Effects of food quality, starvation and life stage on stable isotope fractionation in Collembola

Naturally occurring stable isotopes of carbon and nitrogen are powerful tools to investigate food webs, where the ratio of ¹⁵N/¹⁴N is used to assign trophic levels and of ¹³C/¹²C to determine the food source. A shift in δ¹⁵N value of 3‰ is generally suggested as mean difference between two trophic levels, whereas the carbon isotope composition of a consumer is assumed to reflect the signal of its diet. This study investigates the effects of food quality, starvation and life stage on the stable isotope fractionation in fungal feeding Collembola. The fractionation of nitrogen was strongly affected by food quality, i.e. the C/N ratio of the fungal diet. Collembola showed enrichment in the heavier isotope with increasing N concentration of the food source. Δ¹⁵N varied between 2.4‰, which assigns a shift in one trophic level, and 6.3‰, suggesting a shift in two trophic levels. Starvation up to 4 weeks resulted in an increase in the total δ¹⁵N value from 2.8‰ to 4.0‰. Different life stages significantly affected the isotope discrimination by Collembola with juveniles showing a stronger enrichment (Δ¹⁵N=4.9‰) compared to adults (Δ¹⁵N=3.5‰). Δ¹³C varied between −2.1‰ and −3.3‰ depending on the food quality, mainly due to compensational feeding on low quality diet. During starvation δ¹³C value decreased by 1.1‰, whereas the life stage of Collembola had no significant effect on isotopic ratios. The results indicate that the food resource and the physiological status of the consumer have important impact on stable isotope discrimination. They may cause differences in fractionation rate comparable to trophic level shifts, a fact to consider when analysing food web structure.     

Trophic shift of stable isotopes and fatty acids in Collembola on bacterial diets

Fatty acid (FA) analysis is a promising tool to study trophic relationships in soil food webs. We determined FA biomarkers to trace bacterial food sources (Bacillus megaterium, Pseudomonas putida, Enterobacter aerogenes) of Collembola (Heteromurus nitidus, Protaphorura fimata, Folsomia candida). In addition, δ¹⁵N, δ¹³C, C/N ratio, body weight and NLFA/PLFA ratio (neutral lipid/phospholipid fatty acids) of Collembola were assessed. These measures indicated that P. putida ranked first, B. megaterium second and E. aerogenes third in food quality. FAs specific for bacteria were found in the NLFAs of the Collembola reflecting the respective bacterial diet. Biomarker FAs for gram-positive bacteria were methyl branched i14:0, i15:0, a15:0 and i17:0. Consumption of gram-negative bacteria was reflected by the cyclic form cy17:0 (E. aerogenes, P. putida) and by 16:1ω5 (P. putida).     

Compartmentalization of the soil animal food web as indicated by dual analysis of stable isotope ratios (N/N and C/C)

The soil animal food web has become a focus of recent ecological research but trophic relationships still remain enigmatic for many taxa. Analysis of stable isotope ratios of N and C provides a powerful tool for disentangling food web structure. In this study, animals, roots, soil and litter material from a temperate deciduous forest were analysed. The combined measurement of δ¹⁵N and δ¹³C provided insights into the compartmentalization of the soil animal food web. Leaf litter feeders were separated from animals relying mainly on recent belowground carbon resources and from animals feeding on older carbon. The trophic pathway of leaf litter-feeding species appears to be a dead end, presumably because leaf litter feeders (mainly diplopods and oribatid mites) are unavailable to predators due to large size and/or strong sclerotization. Endogeic earthworms that rely on older carbon also appear to exist in predator-free space. The data suggest that the largest trophic compartment constitutes of ectomycorrhizal feeders and their predators. Additionally, there is a smaller trophic compartment consisting of predators likely feeding on enchytraeids and potentially nematodes.     

Stable isotopes revisited: Their use and limits for oribatid mite trophic ecology

In this review we summarize our knowledge of using stable isotopes (¹⁵N/¹⁴N, ¹³C/¹²C) to better understand the trophic ecology of oribatid mites. Our aims are (a) to recapitulate the history of stable isotope research in soil animals with a focus on oribatid mites, (b) to present new stable isotope data for oribatid mites and overview the current state of knowledge of oribatid mite trophic niche differentiation, (c) to compile problems and limitations of stable isotope based analyses of trophic relationships and (d) to suggest future challenges, questions and problems that may be solved using stable isotope analyses and other novel techniques for improving our understanding on the trophic ecology of soil invertebrates. We conclude that (1) in addition to ¹⁵N/¹⁴N ratios, ¹³C/¹²C ratios contribute to our understanding of the trophic ecology of oribatid mites, allowing, e.g. separation of lichen- and moss-feeding species, (2) there likely are many lichen but few moss feeding oribatid mite species, (3) oribatid mite species that are endophagous as juveniles are separated by their stable isotope signatures from all other oribatid mite species, (4) fungivorous oribatid mite species cannot be separated further, e.g. the fungal taxa they feed on cannot be delineated. A particular problem in using stable isotope data is the difficulty in determining signatures for basal food resources, since decomposing material, fungi and lichens comprise various components differing in stable isotope signatures; ¹³C/¹²C ratios and potentially other isotopes may help in identifying the role of these resources for decomposer animal nutrition.     

Trophic niche differentiation in soil microarthropods (Oribatida, Acari): evidence from stable isotope ratios (N/N)

The large number of animals that coexist in soil without any clear niche differentiation has puzzled biologists for a long time. We investigated stable isotope ratios (¹⁵N/¹⁴N) in a diverse group of soil microarthropods, oribatid mites, to evaluate trophic niche differentiation. The natural variation of the stable isotopes ¹⁵N/¹⁴N was measured in 36 species/taxa from four beech and beech-oak forests. Signatures of δ¹⁵N formed a gradient spanning over 12 δ units suggesting that (a) different species occupy different trophic niches and (b) oribatid mites span three to four trophic levels. This study for the first time documented strong trophic niche differentiation in decomposer microarthropods. The results suggest that trophic niche differentiation within taxonomic groups significantly contributes to the high diversity of soil animal taxa.     

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.     

Origin of the nitrogen assimilated by soil fauna living in decomposing beech litter

We investigated the nitrogen source for main taxa of soil fauna in two beech forests of contrasted humus type using ¹⁵N-labelled beech litter and ¹⁵N analysis of soil fauna. ¹⁵N-labelled beech litter was deposited on the topsoil in December 2000 in four stands of different ages at Leinefelde (Germany) with mull humus and in one mature stand at Sorø (Denmark) with moder humus. The fate of the tracer isotope was measured in litter and soil, as well as in the soil fauna, and for each taxa, we calculated the proportion of N in the animal derived from the labelled substrate. Of the original N contained in the litter, 20-41% was lost after 9 months at Leinefelde, and only 10% at Sorø. This loss was counterbalanced by the incorporation of 24-31% external N at Leinefelde, and 31% at Sorø, partly originating from fungal colonisation of the added litter. The proportion of N assimilated from the labelled litter by the different soil animals varied in relation to their mobility and feeding preferences. Large and mobile soil animals, especially predators, derived on average less ¹⁵N because they were also able to feed outside the labelled litter boxes. Detritivores assimilated at most 15% of their nitrogen content at Leinefelde and 11% at Sorø from the decomposing labelled litter. The most labelled taxa at Leinefelde were small fungivorous and coprophagous species, mainly isotomid Collembola such as Isotomiella and Folsomia. At Sorø, best labelled taxa were saprophagous species such as Enchytraeidae, Glomeridae and Phthiracaroidea. These low rates of ¹⁵N assimilation indicate that fresh litter is not directly the main N source for soil animals. The results obtained suggest that soil fauna fed preferentially upon microorganisms colonising the litter at Leinefelde (mull) and from litter itself at Sorø (moder).     

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.     

The feeding ecology of elaterid larvae in central European arable land: New perspectives based on naturally occurring stable isotopes

To understand soil food webs, empirically generated data on the trophic connections and the feeding ecology of the major below-ground animal taxa are needed. Here we used stable isotope analysis to assess the trophic ecology of wireworms, the larvae of click beetles, in Central European arable land. Wireworms are amongst the major soil macroinvertebrates and are of practical importance in arable soils. Besides feeding on crops, they are thought to feed on weeds, soil organic matter (SOM), and even animal prey, but their feeding ecology is poorly studied under natural conditions. Elaterid larvae and their putative feeding substrates—plant roots, SOM, and litter—were sampled at 17 locations in Austria, Germany, and Italy and their isotope ratios of carbon (¹²C/¹³C) and nitrogen (¹⁴N/¹⁵N) measured to determine the wireworms’ trophic level, the importance of SOM and weeds within the diet of Agriotes larvae, as well as the individual diet variation in Agriotes obscurus larvae. δ¹⁵N signatures suggested that Agriotes larvae are predominately herbivorous, whereas the other wireworm species primarily fed on animal prey. In contrast to SOM, weeds were readily eaten by Agriotes larvae: their dietary contribution ranged between 28% and 67% in weedy maize fields. Most A. obscurus larvae fed on a mixed diet of weeds and maize, although ～15% of the larvae fed primarily on one of the two food sources only. δ¹⁵N signatures indicated that ～10% of the “herbivorous” A. obscurus larvae fed primarily on animal prey, revealing high intraspecific trophic plasticity in these soil insects. Wireworm feeding behaviour is apparently complex at the individual level: the population consists of types A and B generalists, a phenomenon which needs further assessment.     

Evaluating N/N and C/C isotope ratio analysis to investigate trophic relationships of elaterid larvae (Coleoptera: Elateridae)

Carbon and nitrogen isotope ratios in consumer tissues can be used to analyse the diet and trophic level of soil animals. However, life history traits may significantly influence stable isotope patterns. We evaluated in a series of experiments how stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) at natural abundance can be used to study the diet and trophic position of long-lived macro-invertebrates, elaterid larvae, which are major below-ground herbivores. Small, but significant differences in δ¹³C signatures were found between the larvaes’ anterior and posterior body segments, whereas exuvia reflected the body's overall isotopic composition. The species-specific trophic shift (±SE) in δ¹⁵N for Agriotes obscurus and Agriotes sputator (1.62±0.24‰ and 1.08±0.27‰, respectively) was significantly lower than “mean enrichment estimates” reported in the literature, showing the limited applicability of such generalised estimates in studies of invertebrate trophic ecology. To avoid false-positive assignments to two trophic levels due to variation in δ¹⁵N values, a minimum sample size of three and five individuals for A. obscurus and A. sputator, respectively, was needed to reduce this risk to below α=5%. Keeping elaterid larvae for up to 128 days without food did not affect their isotopic signatures, in contrast to previous studies on starving animals. Switching wireworms to isotopically different diets induced changes in their isotopic signatures within 2 weeks. Changes, however, were significant only when the isotopic difference between diets was large. We conclude that experimental studies evaluating how specific life history traits affect stable isotope signatures in consumers have to precede any interpretation of stable isotope data gathered in the field.     

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.     

Plant nitrate use in deciduous woodland: the relationship between leaf N, N natural abundance of forbs and soil N mineralisation

Our aim was to study whether the in situ natural abundance ¹⁵N (δ¹⁵N)-values and N concentration of understory plants were correlated with the form and amount of mineral N available in the soil. Also to determine whether such differences were related to earlier demonstrations of differences in biomass increase in the same species exposed to nutrient solutions with both and or to alone. Several studies show that the δ¹⁵N of in soil solution generally is isotopically lighter than the δ¹⁵N of due to fractionation during nitrification. Hence, it is reasonable to assume that plant species benefiting from in ecosystems without significant leaching or denitrification have lower δ¹⁵N-values in their tissues than species growing equally well, or better, on We studied the δ¹⁵N of six understory species in oak woodlands in southern Sweden at 12 sites which varied fivefold in potential net N mineralisation rate The species decreased in benefit from in the following order: Geum urbanum, Aegopodium podagraria, Milium effusum, Convallaria majalis, Deschampsia flexuosa and Poa nemoralis. Four or five species demonstrated a negative correlation between and leaf δ¹⁵N and a positive correlation between and leaf N concentration. In wide contrast, only D. flexuosa, which grows on soils with little nitrification, showed a positive correlation between and the leaf N concentration and δ¹⁵N-value. Furthermore, δ¹⁵N of plants from the field and previously obtained indices of hydroponic growth on relative to were closely correlated at the species level. We conclude that δ¹⁵N may serve as a comparative index of uptake of among understory species, preferably in combination with other indices of N availability. The use of δ¹⁵N needs careful consideration of known restrictions of method, soils and plants.     

Dissipation of bacterially derived C and N through the meso- and macrofauna of a grassland soil

Feeding relationships between organisms may be determined by observations of behaviour in manipulative experiments or, as in more recent times, by the use of stable isotope labelling to trace the passage of ¹³C and ¹⁵N through food webs. Here we introduce living bacteria, labelled with both ¹³C and ¹⁵N into intact soil cores to understand further the movement of bacterially sourced C and N into the meso- and macrofauna of a grassland soil. We found that these groups showed a range of isotope levels which relate to their feeding strategies. Some had no label (e.g. dipterous larvae), whilst others were highly labelled which may indicate a preference for the added bacteria. This latter group included Collembola, generally perceived as being predominantly fungal feeders. This work describes a novel technique which has the potential to provide critical information about the dissipation of bacterially derived C and N through the soil food web.     

Natural N abundances in a tallgrass prairie ecosystem exposed to 8-y of elevated atmospheric CO2

After 8-y of elevated CO₂, we previously detected greater amounts of total soil nitrogen, suggesting that rates of ecosystem N flux into or out of tallgrass prairie had been altered. Denitrification and associative N fixation rates are the two primary biological processes that are known to control N loss and accumulation in tallgrass prairie soil. Therefore, our objective was to assess the natural abundance of plant and soil ¹⁵N isotopes as a cumulative index of potential change in efflux or influx of N into and out of the tallgrass prairie after 8-y of exposure to elevated CO₂. Aboveground plant delta ¹⁵N values of Andropogon gerardii were close to zero and more positive as a result of elevated CO₂, but whole-soil values at the 5-30 cm depth were significantly reduced (6.8 vs 7.3; P<0.05) under elevated CO₂-chamber (EC) relative to ambient CO₂- chamber (AC). Total, aboveground plant biomass, root-in-growth, extractable N, microbial biomass N, and soil pools collectively exhibited a range of delta ¹⁵N values from −2.8 to 7.3. Measurements of surface soil ¹⁵N indicate that a change in N inputs and outputs has occurred as a result of elevated atmospheric CO₂. In addition to possible changes in denitrification and N₂ fixation, other sources of N such as the re-translocation of N to the surface from deeper soil layers are needed to explain how soil N accrues in surface soils as a consequence of elevated CO₂. Our results support the notion that C accrual may promote N accrual, possibly driven by high plant and microbial N demand amplified by soil N limitation.     

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.     

Savanna-derived organic matter remaining in arable soils of the South African Highveld long-term mixed cropping: Evidence from C and N natural abundance

Sustainable agriculture requires the formation of new humus from the crops. We utilized ¹³C and ¹⁵N signatures of soil organic matter to assess how rapidly wheat/maize cropping contributed to the humus formation in coarse-textured savanna soils of the South African Highveld. Composite samples were taken from the top 20 cm of soils (Plinthustalfs) cropped for lengths of time varying from 0 to 98 years, after conversion from native grassland savanna (C4). We performed natural ¹³C and ¹⁵N abundance measurements on bulk and particle-size fractions. The bulk soil δ¹³C values steadily decreased from −14.6 in (C4 dominated) grassland to −16.5‰ after 90 years of arable cropping. This δ¹³C shift was attributable to increasing replacement of savanna-derived C by wheat crop (C3) C which dominated over maize (C4) inputs. After calculating the annual C input from the crop yields and the output from literature data, by using a stepwise C replacement model, we were able to correct the soil δ¹³C data for the irregular maize inputs for a period of about one century. Within 90 years of cropping 41-89% of the remaining soil organic matter was crop-derived in the three studied agroecosystems. The surface soil C stocks after 90 years of the wheat/maize crop rotation could accurately be described with the Rothamsted Carbon Model, but modelled C inputs to the soil were very low. The coarse sand fraction reflected temporal fluctuations in ¹³C of the last C₃ or C₄ cropping and the silt fraction evidenced selective erosion loss of old savanna-derived C. Bulk soil ¹⁵N did not change with increasing cropping length. Decreasing δ¹⁵N values caused by fertilizer N inputs with prolonged arable cropping were only detected for the coarse sand fraction. This indicated that the present N fertilization was not retained in stable soil C pool. Clearly, conventional cropping practices on the South African highlands neither contribute to the preservation of old savanna C and N, nor the effective humus reformation by the crops.     

Quantification of the contribution of biological nitrogen fixation to Cratylia mollis using the N natural abundance technique in the semi-arid Caatinga region of Brazil

This study was performed to investigate the capacity of the woody perennial Cratylia mollis, a legume endemic to the semi-arid region of the North-East of Brazil, to nodulate, and obtain N from BNF using the ¹⁵N natural abundance technique. To estimate the ¹⁵N abundance of the N-derived from soil, the leaves of several (4-6) non-legume and non-nodulating legume species growing in close proximity to the Cratylia were analysed for δ¹⁵N. The δ¹⁵N values of these reference plants were high (from +9 to +16‰) and showed relatively small differences between species at each site/sampling time. At the irrigated site at both samplings, and at the non-irrigated site sampled in the rainy season, the ¹⁵N abundance of the Cratylia mollis leaves was far lower (+1 to +5‰) strongly suggesting that the legume obtained large proportional contributions from BNF. As was to be expected, no nodules were found on the Cratylia plants at the non-irrigated site in the dry season, and the ¹⁵N abundance of the Cratylia mollis plants were very similar to that of the reference plants, consistent and nodulation and BNF being limited by water deficit at this time.     

Natural 15N and 13C abundance in Andisols influenced by long-term fertilization management in Japan

Two field experiments were conducted on Andisols in Japan to evaluate the changes in the natural ¹⁵N and ¹³C abundance in the soil profile and to determine whether the values of δ¹⁵N could be used as an indicator of fertilizer sources or fertilizer fate. The 6-year experiment conducted at the National Agricultural Research Center (NARC) consisted of the following treatments: application of swine compost (COMPOST), slow-release nitrogen fertilizer (SRNF), readily available nitrogen fertilizer (RANF), and absence of fertilization (CONTROL). Experimental plots located at the Nippon Agricultural Research Institute (NARI) received cattle compost at different rates for 12 years; a forest soil at this site was sampled for comparison. Swine compost application led to a considerable change in the δ¹⁵N distribution pattern in the soil profile, with the highest δ¹⁵N values recorded in the top 20 cm layers of the COMPOST plot, decreasing in the sequence of CONTROL >- RANF > SRNF, mainly due to the relatively high δ¹⁵N value of swine compost and its subsequent decomposition. In contrast, SRNF application resulted in the lowest δ¹⁵N values in soil, indicating the presence of negligible nitrogen losses relative to input and low nitrogen cycling rates. Values of δ¹⁵N increased with compost application rates at NARI. In the leachate collected at 1-m depth, the δ¹⁵N values decreased in the sequence of COMPOST > RANF ≥ CONTROL > SRNF. The δ¹³C values in soil peaked in the 40–60 cm layers for all the fertilizers. The δ¹³C value was lowest in forest soil due to the presence of plant residues in soil organic matter. These results indicated that the δ¹⁵N values in the upper soil layers or leachate may enable to detect pollution sources of organic or inorganic nitrogen qualitatively in Andisols.     

Leaf litter C and N cycling from a deciduous permanent crop

Our understanding of leaf litter carbon (C) and nitrogen (N) cycling and its effects on N management of deciduous permanent crops is limited. In a 30-day laboratory incubation, we compared soil respiration and changes in mineral N [ammonium (NH₄⁺-N) + nitrate (NO₃^–-N)], microbial biomass nitrogen (MBN), total organic carbon (TOC) and total non-extractable organic nitrogen (TON) between a control soil at ¹⁵N natural abundance (δ¹⁵N = 1.08‰) without leaf litter and a treatment with the same soil, but with almond (Prunus dulcis (Mill.) D.A. Webb) leaf litter that was also enriched in ¹⁵N (δ¹⁵N = 213‰). Furthermore, a two-end member isotope mixing model was used to identify the source of N in mineral N, MBN and TON pools as either soil or leaf litter. Over 30 d, control and treatment TOC pools decreased while the TON pool increased for the treatment and decreased for the control. Greater soil respiration and significantly lower (p < 0.05) mineral N from 3 to 15 d and significantly greater MBN from 10 to 30 d were observed for the treatment compared to the control. After 30 d, soil-sourced mineral N was significantly greater for the treatment compared to the control. Combined mineral N and MBN pools derived from leaf litter followed a positive linear trend (R² = 0.75) at a rate of 1.39 μg N g^?1 soil day^?1. These results suggest early-stage decomposition of leaf litter leads to N immobilization followed by greater N mineralization during later stages of decomposition. Direct observations of leaf litter C and N cycling assists with quantifying soil N retention and availability in orchard N budgets.