Non-symbiotic N2 Fixation Research Articles

Competitive fitness and stability of ammonium-excreting Azotobacter vinelandii strains in the soil

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture.Key points• Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium• Large phenotypic instability for NH4+-excreting bacteria in culture medium• Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil

N2 fixation is less sensitive to changes in soil water content than carbon and net nitrogen mineralization

Biogeochemical processes catalyzed by soil microorganisms depend on the soil water content (SWC). Yet, little is known about the sensitivity of non-symbiotic N2 fixation to changes in SWC in comparison to carbon (C) and net nitrogen (N) mineralization. Therefore, we determined the rates of N2 fixation, C mineralization, and net N mineralization in soils at five SWCs that were created by water addition to the field-moist soil (rewetting), resulting in SWCs of 20, 40, 60 and 80% of the soil water holding capacity (WHC) which correspond to 14, 28, 42, and 57% water-filled pore space (WFPS). The soils originated from an arid, semiarid, Mediterranean, and humid site located in the Coastal Cordillera of Chile. The N2 fixation rate was measured immediately after the adjustment of the SWC and additionally after a five-day pre-incubation period. We found that the rates of all three processes where higher in the rewetted soils compared to the field-moist soils, but the sensitivity to changes in SWC differed between the processes. N2 fixation tended not to increase with increasing SWC beyond 20% WHC. Furthermore, the N2 fixation rate increased faster after rewetting in the soils of the semiarid and Mediterranean zone than in the soil of the humid zone. In contrast, C mineralization reached the highest rate at 80% WHC in the soil of the humid zone and at 60% and 80% WHC in the soils of the other three climate zones. The net N mineralization rate was highest at 40% and 60% WHC in the soils of the semiarid and Mediterranean zone, and was significantly decreased at 80% WHC compared to 60% WHC. Our study shows, first, that N2 fixation is less sensitive to changes in SWC than C and net N mineralization and tends to be already at its maximum in a given soil at low SWC, i.e., 20% WHC. This is likely because the diffusion of N2 in soil does not increase with the SWC, in contrast to the diffusion of organic solutes which are the substrate of C and N mineralization. Second, our results indicate that the N2 fixation rate increases more quickly in response to rewetting of soils of the more arid climate zones than of the humid zone, which might indicate a microbial adaptation to the climate conditions. The results have important implications since they suggest that the N2 fixation rate is likely less affected than the C and net N mineralization rates by decreases in the SWC that might occur more frequently in the future due to climate change.

Microbial substrate stoichiometry governs nutrient effects on nitrogen cycling in grassland soils

Human activities have increased nitrogen (N) and phosphorus (P) inputs in terrestrial ecosystems and altered carbon (C) availability, shifting the stoichiometry of microbial substrates in soils, such as the C:N:P ratios of the dissolved organic matter pool. These stoichiometric deviations between microbial biomass and its substrate may control microbial processes of N cycling. We studied the effects of this stoichiometric mismatch using a full factorial N and P addition experiment replicated in six grassland ecosystems in South Africa, the USA, and the UK. We found that N and P addition changed the dissolved organic matter C:N ratio, but not the C:N ratio of the soil microbial biomass. Compared to P addition, N addition decreased microbial N acquisition via non-symbiotic N2 fixation by −55% and increased microbial N release via net N mineralization by +134%. A possible explanation is that the dissolved elements, e.g., dissolved organic C (DOC) and dissolved total N (DN), serve as the main microbial substrate and its C:N ratio defines whether N is scarce or abundant with respect to microbial demands. If N is available in excess relative to microbial demands, net N mineralization increases. In contrast, when N is scarce, immobilization outweighs release decreasing net N mineralization. However, the activity of leucine aminopeptidases, which decompose peptides, was not affected by nutrient additions. Further, C rather than P availability may control the rates of non-symbiotic N2 fixation in the six studied grassland sites. In conclusion, globally increasing nutrient inputs change processes of microbial N acquisition and release in grassland ecosystems and these changes are largely driven by shifts in substrate stoichiometry.

Urochloa Grasses Swap Nitrogen Source When Grown in Association with Legumes in Tropical Pastures

The degradation of tropical pastures sown with introduced grasses (e.g., Urochloa spp.) has dramatic environmental and economic consequences in Latin America. Nitrogen (N) limitation to plant growth contributes to pasture degradation. The introduction of legumes in association with grasses has been proposed as a strategy to improve N supply via symbiotic N2 fixation, but the fixed N input and N benefits for associated grasses have hardly been determined in farmers’ pastures. We have carried out on-farm research in ten paired plots of grass-alone (GA) vs. grass-legume (GL) pastures. Measurements included soil properties, pasture productivity, and sources of plant N uptake using 15N isotope natural abundance methods. The integration of legumes increased pasture biomass production by about 74%, while N uptake was improved by two-fold. The legumes derived about 80% of their N via symbiotic N2 fixation. The isotopic signature of N of grasses in GA vs. GL pastures suggested that sources of grass N are affected by sward composition. Low values of δ15N found in some grasses in GA pastures indicate that they depend, to some extent, on N from non-symbiotic N2 fixation, while δ15N signatures of grasses in GL pastures pointed to N transfer to grass from the associated legume. The role of different soil–plant processes such as biological nitrification inhibition (BNI), non-symbiotic N2 fixation by GA pastures and legume–N transfer to grasses in GL pastures need to be further studied to provide a more comprehensive understanding of N sources supporting the growth of grasses in tropical pastures.

Effects of nitrogen and phosphorus addition on microbial community composition and element cycling in a grassland soil

Microorganisms mediate nutrient cycling in soils, and thus it is assumed that they largely control responses of terrestrial ecosystems to anthropogenic nutrient inputs. Therefore, it is important to understand how increased nitrogen (N) and phosphorus (P) availabilities, first, affect soil prokaryotic and fungal community composition and second, if and how changes in the community composition affect soil element cycling. We measured soil microbial communities and soil element cycling processes along a nine-year old experimental N-addition gradient partially crossed with a P-addition treatment in a temperate grassland. Nitrogen addition affected microbial community composition, and prokaryotic communities were less sensitive to N addition than fungal communities. P addition only marginally affected microbial community composition, indicating that P is less selective than N for microbial taxa in this grassland. Soil pH and total organic carbon (C) concentration were the main factors associated with prokaryotic community composition, while the dissolved organic C-to-dissolved N ratio was the predominant driver of fungal community composition. Against our expectation, plant biomass and plant community structure only explained a small proportion of the microbial community composition. Although microbial community composition changed with nutrient addition, microbial biomass concentrations and respiration rates did not change, indicating functional redundancy of the microbial community. Microbial respiration, net N mineralization, and non-symbiotic N2 fixation were more strongly controlled by abiotic factors than by plant biomass, plant community structure or microbial community, showing that community shifts under increasing nutrient inputs may not necessarily be reflected in element cycling rates. This study suggests that atmospheric N deposition may impact the composition of fungi more than of prokaryotes and that nutrient inputs act directly on element-cycling rates as opposed to being mediated through shifts in plant or microbial community composition.

Interactions of nitrogen and phosphorus cycling promote P acquisition and explain synergistic plant-growth responses.

Plant growth is often co-limited by nitrogen (N) and phosphorus (P). Plants might use one element to acquire another (i.e., trading N for P and P for N), which potentially explains synergistic growth responses to NP addition. We studied a 66-yr-old grassland experiment in South Africa that consists of four levels of N addition with and without P addition. We investigated the response of aboveground net primary production (ANPP) to N and P addition over the last 66yr. Further, we tested whether phosphatase activity and plant P uptake depend on N availability, and vice versa, whether non-symbiotic N2 fixation and plant N uptake depend on P availability. We expected that the interaction of both elements promote processes of nutrient acquisition and contribute to synergistic plant growth effects in response to NP addition. We found synergistic N and P co-limitation of ANPP for the period from 1951 to 2017 but the response to N and P addition diminished over time. In 2017, aboveground P stocks, relative rRNA operon abundance of arbuscular mycorrhizal fungi, and soil organic P storage increased with N fertilization rate when N was added with P compared to the treatment in which only N was added. Further, N addition increased phosphatase activity, which indicates that plants used N to acquire P from organic sources. In contrast, aboveground N stocks and non-symbiotic N2 fixation did not change significantly due to P addition. Taken together, our results indicate that trading N for P likely contributes to synergistic plant-growth response. Plants used added N to mobilize and take up P from organic sources, inducing stronger recycling of P and making the plant community less sensitive to external nutrient inputs. The latter could explain why indications of synergistic co-limitation diminished over time, which is usually overlooked in short-term nutrient addition experiments.

Metabolic profiling of two maize (Zea mays L.) inbred lines inoculated with the nitrogen fixing plant-interacting bacteria Herbaspirillum seropedicae and Azospirillum brasilense.

Maize roots can be colonized by free-living atmospheric nitrogen (N2)-fixing bacteria (diazotrophs). However, the agronomic potential of non-symbiotic N2-fixation in such an economically important species as maize, has still not been fully exploited. A preliminary approach to improve our understanding of the mechanisms controlling the establishment of such N2-fixing associations has been developed, using two maize inbred lines exhibiting different physiological characteristics. The bacterial-plant interaction has been characterized by means of a metabolomic approach. Two established model strains of Nif+ diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense and their Nif- couterparts defficient in nitrogenase activity, were used to evaluate the impact of the bacterial inoculation and of N2 fixation on the root and leaf metabolic profiles. The two N2-fixing bacteria have been used to inoculate two genetically distant maize lines (FV252 and FV2), already characterized for their contrasting physiological properties. Using a well-controlled gnotobiotic experimental system that allows inoculation of maize plants with the two diazotrophs in a N-free medium, we demonstrated that both maize lines were efficiently colonized by the two bacterial species. We also showed that in the early stages of plant development, both bacterial strains were able to reduce acetylene, suggesting that they contain functional nitrogenase activity and are able to efficiently fix atmospheric N2 (Fix+). The metabolomic approach allowed the identification of metabolites in the two maize lines that were representative of the N2 fixing plant-bacterial interaction, these included mannitol and to a lesser extend trehalose and isocitrate. Whilst other metabolites such as asparagine, although only exhibiting a small increase in maize roots following bacterial infection, were specific for the two Fix+ bacterial strains, in comparison to their Fix- counterparts. Moreover, a number of metabolites exhibited a maize-genotype specific pattern of accumulation, suggesting that the highly diverse maize genetic resources could be further exploited in terms of beneficial plant-bacterial interactions for optimizing maize growth, with reduced N fertilization inputs.

Enhancing Non-symbiotic N2 Fixation in Agriculture

Much of the demand for nitrogen (N) in cereal cropping systems is met by using N fertilisers, but the cost of production is increasing and there are also environmental concerns. This has led to a growing interest in exploring other sources of N such as biological N2fixation. Non-symbiotic N2fixation (by free-living bacteria in soils or associated with the rhizosphere) has the potential to meet some of this need especially in the lower input cropping systems worldwide. There has been considerable research on non-symbiotic N2fixation, but still there is much argument about the amount of N that can potentially be fixed by this process largely due to shortcomings of indirect measurements, however isotope-based direct methods indicate agronomically significant amounts of N2fixation both in annual crop and perennial grass systems. New molecular technologies offer opportunities to increase our understanding of N2-fixing microbial communities (many of them non-culturable) and the molecular mechanisms of non-symbiotic N2fixation. This knowledge should assist the development of new plant-diazotrophic combinations for specific environments and more sustainable exploitation of N2-fixing bacteria as inoculants for agriculture. Whilst the ultimate goal might be to introduce nitrogenase genes into significant non-leguminous crop plants, it may be more realistic in the shorter-term to better synchronise plant-microbe interactions to enhance N2fixation when the N needs of the plant are greatest. The review explores possibilities to maximise potential N inputs from non-symbiotic N2fixation through improved management practices, identification of better performing microbial strains and their successful inoculation in the field, and plant based solutions.

Effect of synthetic pyrethroid insecticides on N2-fixation and its mineralization in tea soil

Microorganisms degrade a great variety of chemical substances including pesticide residues in soil. By virtue of low persistence and broad-spectrum efficacy, pyrethroid insecticides are sometimes used in tea garden to combat insect pests but the residual effects of these chemicals on microbial activities in soil have rarely been studied. The present experiment has been conducted under laboratory conditions to investigate the effect of four synthetic pyrethroid insecticides, viz. cypermethrin, deltamethrin, fenvalerate and permethrin at their field application rates (225, 200, 225, 210 g a.i. ha−1, respectively), on growth and activities of microorganisms in relation to non-symbiotic N2-fixation and N mineralization in a tea garden soil of the Himalayan terai (at the lower base) region of West Bengal, India. Application of insecticides, in general, decreased growth and activities of ammonifying and nitrifying bacteria but increased proliferations of non-symbiotic N2-fixing bacteria, resulting in greater fixation of atmospheric N2, more so with permethrin (7.8%) followed by cypermethrin (5.2%). Most of the insecticides had a deleterious effect on the accumulation of oxidizable organic C and total N, more pronounced with fenvalerate and permethrin. The availability of exchangeable NH4+ was also significantly reduced, more prominently with fenvalerate (15.6%) followed by deltamethrin (11.2%), while permethrin followed by cypermethrin accentuated greater accumulation of soluble NO3− in soil. Therefore, the effects of synthetic pyrethroid insecticides on microbial activities in relation to non-symbiotic N2-fixation and its mineralization in tea soil of West Bengal can not be generalized, rather in most cases the effects were inclined more towards detrimental than a few stimulative one.

Global nitrogen budgets in cereals: A 50-year assessment for maize, rice, and wheat production systems.

Industrially produced N-fertilizer is essential to the production of cereals that supports current and projected human populations. We constructed a top-down global N budget for maize, rice, and wheat for a 50-year period (1961 to 2010). Cereals harvested a total of 1551 Tg of N, of which 48% was supplied through fertilizer-N and 4% came from net soil depletion. An estimated 48% (737 Tg) of crop N, equal to 29, 38, and 25 kg ha−1 yr−1 for maize, rice, and wheat, respectively, is contributed by sources other than fertilizer- or soil-N. Non-symbiotic N2 fixation appears to be the major source of this N, which is 370 Tg or 24% of total N in the crop, corresponding to 13, 22, and 13 kg ha−1 yr−1 for maize, rice, and wheat, respectively. Manure (217 Tg or 14%) and atmospheric deposition (96 Tg or 6%) are the other sources of N. Crop residues and seed contribute marginally. Our scaling-down approach to estimate the contribution of non-symbiotic N2 fixation is robust because it focuses on global quantities of N in sources and sinks that are easier to estimate, in contrast to estimating N losses per se, because losses are highly soil-, climate-, and crop-specific.

Non-symbiotic N2-fixation and phosphate-solubility in Gangetic alluvial soil as influenced by pre-emergence herbicide residues

An experiment has been conducted under laboratory conditions to investigate the effect of two pre-emergence herbicides viz., thiobencarb (at 1.5 and 4.5kga.i.ha−1) and pretilachlor (at 0.5 and 1.5kga.i.ha−1), on the changes of growth and activities of aerobic non-symbiotic N2-fixing bacteria and phosphate-solubilizing microorganisms in relation to availability of mineral nitrogen and soluble phosphorus in the Gangetic alluvial soil (Typic Haplustept) of West Bengal, India. Application of herbicides, in general, significantly increased growth and activities of microorganisms, resulting in greater release of available nitrogen and soluble phosphorus in soil; and the stimulation was more pronounced when the herbicides were applied at their lower concentrations (recommended field application rates), more so with thiobencarb, as compared to pretilachlor. As compared to untreated control, application of thiobencarb at lower concentration increased the proliferation of aerobic non-symbiotic N2-fixing bacteria, phosphate-solubilizing microorganisms and non-symbiotic N2-fixing capacity of soil to the extent of 54.0, 44.6 and 31.7%, respectively; and accumulated the highest amount of available nitrogen (37.8%) and phosphorus (54.5%) in soil, while pretilachlor at field application rate highly induced (37.2%) phosphate-solubilizing capacity of soil. At higher concentration, pretilachlor was superior to thiobencarb in augmenting the growth and activities of phosphate-solubilizers. The results of the present study also indicated that gradual increase in concentration of the herbicides over their recommended field application rates was not much conducive for growth and activities of microorganisms, and subsequent release of nutrients in soil.

Significant nonsymbiotic nitrogen fixation in Patagonian ombrotrophic bogs.

Nitrogen (N) nutrition in pristine peatlands relies on the natural input of inorganic N through atmospheric deposition or biological dinitrogen (N2 ) fixation. However, N2 fixation and its significance for N cycling, plant productivity, and peat buildup are mostly associated with the presence of Sphagnum mosses. Here, we report high nonsymbiotic N2 -fixation rates in two pristine Patagonian bogs with diversified vegetation and natural N deposition. Nonsymbiotic N2 fixation was measured in samples from 0 to 10, 10 to 20, and 40 to 50cm depth using the (15) N2 assay as well as theacetylene reduction assay (ARA). The ARA considerably underestimated N2 fixation and can thus not be recommended for peatland studies. Based on the (15) N2 assay, high nonsymbiotic N2 -fixation rates of 0.3-1.4 μmolN2 g(-1) day(-1) were found down to 50cm under micro-oxic conditions (2 vol.%) in samples from plots covered by Sphagnum magellanicum or by vascular cushion plants, latter characterized by dense and deep aerenchyma roots. Peat N concentrations point to greater potential of nonsymbiotic N2 fixation under cushion plants, likely because of the availability of easily decomposable organic compounds and oxic conditions in the rhizosphere. In the Sphagnum plots, high N2 fixation below 10cm depth rather reflects the potential during dry periods or low water level when oxygen penetrates the top peat layer and triggers peat mineralization. Natural abundance of the (15) N isotope of live Sphagnum (5.6 δ‰) from 0 to 10cm points to solely N uptake from atmospheric deposition and nonsymbiotic N2 fixation. A mean (15) N signature of -0.7 δ‰ of peat from the cushion plant plots indicates additional N supply from N mineralization. Our findings suggest that nonsymbiotic N2 fixation overcomes N deficiency in different vegetation communities and has great significance for N cycling and peat accumulation in pristine peatlands.

Field evaluation of the effects of cotton variety and GM status on rhizosphere microbial diversity and function in Australian soils

Despite the high level of adoption of genetically modified (GM) cultivars in the Australian cotton production system, concerns remain over the use of GM technology, particularly with regard to potential non-target effects. To address the hypothesis that GM cotton causes shifts in rhizosphere microbial diversity or function, we assessed rhizosphere soil samples from a range of conventional and GM cotton cultivars for diversity of bacteria and fungi, populations of ammonium oxidisers, rhizosphere basal and selective substrate-induced respiration, and non-symbiotic N2 fixation and nitrification. Comparison of results for GM and conventional cotton cultivars, both between and within seasons, indicated that the cotton rhizosphere plant–microbial interactions are variable in nature and significantly influenced by cultivar type. The GM status of the plant did not result in rhizosphere bacterial or fungal DNA-based grouping, but MicroResp data did show some grouping based on GM status, although this was not consistent by trait, suggesting that the GM trait is not greater than cultivar selection in causing rhizosphere change, especially when measured in the field environment with all the associative management practices.

Nitrogen cycling in summer active perennial grass systems in South Australia: non-symbiotic nitrogen fixation

Non-symbiotic nitrogen (N2) fixation by diazotrophic bacteria is a potential source for biological N inputs in non-leguminous crops and pastures. Perennial grasses generally add larger quantities of above- and belowground plant residues to soil, and so can support higher levels of soil biological activity than annual crops. In this study, the hypothesis is tested that summer-active perennial grasses can provide suitable microsites with the required carbon supply for N2 fixation by diazotrophs, in particular during summer, through their rhizosphere contribution. In a field experiment on a Calcarosol at Karoonda, South Australia, during summer 2011, we measured populations of N2-fixing bacteria by nifH-PCR quantification and the amount of 15N2 fixed in the rhizosphere and roots of summer-active perennial grasses. Diazotrophic N2 fixation estimates for the grass roots ranged between 0.92 and 2.35 mg 15N kg–1 root day–1. Potential rates of N2 fixation for the rhizosphere soils were 0.84–1.4 mg 15N kg–1 soil day–1 whereas the amount of N2 fixation in the bulk soil was 0.1–0.58 mg 15N kg–1 soil day–1. Populations of diazotrophic bacteria in the grass rhizosphere soils (2.45 × 106 nifH gene copies g–1 soil) were similar to populations in the roots (2.20 × 106 nifH gene copies g–1 roots) but the diversity of diazotrophic bacteria was significantly higher in the rhizosphere than the roots. Different grass species promoted the abundance of specific members of the nifH community, suggesting a plant-based selection from the rhizosphere microbial community. The results show that rhizosphere and root environments of summer-active perennial grasses support significant amounts of non-symbiotic N2 fixation during summer compared with cropping soils, thus contributing to biological N inputs into the soil N cycle. Some pasture species also maintained N2 fixation in October (spring), when the grasses were dormant, similar to that found in soils under a cereal crop. Surface soils in the rainfed cropping regions of southern Australia are generally low in soil organic matter and thus have lower N-supply capacity. The greater volume of rhizosphere soil under perennial grasses and carbon inputs belowground can potentially change the balance between N immobilisation and mineralisation processes in the surface soils in favour of immobilisation, which in turn contributes to reduced N losses from leaching.

Modelling changes in nitrogen cycling to sustain increases in forest productivity under elevated atmospheric CO<sub>2</sub> and contrasting site conditions

Abstract. If increases in net primary productivity (NPP) caused by rising concentrations of atmospheric CO2 (Ca) are to be sustained, key N processes such as soil mineralization, biological fixation, root uptake and nutrient conservation must also be increased. Simulating the response of these processes to elevated Ca is therefore vital for models used to project the effects of rising Ca on NPP. In this modelling study, hypotheses are proposed for changes in soil mineralization, biological fixation, root nutrient uptake and plant nutrient conservation with changes in Ca. Algorithms developed from these hypotheses were tested in the ecosystem model ecosys against changes in N and C cycling measured over several years under ambient vs. elevated Ca in Free Air CO2 Enrichment (FACE) experiments in the USA at the Duke Forest in North Carolina, the Oak Ridge National Laboratory forest in Tennessee, and the USDA research forest in Wisconsin. More rapid soil N mineralization was found to be vital for simulating sustained increases in NPP measured under elevated vs. ambient Ca at all three FACE sites. This simulation was accomplished by priming decomposition of N-rich humus from increases in microbial biomass generated by increased litterfall modelled under elevated Ca. Greater nonsymbiotic N2 fixation from increased litterfall, root N uptake from increased root growth, and plant N conservation from increased translocation under elevated Ca were found to make smaller contributions to simulated increases in NPP. However greater nutrient conservation enabled larger increases in NPP with Ca to be modelled with coniferous vs. deciduous plant functional types. The effects of these processes on productivity now need to be examined over longer periods under transient rises in Ca and a greater range of site conditions.

Potential for non-symbiotic N2-fixation in different agroecological zones of southern Australia

Nitrogen fixation by symbiotic and non-symbiotic bacteria can be a significant source of nitrogen in cropping systems. However, contributions from non-symbiotic nitrogen fixation (NSNF) are dependent on available carbon in the soil and environmental conditions (soil moisture and temperature). In Australia, measurements of NSNF have been made in the field by quantifying nitrogenase activity. These studies have included determinations of the moisture and temperature requirements for NSNF and for crop residue decomposition that supplies carbon to NSNF bacteria. Other studies have determined the N input by NSNF using N budget calculations. These data together with information about carbon supply and environmental conditions were used to estimate potential NSNF in the cropping zones of southern Australia. Using the ArcviewGIS Spatial Analyst (v3.1), maps of Australia showing estimates of NSNF in different cropping zones as determined by rainfall and temperature or carbon availability were generated. In Western Australia (represented by Wongan Hills) and South Australia (represented by Avon), where summers are dry, estimates of NSNF were generally low (10–15 kg N/ha from January to June) due to limitations of soil moisture. In New South Wales, particularly in the north where summer rainfall patterns develop (represented by Gunnedah), the warm, moist conditions produced higher estimates of NSNF (totaling 32–38 kg N/ha from January to June). In this region, the majority of estimated NSNF occurred in January and February leading to the depletion of carbon supplies and reduced NSNF in autumn (March–June). Information about potential supplies of N from NSNF across the cropping zones should be useful for researchers to select and study areas that are most likely to benefit from NSNF. It should also help agronomists and extension officers explain changes in N status within paddocks or within specific farming systems and to provide more accurate advice on N fertiliser requirements, particularly in low-input farming systems.

Nonsymbiotic nitrogen fixation in 3-year-old Jeffrey pines and the role of elevated [CO2

Increased belowground labile C inputs under elevated [CO2] could stimulate nonsymbiotic N2 fixation, thereby enhancing growth responses of vegetation to elevated [CO2] on nutrient-poor sites. To test this hypothesis, nonsymbiotic N2 fixation rates in soils planted with 3-year-old Jeffrey pine (Pinus jeffreyi Grev. & Balf.) trees grown under 365 and 700 µL·L–1 atmospheric [CO2] were measured by exposing the soil to 15N2-enriched air for 78 d. Nitrogen fixation rates were estimated by measuring 15N content of trees and soil. Compared with the ambient CO2 treatment, the elevated CO2 treatment did not affect biomass, N content, or δ15N of individual plant parts and soils, indicating that elevated [CO2] did not stimulate nonsymbiotic N2 fixation. Because belowground C inputs did not increase under elevated [CO2], the initial hypothesis could not be accepted or rejected. The results from the 15N2 labeling study agree with other studies showing that nonsymbiotic N2 fixation is not likely to provide a large input of N in forest ecosystems. The 15N2 labeling technique was promising for studying N2 fixation in plant–soil systems, but the preliminary nature of this study did not allow for firm conclusions with regard to the effects of elevated [CO2].

Relationships between soil physico-chemical properties and microbial activity along a restoration chronosequence of alpine grasslands following ski run construction

Rapid establishment of herbaceous cover after ski run construction is fundamental to limit soil erosion and restore initial ecosystem functioning. This paper reports the disturbance and development of relationships between soil physico-chemical properties and microbial activities following ski run construction and along a chronosequence of restored alpine grasslands (1 month and 1, 4 and 13 years). Several measurements were made on vegetation (species composition, root biomass), soil (aggregate stability, particle size distribution, total carbon and nitrogen content, porosity) and soil microbial activity (heterotrophic microbial diversity and functional structure, nitrogenase activity). During the restoration chronosequence, effective porosity was found to be positively correlated with rock fragments (>2 mm) proportion ( r=0.96; P<0.05) while capillary porosity was positively correlated with clay content ( r=0.86; P<0.05). A specific microbial pattern of native alpine soil was evident with low non-symbiotic N2-fixation (6.61 E-11 mol C 2H 4 per gram), low heterotrophic functional diversity and low metabolic quotient (qCO 2). The increase in potential nitrogen fixation along the chronosequence was attributed to labile C input by root exudation and favourable soil moisture. High catabolic diversity in older restored soils (4 and 13 years old) was also sustained by the constant evolution of soil physico-chemical properties, especially heterogeneous C resource input. In accordance with the positive relationship between catabolic evenness and ecosystem function, our data suggests that the microbial community remained very unstable during the early stage of ski run restoration (<13 years old) because of the dominance of opportunist micro-organisms.

Long‐Term Effects of Urea and Green Manure on Rice Yields and Nitrogen Balance

Data from a 14‐yr double‐crop rice (Oryza sativa L.) experiment allowed comparison of the long‐term effects of N fertilizer from different sources (urea and in situ grown azolla [Azolla microphylla Kaulf.] and sesbania [Sesbania rostrata Bremek. &amp; Oberm.]) on N balances, soil N pools (both total and available), and yields. Although data show that plant‐available N was maintained over time in both wet (WS) and dry seasons (DS), yields declined significantly, indicating a decline in physiological N use efficiency. The yield declines were generally similar regardless of N source in both seasons. The WS decline averaged 150 kg ha−1 yr−1 in the three added‐N treatments, while the DS decline averaged 185 kg ha−1 yr−1. After 27 crops, the cumulative positive N balance was estimated at 1244, 348, 646, and 1039 kg N ha−1 in control, urea, sesbania, and azolla treatments, respectively. There was no significant change in soil total N content in the control and urea treatments, whereas it increased to 344 to 541 kg after 27 crops in the sesbania and azolla treatments. Conservation of the soil N status and positive N balance, in spite of the high amounts of N removed through grain and straw, reflect the N contribution (13–46 kg ha−1 crop−1) from nonsymbiotic N2 fixation. In addition, sesbania and azolla were estimated to add ≈57 to 64 kg ha−1 crop−1 through symbiotic N2 fixation. These results demonstrate that in rice–rice cropping systems biological N fixation plays a vital role in replenishing the soil N pool. However, continuous application of green manure N (GM‐N) did not increase crop N availability, perhaps because of the presence of a recalcitrant soil organic matter fraction. Residual effects on rice grain yield and N uptake were observed only with GM‐N sources.

Dynamics of mineral N availability in grassland ecosystems under increased [CO2]: hypotheses evaluated using the Hurley Pasture Model.

The following arguments are outlined and then illustrated by the response of the Hurley Pasture Model to [CO2] doubling in the climate of southern Britain. 1. The growth of N-limited vegetation is determined by the concentration of N in the soil mineral N pools and high turnover rates of these pools (i.e., large input and output fluxes) contribute positively to growth. 2. The size and turnover rates of the soil mineral N pools are determined overwhelmingly by N cycling into all forms of organic matter (plants, animals, soil biomass and soil organic matter — `immobilisation' in a broad sense) and back again by mineralisation. Annual system N gains (by N2 fixation and atmospheric deposition) and losses (by leaching, volatilisation, nitrification and denitrification) are small by comparison. 3. Elevated [CO2] enriches the organic matter in plants and soils with C, which leads directly to increased removal of N from the soil mineral N pools into plant biomass, soil biomass and soil organic matter (SOM). ‘Immobilisation’ in the broad sense then exceeds mineralisation. This is a transient state and as long as it exists the soil mineral N pools are depleted, N gaseous and leaching losses are reduced and the ecosystem gains N. Thus, net immobilisation gradually increases the N status of the ecosystem. 4. At the same time, elevated [CO2] increases symbiotic and non-symbiotic N2 fixation. Thus, more N is gained each year as well as less lost. Effectively, the extra C fixed in elevated [CO2] is used to capture and retain more N and so the N cycle tracks the C cycle. 5. However, the amount of extra N fixed and retained by the ecosystem each year will always be small (ca. 5–10 kg N ha-1 yr-1) compared with amount of N in the immobilisation-mineralisation cycle (ca. 1000 kg N ha-1 yr-1). Consequently, the ecosystem can take decades to centuries to gear up to a new equilibrium higher-N state. 6. The extent and timescale of the depletion of the mineral N pools in elevated [CO2] depends on the N status of the system and the magnitude of the overall system N gains and losses. Small changes in the large immobilisation—mineralisation cycle have large effects on the small mineral N pools. Consequently, it is possible to obtain a variety of growth responses within 1–10 year experiments. Ironically, ecosystem models — artificial constructs — may be the best or only way of determining what is happening in the real world.