Use of the 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials

Biological nitrogen fixation (BNF) associated with trees and shrubs plays a major role in the functioning of many ecosystems, from natural woodlands to plantations and agroforestry systems, but it is surprisingly difficult to quantify the amounts of N₂ fixed. Some of the problems involved in measuring N₂ fixation by woody perennials include: (a) diversity in occurrence, and large plant-to-plant variation in growth and nodulation status of N₂-fixing species, especially in natural ecosystems; (b) long-term, perennial nature of growth and the seasonal or year-to-year changes in patterns of N assimilation; and (c) logistical limitations of working with mature trees which are generally impossible to harvest in their entirety. The methodology which holds most promise to quantify the contributions of N₂ fixation to trees is the so-called `¹⁵N natural abundance' technique which exploits naturally occurring differences in ¹⁵N composition between plant-available N sources in the soil and that of atmospheric N₂. In this review we discuss probable explanations for the origin of the small differences in ¹⁵N abundance found in different N pools in both natural and man-made ecosystems and utilise previously published information and unpublished data to examine the potential advantages and limitations inherent in the application of the technique to study N₂ fixation by woody perennials. Calculation of the proportion of the plant N derived from atmospheric N₂ (%Ndfa) using the natural abundance procedure requires that both the ¹⁵N natural abundance of the N derived from BNF and that derived from the soil by the target N₂-fixing species be determined. It is then assumed that the ¹⁵N abundance of the N₂-fixing species reflects the relative contributions of the N derived from these two sources. The ¹⁵N abundance of the N derived from BNF (B) can vary with micro-symbiont, plant species/provenance and growth stage, all of which create considerable difficulties for its precise evaluation. If the%Ndfa is large and the ¹⁵N abundance of the N acquired from other sources is not several ¹⁵N units higher or lower than B, then this can be a major source of error. Further difficulties can arise in determining the ¹⁵N abundance of the N derived from soil (and plant litter, etc.) by the target plant as it is usually impossible to predict which, if any, non-N₂-fixing reference species will obtain N from the same N sources in the same proportions with the same temporal and spatial patterns as the N₂-fixing perennial. The compromise solution is to evaluate the ¹⁵N abundance of a diverse range of neighbouring non-N₂-fixing plants and to compare these values with that of the N₂-fixing species and the estimate of B. Only then can it be determined whether the contribution of BNF to the target species can be quantified with any degree of confidence. This review of the literature suggests that while the natural abundance technique appears to provide quantitative measures of BNF in tree plantation and agroforestry systems, particular difficulties may arise which can often limit its application in natural ecosystems.     

Alternatives for nitrogen nutrition of crops in tropical agriculture

The development of sustainable agricultural systems for the tropics requires among other technologies, alternatives for nitrogen fertilizers which are often limited in availability for financial reasons and also represent a major source of groundwater and air pollution. There are many new alternatives for the development of agricultural systems which make use of biological processes in soil. Biological nitrogen fixation (BNF), that is, the biological conversion of atmospheric dinitrogen into mineral N, is the most important alternative among them. Examples are given of the impact of various technologies used in Brazil. Soybean, introduced into the country 30 years ago, is now the second most important export crop, reaching 24 million tons annually with no N fertilizer application. Consequently, Brazil today is the country in the world which uses the lowest amounts of nitrogen fertilizers in relation to phosphate. Alternatives for crop rotations and pastures are also discussed. Possibilities of expanding BNF to cereals and other non-legume crops are gaining new credibility due to the identification of endophytic associations with diazotropic bacteria. The definite proof of substantial BNF in sugar cane with N balance and¹⁵N methods in certain genotypes selected under low N fertilizer applications opens up new alternatives for sustainable agriculture and will be the key to viable bio-fuel programmes.     

Towards a Revised Coefficient for Estimating N2O Emissions from Legumes

The Intergovernmental Panel on Climate Change (IPCC) standard methodology to conduct national inventories of soil N₂O emissions is based on default (or Tier I) emission factors for various sources. The objective of our study was to summarize recent N₂O flux data from agricultural legume crops to assess the emission factor associated with rhizobial nitrogen fixation. Average N₂O emissions from legumes are 1.0 kg N ha⁻¹ for annual crops, 1.8 kg N ha⁻¹ for pure forage crops and 0.4 kg N ha⁻¹ for grass legume mixes. These values are only slightly greater than background emissions from agricultural crops and are much lower that those predicted using 1996 IPCC methodology. These field flux measurements and other process-level studies offer little support for the use of an emission factor for biological N fixation (BNF) by legume crops equal to that for fertiliser N. We conclude that much of the increase in soil N₂O emissions in legume crops may be attributable to the N release from root exudates during the growing season and from decomposition of crop residues after harvest, rather than from BNF per se. Consequently, we propose that the biological fixation process itself be removed from the IPCC N₂O inventory methodology, and that N₂O emissions induced by the growth of legume crops be estimated solely as a function of crop residue decomposition using an estimate of above- and below-ground residue inputs, modified as necessary to reflect recent findings on N allocation.     

Nitrogen fixation by trees in relation to soil nitrogen economy

The N₂-fixing potential (NFP) (i.e. the amount of fixed N₂ in a constraint-free environment) of N₂-fixing trees (NFTs) varies with the genotype. The NFP can be higher than 30-50 g N₂ fixed tree^–1 year^–1 in the most active species, be they leguminous trees such asAlbizia lebbeck, Gliricidia sepium andLeucaena leucocephala, or actinorhizal trees such asCasuarina equisetifolia. The actual amount of nitrogen fixed (ANF) (i.e. the amount of N₂ fixed in the field) is lower than the NFP or even nil because of various constraints, especially drought, nutrient deficiencies, excess of available N and pathogenic nematodes. As tree litters are mineralized, the amount of available N in the soil increases with time, this process leading to the cessation of N₂ fixation in aging plantations. When the mineralization rate is slowed down or inhibited, N₂ fixation can continue. NFTs improve the N status of soils, but the transfer of fixed N to associated plants is not always ensured. Three main approaches are appropriate to increase N₂ fixation: clonal selection of trees combined with vegetative propagation, inoculation with effective rhizobium orFrankia strains, and proper fertilization (especially P). In the absence of major environmental constraints, a positive response to inoculation is expected only when specific (non-promiscuous) NFTs are grown in sites where the density of compatible rhizobia is low or nil. The potentialities of NFTs are far from being fully exploited. Further investigations are proposed and the economics of NFT management is briefly discussed.     

Biological N2-fixation and its management in wetland rice cultivation

The review summarizes the current status of the utilization of N₂-fixing organisms as biofertilizer in rice cultivation. Heterotrophic bacteria, free-living cyanobacteria,Azolla, and legume green manures are considered with regard to their potential for increasing yield, their current use and the prospects for their use with regard to the identified limiting factors.Biological N₂ fixation has been the most effective system for sustaining production in low-input traditional rice cultivation. On the other hand, the utilisation of N₂-fixing organisms in intensified rice production encounters serious limitations. The utilization of free-living bacteria and cyanobacteria is refrained by their modest potential and the non establishment of inoculated strains.Azolla and legumes used as green manures have a high potential as N source, but their utilization is severely limited by socio-economic factors.     

Transfer of Nitrogen Fixation (nif) Genes to Non-diazotrophic Hosts

Nitrogen is one of the most important nutrients for plant growth. To enhance crop productivity, chemical nitrogen fertilizer is commonly applied in agriculture. Biological nitrogen fixation, the conversion of atmospheric N₂ to NH₃, is an important source of nitrogen input in agriculture and represents a promising substitute for chemical nitrogen fertilizers. However, nitrogen fixation is only sporadically distributed within bacteria and archaea (diazotrophs). Thus, many biologists hope to reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host, with the final aim of developing N₂-fixing cereal crops. With the advent of synthetic biology and a deep understanding of the fundamental genetic determinants necessary to sustain nitrogen fixation in bacteria, much progress has been made toward this goal. Transfer of native and refactored nif (nitrogen fixation) genes to non-diazotrophs has been attempted in model bacteria, yeast, and plants. Specifically, nif genes from Klebsiella oxytoca, Azotobacter vinelandii, and Paenibacillus polymyxa have been successfully transferred and expressed in Escherichia coli, Saccharomyces cerevisiae, and even in the tobacco plant. These advances have laid the groundwork to enable cereal crops to “fix” nitrogen themselves to sustain their growth and yield.     

Assessment of biological nitrogen fixation

The four commonly used methods for measuring biological nitrogen fixation (BNF) in plants are: the total nitrogen difference (TND) method, acetylene reduction assay (ARA) technique, xylem-solute (or ureide production) method and the use of¹⁵N labelled compounds.The TND method relies on a control non-N₂-fixing plant to estimate the amount of N absorbed by the fixing plant from soil. It is one of the simplest and least expensive methods, but works best under low soil N conditions. The ARA technique measures the rate of acetylene conversion to ethylene by the N₂-fixing enzyme, nitrogenase. The ethylene produced can then be converted into N₂ fixed, using a conversion ratio, originally recommended as 3. Although the method is inexpensive and highly sensitive, its major disadvantages are, the short-term nature of the assays, the doubtful validity of always using a conversion ratio of 3 and the auto-inhibition of acetylene conversion to ethylene. The ARA technique is therefore not a method of choice for measuring BNF.The xylem-solute technique can be used to measure BNF for those species that produce significant quantities of ureide as product of BNF. Although simple and relatively inexpensive, it is an instantaneous assay and also needs to be calibrated against a known method. The most serious limitation is, that only a small proportion of N₂-fixing plants examined are ureide exporters, and the method is therefore not widely applicable.The¹⁵N methods, classified into the isotope dilution and A-value methods, appear to be the most accurate, but also the most expensive. They involve labelling soil with¹⁵N fertilizer and using a non-N₂-fixing reference plant to measure the¹⁵N/¹⁴N ratio in the soil. The¹⁵N isotope dilution approach is both operationally and mathematically simpler than the A-value approach. To limit potential errors in the selection of reference crops, it is recommended to use¹⁵N labelled compounds or soil labelling methods that result in the slow release of¹⁵N or the slow decline of¹⁵N/¹⁴N ratio in the soil. Additionally, the use of several reference plants rather than a single one can improve the accuracy of the results.     

Unraveling Nitrogen Fixing Potential of Endophytic Diazotrophs of Different Saccharum Species for Sustainable Sugarcane Growth

Sugarcane (Saccharum officinarum L.) is one of the world’s highly significant commercial crops. The amounts of synthetic nitrogen (N₂) fertilizer required to grow the sugarcane plant at its initial growth stages are higher, which increases the production costs and adverse environmental consequences globally. To combat this issue, sustainable environmental and economic concerns among researchers are necessary. The endophytic diazotrophs can offer significant amounts of nitrogen to crops through the biological nitrogen fixation mediated nif gene. The nifH gene is the most extensively utilized molecular marker in nature for studying N₂ fixing microbiomes. The present research intended to determine the existence of novel endophytic diazotrophs through culturable and unculturable bacterial communities (EDBCs). The EDBCs of different tissues (root, stem, and leaf) of five sugarcane cultivars (Saccharum officinarum L. cv. Badila, S. barberi Jesw.cv Pansahi, S. robustum, S. spontaneum, and S. sinense Roxb.cv Uba) were isolated and molecularly characterized to evaluate N₂ fixation ability. The diversity of EDBCs was observed based on nifH gene Illumina MiSeq sequencing and a culturable approach. In this study, 319766 operational taxonomic units (OTUs) were identified from 15 samples. The minimum number of OTUs was recorded in leaf tissues of S. robustum and maximum reads in root tissues of S. spontaneum. These data were assessed to ascertain the structure, diversity, abundance, and relationship between the microbial community. A total of 40 bacterial families with 58 genera were detected in different sugarcane species. Bacterial communities exhibited substantially different alpha and beta diversity. In total, 16 out of 20 genera showed potent N₂-fixation in sugarcane and other crops. According to principal component analysis (PCA) and hierarchical clustering (Bray–Curtis dis) evaluation of OTUs, bacterial microbiomes associated with root tissues differed significantly from stem and leaf tissues of sugarcane. Significant differences often were observed in EDBCs among the sugarcane tissues. We tracked and validated the plethora of individual phylum strains and assessed their nitrogenase activity with a culture-dependent technique. The current work illustrated the significant and novel results of many uncharted endophytic microbial communities in different tissues of sugarcane species, which provides an experimental system to evaluate the biological nitrogen fixation (BNF) mechanism in sugarcane. The novel endophytic microbial communities with N₂-fixation ability play a remarkable and promising role in sustainable agriculture production.     

Estimation of N2 fixation in Desmodium ovalifolium from the relative ureide abundance of stem solutes: Comparison with the 15N-dilution and an in situ soil core technique

Many, but not all, legumes of tropical origin, transport fixed N from the nodules to the shoot tissue in the form of ureides, and the mineral N absorbed from the soil is principally transported in the form of nitrate. The analysis of stem xylem sap, or hot-water extracts of stem tissue, for ureide and nitrate has been used successfully to quantify BNF contributions to several grain legumes and more recently to some shrub and forage legumes. The objective of this study was to investigate the application of this technique to the quantification of the contribution of BNF to the forage legume Desmodium ovalifolium by comparing the relative ureide abundance (RUA) of stem extracts of this plant with simultaneous estimates of BNF obtained using the ¹⁵N isotope dilution technique. The first experiment was performed in pots of soil, taken from a grazing study, amended with ¹⁵N-labelled organic matter at four different application rates. The ureide concentration in the stem extracts reflected the changes in BNF activity during plant growth and the RUA was closely correlated with the proportion of N derived from BNF as determined from the ¹⁵N technique (r ² = 0.86 and 0.88 for inoculated and non-inoculated plants, respectively). The use of a calibration curve derived from a previous study where the same legume was fed increasing concentrations of ¹⁵N labelled nitrate in sand/vermiculite culture, resulted in an over-estimation of the BNF contribution which may have been due to a significant uptake of ammonium from this acidic soil. The second experiment was performed in field plots and a good agreement was found between the estimates of BNF derived from using the ureide and ¹⁵N dilution techniques at two harvests six months apart. The uptake of soil N by the D. ovalifoliumand two forage grasses (Brachiaria humidicola and Panicum maximum) was estimated using an in situ soil core technique, and, while the uptake of N by the grasses was successfully estimated, this technique underestimated the N derived from the soil by the legume as determined by the ureide and ¹⁵N dilution techniques.     

Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.     

How much nitrogen is fixed by biological symbiosis in tropical dry forests? 2. Herbs

Although biological nitrogen fixation (BNF) is considered the main input of N in mature and regenerating native tropical vegetation, it has seldom been quantified. Biomass and N accumulation and fixation were determined for spontaneously occurring herbaceous species in caatinga areas in four regeneration stages (2, 17, 39 and >50?years after abandonment from agricultural use). BNF was estimated using the ¹⁵N natural-abundance method. The 2-year regeneration area had the highest total herb (6,355?kg?ha^?1) and legume (262?kg?ha^?1) biomass production, N stocks (82?kg?ha^?1) and fixed N (5.0?kg?ha^?1). N₂-fixing legumes (nine species in the sampled area) contributed over 97?% of legume biomass in all areas. Macroptilium gracile added the largest amount of N (3.9?kg?ha^?1 in the 2-year regeneration area) because of its large biomass production (205?kg?ha^?1), although it was not the species with the highest proportion of fixed N (76?%). All of the N₂-fixing species obtained large proportions of their N from symbiosis, most of them more than 50?%.However, the amounts of fixed N per unit area were relatively low (0.22?C5.00?kg?ha^?1) because the biomass of N₂-fixing species was always less than 5?% of the total herb biomass.     

Short-term induction of alkaloid production in lupines Differences between N2-fixing and nitrogen-limited plants

We used N₂-fixing and nonfixing lupines to examine the effects of plant nutrition on short-term alkaloid production in damaged leaves. Three different treatments were used: damaged leaves from N₂-fixing plants; undamaged leaves from these damaged, N₂-fixing plants; and damaged leaves on nitrogen-limited, nonfixing plants. Relative to controls, alkaloids increased in concentration more quickly in the N₂-fixing than in the nitrogen-limited plants. The magnitude of this increase in alkaloids was correlated with the initial alkaloid concentration. These results suggest that nitrogen-rich plants may benefit from faster and higher alkaloid induction than nitrogen-limited plants. In addition, the detailed dynamics of individual alkaloids are consistent with earlier proposals for the mechanism of lupine alkaloid induction.     

Nitrogen fixation of red clover interseeded with winter cereals across a management-induced fertility gradient

The incorporation of legume cover crops into annual grain rotations remains limited, despite extensive evidence that they can reduce negative environmental impacts of agroecosystems while maintaining crop yields. Diversified grain rotations that include a winter cereal have a unique niche for interseeding cover crops. To understand how management-driven soil fertility differences and inter-seeding with grains influenced red clover (Trifolium pratense) N₂ fixation, we estimated biological N₂ fixation (BNF) in 2006 and 2007, using the ¹⁵N natural abundance method across 15 farm fields characterized based on the reliance on BNF derived N inputs as a fraction of total N inputs. Plant treatments included winter grain with and without interseeded red clover, monoculture clover, monoculture orchardgrass (Dactylis glomerata), and clover-orchardgrass mixtures. Fields with a history of legume-based management had larger labile soil nitrogen pools and lower soil P levels. Orchardgrass biomass was positively correlated with the management-induced N fertility gradient, but we did not detect any relationship between soil N availability and clover N₂ fixation. Interseeding clover with a winter cereal did not alter winter grain yield, however, clover production was lower during the establishment year when interseeded with taller winter grain varieties, most likely due to competition for light. Interseeding clover increased the % N from fixation relative to the monoculture clover (72% vs. 63%, respectively) and the average total N₂ fixed at the end of the first growing season (57 vs. 47 kg N ha⁻¹, respectively). Similar principles could be applied to develop more cash crop-cover crop complementary pairings that provide both an annual grain harvest and legume cover crop benefits.     

Symbiotic dinitrogen fixation by trees: an underestimated resource in agroforestry systems?

We compiled quantitative estimates on symbiotic N₂ fixation by trees in agroforestry systems (AFS) in order to evaluate the critical environmental and management factors that affect the benefit from N₂ fixation to system N economy. The so-called ??N₂-fixing tree?? is a tripartite symbiotic system composed of the plant, N₂-fixing bacteria, and mycorrhizae-forming fungi. Almost 100 recognised rhizobial species associated with legumes do not form an evolutionary homologous clade and are functionally diverse. The global bacterial diversity is still unknown. Actinorrhizal symbioses in AFS remain almost unstudied. Dinitrogen fixation in AFS should be quantified using N isotopic methods or long-term system N balances. The general average?±?standard deviation of tree dependency on N₂ fixation (%Ndfa) in 38 cases using N isotopic analyses was 59?±?16.6?%. Under humid and sub-humid conditions, the percentage was higher in young (69?±?10.7?%) and periodically pruned trees (63?±?11.8?%) than in free-growing trees (54?±?11.7?%). High variability was observed in drylands (range 10?C84?%) indicating need for careful species and provenance selection in these areas. Annual N₂ fixation was the highest in improved fallow and protein bank systems, 300?C650?kg?[N]?ha^?1. General average for 16 very variable AFS was 246?kg?[N]?ha^?1, which is enough for fulfilling crop N needs for sustained or increasing yield in low-input agriculture and reducing N-fertiliser use in large-scale agribusiness. Leaf litter and green mulch applications release N slowly to the soil and mostly benefit the crop through long-term soil improvement. Root and nodule turnover and N rhizodeposition from N₂-fixing trees are sources of easily available N for the crop yet they have been largely ignored in agroforestry research. There is also increasing evidence on direct N transfer from N₂-fixing trees to crops, e.g. via common mycelial networks of mycorrhizal fungi or absorption of tree root exudates by the crop. Research on the below-ground tree-crop-microbia interactions is needed for fully understanding and managing N₂ fixation in AFS.     

Field application of the15N isotope dilution technique for the reliable quantification of plant-associated biological nitrogen fixation

To apply the isotope dilution (ID) technique, it is necessary to grow the N₂-fixing crop in a soil where the mineral N is labelled with¹⁵N. Normally the N₂-fixing crop and a suitable non-N₂-fixing control crop are grown in the same labelled soil and the¹⁵N enrichment of the control crop is assumed to be equal to the¹⁵N enrichment of the nitrogen (N) derived from the soil in the N₂-fixing crop. In this case the proportion of unlabelled N being derived from the air via biological N₂ fixation (BNF) in the N₂-fixing crop will be proportional to the dilution of the enrichment of the N derived from the labelled soil.To label the soil, the technique most often used is to add a single addition of¹⁵N-labelled N fertilizer shortly before, at, or shortly after, the planting of the crops. Data in the literature clearly show that this technique results in a rapid fall in the¹⁵N enrichment of soil mineral N with time. Under these conditions, if the control and the N₂-fixing crops have different patterns of N uptake from the soil they will inevitably obtain different¹⁵N enrichments in the soil-derived N. In this case the isotope dilution technique cannot be applied, or if it is, there will be an error introduced into, the estimate of the contribution of N derived from BNF.Several experiments are described which explore different strategies of application of the ID technique to attempt to attenuate the errors involved. The results suggest that it is wise to use slow-release forms of labelled N, or in some cases, multiple additions, to diminish temporal changes in the¹⁵N enrichment of soil mineral N. The use of several control crops produces a range of different estimates of the BNF contributions to the N₂-fixing crops, and the extent of this range gives a measure of the accuracy of the estimates. Likewise the use of more than one¹⁵N enrichment technique in the same experiment will also give a range of estimates which can be treated similarly. The potential of other techniques, such as sequential harvesting of both control and test crops, are also discussed.     

Estimation of the contribution of biological N2 fixation to twoPhaseolus vulgaris genotypes using simulation of plant nitrogen uptake from15N-labelled soil

A technique for the application of the¹⁵N isotope dilution technique for the quantification of plant associated biological nitrogen fixation (BNF) was tested and applied to quantify the BNF contribution to two genotypes ofPhaseolus vulgaris. The technique makes use of sequential measurements of the¹⁵N enrichment of soil mineral N, and the uptake of labelled N by the N₂-fixing plant, to simulate its uptake of soil N (the soil to plant simulation technique). The test was made with two non-N₂-fixing crops (non-nodulating beans and wheat) and two bean genotypes (PR 923450 and Puebla 152), at two levels of N fertilizer addition (10 and 40 kg N ha^–1), to compare the actual N uptake with that simulated from the soil and crop¹⁵N data. The simulation of the soil N uptake by the non-nod bean crop using this soil to plant simulation technique underestimated by 20 to 30% the true N uptake, suggesting that the mineral N extracted from soil samples taken from the 0–15cm layer had a higher¹⁵N enrichment than that N sampled by the roots of this crop. In the case of the wheat crop the simulation resulted in a much greater underestimation of actual N uptake. In general the results using this technique suggested that BNF inputs to the bean cultivars was higher than would be expected from the nodulation and acetylene reduction data, except for the early PR beans in the 40 kg N ha^–1 treatment. In this case the total N and simulated soil N accumulation were well matched suggesting no BNF inputs. An allied technique (the plant to plant simulation technique) was proposed where the¹⁵N enrichrnent of soil mineral N was simulated from the data for total N and labelled N accumulation taken from sequential harvests of either of the non-N₂ -fixing control crops. This was then utilized in combination with the labelled N uptake data of the other crop to simulate its soil N uptake. However, the results using either technique indicated that the wheat and non-nod or nodulating beans exploited pools of N in the soil with completely different¹⁵N enrichments probably due to differences in exploitation of the soil N with depth.     

The role of <Superscript>15</Superscript>N-depleted fertilizers as tracers in N cycling studies in agroecosystems

A search of the literature (principally Google Scholar, using the keywords given below) revealed that 120 research papers have been published where ¹⁵N-depleted (¹⁴N-enriched) fertilizers, including ammonium sulfate, ammonium nitrate, urea and ammonia forms were used as tracers. The studies included annual cereals, vegetables, a grain legume and a fibre crop, and perennial crops including grasses, shrubs (berries), trees (fruits and nuts) and N₂-fixing forage, grain and woody legumes and an actinorhizal species. Other minor examples include perennial ornamentals and trees harvested for wood. Applications included estimation of fertilizer recovery in crops, in post-harvest soil and estimation of N losses in the soil–plant system by mass balance. The residual value of ¹⁵N-depleted fertilizers and the recovery of ¹⁵N-depleted legume residues have also been reported. ¹⁵N depleted fertilizers have played an important role in differentiating the relative uptake of fertilizer N to N mobilized in storage tissues with respect to the N nutrition of deciduous horticultural trees, shrubs and vines in spring. The symbiotic dependence of N₂-fixing species and transfer to companion non-legumes has been indirectly determined using ¹⁵N-depleted fertilizers. Thus ¹⁵N-depleted fertilizers have shared the same applications as their ¹⁵N-enriched counterparts. Because of their relative cheapness compared with ¹⁵N-enriched fertilizers, larger and more representative unconfined plots can be employed in field studies.     

The Oxidative Fermentation of Ethanol in Gluconacetobacter diazotrophicus Is a Two-Step Pathway Catalyzed by a Single Enzyme: Alcohol-Aldehyde Dehydrogenase (ADHa)

Gluconacetobacter diazotrophicus is a N₂-fixing bacterium endophyte from sugar cane. The oxidation of ethanol to acetic acid of this organism takes place in the periplasmic space, and this reaction is catalyzed by two membrane-bound enzymes complexes: the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH). We present strong evidence showing that the well-known membrane-bound Alcohol dehydrogenase (ADHa) of Ga. diazotrophicus is indeed a double function enzyme, which is able to use primary alcohols (C2–C6) and its respective aldehydes as alternate substrates. Moreover, the enzyme utilizes ethanol as a substrate in a reaction mechanism where this is subjected to a two-step oxidation process to produce acetic acid without releasing the acetaldehyde intermediary to the media. Moreover, we propose a mechanism that, under physiological conditions, might permit a massive conversion of ethanol to acetic acid, as usually occurs in the acetic acid bacteria, but without the transient accumulation of the highly toxic acetaldehyde.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

Effects of decomposing rice straw on growth of and nitrogen fixation byRhizobium

Five phenolic compounds produced by decomposing rice straw and sterile extracts of decomposing rice straw in soil were very inhibitory to growth of three strains ofRhizobium. The effects were additive and in several instances synergistic. The phenolic compounds also reduced nodule numbers and hemoglobin content of the nodules in two bean (Phaseolus vulgaris) varieties. Extracts of decomposing rice straw in soil (same concentration as in the soil) significantly reduced N₂ fixation (acetylene reduction) in Bush Black Seeded beans. This may explain in part the great reduction in soybean yields in Taiwan following rice crops when the rice stubble is left in the field.