Isotopic fractionation during N2 fixation by four tropical legumes

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the ¹⁵N natural abundance technique, it is necessary to determine the ¹⁵N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N₂-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with ¹⁵N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the ¹⁵N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ¹⁵N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (B_s) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (B_wp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.     

Natural N abundances of inorganic nitrogen in soil treated with fertilizer and compost under changing soil moisture regimes

This study was conducted to examine whether the applications of N-inputs (compost and fertilizer) having different N isotopic compositions (δ¹⁵N) produce isotopically different inorganic-N and to investigate the effect of soil moisture regimes on the temporal variations in the δ¹⁵N of inorganic-N in soils. To do so, the temporal variations in the concentrations and the δ¹⁵N of NH₄⁺ and NO₃⁻ in soils treated with two levels (0 and 150 mg N kg⁻¹) of ammonium sulfate (δ¹⁵N=−2.3‰) and compost (+13.9‰) during a 10-week incubation were compared by changing soil moisture regime after 6 weeks either from saturated to unsaturated conditions or vice versa. Another incubation study using ¹⁵N-labeled ammonium sulfate (3.05 ¹⁵N atom%) was conducted to estimate the rates of nitrification and denitrification with a numerical model FLUAZ. The δ¹⁵N values of NH₄⁺ and NO₃⁻ were greatly affected by the availability of substrate for each of the nitrification and denitrification processes and the soil moisture status that affects the relative predominance between the two processes. Under saturated conditions for 6 weeks, the δ¹⁵N of NH₄⁺ in soils treated with fertilizer progressively increased from +2.9‰ at 0.5 week to +18.9‰ at 6 weeks due to nitrification. During the same period, NO₃⁻ concentrations were consistently low and the corresponding δ¹⁵N increased from +16.3 to +39.2‰ through denitrification. Under subsequent water-unsaturated conditions, the NO₃⁻ concentrations increased through nitrification, which resulted in the decrease in the δ¹⁵N of NO₃⁻. In soils, which were unsaturated for the first 6-weeks incubation, the δ¹⁵N of NH₄⁺ increased sharply at 0.5 week due to fast nitrification. On the other hand, the δ¹⁵N of NO₃⁻ showed the lowest value at 0.5 week due to incomplete nitrification, but after a subsequence increase, they remained stable while nitrification and denitrification were negligible between 1 and 6 weeks. Changing to saturated conditions after the initial 6-weeks incubation, however, increased the δ¹⁵N of NO₃⁻ progressively with a concurrent decrease in NO₃⁻ concentration through denitrification. The differences in δ¹⁵N of NO⁻₃ between compost and fertilizer treatments were consistent throughout the incubation period. The δ¹⁵N of NO₃⁻ increased with the addition of compost (range: +13.0 to +35.4‰), but decreased with the addition of fertilizer (−10.8 to +11.4‰), thus resulting in intermediate values in soils receiving both fertilizer and compost (−3.5 to +20.3‰). Therefore, such differences in δ¹⁵N of NO₃⁻ observed in this study suggest a possibility that the δ¹⁵N of upland-grown plants receiving compost would be higher than those treated with fertilizer because NO₃⁻ is the most abundant N for plant uptake in upland soils.     

N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The ¹⁵N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N₂ fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the ¹⁵N abundance of the N derived from N₂ fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched ¹⁵N ammonium sulphate was added. The %Ndfa was then used with the ¹⁵N natural abundance data of the nodulated soybean and non-N₂-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘B_s’) was higher. The differences in ‘B_s’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.     

Interactive effects of N fertilizer source and timing of fertilization leave specific N isotopic signatures in Chinese cabbage and soil

Natural ¹⁵N abundances (δ¹⁵N) in plant and soil can be used as a powerful marker to reveal the history of N fertilization. To investigate whether N fertilizer source and timing of fertilization leave specific δ¹⁵N signals in plant tissue and soil inorganic N, Chinese cabbage (Brassica campestris L. cv. Maeryok), one of the most popular vegetables in Asia, was grown in pots for 60 days with a single or split N applications of organic (composted manure; δ¹⁵N=+16.4‰) or inorganic N (urea; δ¹⁵N=−0.7‰). Seven N treatments were studied: (1) a single basal fertilization with compost or (2) urea; (3) a basal urea application followed by an additional (at 40 days after transplant, same below) compost or (4) urea application; (5) a basal compost application followed by an additional compost or (6) urea application; and (7) no N fertilization. Regardless of the time of N application, δ¹⁵N of cabbage treated with compost was higher (>+9.0‰) than that (< +1.0‰) treated with urea, reflecting the effect of isotopically different N sources. In split N fertilization, only the addition of isotopically different N sources in the middle of the growth period significantly affected the δ¹⁵N of the whole plant. Specific δ¹⁵N signals of basal N inputs were detected in outer cabbage parts formed in the early growth stage, while those of additional N inputs were detected in inner cabbage parts formed in the latter growth stage. We conclude that measurements of temporal variations in δ¹⁵N of plant parts formed in different growth stages could reveal the history of N fertilization.     

Nitrate leaching in soil: Tracing the NO3 sources with the help of stable N and O isotopes

Legumes increase the plant-available N pool in soil, but might also increase NO₃⁻ leaching to groundwater. To minimize NO₃⁻ leaching, N-release processes and the contribution of legumes to NO₃⁻ concentrations in soil must be known. Our objectives were (1) to quantify NO₃⁻-N export to >0.3 m soil depth from three legume monocultures (Medicago x varia Martyn, Onobrychis viciifolia Scop., Lathyrus pratensis L.) and from three bare ground plots. Furthermore, we (2) tested if it is possible to apply a mixing model for NO₃⁻ in soil solution based on its dual isotope signals, and (3) estimated the contribution of legume mineralization to NO₃⁻ concentrations in soil solution under field conditions. We collected rainfall and soil solution at 0.3 m soil depth during 1 year, and determined NO₃⁻ concentrations and δ¹⁵N and δ¹⁸O of NO₃⁻ for >11.5 mg NO₃⁻-N l⁻¹. We incubated soil samples to assess potential N release by mineralization and determined δ¹⁵N and δ¹⁸O signals of NO₃⁻ derived from mineralization of non-leguminous and leguminous organic matter.Mean annual N export to >0.3 m soil depth was highest in bare ground plots (9.7 g NO₃⁻-N m⁻²; the SD reflects the spatial variation) followed by Medicago x varia monoculture (6.0 g NO₃⁻-N m⁻²). The O. viciifolia and L. pratensis monocultures had a much lower mean annual N export (0.5 and 0.3 g NO₃⁻-N m⁻²). The averaged NO₃⁻-N leaching during 70 days was not significantly different between field estimates and incubation for the Medicago x varia Martyn monoculture.The δ¹⁵N and δ¹⁸O values in NO₃⁻ of rainfall (δ¹⁵N: 3.3±0.8‰; δ¹⁸O: 30.8±4.7‰), mineralization of non-leguminous SOM (9.3±0.9‰; 6.7±0.8‰), and mineralization of leguminous SOM (1.5±0.6‰; 5.1±0.9‰) were markedly different. Applying a linear mixing model based on these three sources to δ¹⁵N and δ¹⁸O values in NO₃⁻ of soil solution during winter 2003, we calculated 18-41% to originate from rainfall, 38-57% from mineralization of non-leguminous SOM, and 18-40% from mineralization of leguminous SOM.Our results demonstrate that (1) even under legumes NO₃⁻-N leaching was reduced compared to bare ground, (2) the application of a three-end-member mixing model for NO₃⁻ based on its dual isotope signals produced plausible results and suggests that under particular circumstances such models can be used to estimate the contributions of different NO₃⁻ sources in soil solution, and (3) in the 2nd year after establishment of legumes, they contributed approximately one-fourth to NO₃⁻-N loss.     

Natural abundance δN and δC of DNA extracted from soil

We report the first simultaneous measurements of δ¹⁵N and δ¹³C of DNA extracted from surface soils. The isotopic composition of DNA differed significantly among nine different soils. The δ¹³C and δ¹⁵N of DNA was correlated with δ¹³C and δ¹⁵N of soil, respectively, suggesting that the isotopic composition of DNA is strongly influenced by the isotopic composition of soil organic matter. However, in all samples DNA was enriched in ¹³C relative to soil, indicating microorganisms fractionated C during assimilation or preferentially used ¹³C enriched substrates. Enrichment of DNA in ¹⁵N relative to soil was not consistently observed, but there were significant differences between δ¹⁵N of DNA and δ¹⁵N of soil for three different sites, suggesting microorganisms are fractionating N or preferentially using N substrates at different rates across these contrasting ecosystems. There was a strong linear correlation between δ¹⁵N of DNA and δ¹⁵N of the microbial biomass, which indicated DNA was depleted in ¹⁵N relative to the microbial biomass by approximately 3.4‰. Our results show that accurate and precise isotopic measurements of C and N in DNA extracted from the soil are feasible, and that these analyses may provide powerful tools for elucidating C and N cycling processes through soil microorganisms.     

Natural N abundance of plants and soils under different management practices in a montane grassland

The natural ¹⁵N abundance (δ¹⁵N) of different ecosystem compartments is considered to be an integrator of nitrogen (N) cycle processes. Here we investigate the extent to which patterns of δ¹⁵N in grassland plants and soils reflect the effect of different management practices on N cycling processes and N balance. Investigations were conducted in long-term experimental plots of permanent montane meadows with treatments differing in the amount and type of applied fertilizer (0-200 kg N ha⁻¹ yr⁻¹; mineral fertilizer, cattle slurry, stable manure) and/or the cutting frequency (1-6 cuts per season). The higher δ¹⁵N values of organic fertilizers compared to mineral fertilizer were reflected by higher δ¹⁵N values in soils and harvested plant material. Furthermore, δ¹⁵N of top soils and plant material increased with the amount of applied fertilizer N. N balances were calculated from N input (fertilization, atmospheric N deposition and symbiotic N₂ fixation) and N output in harvest. ‘Excess N’—the fraction of N input not harvested—was assumed to be lost to the environment or accumulated in soil. Taking fertilizer type into account, strong positive correlations between δ¹⁵N of top soils and the N input-output balance were found. In plots receiving mineral N fertilizer this indicates that soil processes which discriminate against ¹⁵N (e.g. nitrification, denitrification, ammonia volatilization) were stimulated by the increased supply of readily available N, leading to loss of the ¹⁵N depleted compounds and subsequent ¹⁵N enrichment of the soils. By contrast, in plots with organic fertilization this correlation was partly due to accumulation of ¹⁵N-enriched fertilizer N in top soils and partly due to the occurrence of significant N losses. Cutting frequency appeared to have no direct effect on δ¹⁵N patterns. This study for the first time shows that the natural abundance of ¹⁵N of agricultural systems does not only reflect the type (organic or mineral fertilizer) or amount of annual fertilizer amendment (0-200 kg ha⁻¹ yr⁻¹) but that plant and soil δ¹⁵N is better described by N input-output balances.     

Patterns of natural N in soils and plants from chemically and organically fertilized uplands

Diagnostic tests for organic production of crops would be useful. In this study, the difference in natural ¹⁵N abundances (δ¹⁵N) of soils and plants between fertilizer-applied upland (FU) and compost-applied upland (CU) fields was investigated to study using δ¹⁵N as a marker of organic produce. Twenty samples each of soils and plants were collected from each field in early summer after applying fertilizer or compost. The δ¹⁵N of fertilizers and composts was −1.6±1.5‰ (n=8) and 17.4±1.2‰ (n=10), respectively. The δ¹⁵N of total soil-N was significantly (P<0.05) higher in CU fields (8.8±2.0‰) than in FU fields (5.9±0.7‰) due to long-term continuous application of ¹⁵N-enriched compost, as indicated by a positive correlation (r=0.62) between N content and δ¹⁵N of total soil-N. The NO₃⁻ pool of CU soils (11.6±4.5‰) was also significantly (P<0.05) enriched in ¹⁵N compared to FU soils (4.7±1.1‰), while the ¹⁵N contents of NH₄⁺ pool were not different between both soils. Compost application resulted in ¹⁵N enrichment of plants; the δ¹⁵N values were 14.6±3.3‰ for CU and 4.1±1.7‰ for FU fields. These results showed that long-term application of compost resulted in a significant ¹⁵N-enrichment of soils and plants relative to fertilizer. Therefore, this study suggested that δ¹⁵N could serve as promising indicators of organic fertilizers application when used with other independent evidence. However, further studies under many conditions should be conducted to prepare reliable δ¹⁵N guidelines for organic produce, since the δ¹⁵N of inorganic soil-N and plant-N are influenced by various factors such as soil type, plant species, the rate of N application, and processes such as mineralization, nitrification, and denitrifcation.     

Effects of Multiple Reference Plants,Season, and Irrigation on Biological Nitrogen Fixation by Pasture Legumes using the Isotope Dilution Method

Abstract

The popular and widely used ¹⁵nitrogen (N)–isotope dilution method for estimating biological N fixation (BNF) of pasture and tree legumes relies largely on the ability to overcome the principal source of error due to the problem of selecting appropriate reference plants. A field experiment was conducted to evaluate the suitability of 12 non‐N₂‐fixing plants (i.e., nonlegumes) as reference plants for estimating the BNF of three pasture legumes (white clover, Trifolium repens L.; lucerne, Medicago sativa; and red clover, Trifolium pratense L.) in standard ryegrass–white clover (RWC) and multispecies pastures (MSP) under dry‐land and irrigation systems, over four seasons in Canterbury, New Zealand. The ¹⁵N‐isotope dilution method involving field ¹⁵N‐microplots was used to estimate BNF. Non‐N₂‐fixing plants were used either singly or in combination as reference plants to estimate the BNF of the three legumes. Results obtained showed that, on the whole, ¹⁵N‐enrichment values of legumes and nonlegumes varied significantly according to plant species, season, and irrigation. Grasses and herb species showed higher ¹⁵N‐enrichment than those of legumes. Highest ¹⁵N‐enrichment values of all plants occurred during late summer under dry‐land and irrigation conditions. Based on single or combined non‐N₂‐fixing plants as reference plants, the proportion of N derived from the atmosphere (% Ndfa) values were high (50 to 90%) and differed between most reference plants in the MSP pastures, especially chicory (Cichorium intybus), probably because it is different in phenology, rooting depth, and N‐uptake patterns compared to those of legumes. The percent Ndfa values of all plants studied also varied according to plant species, season, and irrigation in the MSP pastures. Estimated daily amounts of BNF varied according to pasture type, time of plant harvest, and irrigation, similar to those shown by percent Ndfa results as expected. Irrigation increased daily BNF more than 10‐fold, probably due to increased dry‐matter yield of pasture under irrigation compared to dry‐land conditions. Seasonal and irrigation effects were more important in affecting estimates of legume BNF than those due to the appropriate matching of N₂‐fixing and non‐N₂‐fixing reference plants.     

Paddy System with a Hybrid Rice Enhances Cyanobacteria Nostoc and Increases N2 Fixation

Biological nitrogen(N) fixation(BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight,field-based ~(15)N_2-labelling growth chamber experiments were used to assess the BNF capacity of different rice varieties. In addition,both the 16 S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based ~(15)N_2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha~(-1) in inbred japonica(W23) and hybrid indica(IIY) rice cultivars planted in the rice-soil systems, respectively, and only 1%–2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil(0–1 cm) under IIY than under W23. Sequencing of the 16 S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23.Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar(IIY) promoted BNF to a greater extent than the inbred rice cultivar(W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.     

C signature of CO2 evolved from incubated maize residues and maize-derived sheep faeces

Analyses of the spatial and temporal variations in the natural abundance of ¹³C are frequently employed to study transformations of plant residues and soil organic matter turnover on sites where long continued vegetation with the C₃-type photosynthesis pathway has been replaced with a C₄-type vegetation (or vice versa). One controversial issue associated with such analyses is the significance of isotopic fractionation during the microbial turnovers of C in complex substrates. To evaluate this issue, C₃-soil and quartz sand were amended with maize residues and with faeces from sheep feed exclusively on maize silage. The samples were incubated at 15 °C for 117 days (maize residues) or 224 days (sheep faeces). CO₂ evolved during incubation was trapped in NaOH and analysed for C isotopic contents. At the end of incubation, 63 and 50% of the maize C was evolved as CO₂ in the soil and sand, respectively, while 32% of the faeces C incubated with soil and with sand was recovered as CO₂. Maize and faeces showed a similar decomposition pattern but maize decomposed twice as fast as faeces. The δ¹³C of faeces was 0.3‰ lower than that of the maize residue (δ¹³C −13.4‰), while the δ¹³C of the C₃-soil used for incubation was −31.6‰. The δ¹³C value of the CO₂ recovered from unamended C₃-soil was similar or slightly lower (up to −1.5‰) than that of the C₃-soil itself except for an initial flush of ¹³C enriched CO₂. The δ¹³C values of the CO₂ from sand-based incubations typically ranged −15‰ to −17‰, i.e. around −3‰ lower than the δ¹³C measured for maize and faeces. Our study clearly demonstrates that the decomposition of complex substrates is associated with isotopic fractionation, causing evolved CO₂ to be depleted in ¹³C relative to substrates. Consequently the microbial products retained in the soil must be enriched in ¹³C.     

The role of arbuscular mycorrhiza in legume symbiotic performance

Legumes may respond to non-rhizobial inoculants such as arbuscular mycorrhizal (AM) fungi either through an effect on plant growth or, in addition, through an effect on the function of the legume-Rhizobium symbiosis. We have examined the literature where the application of ¹⁵N isotope dilution methodology permits the effect of indigenous AM and AM inoculants to be quantitatively separated into plant-growth-mediated and biological N₂ fixation (BNF)-mediated components. These studies clearly demonstrate the beneficial effects that both indigenous and inoculated AM have on legume growth, N uptake and the proportional dependence of the legume on atmospheric N₂. While the published data allow an assessment of various biological, edaphic and environmental factors that affect the response of various legumes to AM inoculation, they also highlight the paucity of quantitative field data and the lack of understanding of the interaction of legume genotype with AM species with respect to legume symbiotic performance.     

Compound-specific δC and δH analyses of plant and soil organic matter: A preliminary assessment of the effects of vegetation change on ecosystem hydrology

Here we present δ¹³C and δ²H data of long-chained, even-numbered (C₂₇-C₃₁) n-alkanes from C3 (trees) and C4 (grasses) plants and from the corresponding soils from a grassland-woodland vegetation sequence in central Queensland, Australia. Our data show that δ¹³C values of the C4 grassland species were heavier relative to those of C3 tree species from the woodland (Acacia leaves) and woody grassland (Atalaya leaves). However, n-alkanes from the C4 grasses had lighter δ²H values relative to the Acacia leaves, but showed no significant difference in δ²H values when compared with C3 Atalaya leaves. These results differ from those of previous studies, showing that C4 grasses had heavier δ²H values relative to C3 grasses and trees. Those observations have been explained by C4 plants accessing the more evaporation-influenced and isotopically heavier surface water and tree roots sourcing deeper, isotopically lighter soil water (“Two-layered soil-water system”). By comparison, our data suggest that ecosystem changes (vegetation “thickening”) can significantly alter the soil hydrological characteristics. This is shown by the heavier δ²H values in the woodland soil compared with lighter δ²H values in the grassland soil, implying that the recent vegetation change (increased tree biomass) in the woodland had altered soil hydrological conditions. Estimated δ²H values of the source-water for vegetation in the grassland and woodland showed that both trees and grasses in open settings accessed water with lighter δ²H values (avg. −46‰) compared with water accessed by trees in the woodland vegetation (avg. −7‰). These data suggest that in semi-arid environments the “two-layer” soil water concept might not apply. Furthermore, our data indicate that compound-specific δ²H and δ¹³C analyses of n-alkanes from soil organic matter can be used to successfully differentiate between water sources of different vegetation types (grasses versus trees) in natural ecosystems.     

Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system

Below-ground transfer of nitrogen (N) fixed by legume trees to associated non-N₂-fixing crops has received little attention in agroforestry, although the importance of below-ground interactions is shown in other ecosystems. We used ¹⁵N natural abundance to estimate N transfer from the legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. to C₄ grass Dichanthium aristatum (Poir.) C.E. Hubb. in a silvopastoral system, where N was recycled exclusively by below-ground processes and N₂ fixation by G. sepium was the sole N input to the system. Finding a suitable reference plant, a grass without contact with tree roots or litter, was problematic because tree roots invaded adjacent grass monocrop plots and soil isotopic signature in soil below distant grass monocrops differed significantly from the agroforestry plots. Thus, we used grass cultivated under greenhouse conditions in pots filled with agroforestry soil as the reference. A model of soil ¹⁵N fractionation during N mineralization was developed for testing the reliability of that estimate. Experimental and theoretical results indicated that 9 months after greenhouse transplanting, the percentage of fixed N in the grass decreased from 35% to <1%, due to N export in cut grass and dilution of fixed N with N taken up from the soil. The effect of soil ¹⁵N fractionation on the estimate of the reference value was negligible. This indicates that potted grass is a suitable reference N transfer studies using ¹⁵N natural abundance. About one third of N in field-grown grass was of atmospheric origin in agroforestry plots and in adjacent D. aristatum grassland invaded by G. sepium roots. The concentration of fixed N was correlated with fine root density of G. sepium but not with soil isotopic signature. This suggests a direct N transfer from trees to grass, e.g. via root exudates or common mycorrhizal networks.     

Soil organic matter dynamics and land use change at a grassland/forest ecotone

In the grassland/forest ecotone of North America, many areas are experiencing afforestation and subsequent shifts in ecosystem carbon (C) stocks. Ecosystem scientists commonly employ a suite of techniques to examine how such land use changes can impact soil organic matter (SOM) forms and dynamics. This study employs four such techniques to compare SOM in grassland (Bromus inermis) and recently forested (∼35 year, Ulmus spp. and Quercus spp.) sites with similar soil types and long-term histories in Kansas, USA. The work examines C and nitrogen (N) parameters in labile and recalcitrant SOM fractions isolated via size and density fractionation, acid hydrolysis, and long-term incubations. Size fractionation highlighted differences between grassland and forested areas. N concentration of forested soils’ 63-212 μm fraction was higher than corresponding grassland soils’ values (3.0±0.3 vs. 2.3±0.3 mg g_fraction⁻¹, P<0.05), and N concentration of grassland soils’ 212-2000 μm fraction was higher than forested soils (3.0±0.4 vs. 2.3±0.2 mg g_fraction⁻¹, P<0.05). Similar trends were observed for these same fractions for C concentration; forested soils exhibited 1.3 times the C concentration in the 63-212 μm fraction compared to this fraction in grassland soils. Fractions separated via density separation and acid hydrolysis exhibited no differences in [C], [N], δ¹⁵N, or δ¹³C when compared across land use types. Plant litterfall from forested sites possessed significantly greater N concentrations than that from grassland sites (12.41±0.10 vs. 11.62±0.19 mg g_litter⁻¹). Long-term incubations revealed no differences in C or N dynamics between grassland and forested soils. δ¹³C and δ¹⁵N values of the smallest size and the heavier density fractions, likely representing older and more recalcitrant SOM, were enriched compared to younger and more labile SOM fractions; δ¹⁵N of forested soils’ 212-2000 μm fraction were higher than corresponding grassland soils (1.7±0.3‰ vs. 0.5±0.4‰). δ¹³C values of acid hydrolysis fractions likely reflect preferential losses of ¹³C-depleted compounds during hydrolysis. Though C and N data from size fractions were most effective at exhibiting differences between grassland and forested soils, no technique conclusively indicates consistent changes in SOM dynamics with forest growth on these soils. The study also highlights some of the challenges associated with describing SOM parameters, particularly δ¹³C, in SOM fractions isolated by acid hydrolysis.     

Nitrogen fixation in six forage legumes in Mediterranean central Chile

Abstract

The contribution of biological nitrogen fixation (BNF) to the N nutrition of six annual forage legumes, subterranean clover (Trifolium subterraneum), burr medic (Medicago polymorpha), balansa clover (Trifolium michelianum), Persian clover (Trifolium resupinatum), yellow serradela (Ornithopus compressus), and pink serradela (Ornithopus sativus) was evaluated by the ¹⁵N natural abundance technique, using four grass species (Briza máxima, Bromus mollis, Hordeum berteroanum, Avena barbata) and two composite species (Leontodon leysseri and Hedipnois cretica) as reference plants. An additional objective was to determine whether alternative legume species to those in common use (T. subterraneum and M. polymorpha) in the area, could improve BNF. The field studies were conducted in two edaphic conditions, granitic (Entisol) and clay (Vertisol) soil, located in Cauquenes, VII Region, in the sub-humid Mediterranean zone of Chile. In the granitic soil the percentages of N derived from fixation were high in all species (74 to 94%); yellow serradela cv. Tauro presented the greatest N content in dry matter and N fixation, equivalent to 91 kg N ha^?1. In contrast, pink serradela cv. Cádiz and subterranean clover cv. Gosse presented the lowest N fixation. In the clay soil, under periodically waterlogged conditions, balansa clover cv. Paradana and persian clover cv. Prolific had high percentage values of BNF (>95%) and fixed more N (100.2 and 82.5 kg N ha^?1, respectively) than burr medic and subterranean clover cv. Gosse. The present study allowed the identification of new germplasm of high capacity of N fixation which is an additional criterion for selecting species for infertile and waterlogged soil conditions in the Mediterranean area of Chile.     

Arbuscular mycorrhizae affect the N and C economy of nodulated Phaseolus vulgaris (L.) during NH4 nutrition

In the symbiosis between nodulated legume roots and arbuscular mycorrhizal (AM) fungi, the C and N economy can be influenced by the source of N-supply from either AM-derived NH₄⁺ uptake or nodule-derived biological nitrogen fixation (BNF). This relationship was investigated in terms of NH₄⁺ supply and BNF by the two symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were hydroponically grown with either 0 N or 1 mM NH₄⁺ supply. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, CO₂ and O₂ root respiration, calculated C and N economy. AM roots had higher NH₄⁺ uptake and this was associated with the suppression of BNF and nodule growth. The higher NH₄⁺ uptake in AM roots occurred with lower root maintenance respiration, compared to when N was derived from BNF. There was also an increase in the below-ground sink strength of NH₄⁺ fed AM roots compared to NH₄⁺ fed non-AM roots, as evidenced by the increases in root CO₂ and O₂ respiration and photosynthetic stimulation. These results indicate that although the AM root had higher total below-ground respiratory costs during NH₄⁺ nutrition, there were lower respiratory C costs associated with N derived from AM symbionts in comparison to N from BNF.     

Immobilization of avian excreta-derived nutrients and reduced lignin decomposition in needle and twig litter in a temperate coniferous forest

The possible effects of excreta of the Great Cormorant Phalacrocorax carbo on decomposition processes and dynamics of nutrients (N, P, Ca, K, Mg) and organic chemical components (lignin, total carbohydrates) were investigated in a temperate evergreen coniferous forest near Lake Biwa in central Japan. Two-year decomposition processes of needles and twigs of Chamaecyparis obtusa were examined at two sites, control site never colonized by the cormorants (site C) and colonizing site (site 2). Mass loss was faster in needles than in twigs. Mass loss of these litter types was faster at site C than at site 2, which was ascribed to the decreased mass loss rate of acid-insoluble ‘lignin’ at site 2. Net immobilization of N, P, and Ca occurred in needles and twigs at site 2; whereas at site C, mass of these elements decreased without immobilization during decomposition. Duration of immobilization phase of these nutrients at site 2 was estimated to be 1.6 to 2.5 years in needles and 19.6 to 23.5 years in twigs. Immobilization potential (maximum amount of exogenous nutrient immobilized per gram initial material) was similar between needles and twigs for N and Ca but was about 10 times higher in twigs than in needles for P. δ¹³C in needles was relatively constant during the first year and then increased during the second year, whereas δ¹³C in twigs was variable during decomposition. Acid-insoluble fraction was depleted in ¹³C compared to whole needles (1.6-2.1‰) and twigs (2.0-2.5‰). δ¹⁵N of needles and twigs and their acid-insoluble fractions approached to δ¹⁵N of excreta during decomposition at site 2. This result demonstrated the immobilization of excreta-derived N into litter due to the formation of acid-insoluble lignin-like substances complexed with excreta-derived N. No immobilization occurred in K and Mg and their mass decreased during decomposition at both sites. Based on these results of nutrient immobilization during decomposition and on the data of litter fall and excreta amount at site 2, we tentatively calculated stand-level immobilization potential of litter fall and its contribution to total amount of N and P deposited as excreta. Thus, the potential maximum amount immobilized into litter fall (needles and twigs) was estimated to account for 5-7% of total excreta-derived N and P.     

Feeding guilds in Collembola based on nitrogen stable isotope ratios

In soil a high number of species co-exist without extensive niche differentiation, which was assigned as ‘the enigma of soil animal species diversity’. In particular, the detritivores are regarded as food generalists. We have investigated nitrogen stable isotope ratios (¹⁵N/¹⁴N) of a major decomposer group, the Collembola, to evaluate trophic relationship and determine feeding guilds. Additionally, the δ¹⁵N values of potential food sources such as mosses, lichens and other plant derived material (bark, nuts, leaves) were analysed. The natural variation in nitrogen isotopes was assessed in 20 Collembola taxa from three deciduous forest stands. The δ¹⁵N signature formed a continuum from phycophages/herbivores to primary and secondary decomposers, reflecting a gradual shift from more detrital to more microbial diets. The δ¹⁵N gradient spanned over 9 δ units, which implies a wide range in food sources used. Assuming a shift in ¹⁵N of about 3 ‰ per trophic level, the results indicate a range of three trophic levels. These variations in ¹⁵N/¹⁴N ratios suggest that trophic niches of Collembola species differ and this likely contributes to Collembola species diversity.     

Seasonal variations in natural 13C abundances in C3 and C4 plants collected in Thailand and the Philippines

Abstract

The natural ¹³C abundance (δ ¹³C) of plant leaves collected from fields in Thailand and the Philippines (Asian Monsoon tropics) was analyzed, and changes in the δ ¹³C values of C₃ and C₄ plants in wet and dry seasons were characterized. In Thailand, the δ ¹³C values of C₃ plants were ?29.2?±?1.04 (mean?±?standard deviation) ‰ in July and August (wet season) and ?28.6?±?1.05‰ in February and March (dry season): these values are not significantly different, whereas the values of C₄ plants were ?12.7?±?0.56‰ in the wet season and ?14.5?±?0.68‰ in the dry season (P?<?0.01, t-test). In the Philippines, where plants were collected only in October (late wet season), the δ ¹³C values of C₃ plants were ?29.5?±?1.28‰, whereas those of C₄ plants were ?12.6?±?1.11‰. These results suggest that under an Asian Monsoon climate, C₄ plants exhibit more negative δ ¹³C values in the dry season than in the wet season, whereas C₃ plants as a whole show no clear seasonal changes in δ ¹³C values.