Nitrous oxide emissions associated with nitrogen fixation by grain legumes

Nitrous oxide (N₂O) emissions and biological nitrogen (N₂) fixation by grain legumes are two major processes of N transformation in agroecosystems. However, the relationship between these two processes is not well understood. The objective of this study was to quantify N₂O emissions associated with N₂ fixation by grain legumes under controlled conditions. The denitrifying capability of two Rhizobium leguminosarum biovar viciae strains, 99A1 and RGP2, was tested in pure culture in the presence of nitrate and in symbiosis with lentil (Lens esculenta Moench) and pea (Pisum sativum L.), respectively, in sterile Leonard jars. Lentil and pea, either inoculated or N-fertilized, were grown in soil boxes under controlled conditions. Profile N₂O concentration and surface N₂O emissions were measured from soil–crop systems, and were compared with that of a cereal – spring wheat (Triticum aestivum L. ac. Barrie). Results indicated that: 1) neither R. leguminosarum strain, 99A1 or RGP2 was capable of denitrification in pure culture, nor in symbiosis with lentil and pea in sterile Leonard jars, suggesting that introducing these Rhizobium into soils through rhizobial inoculation onto lentil and pea will not increase denitrification or N₂O emissions; 2) soil-emitted N₂O from well-nodulated lentil and pea crops grown under controlled conditions was not significantly different than that from the check treatments, indicating that biological N₂ fixation by lentil and pea was not a direct source of N₂O emissions.     

Can differences in 15N natural abundance be used to quantify the transfer of nitrogen from legumes to neighbouring non-legume plant species?

Natural variations in the stable isotope ¹⁵N are often exploited in studies of N cycling in ecosystems. Lower ¹⁵N natural abundance in non-legume plants growing in association with legumes, compared with the non-legume grown alone in pure stands have been observed in cropping, forage, and agroforestry systems. Such observations have frequently been attributed to the transfer of biologically-fixed nitrogen (N) from the legume to the companion non-legume, and various methodologies have been employed to calculate the extent of the N transfer. While some of these ¹⁵N natural abundance-based estimates of N transfer were within the range previously reported using equivalent ¹⁵N-enriched techniques (<20% of non-legume plant N and <10 kg N ha⁻¹ derived from fixed N contributed by neighbouring legumes), many of the values obtained using natural abundance were much higher (30%–83% of the non-legume N derived from fixed N representing up to 30–40 kg N ha⁻¹) than generally measured by ¹⁵N-enriched methods; with even greater estimates being determined where data were available to allow N transfer to be re-calculated on the basis of total legume N rather than fixed N (42% to >100%, and up to 110 kg N ha⁻¹ per year). This review raises concerns about the assumptions behind the natural abundance approach, and provides some alternative interpretations for the observed differences in natural ¹⁵N abundance between plants grown in the presence and absence of legumes. It was concluded that simple comparative measures of non-legume δ¹⁵N alone cannot provide a quantitative estimate of N transfer between plant species if the dominant source and the isotopic identity of the transferred N cannot be validated, and if the extent of any isotopic fractionation associated with relevant N transformations occurring during transfer cannot be defined. To date this information is not forthcoming. There is a need to greatly improve our understanding of the transfer processes before the real value of the δ¹⁵N technology can be realized. In the first instance this will primarily be achieved by carefully executed experiments under controlled conditions, and in the field, employing both ¹⁵N natural abundance and enrichment approaches so estimates of transfer can be compared, and the data interrogated using modelling approaches to explore isotopic fractionation.     

Earthworms as drivers of the competition between grasses and legumes

Grasses and legumes are grown together worldwide to improve total herbage yield and the quality of forage, however, the causes of population oscillations of grasses and legumes are poorly understood. Especially in grasslands, earthworms are among the most important detritivore animals functioning as ecosystem engineers, playing a key role in nutrient cycling and affecting plant nutrition and growth. The objectives of the present greenhouse experiment were to quantify the effects of earthworms on grass–legume competition in model grassland systems at two harvesting dates – simulating the widespread biannual mowing regime in Central European grasslands.The presence of earthworms increased the productivity of grasses and legumes after 6 weeks but only that of grasses after another 10 weeks. In mixed treatments, the presence of grasses and earthworms decreased legume shoot biomass, the amount of nitrogen (N) in shoot tissue and the number of legume flowerheads while the presence of legumes and earthworms increased the amount of N in grass shoots and the infestation of grasses with aphids. Analyses of ¹⁵N/¹⁴N ratios indicate that, compared to legumes, grasses more efficiently exploit soil mineral N and benefit from legume presence through reduced “intra-functional group” competition. In contrast to previous experiments, we found no evidence for N transfer from legumes to grasses. However, legume presence improved total herbage and N yield.Earthworms likely modulate the competition between grasses and legumes by increasing soil N uptake by plants and thereby increasing the competitive strength of grasses. Earthworms function as essential driving agents of grass–legume associations by (I) increasing grass yield, (II) increasing the amount of N in grass hay, (III) increasing the infestation rate of grasses with aphids, and (IV) potentially reducing the attractiveness of grass–legume associations to pollinators.     

几种豆科、禾本科植物对多环芳烃复合污染土壤的修复

通过盆栽试验,研究了几种豆科植物与禾本科植物对多环芳烃(PAHs)复合污染土壤的修复作用。结果显示,90天后8种植物对土壤中PAHs均有不同程度降解效果,其中紫花苜蓿和多年生黑麦草对土壤PAHs的去除率分别达48.4%、46.8%,且对3环PAHs去除较为彻底,对4环及4环以上的PAHs去除效果较差。8种供试植物对PAHs均有一定的吸收、富集与转运的能力,紫花苜蓿和多年生黑麦草对土壤PAHs的生物富集系数分别为0.096、0.085,其提取修复效率为0.017%和0.013%。可见,紫花苜蓿和多年生黑麦草具有较好的根际修复潜力。     

Arbuscular mycorrhizal fungi-mediated nitrogen transfer from vineyard cover crops to grapevines

Cover crops are often planted in between vineyard rows to reduce soil erosion, increase soil fertility, and improve soil structure. Roots of both grapevines and cover crops form mutualistic symbioses with arbuscular mycorrhizal (AM) fungi, and may be interconnected by AM hyphae. To study nutrient transfer from cover crops to grapevines through AM fungal links, we grew grapevines and cover crops in specially designed containers in the greenhouse that restricted their root systems to separate compartments, but allowed AM fungi to colonize both root systems. Leaves of two cover crops, a grass (Bromus hordeaceus) and a legume (Medicago polymorpha), were labeled with 99 atom% ¹⁵N solution for 24 h. Grapevine leaves were analyzed for ¹⁵N content 2, 5, and 10 days after labeling. Our results showed evidence of AM fungi-mediated ¹⁵N transfer from cover crops to grapevines 5 and 10 days after labeling. N transfer was significantly greater from the grass to the grapevine than from the legume to the grapevine. Possible reasons for the differences between the two cover crops include lower ¹⁵N enrichment in legume roots, higher biomass of grass roots, and/or differences in AM fungal community composition. Further studies are needed to investigate N transfer from grapevines to cover crops and to determine net N transfer between the two crops throughout their growing seasons, in order to understand the significance of AM fungi-mediated interplant nutrient transfers in the field.     

How are nitrogen fixation rates regulated in legumes?

Symbiotic nitrogen fixation by the legume‐Rhizobium symbiosis is a finely regulated process that involves significant carbon and energy metabolism by the plant. At present, there are three competing theories as to how the regulation of the process is achieved at the whole plant level. Theory one (carbon supply regulation) states that nitrogen fixation rates are regulated by either current nodule assimilate supply or through metabolism of assimilates into compounds that can be used by the bacteroids. A second theory (oxygen supply regulation) assumes that oxygen diffusion into the nodules is tightly regulated and is the principal regulatory factor for nitrogen fixation rates. A third theory (N‐feedback regulation) suggests that a product of nitrogen fixation or assimilation exerts a feedback regulatory impact. The paper summarizes experimental data which support or reject the mentioned theories. Moreover, implications of nitrogen fixation under P stress and of phenomena connected with the argon or acetylene induced decline in nitrogenase activity for the understanding of nitrogen fixation regulation are discussed. It is concluded that we currently have no theory that explains all aspects and experimental results concerning the regulation of nitrogen fixation in legumes. With our growing ability to impact the process, e.g., by genetic engineering, this understanding of nitrogen fixation regulation has potential to be translated into agronomically sustainable benefits.     

Winter legumes as a nitrogen source for no-tillage cotton

Winter legumes can serve dual purposes in no-tillage cropping systems. They can provide a no-tillage mulch, and supply a considerable quantity of N for thesummer crops. Cotton (Gossypium hirsutum L.) was no-tillage planted into crimson clover (Trifolium incarnatum L.), common vetch (Vicia sativa L.), and fallowed soil for two years to determine the effects of winter legume mulches on growth, yield, and N fertilizer requirements. The legumes were allowed to mature and reseed prior to planting cotton. The winter legumes produced no measurable changes in soil organic matter, N, or bulk density, but water infiltration was more rapid in the legume plots than in the fallowed soil. In the fallow system, 34 kg ha^?1 N fertilizer was required for near maximum yields. In the clover plots, yields without N fertilizer were higher than when N (34 and 68 kg ha^?1) was applied. In the vetch plots, cotton yields were highest without N fertilizer the first year, but yields were increased with 34 kg ha^?1 N the second year because of a poor vetch seed crop and a subsequently poor legume stand. In the clover plots, a 20–30% cotton seedling mortality occurred in one year, but this stand reduction apparently did not affect cotton yields. Winter legume mulches can provide the N needs for no-tillage cotton without causing an excessive and detrimental quantity of N in sandy soils naturally low in soil N (0.04%). Unless the reseeding legume systems are maintained for at least 3 years, the legumes do not, however, provide an economical N source for cotton when N fertilizer requirementsare low (34 kg ha^?1 in this study). A possible disadvantage of the system for reseeding legumes is that cotton planting is delayed 4–6 weeks beyond the normal planting date, which can reduce yields in some years.     

15N studies on the transfer of legume-fixed nitrogen to associated cereals in intercropping systems

Summary An attempt has been made to estimate quantitatively the amount of N fixed by legume and transferred to the cereal in association in intercropping systems of wheat (Triticum aestivum L.) — gram (Cicer arietinum L.) and maize (Zea mays L.) —cowpea (Vigna unguiculate L.) by labelling soil and fertilizer nitrogen with ¹⁵N. The intercropped legumes have been found to fix significantly higher amounts of N as compared with legumes in sole cropping if the intercropped cereal-legume received the same dose of fertilizer N as the sole cereal crop. But when half of the dose of the fertilizer N applied to sole cereal crop was received by intercropped plants, the amount of N fixed by legumes in association with cereals was significantly less than that fixed by sole legumes. Under field conditions 28% of the total N uptake by maize (21.2 kg N ha^–1) was of atmospheric origin and was obtained by transfer of fixed N by cowpea grown in association with maize. Under greenhouse conditions gram and summer and monsoon season cowpea have been found to contribute 14%–20%, 16% and 32% of the total N uptake by associated wheat and summer and monsoon maize crops, respectively. Inoculation of cowpea seeds with Rhizobium increased both the amount of N fixed by cowpea and transferred to maize in intercropping system.     

Transfer of legume nitrogen to associated grass

In improved hill-peat a rapid increase in rhizosphere soil bacteria occurs following clover defoliation. The increase in bacterial-cell N is equivalent to 19% of the N released from the clover roots in the first 5 days after cutting. Subsequent ammonification of the immobilized N is dependent on the resistance of the microbial fraction to further degradation. In three fertilized hill soils examined, [¹⁵N]labelled bacterial N was mineralized quite rapidly, some 20% of the bacterial N being taken up by grass over 17 days. Under glasshouse conditions the rate of mineralization is different in the three soils examined, the peat providing a more favourable environment than brown earth for bacterial N transfer.     

Indigenous rhizobia associated with native shrubby legumes in Taiwan

Root-nodulating bacteria were isolated and characterized from seven native shrubby legumes growing in Taiwan. Phenotypic characteristics measured included growth rates in various media, colony morphology, and tolerances to extremes of temperature, salt and pH. The isolates were very diverse phenotypically. Among the 83 isolates that were screened, the majority were fast-growing rhizobia. Twenty eight strains tolerated high concentration of salt (4.5% NaCl) and grew well between temperatures of 37 and 45 °C. The majority of the strains also tolerated extreme pH in their medium from 3.5 to 12. All strains formed nitrogen fixing nodules, and the highest activity was detected in the legume Hedysarum crinita L. PCR restriction fragment length polymorphism (PCR-RFLP) and sequencing of the small subunit ribosomal RNAs revealed that the majority of the isolates belonged to the genera Rhizobium, Bradyrhizobium and Agrobacterium. Only a single strain represented the genus Sinorhizobium. In addition, a strain related to Burkholderia from the β-class of the Proteobacteria (CC-CC-5) was found within nodules of the legume Catenaria caudatum. The study contributes to the understanding of symbiotic nitrogen fixation in selected wild legumes that are native to Taiwan and provides insights into the distribution of nodulating and nitrogen-fixing bacteria from other distinct lineages.     

Residual nitrogen effects on wheat following legumes in the southern plains

Increasing nitrogen (N) fertilizer prices give rise to the question of N benefits from legumes in cropping systems in the Southern Great Plains. This study quantified wheat (Triticum aestivium L.) hay production and N uptake over seven years following six years of alfalfa (Medicago sativa L.), cicer milkvetch (Astragalus cicer L.), or grass (Old World bluestem, Bothriochloa ischaemum L.) production in western Oklahoma. Precipitation over the seven years averaged 550 mm·yr^‐1. The major residual N effects were measured within the first five years. On a fine sandy loam soil, wheat hay yields averaged 3,070 kg·ha^‐1·yr^‐1 over five years following alfalfa, 2,580 kg·ha^‐1·yr^‐1 following milkvetch, and 950 kg·ha^‐1·yr^‐1 following grass with N uptake attributed to the residual effect from legumes (calculated by the difference method) averaged 34 kg N ha^‐1·yr^‐1 from alfalfa and 25 kg·ha^‐1·yr^‐1 from milkvetch. On a deep loamy sand soil, wheat hay yields averaged 1,290 kg·ha^‐1·yr^‐1 over five years following alfalfa and 710 kg·ha^‐1·yr^‐1 following grass with N uptake attributed to the residual effect from alfalfa averaged 8 kg N ha^‐1·yr^‐1. Thus, the residual N effect attributed to legumes was substantial on the fine sandy loam soil and relatively small on the deep loamy sand soil.     

Grain legumes differ in nitrogen accumulation and remobilisation during seed filling

In grain legumes, the N requirements of growing seeds are generally greater than biological nitrogen fixation (BNF) and soil N uptake during seed filling, so that the N previously accumulated in the vegetative tissues needs to be redistributed in order to provide N to the seeds. Chickpea, field bean, pea, and white lupin were harvested at flowering and maturity to compare the relative contribution of BNF, soil N uptake, and N remobilisation to seed N. From flowering to maturity, shoot dry weight increased in all crops by approximately 50%, root did not appreciably change, and nodule decreased by 18%. The amount of plant N increased in all crops, however in field bean (17?g?m^?2) it was about twice that in chickpea, pea, and lupin. The increase was entirely due to seeds, whose N content at maturity was 26?g?m^?2 in field bean and 16?g?m^?2 in chickpea, pea, and lupin. The seed N content at maturity was higher than total N accumulation during grain filling in all crops, and endogenous N previously accumulated in vegetative parts was remobilised to fulfil the N demand of filling seeds. Nitrogen remobilisation ranged from 7?g?m^?2 in chickpea to 9?g?m^?2 in field bean, and was crucial in providing N to the seeds of chickpea, pea, and lupin (half of seed N content) but it was less important in field bean (one-third). All the vegetative organs of the plants underwent N remobilisation: shoots contributed to the N supply of seeds from 58% to 85%, roots from 11% to 37%, and nodules less than 8%. Improving grain legume yield requires either reduced N remobilisation or enhanced N supply, thus, a useful strategy is to select cultivars with high post-anthesis N₂ fixation or add mineral N at flowering.     

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N₂-fixing ability was demonstrated by C₂H₂ reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N₂-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N₂-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N₂-fixing bacteria associated with grass roots.     

用15N富积标记和稀释法研究旱作水稻/花生间作系统中氮素固定和转移

采用花生叶片15N富积标记法和15N同位素稀释法两种标记方法研究了水稻 花生间作条件下花生的生物固氮以及花生向水稻的氮素转移。盆栽结果表明,两种方法都证明在水稻花生间作的共生期内发生了氮素转移,花生固氮量的2%～3.5%转移到水稻体内。同位素稀释试验还表明,间作对花生固氮有促进作用,能提高其固氮效率(BNF)。本文还对两种同位素标记方法的优缺点进行了讨论。     

Nitrogenase activity associated with soil cores of grasses in Brazil

The N₂-ase activities of field-grown Brazilian grasses were measured with C₂H₂ reduction by soil cores containing the plants. C₂H₂ and C₂H₄ were observed to diffuse at similar rates through soil and equilibrated across the Brazilian soil in 3 h, but could take up to 30 h or more with some British soils. The diurnal fluctuation in the rates of N₂-ase activity by Brachiaria mutica and Sorghum vulgare were similar and the variation in rate was correlated with soil temperature. Estimates of N₂-fixation by measurement of C₂H₂ reduction by soil cores ranged from 14.7 to 51.4 g N ha^?1 day^?1 and were much lower than with “pre-incubated” excised roots from the cores or taken directly from the field. The merits of the soil core and the “pre-incubated” excised root assays are discussed. ft1|Present address: U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Northeastern Region, Room 309, Building 001, Beltsville, MD 20705, U.S.A.     

Fixation and transfer of nitrogen by white clover to ryegrass

Abstract. ¹⁵N₂ was used in a sealed controlled environment chamber to investigate the transfer of fixed nitrogen from white clover to perennial ryegrass growing in soil in pots. There was no difference in the ¹⁵N content of roots and shoots of clover plants after exposure to ¹⁵N. No labelled fixed nitrogen was detected in ryegrass plants growing with the clover plants for a period of 129 days. There was therefore no evidence of rapid direct transfer (excretion) of fixed nitrogen from clover to ryegrass.     

Nitrogenase activity associated with pasture grasses in Northern New South Wales

Nitrogenase activity associated with roots of grasses was initially examined at 67 sites in New South Wales using an enriched malate medium. Twenty six of the 39 grass species examined produced at least 10 nmol C₂H₄cm^?1 root h^?1—a level accepted as positive presumptive evidence of N₂-fixation: 40 of the 288 samples exceeded 100nmol C₂H₄h^?1. The seasonal N₂ase activity of up to 4 grass species collected in soil cores at 6 sites was measured over 16 months.Activity at field moisture levels, but incubated at 30°C was greatest for cores collected in summer months. Activity was increased in 33.5% of samples by raising soil moisture to field capacity. No single species of grass consistently supported higher activity than any other.Nitrogenase activity was compared in cores of Kikuyu grass (Pennisetum clandestinum) watered to in excess of field capacity and allowed to drain for between 7 and 28 days before re-watering. Activity declined rapidly in the first 7 days and although recovery was also rapid, integration of N₂ase activity over time showed a loss of 20 and 61% for 7 and 28 days drainage respectively. N₂ase activity was greatest at 30°C.Maximum N₂ase activity in field samples was only 246 nmol C₂H₄ core^?1 h^?1 indicating that fixation of N₂ would not be of agronomic significance.     

Assessing risks to groundwater from contaminated soils

Abstract. The regulatory regime for contaminated soils and groundwater in the UK is risk based. Indeed, the definition of contaminated land in UK law requires the presence, or likelihood, of significant pollution or harm before affected land meets the statutory test of being contaminated land. In dealing with contamination, a risk management approach is used that also requires the consideration of wider land-use planning priorities, the costs and benefits of remedial action, and the goal of achieving sustainable development. A parallel risk-based framework is adopted for polluted groundwater. The risk assessment process adopted for contaminated groundwater in the UK follows a tiered structure. A simple conceptual model is developed from desk study and site reconnaissance. This model is then used as the basis for designing a site investigation programme, the sampling being undertaken to test assumptions within the model as well as to develop it further. The Environment Agency (England & Wales) has developed a package of tools to aid this tiered approach. This paper describes the integrated risk assessment and risk management processes for contaminated groundwater in the UK, with emphasis on the tools and methods developed. It explains the importance of a conceptual understanding (or model) to all tiers of risk assessment and subsequent risk management.     

Non-symbiotic nitrogen fixation in the rhizospheres of rice,maize and different tropical grasses

Nitrogenase activity estimated in the rhizospheres of rice, maize and different tropical grasses grown under controlled laboratory conditions was shown to depend upon plant species. High nitrogenase activity (2000–6000 nmoles C₂H₄ h^?1 g^?1 dry root) occurred in rice rhizosphere, this activity being only 10 times lower than that of symbiotic systems; in the rhizosphere of many other grasses grown in a similar way nitrogenase activity was as low as 10 nmoles C₂H₄ h^?1 g^?1 dry root. The influence of soil type on nitrogenase activity was impressive; but the exact nature of the factors implicated could not be established. A rather weak flush of nitrogenase activity in the rhizosphere occurred in the early stage of the plant growth; it was probably due to the exudation of compounds from the seed and lasted 2 or 4 days according to the size of the seed. When the plant entered into its intense photosynthetic phase, the nitrogenase activity gradually increased. When the shoots were severed, nitrogenase activity in the rhizosphere ceased. Nitrogenase activity in the rhizosphere responded greatly to light intensity. Extrapolation of these laboratory findings to the field is discussed.