Growth on nitrate and occurrence of nitrate reductaseencoding genes in a phylogenetically diverse range of

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1 Growth on nitrate and occurrence of nitrate reductaseencoding genes in a phylogenetically diverse range of Blackwell Publishing Ltd ectomycorrhizal fungi Cajsa M. R. Nygren 1, Ursula Eberhardt 2, Magnus Karlsson 1, Jeri L. Parrent 1,3, Björn D. Lindahl 1 and Andy F. S. Taylor 1, 1 Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, PO Box 7026, SE Uppsala, ; 2 Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures, PO Box 85167, NL-3508 AD Utrecht, the Netherlands; 3 Department of Integrative Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada; The Macaulay Institute, Craigiebuckler, Aberdeen, AB15 8QH, UK Summary Author for correspondence: Cajsa M. R. Nygren Tel: Fax: Cajsa.Nygren@mykopat.slu.se Received: 25 June 2008 Accepted: 21 July 2008 Ectomycorrhizal (ECM) fungi are often considered to be most prevalent under conditions where organic sources of N predominate. However, ECM fungi are increasingly exposed to nitrate from anthropogenic sources. Currently, the ability of ECM fungi to metabolize this nitrate is poorly understood. Here, growth was examined among 106 isolates, representing 68 species, of ECM fungi on nitrate as the sole N source. In addition, the occurrence of genes coding for the nitrate reductase enzyme (nar gene) in a broad range of ectomycorrhizal fungi was investigated. All isolates grew on nitrate, but there was a strong taxonomic signature in the biomass production, with the Russulaceae and Amanita showing the lowest growth. Thirty-five partial nar sequences were obtained from 3 tested strains comprising 31 species and 10 genera. These taxa represent three out of the four clades of the Agaricales within which ECM fungi occur. No nar sequences were recovered from the Russulaceae and Amanita, but Southern hybridization showed that the genes were present. The results demonstrate that the ability to utilize nitrate as an N source is widespread in ECM fungi, even in those fungi from boreal forests where the supply of nitrate may be very low. Key words: boreal forest, ectomycorrhizal fungi, nitrate reductase-encoding genes, nar1, nitrate assimilation, nitrate reductase, nitrogen fertilization, nitrogen nutrition. New Phytologist (2008) doi: /j x The Authors (2008). Journal compilation New Phytologist (2008) Introduction 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 3kgNha 1 yr 1 (Binkley et al., 2000; Persson et al., 2000; Brenner et al., 2005), mainly in the form of nitrate ( NO 3 ) 1

2 2 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 times as a result of forest fertilization with ammonium nitrate (c kg ha 1 ). This amount of application is a common forest practice in the boreal region carried out to increase timber yield (Pettersson, 199). 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 ( NH+ ) 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, 198; Ho & Trappe, 1987; Anderson et al., 1999). A few ECM species (e.g. some Pisolithus isolates) seem to prefer to grow on NO 3 rather than on NH+ (Scheromm et al., 1990; Aouadj et al., 2000; Sangtiean & Schmidt, 2002), while others show limited (Sawyer et al., 2003) or no growth (Norkrans, 199) 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 ) 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, NO and + 2 NH 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. Materials and Methods Fungal isolates used in the growth experiment One hundred and six isolates representing 68 species of ECM fungi were used in this study to examine growth on nitrate (Table 1). Cultures were obtained from fresh fruitbody material, except Piloderma spp. and Meliniomyces bicolor Hambleton and Sigler (formerly Piceirhiza bicolorata), which were isolated The Authors (2008). Journal compilation New Phytologist (2008)

3 3 Table 1 Details of 106 isolates representing 68 ectomycorrhizal fungal species used in a study of growth on nitrate as the sole N source Species Abbreviation Collection code ID number Host and origin GenBank accession number Lactarius mitissimus (Fr.:Fr.) Fr. Lac mit UP562 1 Mixed forest, Trento, Italy EF93295 Lactarius blennius (Fr.:Fr.) Fr. Lac ble UP550 2 Mixed forest, Uppsala, EF93303 Lactarius tabidus Fr. Lac tab UP571 3 Mixed forest, Uppsala, EF93293 Amanita muscaria (L.:Fr.) Hook Ama mus UP538 Mixed forest, Uppsala, EF93267 Amanita spissa (Fr.) Kumm Ama spi UP502 5 Mixed forest, Gusum, DQ Lactarius rufus (Scop.:Fr.) Fr. Lac ruf UP521 6 Mixed forest, Uppsala, DQ Lactarius acerrimus Britzelm. Lac ace UP58 7 Quercus sp., Drottningholm, EF93285 Lactarius quietus (Fr.:Fr.) Fr. Lac quie UP568 8 Mixed forest, Aberdeen Scotland, EF93299 UK Lactarius acerrimus Lac ace UP507 9 Quercus sp., Belleme, DQ Provence, France Amanita muscaria Ama mus UP Mixed forest, Uppsala, DQ Lactarius salmonicolor R. Lac sal UP P. abies, Trento, Italy EF93309 Heim & Leclair Lactarius cf. lacunarum Lac lac UP Mixed forest, Uppsala, EF (Romagn.) ex Hora Lactarius chryssoreus Fr. Lac chr UP Quercus forest, Trento, Italy DQ Lactarius glyciosmus Lac gly UP559 1 Mixed forest, Uppsala, EF93307 (Fr.:Fr.) Fr. Lactarius evosmus Lac evo UP Mixed forest, Belleme, Provence, EF93283 Kühner & Romagn France Lactarius deterrimus Gröger Lac det UP Mixed forest, Uppsala, EF Amanita muscaria Ama mus UP Pinus sylvestris, Riddarhyttan, DQ Lactarius trivialis Lac tri UP57 18 Mixed forest, Trento, Italy EF93290 (Fr.:Fr.) Fr. Lactarius evosmus Lac evo UP Q. robur, Stockholm, DQ Lactarius quieticolor Lac qui UP P. sylvestris, Baden-Wuerttemberg, EF93287 Romagn. Tübingen, Germany Lactarius evosmus Lac evo UP55 21 Quercus sp., Drottningholm, EF93282 Lactarius blennius Lac ble UP59 22 Mixed forest, Uppsala, EF93301 Lactarius acerrimus Lac ace UP57 23 Quercus sp., Uppsala, EF9328 Lactarius tabidus Lac tab UP572 2 Mixed forest, Betsele, EF9329 Lactarius subdulcis Lac sub UP Fagus sylvatica, Lahnberge, DQ65887 (Pers.:Fr.) Gray Marburg, Germany Russula integra (L.) Rus int UP Betula sp., Ramsele, EF93310 Fr. ss. Maire Cortinarius purpurascens Fr. Cor pur UP53 27 Mixed forest, Uppsala, DQ Piloderma sp. 2 Pil sp2 UP58 28 Mixed forest, Nyänget, AY8822 Lactarius fulvissimus Lac ful UP Quercus sp., Uppsala, EF93297 Romagn. Lactarius pallidus Pers.: Fr. Lac pal UP Mixed forest, Belleme, Provence, EF9330 France Lactarius controversus Pers.:Fr. Lac con UP Mixed forest, Flen, DQ Lactarius helvus (Fr.:Fr.) Fr. Lac hel UP Mixed forest, West of Örrasjön, EF93300 Amanita spissa Ama spi UP51 33 Mixed forest, Uppsala, EF93270 Lactarius evosmus Lac evo UP556 3 Mixed forest, Belleme, Provence, EF93291 France Amanita pantherina Ama pan UP Mixed forest, Knivsta, EF93269 (DC.:Fr.) Krombh. Hebeloma sp. Heb sp. UP56 36 P. sylvestris, P. abies, central EF93265 Lithuania Lactarius semisanguifluus R. Heim & Leclair Lac sem UP P. sylvestris, Schlossberg, Tübingen, Germany DQ Russula sanguinea (Bull.) Fr Rus san UP Mixed forest, Uppsala, DQ The Authors (2008). Journal compilation New Phytologist (2008)

4 Table 1 continued Species Abbreviation Collection code ID number Host and origin GenBank accession number Lactarius deliciosus Lac del UP P. sylvestris, Riddarhyttan, DQ (L.:Fr.) Gray Lactarius quieticolor Lac qui UP565 0 P. sylvestris, Baden-Wuerttemberg, EF93286 Tübingen, Germany Lactarius tuomikoskii Kytöv. Lac tuo UP576 1 Mixed forest, Uppsala, EF93292 Lactarius quietus Lac quie UP520 2 Quercus sp., Aberdeen, Scotland DQ Lactarius deliciosus Lac del UP552 3 P. sylvestris, Uppsala, EF93289 Tricholoma cf. equestre Tri equ UP533 P. sylvestris, Riddarhyttan, DQ (L.:Fr.) Kummer Amanita regalis (Fr.) Michael Ama reg UP50 5 Mixed forest, Uppsala, EF93268 Piloderma fallax (Lib.) Stalpers Pil fal UP583 6 P. sylvestris, P. abies, EF93276 central Lithuania Lactarius trivialis Lac tri UP575 7 Mixed forest, Jämtland, EF93308 Lactarius pubescens Fr. Lac pub UP56 8 Betula sp., Ramsele, EF93305 Suillus granulatus (L.:Fr.) Roussel Sui gra UP59 9 Mixed forest, Uppsala, EF93252 Piloderma sphaerosporum Jülich Pil sph UP Mixed forest, Nyänget, EF93279 Amanita spissa Ama spi UP52 52 Fagus sylvatica, Uppsala, EF93271 Lactarius torminosus Lac tor UP Betula sp., Uppsala, EF93306 (Schaeff.:Fr.) Pers. Tricholoma fulvum (DC.:Fr.) Sacc. Tri ful UP602 5 Mixed forest, Uppsala, EF93258 Tricholoma pardinum (Pers.) Quel. Tri par UP Mixed forest, Munich, Germany EF93302 Hydnum rufescens Schaeff.:Fr. Hyd ruf UP50 56 P. abies, P. sylvestris, DQ Uppsala, Piloderma sp. 1 Pil sp1 UP Mixed forest, Nyänget, AY8823 Tricholoma scalpturatum Tri sca UP93 58 Betula pendula, Uppsala, DQ (Fr.) Quél. Lactarius quieticolor Lac qui UP Mixed forest, Uppsala,. DQ Lactarius controversus Lac con UP Populus sp., Monclus, France DQ Tricholoma equestre Tri equ UP P. sylvestris, P. abies, central EF93263 Lithuania Lactarius quietus Lac quie UP Mixed forest, Baden-Wuerttemberg, EF93298 Germany Lactarius deterrimus Lac det UP51 63 P. abies, P. sylvestris Steinenberg, DQ Tübingen, Germany Lactarius rufus Lac ruf UP569 6 P. sylvestris, Gusum, EF93296 Cf. Otidea Cf Oti UP Mixed forest, Nyänget, EF93312 Tricholoma fulvum Tri ful UP88 66 Mixed forest, Uppsala, DQ Piloderma fallax Pil fal UP P. sylvestris, P. abies, central DQ65886 Lithuania Cortinarius glaucopus Cor gla UP55 68 Mixed forest, Uppsala, EF93266 (Schaeff.:Fr.) Fr. Tricholoma populinum Lange Tri pop UP Mixed forest, Uppsala, EF93259 Lactarius controversus Lac con UP Salix repens, Newborough DQ Warren, Wales. Lactarius zonarius (Bull.) Fr. Lac zon UP Quercus sp., Belleme, DQ Provence, France Piloderma byssinum Pil bys UP Mixed forest, Nyänget, EF93281 (P. Karst.) Jülich Piloderma byssinum Pil bys UP P. sylvestris, P. abies, DQ central Lithuania Tricholoma stans (Fr.) Sacc. Tri sta UP60 7 P. sylvestris, Riddarhyttan, EF93261 Cf. Otidea Cf. Oti UP Mixed forest, Nyänget, EF93313 Piloderma. sphaerosporum Pil sph UP Mixed forest, Nyänget, EF93278 Cenococcum geophilum Fr. Cen geo UP5 77 P. sylvestris, P. abies, Umeå, EF Sarcodon imbricatus Sar imb UP Mixed forest, Uppsala, EF93311 (L.:Fr.) Karst. Piloderma sp. 1 Pil sp1 UP Mixed forest, Nyänget, AY The Authors (2008). Journal compilation New Phytologist (2008)

5 5 Table 1 continued Species Abbreviation Collection code ID number Host and origin GenBank accession number Amphinema byssoides Amp bys UP53 80 P. sylvestris, Picea abies, EF93272 (Pers.) J. Erikss central Lithuania Piloderma sp. 1 Pil sp1 UP Mixed forest, Nyänget, AY88239 Hebeloma sp. Kumm. Heb sp. UP95 82 P. abies, Flakaliden,. EF9326 Suillus luteus (L.:Fr.) Roussel Sui lut UP P. sylvestris, Riddarhyttan, EF93253 Suillus bovinus (L.:Fr.) Roussel Sui bov UP591 8 P. sylvestris, Kiruna, N. EF9329 Cenococcum geophilum Cen geo UP P. sylvestris, P. abies, EF9331 central Lithuania Suillus luteus Sui lut UP P. sylvestris, Riddarhyttan, EF9328 Tricholoma album (Fr.) Kumm. Tri alb UP Betula sp., Abisko, N. EF93262 Cortinarius glaucopus Cor gla UP21 88 P. abies, Flakaliden, DQ65885 Suillus grevillei Sui gre UP71 89 Mixed forest, Uppsala, EF93260 (Klotzsch:Fr.) Sing. Suillus variegatus Sui var UP P. sylvestris, Tömte Field EF93256 (Sw.:Fr.) O. Kuntze Station, Norway Suillus granulatus Sui gra UP Mixed forest, Uppsala, EF93251 Cenococcum geophilum Cen geo UP P. abies, Vedby, DQ Suillus luteus Sui lut UP P. sylvestris, Kiruna, N. DQ Pisolithus arhizus Pis arh UP587 9 P. sylvestris, Umeå, N. EF93273 (Scop.:Pers.) Rauschert Tomentella sublilacina Tom sub UP Mixed forest, Lund, EF93288 (Ellis & Holw.) Wakef. Suillus bovinus Sui bov UP P. sylvestris, Riddarhyttan, EF93250 Suillus variegatus Sui var UP P. sylvestris, Kiruna, DQ Suillus viscidus (L.) Sui vis UP Larix sp., Uppsala, EF9325 Roussel ss. Fries Suillus variegatus Sui var UP P. sylvestris, Bispgården, EF93257 Paxillus involutus (Batsch:Fr.) Fr. Pax inv UP Mixed forest, Uppsala, EF9325 Laccaria bicolor (Maire) Orton Lac bic UP P. sylvestris, P. abies, DQ central Lithuania Xerocomus communis Bull. Xer com UP Quercus sp., S. Italy EF9327 Paxillus involutus Pax inv UP Mixed forest, Monclus, France EF9326 Rhizopogon roseolus Rhi ros UP P. sylvestris, Kiruna, N. EF93255 (Corda) Th. M. Fr. Rhizoscyphus ericae Rhi eri UP Highly acidic stagnohumic gley, DQ (D.J. Read) W.Y. Zhuang & Korf North York Moors, UK Meliniomyces bicolor Hambleton & Sigler Mel bic UP P. abies, Uppsala, DQ The ID numbers refer to the ranked order of the isolates based on increasing biomass production. See Fig. 1 for more details on growth rates. from sterilized mycorrhizal root tips. Stock cultures are maintained on modified half-strength Melin-Norkrans (MMN) media (Marx, 1969) in darkness at 25 C. A single isolate of the ericoid mycobiont Rhizoscyphus ericae (formerly Hymenoscyphus ericae: the type culture, used in Leake & Read, 1990) was also included as a comparison with ECM taxa. To confirm the identities of the isolates, the internal transcribed spacer ribosomal RNA gene region (ITS) was sequenced using the method of Rosling et al. (2003) and sequence similarity was compared with known mycorrhizal taxa in the UNITE (Kõljalg et al., 2005) and GenBank (Benson et al., 2005) databases using the BLASTN algorithm (Altschul et al., 1997). For species identification within Russulaceae, Tricholoma and Piloderma, comparisons were made with the personal databases of Ursula Eberhardt (Fungal Biodiversity Centre, Utrecht, the Netherlands), Rasmus Kjøller (Department of Microbiology, Institute of Biology, University of Copenhagen, Denmark) and Karl-Henrik Larsson (Department of Plant and Environmental Sciences, University of Gothenburg, ), respectively. The Genbank accession numbers of the sequences obtained from the isolates are listed in Table 1. The Authors (2008). Journal compilation New Phytologist (2008)

6 6 Growth on nitrate Square plugs (5 5 mm) were cut from the actively growing edge of mycelia and placed on a new half-strength MMN plate. After 1 2 wk, when regrowth of the mycelium was visible, the plugs were transferred to conical 100 ml flasks containing 25 ml of sterilized, modified liquid Norkrans medium (Norkrans, 199), where the ammonia was replaced by KNO 3 (1.79 g l 1 ) as the sole nitrogen source and the amount of glucose was 2.5 g l 1 (C : N ratio = :1). The initial ph of the medium was adjusted to.5. There were three to five replicates of each isolate. Cultures were harvested after 2 wk (fast-growing isolates) or wk (slow growing). Harvest times were chosen to avoid sampling mycelia experiencing either C or N limitations. The mycelia were removed by filtering, dried for 8 h at 65 C and weighed. Concentrations of NO 2 and NH+ in the culture filtrate were measured using a flow injection analyser (FiaSTAR, Foss Tecator, Höganäs, ). Determination of NO and + 2 NH was carried out using the methods of Henriksen & Selmer-Olsen (1970) and Svensson & Anfält (1982), respectively. The final ph in the culture filtrate was determined. The initial weight of the inoculum plugs was determined using five replicate plugs from 20 randomly selected isolates. Plugs were dried and weighed in the same manner as the mycelia. The average dry weight of these inoculum plugs was then subtracted from the final biomass of each of the isolates. To estimate the potential growth of the isolates on residual N in the inoculum plug, we calculated the amount of N in the inoculum plug and then estimated the growth that this could support. The 5 5 mm inoculum plug could contain a maximum of 2.6 μg of N, based on the concentration of N in ½ MMN. The average weight of the fungus in the inoculum plugs was 0.3 mg, and with an estimated N content of 3% (Colpaert et al., 1992) this would give a total N content of the fungus of c. 9 μg N. The total N content of the plug would therefore be c μg. If this was entirely mobilized for new growth, the biomass of new mycelium that could be produced, assuming a much reduced N content of 1%, would be 1.16 mg. Biomass increments for each isolate are reported as mg d 1 increase. When species were represented by multiple isolates, mean species values were calculated for that species. These single mean values were used for the intergeneric comparisons, which were carried out for genera represented by > two species. Mean growth rates were compared using one-way ANOVA (Minitab Inc., 1998). Primer construction The amino acid sequence for the nar gene in the ECM fungus H. cylindrosporum (Jargeat et al., 2000) was used to identify homologues within the whole-genome sequences of Phanerochaete chrysosporium (strain RP 78, DOE Joint Genome Institute, Fig. 1 A fragment of the nitrate reductase gene in ectomycorrhizal fungi amplified with degenerate primers. The sequenced region was c. 700 bp long. The primers nara and narb are located in the region coding for the molybdopterin binding domain (indicated in grey) and the reverse primer narc is located in the region coding for cytochrome b5-like haem/steroid binding domain (striped). The black areas indicate the approximate location of the three introns common for all fungi screened in this study. Walnut Creek, CA, USA), Laccaria bicolor (access kindly provided by Francis Martin, now published as strain H82, DOE Joint Genome Institute, Walnut Creek, CA, USA, see Martin et al., 2008) and Coprinopsis cinerea (strain Okayama 7, Fungal Genome Initiative, USA) using the BLASTN algorithm (Altschul et al., 1997). Only one homologous locus was found within each genome. The four fungal sequences were then aligned, and conserved gene regions were targeted for primer design. Three different degenerate primers were designed, two forward primers, nara (5 -CTTCTSYTGGTGCTT- YTGG-3 ) and narb (5 -GGIATGATGAAYAAYTGGT-3, located in the region coding for the molydopterin binding domain where the nitrate binds and the reducing active site is located) and one reverse primer, narc (5 -GAIGAYGCIAC- IGARGAYTTYATSGC-3, located in the region coding for cytochrome B5 where haem-fe is bound; Campbell & Kingshorn, 1990). The location of the primer sites and the amplified region of the nar gene are shown in Fig. 1. NarA and narc amplified a bp-long fragment and narb (located closer to narc) and narc amplified a fragment of c. 500 bp. DNA extraction and amplification Forty-three fungal samples (2 ECM isolates and one saprotrophic fungus, Coprinellus micaeus), representing 1 species, were screened for the nar gene using the degenerate primers (Table 2). DNA was extracted from dried sporocarp material, pure cultures or from whole, fresh sporocarps of fungi collected mainly in boreal forests in central. The material was homogenized in a buffer containing 3% (w/v) hexadecyl-tri-methyl-ammonium bromide (CTAB), 2.5 m NaCl, 0.15 m Tris and 2 mm EDTA and incubated at 65 C for 1 h. After centrifugation, the supernatant was extracted once with chloroform. The DNA was precipitated with 1.5 volumes of isopropanol and the pellet was washed with 70% ice-cold ethanol and resuspended in water. Where whole sporocarps were used, the fungal material was first ground in liquid nitrogen before being mixed with the buffer. The PCR reactions were performed using the following programmes: an initial 9 C for 5 min (9 C for 30 s, X C for 30 s, 72 C for 1 min) for Y cycles, and finally 72 C for 10 min; where X varied between and 60 C and Y was 35 The Authors (2008). Journal compilation New Phytologist (2008)

7 7 Table 2 Fungal taxa screened for the presence of nitrate reductase-encoding genes Species Strain ID GenBank accession number PCR programme Phylogenetic clade Amanita porphyria Alb. & Schwein.: Fr. AT a a, b b, c c Pluteoid A. muscaria (L.:Fr.) Pers. AT a, b, c Pluteoid Coprinellus micaceus (Bull.) Vilgalys, AT EU20100 a Agaricoid Hopple & Jacq. Johnson Cortinarius anthracinus (Fr.) Fr. AT EU20108 a Agaricoid C. obtusus (Fr.) Fr. AT EU20107 a Agaricoid C. olearioides Rob. Henry AT20028 EU20106 a Agaricoid C. triumphans Fr. AT EU20109 a Agaricoid C. variicolor (Pers.) Fr. AT EU20110 a Agaricoid Gyrodon lividus (Bull.:Fr.) P. Karst. UP179 EU20096 c Boletoid Hebeloma birrus (Fr.) Gill. HJB5118 EU20119 a Agaricoid H. cf velutipes. Bruchet AT EU20123 a Agaricoid H. cylindrosporum Romagnesi HJB10527 EU20125 a Agaricoid H. cylindrosporum Romagnesi HJB1052 EU2012 a Agaricoid H. sacchariolens Quél. AT EU20121 a Agaricoid H. sinapizans (Paulet:Fr.) Gillet HJB96 EU20122 a Agaricoid Hebeloma mesophaeum (Pers.) Quél. AT EU20120 a Agaricoid Hygrophorus agathosmus (Fr.) Fr. AT EU20101 a Hygrophoroid H. erubescens (Pers.:Fr.) Fr. AT EU20102 a Hygrophoroid H. hypothejus (Fr.:Fr.) Fr. AT EU20103 a Hygrophoroid Lactarius flexuosus (Pers.:Fr.) S. F. Gray AT b Russuloid L. fulvissimus Romagnesi AT a, b, c Russuloid L. pubescens (Fr.) Fr. AT a, b, c Russuloid L. scrobiculatus (Scop.Fr.) Fr. AT a, b, c Russuloid Laccaria amethystina (Huds.) Cooke. AT20009 EU20105 a Agaricoid Laccaria bicolor (Maire) Orton UP506 EU2010 a Agaricoid Piloderma sphaerosporum Jülich UP585 EU20097 a Atheloid P. sphaerosporum UP586 EU20098 a Atheloid P. byssinum (P.Karst.) Jülich UP527 EU20099 a Atheloid Rhizopogon roseolus (Corda) Th. M. Fr. UP588 EU20092 b Boletoid Russula postiana Romell AT a, b, c Russuloid R. sardonia Fr. AT a, b, c Russuloid R. xerampelina (Schaeff.) Fr. AT a, b, c Russuloid Suillus granulatus (L.:Fr.) Kuntze UP59 EU20095 c Boletoid S. luteus (L.) Roussel UP595 EU2009 c Boletoid Tricholoma album (Fr.) Kumm. UP600 EU20112 c Tricholomatoid T. equestre (L.:Fr.) Kumm. UP601 EU20117 c Tricholomatoid T. cf. equestre UP533 EU20111 c Tricholomatoid T. fulvum (Bull.) Sacc. UP88 EU20118 c Tricholomatoid T. scalpturatum (Bull.) Gill. AT EU20113 a Tricholomatoid T. terreum (Schaeff.:Fr.) Kumm. AT EU2011 a Tricholomatoid T. cf stans (Fr.) Sacc. AT EU20115 a Tricholomatoid T. vaccinum (Schaeff.) P. Kumm. AT EU20116 a Tricholomatoid Xerocomus communis (Bull.) Bon UP10 EU20093 c Boletoid ID numbers starting with UP are extracted from cultures, while numbers starting with AT or HJB originate from sporocarps. a 5min; 9 C, (30 s; 9 C, 30 s; 8 C, 1 min; 72 C) for 35 cycles, 10 min; 72 C, ; C. b 5min; 9 C, (30 s; 9 C, 30 s; 8 C, 1 min; 72 C) for 0 cycles, 10 min; 72 C, ; C. c 5min; 9 C, (30 s; 9 C, 30 s; 50 C, 1 min; 72 C) for 35 cycles, 10 min; 72 C, ; C. or 0 cycles, in all combinations. The three programmes optimized to generated nar products can be found in Table 2. When amplification was unsuccessful using genomic DNA samples, PCR amplification was performed on cdna to overcome possible PCR inhibition as a result of introns located in the primer site. Isolates from the species Amanita muscaria (culture number UP501), A. regalis (UP50), A. spissa (UP51), Hebeloma velutipes (UP18, used as a positive control), Lactarius tabidus (UP571), L. deterrimus (UP51), L. quietus (UP520), Russula sanguinea (UP529) and R. chloroides (UP528) were grown in liquid culture on Norkrans media (Norkrans, 199) with nitrate as sole N source. The mycelia were harvested and RNA extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNA was treated The Authors (2008). Journal compilation New Phytologist (2008)

8 8 with DNAase 1 (Sigma, St. Louis, MO, USA) and cdna was constructed using the iscript cdna Synthesis Kit (Bio-Rad, Hercules, CA, USA). The cdna was then used to generate nar products as discussed earlier. Cloning PCR products were ligated into the PCR 2.1 Topo vector, which was used to transform chemically competent Escherichia coli strain TOP 10 cells (Invitrogen, Carlsbad, CA, USA) following the manufacturer s instructions. Batches of 10 colonies were repeatedly collected and colony PCR was performed using the forward and reverse primers M13F and M13R. This was repeated until either at least 10 products of the correct size were found (evaluated by agarose gel electrophoresis, samples between 500 and 1000 bp were sequenced) or 100 colonies had been sampled. Selected products were purified using the QIAquick PCR Purification Kit (250) (Qiagen, GmbH, Germany) and sequenced using an automated multicapillary sequencer CEQ 2000XL (Beckman Coulter, Fullerton, CA, USA). Southern hybridization For species where no nar PCR products could be amplified with the degenerate primers, either from DNA or cdna, a Southern blot hybridization was performed to further investigate the presence of nar gene homologues. DNA from Cortinarius varius (used as a positive control), Amanita muscaria, A. pantherina, Lactarius pubescens, L. fulvissimus, L. scrobiculatus, L. flexuosus, Russula postiana, R. xerampelina and R. sardonia was digested with the restriction enzymes BamHI and HindIII. Fifteen nanograms of digested DNA from each species was separated on a 1.0% agarose gel in 1 TBE buffer overnight at 30 V. The gel was trimmed and washed in 0.25 m HCl for 15 min, rinsed in deionized water and placed in 0. m of NaOH for 15 min. The DNA in the gel was transferred onto a Hybond-N+ nitrocellulose membrane (Amersham Biosciences Corp., Piscataway, NJ, USA). The nar PCR product from H. mesophaeum was used as a probe and was radioactively labelled with α- 32 P dctp, 3000 Ci mmol 1 (Perkin Elmer, Norwalk, CT, USA) using the Random Primed DNA Labelling Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA). The blots were hybridized at 65 C overnight and washed for 5 min in 2 SSC/0.1% SDS, 2 10 min in 1 SSC/0.1% SDS and finally for 2 10 min in 0.1 SSC/ 0.1% SDS. The membrane was wrapped in plastic film and exposed to a phosphor screen for 3 h before being scanned with a Molecular Imager FX (Bio-rad, Hercules, CA, USA). Sequence analysis To get an overview of the nar genes, the results are presented in a neighbour-joining tree. The sequences were roughly aligned using ClustalW (Thompson et al., 1997) and then manually edited with BioEdit Version 7 (Hall, 1999). Potential introns were identified by comparing the sequences to the cdna sequence as predicted from the H. cylindrosporum genomic sequence (NCBI accession number AJ23866; Jargeat et al., 2000). The potential introns were removed and a neighbour-joining tree was constructed from the coding sequence using PAUP*.0b10 (Swofford, 2002) with the ascomycetes Aspergillus niger and T. borchii as outgroups. Statistical support was estimated with a bootstrap analysis of 1000 replicates. Results Growth on nitrate The N-content of the inoculum plugs was c μg, which we estimated could support c mg of growth in mycelia with a 1% N content. This value is considerably lower than the minimum total biomass of 5.7 mg produced by the slowest-growing isolate (Lactarius mitissimus) in the growth experiment. It is therefore unlikely that the observed biomass increments were solely the result of residual growth on the N in the inoculum plug. Biomass production varied considerably between the isolates, but all isolates had some ability to grow on nitrate (Fig. 2, Table 1). Daily growth rates spanned one order of magnitude, from a minimum of 0.2 (L. mitissimus) to a maximum of 2. mg d 1 (Meliniomyces bicolor), with a mean of 0.80 ± 0.06 mg d 1 (n = 68). In general, species of Amanita, Lactarius and Russula had comparable and the lowest rates of mycelial growth, while M. bicolor, Rhizoscyphus ericae, Rhizopogon roseolus, Paxillus involutus, Xerocomus communis and Laccaria bicolor had the highest rates (Fig. 2, Table 1). Suillus grew well on NO 3 and, along with Piloderma and Tricholoma, produced significantly greater biomass than Lactarius and Amanita. Average growth rates differed significantly (F-value = 21.55, P < 0.001) among the genera that were represented by > two species in the following order Lactarius = Amanita = R ussula < Tricholoma = Piloderma < Suillus, where < indicates significant differences at P = The majority of the species increased the ph of the culture medium (Fig. 3, Table 1) with the greatest increases associated with Cenococcum geophilum, M. bicolor, Tricholoma album, R. ericae and Hebeloma sp. There was a significant correlation between biomass production and ph of the medium (total biomass, r = 0.62, P < 0.001; growth rate, r = 0.395, P < 0.001). However, a number of isolates, in particular Tricholoma spp. and suilloid fungi, either maintained or lowered the original ph of the medium. In general, the concentrations of NH+ and NO 2 in the culture filtrates were low (Fig., Table 1), but many isolates from the genus Lactarius produced high concentrations NH + The Authors (2008). Journal compilation New Phytologist (2008)

9 9 Fig. 2 Growth of ectomycorrhizal fungi on nitrate as the sole N source (n = 3 5, ± SE). Numbers refer to details of isolates in Table 1. Fig. 3 Culture filtrate ph after growth of ectomycorrhizal fungi on nitrate as the sole N source (n = 3 5, ± SE). The original ph was.5. See Table 1 for isolate identity. relative to the majority. This produced a strong taxonomic signal with respect to NH+ accumulation, with 23 of the 25 highest values being associated with Lactarius isolates (Fig. ). Most NO 2 values in the culture filtrate were close to the lower detection limits of the analysis. But some isolates, especially C. geophilum, L. acerrimus, Suillus granulatus, P. involutus and Pisolithus arhizus, produced much higher amounts of NO 2. This would suggest differential rates of the nitrate reductase and nitrite reductase activities. Nar sequences Sequencing of the cloned products and comparison with the NCBI sequence database using the BLASTN algorithm showed that the obtained sequences were homologous with identified genes coding for nitrate reductase. We obtained 3 nar amplicon sequences from the 3 tested strains with the primers nara and narc (Table 2). A PCR product could only be obtained from R. roseolus with the primers narb and narc. The Authors (2008). Journal compilation New Phytologist (2008)

10 10 Fig. Accumulation of nitrite and ammonia in culture filtrate after growth of ectomycorrhizal isolates on nitrate as the sole N source (n = 3 5, ± SE). See Table 1 for isolate identity. The GenBank accession numbers of the sequences are given in Table 2. The obtained sequences represent most of the major ECM-forming clades: the agaricoid, athelioid, boletoid, hygrophoroid and tricholomatoid clades. Three putative introns were found in the amplified nar fragment in all species. In addition, Gyrodon lividus, Suillus luteus and S. granulatus may possess one extra intron and R. roseolus two extra noncoding regions. Only a single sequence of the nar gene was amplified from each fungal species. No sequences corresponding to nar genes were recovered from taxa within Amanita and Russulaceae with any combination of PCR programmes or primers. Amplification from cdna from fungal growth in liquid culture yielded a nar product from the control fungus Hebeloma velutipes, but not from any isolate of Amanita or Russulaceae. Southern blot and sequence analysis The Southern hybridization using the Hebeloma mesophaeum nar PCR product as a probe, yielded bands in all Amanita, Lactarius and Russula species (Fig. 5), indicating the presence of a nar gene in these taxa. The results also indicate that the nar gene exists only as a single copy in these taxa (and in the control, C. varius, data not shown). The neighbour-joining analysis of the obtained nar gene fragments successfully grouped species of the same genera together and all genera represented by multiple species received high bootstrap support (Fig. 6). In addition, there was sufficient variation within the gene fragment to clearly Fig. 5 Southern blot of genomic DNA from the genera Lactarius, Russula and Amanita digested with BamHI (lanes with uneven numbers) and HindIII (lanes with even numbers). Hybridization was carried out using a 700 bp fragment of the nitrate reductase gene of Hebeloma mesophaeum as a probe. Lanes 1 2, Amanita muscaria; 3, Lactarius pubescens; 5 6, Russula postiana; 7 8, Amanita pantherina; 9 10, Lactarius fulvissimus; 11 12, Russula xerampelina; 13 1, Lactarius scrobiculatus; 15 16, Russula sardonia; 17 18, Lactarius flexuosus. distinguish the taxa at the species level, with the exception of Hebeloma sacchariolens and H. velutipes. Higher taxonomic groupings were also upheld within the tree, with the bolete taxa forming a well supported group separated from the other ECM taxa. Discussion The roots of boreal forest trees and their associated ECM fungi proliferate most extensively in the organic soil horizons The Authors (2008). Journal compilation New Phytologist (2008)

11 11 Fig. 6 A rooted neighbour-joining tree of amplified coding regions (c. 550 bp) of the nitrate reductase gene from a range of ectomycorrhizal fungi. The numbers on the branches refer to a bootstrap analysis carried out with 1000 replicates. The sequences were obtained by cloning and sequencing PCR products obtained with degenerate primers. Sequences for Phanerochaete chrysosporium, Laccaria bicolor and Coprinopsis cinerea were obtained from whole-genome sequences. The sequences from Tuber borchii, Aspergillus niger (chosen as an outgroup) and one of the Hebeloma cylindrosporum sequences were acquired from GenBank. (Perez-Moreno & Read, 2000; Lindahl et al., 2007), where organic sources of N predominate (Tamm, 1991). The results from the present study show that many of the same fungi that utilize organic N are also able to grow on nitrate as a sole N source even though this nutrient is usually present in only trace concentrations in boreal systems (Andersen & Gundersen, 2000; Persson et al., 2000). The results also demonstrate that the majority of basidiomycete ECM fungi are likely to contain the gene necessary for the initial step in the metabolism of nitrate. Before this study, our knowledge of the occurrence of the nar gene in ECM fungi was limited to two published sequences from ECM fungi, H. cylindrosporum (Jargeat et al., 2000) and T. borchii (Guescini et al., 2003). It is now clear that the gene occurs widely in ECM boletales and in all four of the clades within the agaricales that contain ECM-forming taxa (Matheny et al., 2006). Chronic (atmospheric deposition) or drastic (forest fertilization) additions of N into forest ecosystem usually result in significant changes in ECM communities (Lilleskov et al., 2001, 2002; Peter et al., 2001; Avis et al., 2003). In the introduction, we hypothesized that taxa which proliferate with elevated N concentrations (e.g. Lactarius) may be those The Authors (2008). Journal compilation New Phytologist (2008)

12 12 that are able to utilize nitrate more efficiently than those taxa that are negatively influenced (e.g. Cortinarius, Piloderma and Suillus). There was however, little support for this idea from the growth study, with Lactarius isolates comprising the main bulk of the species with the slowest biomass increments. Although the observed growth patterns may reflect intrinsic growth rates, many of the Lactarius, Russula and Amanita isolates grow more rapidly on MMN with ammonium as the N source than do the Piloderma isolates (data not shown). An alternative explanation for the proliferation of some ECM taxa under elevated N may relate to the nature of the mycorrhizas which they form and the pathways of mineral N uptake and metabolism. The uptake of NH+ uptake occurs via passive transport along an electrical potential difference across the plasma membrane through specific membranebound proteins (Boeckstaens et al., 2007), while NO 3 uptake is energy-consuming involving a nitrate transporter protein (Javelle et al., 200; Slot et al., 2007). The high energetic cost of nitrate uptake and subsequent reduction mean that any mechanism that enabled the nitrate to enter directly into the host tissue without any metabolic processing by the fungus would be advantageous to ECM fungi. Most Lactarius and Russula taxa form hydrophilic, smooth mantles with few emanating hyphae (contact exploration types, sensu Agerer, 2001). The hydrophilic nature of these structures may allow nitrate to diffuse through the mantles and pass directly into the host plant. Movement of nitrate through the apoplast could deliver N to the fungal/plant interface in the Hartig net without a need for the fungus to process the mineral N and thus avoid the carbon drain that this would entail. This enhanced N supply could result in down-regulation of monosaccharide uptake back into the root cortical cells (Nehls et al., 2007) and to the fungus receiving additional C, leading to increased growth and greater fruit body production. By contrast, Cortinarius, Piloderma, Suillus and Tricholoma species produce mycorrhizas with hydrophobic mantles and extensive mycelial systems in which nutrient uptake takes place some distance from the mycorrhizal root tips (Agerer, 2001). In order to avoid potential toxicity effects of NO 3, + NO 2 and NH (Stöhr, 1999), these taxa must first metabolize these compounds before translocating them to the host. This would create a significant C drain, thereby leading to a reduction in mycelial growth and reduced fruiting. One notable exception among ECM fungi to this potential link between mycorrhizal morphology and the negative effects of N additions is P. involutus, which often responds to N addition by producing large numbers of fruit bodies (Shubin, 1988). Paxillus involutus mycorrhizas develop an extensive soil mycelium, but the species grows well on nitrate. Intriguingly, Ek et al. (199) found that P. involutus was able to transfer N as nitrate through the mycelium to the host plant, suggesting a mechanism for avoiding potential toxic effects of nitrate. Many of the Lactarius isolates produced measurable quantities of NH+ in the culture media, indicating that they posses the necessary enzymatic capabilities to reduce the NO 3 to + NH. Boeckstaens et al. (2007) recently examined loss of ammonium from Saccharomyces cerevisiae cells when growing on different N sources. They suggested that cells may control the internal ammonium concentrations by releasing it through nonselective cation channels. But Boeckstaens et al. (2007) also suggested that cells were unable to prevent ammonia (NH 3 ) diffusing through the plasma membrane and that released NH 3 was reabsorbed as ammonium by the nonselective cation transporters involved in its release. Al Kubisi et al. (1996) found that the yeast Candida nitratophila was able to reduce nitrite to ammonium but released it into the growth media without further assimilation. It is possible that uncontrolled loss of NH+ or NH 3 by Lactarius species in our study may have restricted biomass production. However, it is questionable if ammonia would be released from the mycelium of Lactarius spp. in a natural system where the host root would act as a strong sink for ammonia (Chalot et al., 2006). Nitrate uptake into cells is accompanied by the influx of two protons, resulting in an increase in the ph of the local environment (Galvan & Fernández, 2001). In contrast to most isolates that raised the ph of the media as expected, there was a significant number of isolates that had either lowered or maintained the original ph despite considerable biomass production (Fig. 2). There was a strong taxonomic signature to this phenomenon, with most of these isolates belonging to the genus Tricholoma or the suilloid group. Many ECM fungi, including Suillus, are known to produce a wide range of low-molecular-mass organic acids as weathering agents (Landweert et al., 2001; Machuca et al., 2007). One possible explanation for the anomalous ph values is that these fungi are releasing large quantities of low-molecular-mass organic acids into the culture medium and thus counteracting the rise in ph associated with nitrate uptake. The accumulation of organic acids is a commonly observed phenomenon in plants growing on nitrate (Plassard et al., 1991) and elevated oxalic acid excretion into the growth medium has been observed in P. involutus growing on nitrate (Lapeyrie et al., 1987). The primers developed in this study successfully amplified the nar gene from 3 ECM fungi from a diverse range of taxonomic groups, but did not amplify any nar genes from taxa within the Russulaceae or the genus Amanita. There are several plausible explanations for this. The presence of introns in the primer site could have prevented amplification. However, no gene fragments were amplified from cdna constructed from mrna from any isolates in these groups, suggesting that primer failure was not the result of introns. In additional attempts to obtain gene sequences from these taxa, 10 other primer pairs were designed from different conserved parts of the nar gene (data not shown), but these also all failed to detect the nar gene, even though several could amplify the gene from the genus Hebeloma. The Authors (2008). Journal compilation New Phytologist (2008)

13 13 Although no amplification was achieved in this study, the results from the Southern hybridization show that the selected species from Amanita, Lactarius and Russula contained a nar gene. This would suggest that amplification failure is the result of mutations in the primer binding sites. The primers were designed from highly conserved regions with nara and narb located in the region coding for the reducing active site, and narc located in the region coding for cytochrome B5 where haem-fe is bound (Campbell & Kingshorn, 1990). The conserved nature of these primer binding sites in all other investigated taxa suggests that there has been a loss of selective constraints in the nar gene in taxa within the Russulaceae and Amanita, and that this has resulted in accumulations of mutations in the primer binding sites. The probe developed from H. mesophaeum used in the Southern hybridization yielded only a single band in the control (C. varius). A potential for false positives could exist with other genes coding for other enzymes (e.g. sulphite oxidase) with similar regions (i.e. the molydopterin binding domain) to nar. However, only a single band was observed with the C. varius, suggesting that the probe was not sufficiently similar to other genes to create false positives. The single bands observed in the Russulaceae and Amanita taxa would therefore suggest that these taxa posses a nar gene, even though no amplification was possible with the developed primers. In conclusion, even though ECM species are generally considered to be adapted to ecosystems where mineral N, especially nitrate, is present in trace quantities, many of them appear to readily metabolize nitrate as an N source. The widespread abilities to use both organic and mineral N in ECM fungi supports the view that in the nutrient-poor conditions, the fungi have the ability to acquire N from a wide range of potential sources. All of the isolates used in the present study showed some growth on NO 3, although growth rates differed markedly among taxa. We hypothesize that under field conditions, certain ECM taxa (e.g. members of the Russulaceae) may be able to avoid both the carbon cost of nitrate assimilation and the potential nitrate toxicity under elevated nitrate concentrations by having a mycorrhizal morphology that allows nitrate to pass directly into the host tissue by diffusion. This mechanism, which remains to be verified, could explain why these taxa have been found to proliferate following forest fertilization. Acknowledgements The authors would like to thank Ingrid Eriksson and Malin Elfstrand for their help with the Southern blot hybridization and Timothy James for his advice on primer design. Rasmus Kjøller and Karl-Henrik Larsson are gratefully acknowledged for the use of their private sequence databases. Three anonymous reviewers are acknowledged for their helpful advice on an earlier version of the manuscript. This work was financially supported by the Swedish Council for Environment, Agricultural Sciences and Spatial Planning, FORMAS (AT) and FORMAS (MK, Uppsala Microbiomics Center). References Agerer R Exploration types of ectomycorrhizae a proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza 11: Al Kubisi A, Ali AH, Hipkin AR Nitrite assimilation by the yeast Candida nitratophila. New Phytologist 132: Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller ZW, Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids 25: Andersen BR, Gundersen P Nitrogen and carbon interactions of forest soil water. In: Schulze E-D, ed. Carbon and nitrogen cycling in European forest ecosystems. Ecological studies, Vol. 12. Berlin, Germany: Springer-Verlag, Anderson IC, Chambers SM, Cairney JWG Intra- and interspecific variation in patterns of organic and inorganic nitrogen utilization by three Australian Pisolithus species. Mycological 103: Aneja VP, Bunton B, Walker JT, Malik BP Measurement and analysis of atmospheric ammonia emissions from anaerobic lagoons. Atmospheric Environment 35: Aouadj R, Es-Sgaouri A, Botton B A study of the stability and properties of nitrate reductase from the ectomycorrhizal fungus Pisolithus tinctorius. Cryptogamie Mycologie 21: Avis PG, McLaughlin DJ, Dentinger BC, Reich PB Long-term increase in nitrogen supply alters above- and below-ground ectomycorrhizal communities and increases the dominance of Russula spp. in a temperate oak savanna. New Phytologist 160: Barbour MG, Burk JH, Pitts WD Terrestrial plant ecology. Menlo Park, CA, USA: Benjamin/Cummins. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL GenBank. Nucleic Acids 33: D3 D38. Binkley D, Son Y, Valentine DW Do forests receive occult inputs of nitrogen? Ecosystems 3: Boeckstaens M, Andre B, Marini AM The yeast ammonium transport protein Mep2 and its positive regulator, the Npr1 kinase, play an important role in normal and pseudohyphal growth on various nitrogen media through retrieval of excreted ammonium. Molecular Microbiology 6: Brandrud TE The effects of experimental nitrogen addition on the ectomycorrhizal fungus flora in an oligotrophic spruce forest at Gårdsjön,. Forest Ecology and Management 71: Brenner RE, Boone RD, Ruess RW Nitrogen additions to pristine, high-latitude, forest ecosystems: consequences for soil nitrogen transformations and retention in mid and late succession. Biogeochemistry 72: Campbell WH, Kingshorn J Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends in Biochemical Sciences 15: Chalot M, Bernard D, Botton B, Martin F Nitrogen assimilation enzymes in mycorrhizas of Norway spruce, Douglas fir and beech. Agriculture, Ecosystems & Environment 28: Chalot M, Blaudez D, Brun A Ammonia: a candidate for nitrogen transfer at the mycorrhizal interface. Trends in Plant Science 11: Chalot M, Stewart GR, Brun A, Martin F, Botton B Ammonium assimilation by spruce Hebeloma sp. ectomycorrhizas. New Phytologist 119: Colpaert JV, Van Assche JA, Luijtens K The growth of the The Authors (2008). Journal compilation New Phytologist (2008)