Relationships between fungal uptake of ammonium, fungal growth and nitrogen availability in ectomycorrhizal Pinus sylvestris seedlings

 Nitrogen deposition and intentional forest fertilisation with nitrogen are known to affect the species composition of ectomycorrhizal fungal communities. To learn more about the mechanisms responsible for these effects, the relations between fungal growth, nitrogen uptake and nitrogen availability were studied in ectomycorrhizal fungi in axenic cultures and in symbiosis with pine seedlings. Effects of different levels of inorganic nitrogen (NH₄) on the mycelial growth of four isolates of Paxillus involutus and two isolates of Suillus bovinus were assessed. With pine seedlings, fungal uptake of ¹⁵N-labelled NH₄ was studied in short-term incubation experiments (72 h) in microcosms and in long-term incubation experiments (3 months) in pot cultures. For P. involutus growing in symbiosis with pine seedlings, isolates with higher NH₄ uptake were affected more negatively at high levels of nitrogen availability than isolates with lower uptake. More NH₄ was allocated to shoots of seedlings colonised by a high-uptake isolate, indicating transfer of a larger fraction of assimilated NH₄ to the host than with isolates showing lower NH₄ uptake rates. Thus low rates of N uptake and N transfer to the host may enable EM fungi avoid stress induced by elevated levels of nitrogen. Seedlings colonised by S. bovinus transferred a larger fraction of the ¹⁵N label to the shoots than seedlings colonised by P. involutus. Seedling shoot growth probably constituted a greater carbon sink in pot cultures than in microcosms, since the mycelial growth of P. involutus was more sensitive to high NH₄ in pots. There was no homology in mycelial growth rate between pure culture and growth in symbiosis, but N uptake in pure culture corresponded to that during growth in symbiosis. No relationship was found between deposition of antropogenic nitrogen at the sites of origin of the P. involutus isolates and their mycelial growth or uptake of inorganic nitrogen. Accepted: 18 September 1998     

Bryophytes attenuate anthropogenic nitrogen inputs in boreal forests

Productivity in boreal ecosystems is primarily limited by available soil nitrogen (N), and there is substantial interest in understanding whether deposition of anthropogenically derived reactive nitrogen (N_r) results in greater N availability to woody vegetation, which could result in greater carbon (C) sequestration. One factor that may limit the acquisition of N_r by woody plants is the presence of bryophytes, which are a significant C and N pool, and a location where associative cyanobacterial N‐fixation occurs. Using a replicated stand‐scale N‐addition experiment (five levels: 0, 3, 6, 12, and 50 kg N ha^?1 yr^?1; n=6) in the boreal zone of northern Sweden, we tested the hypothesis that sequestration of N_r into bryophyte tissues, and downregulation of N‐fixation would attenuate N_r inputs, and thereby limit anthropogenic N_r acquisition by woody plants. Our data showed that N‐fixation per unit moss mass and per unit area sharply decreased with increasing N addition. Additionally, the tissue N concentrations of Pleuorzium schreberi increased and its biomass decreased with increasing N addition. This response to increasing N addition caused the P. schreberi N pool to be stable at all but the highest N addition rate, where it significantly decreased. The combined effects of changed N‐fixation and P. schreberi biomass N accounted for 56.7% of cumulative N_r additions at the lowest N_r addition rate, but only a minor fraction for all other treatments. This ‘bryophyte effect’ can in part explain why soil inorganic N availability and acquisition by woody plants (indicated by their δ¹⁵N signatures) remained unchanged up to N addition rates of 12 kg ha^?1 yr^?1 or greater. Finally, we demonstrate that approximately 71.8% of the boreal forest experiences N_r deposition rates at or below 3 kg ha^?1 yr^?1, suggesting that bryophytes likely limit woody plant acquisition of ambient anthropogenic N_r inputs throughout a majority of the boreal forest.     

Limited transfer of nitrogen between wood decomposing and ectomycorrhizal mycelia when studied in the field

Transfer of ¹⁵N between interacting mycelia of a wood-decomposing fungus (Hypholoma fasciculare) and an ectomycorrhizal fungus (Tomentellopsis submollis) was studied in a mature beech (Fagus sylvatica) forest. The amount of ¹⁵N transferred from the wood decomposer to the ectomycorrhizal fungus was compared to the amount of ¹⁵N released from the wood-decomposing mycelia into the soil solution as ¹⁵N-NH₄. The study was performed in peat-filled plastic containers placed in forest soil in the field. The wood-decomposing mycelium was growing from an inoculated wood piece and the ectomycorrhizal mycelium from an introduced root from a mature tree. The containers were harvested after 41 weeks when physical contact between the two foraging mycelia was established. At harvest, ¹⁵N content was analyzed in the peat (total N and ¹⁵NH₄ ⁺) and in the mycorrhizal roots. A limited amount of ¹⁵N was transferred to the ectomycorrhizal fungus and this transfer could be explained by ¹⁵NH₄ ⁺ released from the wood-decomposing fungus without involving any antagonistic interactions between the two mycelia. Using our approach, it was possible to study nutritional interactions between basidiomycete mycelia under field conditions and this and earlier studies suggest that the outcomes of such interactions are highly species-specific and depend on environmental conditions such as resource availability.     

Role of nutrient level and defoliation on symbiotic function: experimental evidence by tracing 14C/15N exchange in mycorrhizal birch seedlings

High nutrient availability and defoliation generally reduce ectomycorrhizal colonization levels in trees, but it is not known how this affects the functional aspects of mycorrhizal symbiosis. It was therefore investigated whether (1) defoliation or increasing substrate N availability reduce C allocation from the plant to the fungus and N allocation from the fungus to the plant (symbiotic resource exchange), (2) symbiotic resource exchange depends on relative N and P availability, and (3) fungal N translocation to plant and plant C allocation to fungus are interdependent. Birch (Betula pendula) seedlings were grown in symbiosis with the ectomycorrhizal fungus Paxillus involutus at five times excess N, or at five times excess N and P for 6 weeks. One-half of the plants were defoliated and the plant shoots were allowed to photosynthesize ¹⁴CO₂ while the fungal compartment was exposed to ¹⁴NH₄. After 3 days, the ¹⁴C of plant origin in fungal tissues and ¹⁵N of fungal origin in plant tissues were quantified. Nutrient availability had no observable effect on symbiotic resource exchange in non-defoliated systems. Defoliation reduced symbiotic N acquisition by plants at all levels of nutrient availability, with the reduction being most marked at higher N availability, indicating an increased tendency in the symbiotic system to discontinue resource exchange after defoliation at higher fertility levels. The concentration of ¹⁴C in extramatrical mycelium correlated significantly with the concentration of ¹⁵N in birch shoots. The results support the assumption that N delivery to the host by the mycorrhizal fungus is dependent on C flow from the plant to the fungus, and that exchanges between the partners are reciprocal. No significant reductions in root ¹⁴C content as a response to defoliation were observed, indicating that defoliation specifically reduced allocation to fungus, but not markedly to roots.     

Nitrogen limitation in mycorrhizal Norway spruce (Picea abies) seedlings induced mycelial foraging for ammonium: implications for Ca and Mg uptake

Although many studies support the importance of the external mycelium for nutrient acquisition of ectomycorrhizal plants, direct evidence for a significant contribution to host nitrogen nutrition is still scarce. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus (Batsch) Fr. in a sand culture system with two compartments separated by a 45-m Nylon mesh. Hyphae, but not roots, can penetrate this net. Nutrient solutions were designed to limit seedling growth by nitrogen. Hyphal density in the hyphal compartment, host N status and shoot growth of mycorrhizal seedlings significantly increased in response to NH₄ ⁺ addition to the hyphal compartment. Labeling the compartment only accessible to hyphae with ¹⁵NH₄ ⁺ showed that the increase in N uptake in the mycorrhizal seedlings was a result of hyphal N acquisition from the hyphal compartment. These results indicate that hyphae of P. involutus may actively forage into N-rich patches and improve host N status and growth. In the mycorrhizal seedlings, which received additional NH₄ ⁺ via their external mycelium, the increase in NH₄ ⁺ supply less negatively affected Ca and Mg uptake than in nonmycorrhizal seedlings, where the additional NH₄ ⁺ was directly supplied to the roots. This was most likely due to the close link of NH₄ ⁺ uptake and H⁺ extrusion, which, in the nonmycorrhizal seedlings, lead to a strong acidification in the root compartment, and subsequently reduced Ca and Mg uptake, whereas in the mycorrhizal seedlings the site of intensive NH₄ ⁺ uptake and acidification was in the hyphal and not in the root compartment. Our data support the idea that the ectomycorrhizal mycelium connected to an N-deficient host may actively forage for N. The mycelium may also be important as a biological buffer system ameliorating negative influence of high NH₄ ⁺ supply on cation uptake.     

Soil conditions and regeneration after clear felling of a Pinus sylvestris L. stand in a nitrogen experiment,Central Sweden

Forest N fertilization is a common practice in areas of Sweden that are not affected by high levels of N deposition. The environmental consequences of high N input to closed forests are fairly well known, but the long-term effects following clear-felling are a lot less well known. Thus, residual effects on soil and planted seedlings of previous N additions at an experimental N gradient 11 years after clear-felling were studied at a naturally nutrient-poor forest site in central Sweden. The experimental N gradient had been established by three repeated applications (in 1967, 1974 and 1981) of six dosages of NH₄NO₃ with increments of 120 kg N ha^–1. Thus, in total, the applied N dose ranged between 0 and 1800 kg N ha^–1. The study examined extractable base cations and P, soil pH, total-N, total-C, net N-mineralization and potential nitrification in four soil horizons (the humus layer, and 0–5, 5–10 and 10–20 cm in the mineral soil). We also measured the survival and growth of planted Pinus sylvestris L. seedlings. The applied N had no effect on the amounts of extractable-P or base cations in the soil. The soil pH decreased with increasing N dose in the deeper soil horizons, while in the humus the pH showed a weak but statistically significant increase due to the N application. Both total-C and total-N increased as a result of the N application, while the C/N ratio decreased. In the humus layer and the uppermost mineral soil layer NH₄ ⁺ was the major inorganic N source, in contrast to the lowest mineral soil horizon where NO₃ ^– dominated. For most of the studied horizons, there was a positive linear relationship between applied N dose and amount of inorganic N. Both net N-mineralization and potential nitrification showed increases with increasing N dose. As for the plants, no difference in survival or growth was found between the different N treatments. For doses generally applied in forest fertilization no significant differences in any of the studied properties were found.     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.     

Nitrogen deposition and forest nitrogen cycling along an urban–rural transect in southern China

There is increasing concern over the impact of atmospheric nitrogen (N) deposition on forest ecosystems in the tropical and subtropical areas. In this study, we quantified atmospheric N deposition and revealed current plant and soil N status in 14 forests along a 150 km urban to rural transect in southern China, with an emphasis on examining whether foliar δ¹⁵N can be used as an indicator of N saturation. Bulk deposition ranged from 16.2 to 38.2 kg N ha^?1 yr^?1, while the throughfall covered a larger range of 11.7–65.1 kg N ha^?1 yr^?1. Foliar N concentration, NO₃^? leaching to stream, and soil NO₃^? concentration were low and NO₃^? production was negligible in some rural forests, indicating that primary production in these forests may be limited by N supply. But all these N variables were enhanced in suburban and urban forests. Across the study transect, throughfall N input was correlated positively with soil nitrification and NO₃^? leaching to stream, and negatively with pH values in soil and stream water. Foliar δ¹⁵N was between ?6.6‰ and 0.7‰, and was negatively correlated with soil NO₃^? concentration and NO₃^? leaching to stream across the entire transect, demonstrating that an increased N supply does not necessarily increase forest δ¹⁵N values. We proposed several potential mechanism that could contribute to the δ¹⁵N pattern, including (1) increased plant uptake of ¹⁵N‐depleted soil NO₃^?, (2) foliage uptake of ¹⁵N‐depleted NH₄⁺, (3) increased utilization of soil inorganic N relative to dissolved organic N, and (4) increased fractionation during plant N uptake under higher soil N availability.     

Effects of planted tree fallows on soil nitrogen dynamics,above-ground and root biomass,N2-fixation and subsequent maize crop productivity in Kenya

The effects of planted fallows of Sesbania sesban (L.) Merr. and Calliandra calothyrsus (Meissner) on soil inorganic nitrogen dynamics and two subsequent maize crops were evaluated under field conditions in the highlands of eastern Kenya. Continuous unfertilised maize, maize/bean rotation and natural regrowth of vegetation (weed fallow) were used as control treatments. The proportion of symbiotic N₂-fixation was estimated by measuring both leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment by the ¹⁵N dilution technique for Sesbania and Calliandra, using Eucalyptus saligna (Sm.) and Grevillea robusta (A. Cunn) as reference species. Above- and below-ground biomass and N contents were examined in Sesbania, Calliandra, Eucalyptus and Grevillea 22 months after planting. Both the content of inorganic N in the topsoil and the quantity of N mineralised during rainy seasons were higher after the Sesbania fallows than after the other treatments. Compared to the continuous unfertilised maize treatment, both residual crop yields were significantly higher when mineral N (one application of 60 kg N ha^–1) was added. Furthermore, the second crop following the Sesbania fallow was significantly higher than the continuous maize crop. The above-ground biomass of the trees at final harvest were 31.5, 24.5, 32.5 and 43.5 Mg ha^–1 for the Sesbania, Calliandra, Grevillea and Eucalyptus, respectively. For the total below-ground biomass the values for these same tree species were 11.1, 15.5, 17.7, and 19.1 Mg ha^–1, respectively, of which coarse roots (>2 mm), including tap roots, amounted to 70–90%. About 70–90% of the N in Sesbania, and 50–70% in Calliandra, was derived from N₂-fixation. Estimates based on leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment were strongly correlated. The N added by N₂-fixation amounted to 280–360 kg N ha^–1 for Sesbania and 120–170 kg N ha^–1 for Calliandra, resulting in a positive N balance after two maize cropping seasons of 170–250 kg N ha^–1 and 90–140 kg N ha^–1, for Sesbania and Calliandra, respectively. All the other treatments gave negative N balances after two cropping seasons. We conclude that Sesbania sesban is a tree species well suited for short duration fallows due to its fast growth, high nutrient content, high litter quality and its ability to fix large amounts of N₂ from the atmosphere.     

Uptake and transport of organic and inorganic nitrogen by arbuscular mycorrhizal fungi

New information on N uptake and transport of inorganic and organic N in arbuscular mycorrhizal fungi is reviewed here. Hyphae of the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) were shown to transport N supplied as ¹⁵N-Gly to wheat plants after a 48 h labelling period in semi-hydroponic (Perlite), non-sterile, compartmentalised pot cultures. Of the ¹⁵N supplied to hyphae in pot cultures over 48 h, 0.2 and 6% was transported to plants supplied with insufficient N or sufficient N, respectively. The increased ¹⁵N uptake at the higher N supply was related to the higher hyphal length density at the higher N supply. These findings were supported by results from in vitro and monoxenic studies. Excised hyphae from four Glomus isolates (BEG 84, 107, 108 and 110) acquired N from both inorganic (¹⁵NH₄ ¹⁵NO₃, ¹⁵NO₃ ⁻ or ¹⁵NH₄ ⁺) and organic (¹⁵N-Gly and ¹⁵N-Glu, except in BEG 84 where amino acid uptake was not tested) sources in vitro during short-term experiments. Confirming these studies under sterile conditions where no bacterial mineralisation of organic N occurred, monoxenic cultures of Glomus intraradices Schenk and Smith were shown to transport N from organic sources (¹⁵N-Gly and ¹⁵N-Glu) to Ri T-DNA transformed, AM-colonised carrot roots in a long-term experiment. The higher N uptake (also from organic N) by isolates from nutrient poor sites (BEG 108 and 110) compared to that from a conventional agricultural field implied that ecotypic differences occur. Although the arbuscular mycorrhizal isolates used contributed to the acquisition of N from both inorganic and organic sources by the host plants/roots used, this was not enough to increase the N nutritional status of the mycorrhizal compared to non-mycorrhizal hosts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and photosynthetic responses of <Emphasis Type="Italic">Fagus crenata</Emphasis> seedlings to O<Subscript>3 </Subscript>under different nitrogen loads

To obtain the basic data for evaluating the critical level of ozone (O₃) to protect Japanese deciduous broad-leaved forest tree species, the growth and photosynthetic responses of Fagus crenata seedlings to O₃ under different nitrogen (N) loads were investigated. The seedlings were grown in potted andisol supplied with N as NH₄NO₃ solution at 0, 20 or 50 kg ha⁻¹ year⁻¹ and were exposed to charcoal-filtered air or O₃ at 1.0, 1.5 and 2.0 times the ambient concentration for two growing seasons. The interactive effect of O₃ and N load on the whole-plant dry mass of the seedlings at the end of the second growing season was significant. The O₃-induced reduction in the whole-plant dry mass of the seedlings was greater in the relatively high N treatment than that in the low N treatment. This interactive effect was mainly due to the difference in the degree of O₃-induced reduction in net photosynthesis among the N treatments. The degree of O₃-induced reduction in N availability to photosynthesis was greater in the relatively high N treatment than that in the low N treatment. In conclusion, the sensitivity of growth and photosynthetic parameters of F. crenata seedlings to O₃ become high with increasing amounts of N added to the soil. Therefore, N deposition from the atmosphere should be taken into account to evaluate the critical level of O₃ to protect Japanese deciduous broad-leaved forest tree species.     

Carbon and nitrogen flow in silver birch and Norway spruce connected by a common mycorrhizal mycelium

 Spruce and birch seedlings were grown together in boxes filled with unsterile peat. Both seedlings were colonized by the ectomycorrhizal fungus Scleroderma citrinum. The two plants thus shared a common external mycelium. ¹⁵N-labelled ammonium was supplied exclusively to the fungus, while the birch or the spruce plant was continuously fed with ¹³C-labelled CO₂ for 72 h. The carbon and nitrogen transfer rates were strikingly different for birch and spruce seedlings. The mycorrhizal mycelium received carbohydrates mainly from the birch plant and the nitrogen transfer by the fungus to the plants was largely directed towards the birch. Carbon assimilates were also transferred in both directions between birch and spruce; however, there was no conclusive evidence for a net transfer of carbon between the plants. Accepted: 20 September 1996     

Dual inoculation withRhizobium sp. andGlomus fasciculatum enhances nodulation,yield and nitrogen fixation in chickpea (Cicer arietinum Linn.)

Summary Seed inoculation with Rhizobium and soil inoculation withGlomus fasciculatum increased nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea (Cicer arietinum) var. BG 212 in pots containing unsterilized soil especially with 50kgP₂O₅ ha⁻¹ in the form of superphosphate. Inoculation with Rhizobium orG. fasciculatum separately or in combination significantly increased the N₂ fixed in straw and grain than uninoculated controls as determined by¹⁵N atom percent excess of plants grown in soil amended with labelled ammonium sulphate (¹⁵NH₄)₂SO₄) at the rate of 20kg N ha⁻¹. These increases were most pronounced when P was applied at 50kgP₂O₅ ha⁻¹.     

Fate of nitrogen fertilizer applied on two main arables crops,winter wheat (Triticum aestivum) and sugar beet (Beta vulgaris) in the loam region of Belgium

Since 1986, the fate of fertilizer N (NH₄NO₃ or NaNO₃) applied in field conditions on two main arable crops, winter wheat (Triticum aestivum) and sugar beet (Beta vulgaris), has been studied using ¹⁵N. Up to a rate of 200 kg ha^-1 of N, mean recovery of fertilizer by winter wheat was 70%, provided it had been split applied. Single application (with or without dicyandiamid) was less effective. For sugar beet, in 1990, 1991 and 1992, 40% of fertilizer N was found in the crop at harvest when NH₄NO₃ had been broadcast at 100 to 160 kg N ha^-1 at sowing time. For the same N rate, recovery was 50% when row applied near the seeds and 60% for 80 kg N ha^-1. For the two experimental crops, residual fertilizer N in soil was exclusively organic. It ranged from 15 to 30% of applied N and was located in the 30 cm upper layer. Losses were generally lower with winter wheat (12%) than with sugar beet (20–40%) and could be ascribed to volatilization and denitrification. Soil derived N taken up by the plant was site and year dependent.     

Extraradical hyphae exhibit more plastic nutrient-acquisition strategies than roots under nitrogen enrichment in ectomycorrhiza-dominated forests

Ectomycorrhizal (ECM) functional traits related to nutrient acquisition are impacted by nitrogen (N) deposition. However, less is known about whether these nutrient-acquisition traits associated with roots and hyphae differentially respond to increased N deposition in ECM-dominated forests with different initial N status. We conducted a chronic N addition experiment (25 kg N ha⁻¹ year⁻¹) in two ECM-dominated forests with contrasting initial N status, that is, a Pinus armandii forest (with relatively low N availability) and a Picea asperata forest (with relatively high N availability), to assess nutrient-mining and nutrient-foraging strategies associated with roots and hyphae under N addition. We show that nutrient-acquisition strategies of roots and hyphae differently respond to increased N addition. Root nutrient-acquisition strategies showed a consistent response to N addition, regardless of initial forest nutrient status, shifting from organic N mining toward inorganic N foraging. In contrast, the hyphal nutrient-acquisition strategy showed diverse responses to N addition depending on initial forest N status. In the Pinus armandii forest, trees increased belowground carbon (C) allocation to ECM fungi thus enhancing hyphal N-mining capacity under increased N availability. By comparison, in the Picea asperata forest, ECM fungi enhanced both capacities of P foraging and P mining in response to N-induced P limitation. In conclusion, our results demonstrate that ECM fungal hyphae exhibit greater plasticity in nutrient-mining and nutrient-foraging strategies than roots do in response to changes of nutrient status induced by N deposition. This study highlights the importance of ECM associations in tree acclimation and forest function stability under changing environments.     

Biological N2 Fixation in wetland rice fields: Estimation and contribution to nitrogen balance

This paper 1) reviews improvements and new approaches in methodologies for estimating biological N₂ fixation (BNF) in wetland soils, 2) summarizes earlier quantitative estimates and recent data, and 3) discusses the contribution of BNF to N balance in wetland-rice culture.Measuring acetylene reducing activity (ARA) is still the most popular method for assessing BNF in rice fields. Recent studies confirm that ARA measurements present a number of problems that may render quantitative extrapolations questionable. On the other hand, few comparative measures show ARA's potential as a quantitative estimate. Methods for measuring photodependent and associative ARA in field studies have been standardized, and major progress has been made in sampling procedures. Standardized ARA measurements have shown significant differences in associative N₂ fixation among rice varieties.The ¹⁵N dilution method is suitable for measuring the percentage of N derived from the atmosphere (% Ndfa) in legumes and rice. In particular, the ¹⁵N dilution technique, using available soil N as control, appears to be a promising method for screening rice varieties for ability to utilize biologically fixed N. Attempts to adapt the ¹⁵N dilution method to aquatic N₂ fixers (Azolla and blue-green algae [BGA]) encountered difficulties due to the rapid change in ¹⁵N enrichment of the water.Differences in natural ¹⁵N abundance have been used to show differences among plant organs and species or varieties in rice and Azolla, and to estimate Ndfa by Azolla, but the method appears to be semi-quantitative.Recent pot experiments using stabilized ¹⁵N-labelled soil or balances in pots covered with black cloth indicate a contribution of 10–30 kg N ha^-1 crop^-1 by heterotrophic BNF in flooded planted soil with no or little N fertilizer used.Associative BNF extrapolated from ARA and ¹⁵N incorporation range from 1 to 7 kg N ha^-1 crop^-1. Straw application increases heterotrophic and photodependent BNF. Pot experiments show N gains of 2–4 mg N g^-1 straw added at 10 tons ha^-1.N₂ fixation by BGA has been almost exclusively estimated by ARA and biomass measurements. Estimates by ARA range from a few to 80 kg N ha^-1 crop^-1 (average 27 kg). Recent extensive measurements show extrapolated values of about 20 kg N ha^-1 crop^-1 in no-N plots, 8 kg in plots with broadcast urea, and 12 kg in plots with deep-placed urea.Most information on N₂ fixed by Azolla and legume green manure comes from N accumulation measurements and determination of % Ndfa. Recent trials in an international network show standing crops of Azolla averaging 30–40 kg N ha^-1 and the accumulation of 50–90 kg N ha^-1 for two crops of Azolla grown before and after transplanting rice. Estimates of % Ndfa in Azolla by ¹⁵N dilution and delta ¹⁵N methods range from 51 to 99%. Assuming 50–80% Ndfa in legume green manures, one crop can provide 50–100 kg N ha^-1 in 50 days. Few balance studies in microplots or pots report extrapolated N gains of 150–250 kg N ha^-1 crop^-1.N balances in long-term fertility experiments range from 19 to 98 kg N ha^-1 crop^-1 (average 50 kg N) in fields with no N fertilizer applied. The problems encountered with ARA and ¹⁵N methods have revived interest in N balance studies in pots. Balances are usually highest in flooded planted pots exposed to light and receiving no N fertilizer; extrapolated values range from 16 to 70 kg N ha^-1 crop^-1 (average 38 kg N). A compilation of balance experiments with rice soil shows an average balance of about 30 kg N ha^-1 crop^-1 in soils where no inorganic fertilizer N was applied.Biological N₂ fixation by individual systems can be estimated more or less accurately, but total BNF in a rice field has not yet been estimated by measuring simultaneously the activities of the various components in situ. As a result, it is not clear if the activities of the different N₂-fixing systems are independent or related. A method to estimate in situ the contribution of N₂ fixed to rice nutrition is still not available. Dynamics of BNF during the crop cycle is known for indigenous agents but the pattern of fixed N availability to rice is known only for a few green manure crops.     

First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass

Fungal succession in rotting wood shows a surprising abundance of ectomycorrhizal (EM) fungi during the late decomposition stages. To better understand the links between EM fungi and saprotrophic fungi, we investigated the potential capacities of the EM fungus Paxillus involutus to mobilize nutrients from necromass of Postia placenta, a wood rot fungus, and to transfer these elements to its host tree. In this aim, we used pure cultures of P. involutus in the presence of labelled Postia necromass (¹⁵N/¹³C) as nutrient source, and a monoxenic mycorrhized pine experiment composed of labelled Postia necromass and P. involutus culture in interaction with pine seedlings. The isotopic labelling was measured in both experiments. In pure culture, P. involutus was able to mobilize N, but C as well, from the Postia necromass. In the symbiotic interaction experiment, we measured high ¹⁵N enrichments in all plant and fungal compartments. Interestingly, ¹³C remains mainly in the mycelium and mycorrhizas, demonstrating that the EM fungus transferred essentially N from the necromass to the tree. These observations reveal that fungal organic matter could represent a significant N source for EM fungi and trees, but also a C source for mycorrhizal fungi, including in symbiotic lifestyle.     

Ectomycorrhizal fungi associated with Pinus sylvestris seedlings respond differently to increased carbon and nitrogen availability: implications for ecosystem responses to global change

The ectomycorrhizal (ECM) symbiosis can cause both positive and negative feedback with trees under elevated CO₂. Positive feedback arises if the additional carbon (C) increases both nutrient uptake by the fungus and nutrient transfer to the plant, whereas negative feedback results from increased nutrient uptake and immobilization by the fungus and reduced transfer to the plant. Because species of ECM fungi differ in their C and nitrogen (N) demand, understanding fungal species‐specific responses to variation in C and N supply is essential to predict impacts of global change. We investigated fungal species‐specific responses of ECM Scots pine (Pinus sylvestris) seedlings under ambient and elevated CO₂ (350 or 700 μL L⁻¹ CO₂) and under low and high mineral N availability. Each seedling was associated with one of the following ECM species: Hebeloma cylindrosporum, Laccaria bicolor and Suillus bovinus. The experiment lasted 103 days. During the final 27 days, seedlings were labeled with ¹⁴CO₂ and ¹⁵N. Most plant and fungal parameters were significantly affected by fungal species, CO₂ level and N supply. Interactions between fungal species and CO₂ were also regularly significant. At low N availability, elevated CO₂ had the smallest impact on the photosynthetic performance of seedlings inoculated with H. cylindrosporum and the largest impact on seedlings with S. bovinus. At ambient CO₂, increasing N supply had the smallest impact on seedlings inoculated with S. bovinus and the largest on seedlings inoculated with H. cylindrosporum. At low N availability, extraradical hyphal length increased after doubling CO₂ level, but this was significant only for L. bicolor. At ambient CO₂, increasing N levels reduced hyphal length for both H. cylindrosporum and S. bovinus, but not for L. bicolor. We discuss the potential interplay of two major elements of global change, elevated CO₂ and increased N availability, and their effects on plant growth. We conclude that increased N supply potentially relieves mycorrhiza‐induced progressive N limitation under elevated CO₂.     

Utilization of organic nitrogen at two different substrate pH by different ectomycorrhizal fungi growing in symbiosis with Pinus sylvestris seedlings

The aim of this study was to test the potential of four isolates of ectomycorrhizal (EM) fungi to utilize organic nitrogen (N) at two different substrate pHs. The organic N source (¹⁵N labelled lyophilised fungal mycelium) was mixed with either untreated peat/sand mixture (pH 4.9) or peat/sand mixture limed to a pH of 5.9 and put in cylindrical containers added to each pot. The content of the containers was separated from the roots of Pinus sylvestris seedlings by a nylon mesh and a 2 mm air gap to reduce diffusion of labelled N to the roots. The mycorrhizal plants (except those colonized by Suillus variegatus 2) took up significantly more ¹⁵N from the labelled mycelium than uncolonized seedlings. Liming significantly reduced the uptake of ¹⁵N by one of the EM fungi (unidentified) but not the other tested species (Paxillus involutus and two isolates of S. variegatus). The EM fungal isolates differed in their influence on the bacterial activity of the soil. This was reduced with P. involutus at both pH levels and increased with one of the two S. variegatus isolates at the high pH and with the other S. variegatus isolate at the low pH level. Liming the soil generally increased bacterial activity. The influence of liming on the proportion of organic N uptake in relation to inorganic N uptake by ectomycorrhizal trees is discussed.     

Segregation of nitrogen use between ammonium and nitrate of ectomycorrhizas and beech trees

Here, we characterized nitrogen (N) uptake of beech (Fagus sylvatica) and their associated ectomycorrhizal (EM) communities from NH₄⁺ and NO₃^?. We hypothesized that a proportional fraction of ectomycorrhizal N uptake is transferred to the host, thereby resulting in the same uptake patterns of plants and their associated mycorrhizal communities. ¹⁵N uptake was studied under various field conditions after short‐term and long‐term exposure to a pulse of equimolar NH₄⁺ and NO₃^? concentrations, where one compound was replaced by ¹⁵N. In native EM assemblages, long‐term and short‐term ¹⁵N uptake from NH₄⁺ was higher than that from NO₃^?, regardless of season, water availability and site exposure, whereas in beech long‐term ¹⁵N uptake from NO₃^? was higher than that from NH₄⁺. The transfer rates from the EM to beech were lower for ¹⁵N from NH₄⁺ than from NO₃^?. ¹⁵N content in EM was correlated with ¹⁵N uptake of the host for ¹⁵NH₄⁺, but not for ¹⁵NO₃^?‐derived N. These findings suggest stronger control of the EM assemblage on N provision to the host from NH₄⁺ than from NO₃^?. Different host and EM accumulation patterns for inorganic N will result in complementary resource use, which might be advantageous in forest ecosystems with limited N availability.