene was found in T. borchii, while in H. cylindrosporum one functional gene (nar1) and one pseudogene (nar2) that was considered to be a nonfunctional duplicate of nar1 were found.
The ability of most ecologically important ECM fungi to use nitrate as an N source is largely unknown because of the difficulty of isolating them into pure culture. However, we have recently obtained isolates from a range of these recalcitrant taxa. These include taxa known to be either negatively (e.g. Cortinarius, Piloderma and Tricholoma) or positively (e.g. Lactarius, Russula) affected by N additions. These cultures represent a considerable investment in time and effort, as only a small percentage of attempts from most genera were successful, but they offer unique possibilities for examining ecological traits in a wide range of ECM fungi. The taxonomic identities of the mycelia have been verified using molecular identification.
In this study, we examined the ability of ECM fungi to use nitrate as an N source by examining the ability of the ECM isolates to grow on nitrate as the sole N source. Biomass production, pH change, and production were used to evaluate nitrate utilization. In addition, we studied the ECM fungal genes coding for nitrate reductase to assess the genetic potential of a taxonomically diverse range of fungi to utilize nitrate as an N source. Degenerate primers, targeted against highly conserved regions of the nar gene, were designed using DNA sequences from H. cylindrosporum in combination with sequence data from fully sequenced basidiomycetes. The primers were then used to investigate ECM fungi from a wide range of taxa for the occurrence of nar genes. The PCR approach was complemented with Southern blot hybridization.