Symbiotic Biological Nitrogen Fixation Research Articles

Overexpression of GmPAP4 Enhances Symbiotic Nitrogen Fixation and Seed Yield in Soybean under Phosphorus-Deficient Condition.

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean.

Enhancing Rhizobium–Legume Symbiosis and Reducing Nitrogen Fertilizer Use Are Potential Options for Mitigating Climate Change

This review article explores the impact of nitrogen fertilizers on the symbiotic relationship between Rhizobium bacteria and legume plants. Nitrogen fixation has the potential to address the global protein shortage by increasing nitrogen supply in agriculture. However, the excessive use of synthetic fertilizers has led to environmental consequences and high energy consumption. To promote sustainable agriculture, alternative approaches such as biofertilizers that utilize biological nitrogen fixation have been introduced to minimize ecological impact. Understanding the process of biological nitrogen fixation, where certain bacteria convert atmospheric nitrogen into ammonia, is crucial for sustainable agriculture. This knowledge helps reduce reliance on synthetic fertilizers and maintain soil fertility. The symbiotic relationship between Rhizobium bacteria and leguminous plants plays a vital role in sustainable agriculture by facilitating access to atmospheric nitrogen, improving soil fertility, and reducing the need for chemical fertilizers. To achieve optimal nitrogen fixation and plant growth, it is important to effectively manage nitrogen availability, soil conditions, and environmental stressors. Excessive nitrogen fertilization can negatively affect the symbiotic association between plants and rhizobia, resulting in reduced soil health, altered mutualistic relationships, and environmental concerns. Various techniques can be employed to enhance symbiotic efficiency by manipulating chemotaxis, which is the ability of rhizobia to move towards plant roots. Plant-specific metabolites called (iso)flavonoids play a crucial role in signaling and communication between legume plants and rhizobia bacteria, initiating the symbiotic relationship and enhancing nitrogen fixation and plant growth. Excessive nitrogen fertilizer application can disrupt the communication between rhizobia and legumes, impacting chemotaxis, root exudation patterns, nodulation, and the symbiotic relationship. High levels of nitrogen fertilizers can inhibit nitrogenase, a critical enzyme for plant growth, leading to reduced nitrogenase activity. Additionally, excessive nitrogen can compromise the energy demands of nitrogen fixation, resulting in decreased nitrogenase activity. This review discusses the disadvantages of using nitrogenous fertilizers and the role of symbiotic biological nitrogen fixation in reducing the need for these fertilizers. By using effective rhizobial strains with compatible legume cultivars, not only can the amounts of nitrogenous fertilizers be reduced, but also the energy inputs and greenhouse gas emissions associated with their manufacturing and application. This approach offers benefits in terms of reducing greenhouse gas emissions and saving energy. In conclusion, this paper provides a comprehensive overview of the current understanding of the impact of nitrogen fertilizers on the symbiotic relationship between Rhizobium and legume plants. It also discusses potential strategies for sustainable agricultural practices. By managing nitrogen fertilizers carefully and improving our understanding of the symbiotic relationship, we can contribute to sustainable agriculture and minimize environmental impact.

Genotypic variation in response to drought stress is associated with biochemical and transcriptional regulation of ureides metabolism in common bean (Phaseolus vulgaris L.)

Ureidic legumes such as common bean (Phaseoulus valgaris L.) plants export nitrogen from the nodules to shoots and leaves as ureides during symbiotic biological nitrogen fixation. Common bean gene encoding allantoinase (allantoin amidohydrolase, EC 3.5.2.5), is a key enzyme that catalyses the hydrolysis of allantoin to allantoic acid. It plays a role in ureide generation for export and ureide catabolism to generate a nitrogen source in sinks tissues. As such, one of the adaptive mechanisms of plants to drought stress, is associated with ureides accumulation. To identify genetic variation of common bean in response to drought stress, changes in the expression of ALLANTONAISE (PvALN) gene and ureides content were examined in the leaf tissues of the three common bean genotypes (CAL96, DAB514 and DAB541) and one tepary bean genotype (Phaseolus acutifolius A.Gray). Amongst all the genotypes, the suggested drought susceptibility in DAB514 common bean genotype, was probably attributed to a repressed PvALN expression rate which were corroborated by an impaired ureides levels, and reduced plant growth. On contrary, drought stress induced an upregulated relative expression of PvALN coupled with an increase in allantoin and allantoate in DAB541 common bean genotype. In addition, the sustained plant growth in CAL96 was probably attributed to a steady amount of allantoin synthesized under drought stress. Taken together, DAB541 and CAL96 common bean genotypes are the promising genotypes with an induced upregulated transcriptional control of catabolism and/or biosynthesis of ureides, hence potential genotypes for selection and introduction under Botswana semi-arid conditions.

Potential Contribution of Groundnut Residues to Soil N and the Influence of Farmer Management in Western Uganda

Through symbiotic biological nitrogen fixation (BNF), grain legumes, such as groundnuts, can enhance soil nitrogen (N) and be an important source of N fertility, as well as a critical component of human nutrition and food security. Because legumes obtain N from soil N stocks as well as BNF, legume residues are key to capturing potential N benefits for soils, which may contribute to increased yields and food production. Here, we conducted a detailed survey at household and field level within a six-village corridor along the western boundary of Kibale National Park (KNP) in western Uganda. We focused on groundnut production and residue management practices and soil organic carbon (SOC) and total N (TN) in fields managed by 100 different households. We also determined SOC and TN in adjacent uncultivated KNP soils. We tested for relationships between socioeconomic factors and farmer groundnut management practices. We calculated a partial N balance and estimated potential N benefits under three scenarios for groundnut BNF. Within the study area, groundnut residue management varied greatly with 51% of surveyed farmers retaining residues on fields through spreading or incorporation, and 49% removing residues, either by transfer to banana groves or burning. Groundnut population density was relatively high with 43% of fields having >30 plants m−2. Despite providing net N inputs of up to 27 kg N ha−1, there was no observed effect of groundnut residue management practices on SOC, TN, or soil C:N ratios. Compared to uncultivated KNP soils, groundnut fields had lower mean levels of SOC and TN and wider C:N ratios. These values are consistent with cultivated soils; however, losses of SOC and TN were lower compared to losses previously reported for conversion from tropical forest to agricultural use. We found that farmer valuation and perception of groundnut residues were influential factors in residue management practices. Overall, we estimated that groundnut residues have the potential to contribute to SOC and TN stocks if retained in the field, but, conversely, removal will result in sizable losses. We find that both environmental and social contexts must be considered when recommending legumes for N provisioning services.

A novel representation of biological nitrogen fixation and competitive dynamics between nitrogen-fixing and non-fixing plants in a land model (GFDL LM4.1-BNF)

Abstract. Representing biological nitrogen fixation (BNF) is an important challenge for coupled carbon (C) and nitrogen (N) land models. Initial representations of BNF in land models applied simplified phenomenological relationships. More recent representations of BNF are mechanistic and include the dynamic response of symbiotic BNF to N limitation of plant growth. However, they generally do not include the competitive dynamics between N-fixing and non-fixing plants, which is a key ecological mechanism that determines ecosystem-scale symbiotic BNF. Furthermore, asymbiotic BNF is generally not included in land models. Here, we present LM4.1-BNF, a novel representation of BNF (asymbiotic and symbiotic) and an updated representation of N cycling in the Geophysical Fluid Dynamics Laboratory Land Model 4.1 (LM4.1). LM4.1-BNF incorporates a mechanistic representation of asymbiotic BNF by soil microbes, a representation of the competitive dynamics between N-fixing and non-fixing plants, and distinct asymbiotic and symbiotic BNF temperature responses derived from corresponding observations. LM4.1-BNF makes reasonable estimations of major carbon (C) and N pools and fluxes and their temporal dynamics, in comparison to the previous version of LM4.1 with N cycling (LM3-SNAP) and to previous representations of BNF in land models generally (phenomenological representations and those without competitive dynamics between N-fixing and non-fixing plants and/or asymbiotic BNF) at a temperate forest site. LM4.1-BNF effectively reproduces asymbiotic BNF rate (13 kgNha-1yr-1) in comparison to observations (11 kgNha-1yr-1). LM4.1-BNF effectively reproduces the temporal dynamics of symbiotic BNF rate: LM4.1-BNF simulates a symbiotic BNF pulse in early succession that reaches 73 kgNha-1yr-1 at 15 years and then declines to ∼0 kgNha-1yr-1 at 300 years, similarly to observed symbiotic BNF, which reaches 75 kgNha-1yr-1 at 17 years and then declines to ∼0 kgNha-1yr-1 in late successional forests. As such, LM4.1-BNF can be applied to project the dynamic response of vegetation to N limitation of plant growth and the degree to which this will constrain the terrestrial C sink under elevated atmospheric CO2 concentration and other global change factors.

A roadmap for sampling and scaling biological nitrogen fixation in terrestrial ecosystems

Abstract Accurately quantifying rates and patterns of biological nitrogen fixation (BNF) in terrestrial ecosystems is essential to characterize ecological and biogeochemical interactions, identify mechanistic controls, improve BNF representation in conceptual and numerical modelling, and forecast nitrogen limitation constraints on future carbon (C) cycling. While many resources address the technical advantages and limitations of different methods for measuring BNF, less systematic consideration has been given to the broader decisions involved in planning studies, interpreting data, and extrapolating results. Here, we present a conceptual and practical road map to study design, study execution, data analysis and scaling, outlining key considerations at each step. We address issues including defining N‐fixing niches of interest, identifying important sources of temporal and spatial heterogeneity, designing a sampling scheme (including method selection, measurement conditions, replication, and consideration of hotspots and hot moments), and approaches to analysing, scaling and reporting BNF. We also review the comparability of estimates derived using different approaches in the literature, and provide sample R code for simulating symbiotic BNF data frames and upscaling. Improving and standardizing study design at each of these stages will improve the accuracy and interpretability of data, define limits of extrapolation, and facilitate broader use of BNF data for downstream applications. We highlight aspects—such as quantifying scales of heterogeneity, statistical approaches for dealing with non‐normality, and consideration of rates versus ecological significance—that are ripe for further development.

Diversity of Rhizobia and Importance of Their Interactions with Legume Trees for Feasibility and Sustainability of the Tropical Agrosystems

Symbiotic biological nitrogen fixation (BNF) is a complex process that involves rhizobia, a diverse group of α and β-proteobacteria bacteria, and legume species. Benefits provided by BNF associated with legume trees in tropical environments include improvements to efficiency of nitrogen (N) use, increase of soil carbon sequestration, stabilization of soil organic matter, decrease of soil penetration resistance, and improvement of soil fertility. All these benefits make BNF a crucial ecosystem service to the sustainability of tropical agriculture. Due to the importance of this ecological process and the high diversity of rhizobia, these bacteria have been extensively characterized worldwide. Currently, over 400 species of rhizobia are known, distributed into seven families. In the humid tropics, Leucaena leucocephala, Acacia mangium, Gliricidia sepium, and Clitoria fairchildiana are four of the most common species used by family farmers to create sustainable agricultural systems. These four legumes perform symbiosis with different groups of rhizobia. Exploring BNF could help to enable sustainable intensification of agriculture in the humid tropics, mainly because it can increase N use efficiency in an environment where N is a limiting factor to plant growth.

Potential of Rhizobium sullae–Sulla coronaria Symbiotic Biological Nitrogen Fixation to Supplement Synthetic Mineral Nitrogen in Olive Tree Fertilization

The aim of the present work is to compare olive tree nitrogen fertilization over two years of trials, using synthetic chemical fertilizers along with organic fertilizers composed of the green manure of sulla (Sulla coronaria) inoculated with the symbiont Rhizobium sullae or left uninoculated. The tests indicated that symbiotic nitrogen fixation promoted by the sulla–R. sullae symbiosis represents an important source of nitrogen that can replace or supplement synthetic nitrogen fertilizers for olive tree cultivation when sulla is inoculated with R. sullae in a soil already populated by the symbiont. Integration of the indigenous population of R. sullae via sulla inoculation with a selected strain yielded nodule formation in 100% of plants and produced a sufficient amount of biomass rich in nitrogen with a low C/N ratio. On the contrary, olive tree fertilization using the green manure of sulla that was not inoculated with the symbiont supplied significantly less organic nitrogen in 2017 and 2018, respectively, compared to the control. Optimal management of the multi-factorial approaches involved in green manure olive fertilization are also reported.

A spatially explicit, empirical estimate of tree-based biological nitrogen fixation in forests of the United States.

Quantifying human impacts on the N cycle and investigating natural ecosystem N cycling depend on the magnitude of inputs from natural biological nitrogen fixation (BNF). Here, we present two bottom-up approaches to quantify tree-based symbiotic BNF based on forest inventory data across the coterminous US plus SE Alaska. For all major N-fixing tree genera, we quantify BNF inputs using (1) ecosystem N accretion rates (kg N ha-1 yr-1) scaled with spatial data on tree abundance and (2) percent of N derived from fixation (%Ndfa) scaled with tree N demand (from tree growth rates and stoichiometry). We estimate that trees fix 0.30-0.88 Tg N yr-1 across the study area (1.4-3.4 kg N ha-1 yr-1). Tree-based N fixation displays distinct spatial variation that is dominated by two genera, Robinia (64% of tree-associated BNF) and Alnus (24%). The third most important genus, Prosopis, accounted for 5%. Compared to published estimates of other N fluxes, tree-associated BNF accounted for 0.59 Tg N yr-1, similar to asymbiotic (0.37 Tg N yr-1) and understory symbiotic BNF (0.48 Tg N yr-1), while N deposition contributed 1.68 Tg N yr-1 and rock weathering 0.37 Tg N yr-1. Overall, our results reveal previously uncharacterized spatial patterns in tree BNF that can inform large-scale N assessments and serve as a model for improving tree-based BNF estimates worldwide. This updated, lower BNF estimate indicates a greater ratio of anthropogenic to natural N inputs, suggesting an even greater human impact on the N cycle.

Biological Nitrogen Fixation Does Not Replace Nitrogen Losses After Forest Fires in the Southeastern Amazon

Tropical forest fires have become more common due to interactions between deforestation, land clearing, and drought. Forest recovery following fires may be limited by nitrogen. Biological nitrogen fixation (BNF) is the main pathway for new nitrogen (N) to enter most ecosystems, but BNF may be constrained by other nutrients, such as molybdenum and phosphorus, which are required for the process. We studied the role of BNF 7 years into the recovery of southeastern Amazon tropical forests that were burned experimentally either annually or every 3 years between 2004 and 2010. We hypothesized that, compared with unburned primary forests, BNF in burned forests would increase due to the depletion of N in the aboveground biomass pool and that soil concentrations of molybdenum and phosphorus from ash inputs would increase, reducing their potential constraints on BNF. Despite the fires depleting about half the aboveground N pool and rapid rates of recovery in leaf biomass and litterfall production, we found low rates of both free-living and symbiotic BNF. Higher concentrations of molybdenum and phosphorus in the mineral soils of the burned forests indicated that these elements were likely not limiting BNF. Our results suggest that despite the N demand for regrowth post-fire, substantial N stored in soils likely downregulates BNF. Overall, we found surprisingly low BNF rates (< 1.2 kg N ha−1 y−1) in this region of the Amazon, which contrasts with the traditional paradigm that (1) tropical forests fix large quantities of N and (2) that BNF increases after forest disturbance.

Evaluation of symbiotic nitrogen fixing ability of legume crops in Central Himalaya, India

Symbiotic biological nitrogen fixation is the major source of nitrogen input in traditional agriculture and plays a significant role in improving soil fertility and productivity, though it has hardly been estimated in Himalayan region. In the present study, nitrogenase activity of selected legumes was estimated in pure-legume and legume-Eleusine coracana mixed cropping systems to assess the potential of legumes in maintaining soil fertility in the mountain region of Central Himalaya. Assessment of nitrogenase activity and effect of legume crop cultivation on soil chemical properties reveals that legume crops possess great potential for nitrogen fixation and soil fertility improvement for sustainable rainfed agriculture system.

Antibiotics Resistance in Rhizobium: Type, Process, Mechanism and Benefit for Agriculture.

The use of high-quality rhizobial inoculants on agricultural legumes has contributed substantially to the N economy of farming systems through inputs from biological nitrogen fixation (BNF). Large populations of symbiotically effective rhizobia should be available in the rhizosphere for symbiotic BNF with host plants. The rhizobial populations should also be able to compete and infect host plants. However, the rhizosphere comprises large populations of different microorganisms. Some of these microorganisms naturally produce antibiotics which are lethal to susceptible rhizobial populations in the soil. Therefore, intrinsic resistance to antibiotics is a desirable trait for the rhizobial population. It increases the rhizobia's chances of growth, multiplication and persistence in the soil. With a large population of rhizobia in the soil, infectivity of host plants and the subsequent BNF efficiency can be guaranteed. This review, therefore, puts together findings by various researchers on antibiotic resistance in bacteria with the main emphasis on rhizobia. It describes the different modes of action of different antibiotics, the types of antibiotic resistance exhibited by rhizobia, the mechanisms of acquisition of antibiotic resistance in rhizobia and the levels of tolerance of different rhizobial species to different antibiotics.

Integration of nitrogen dynamics into the Noah-MP land surface model v1.1 for climate and environmental predictions

Abstract. Climate and terrestrial biosphere models consider nitrogen an important factor in limiting plant carbon uptake, while operational environmental models view nitrogen as the leading pollutant causing eutrophication in water bodies. The community Noah land surface model with multi-parameterization options (Noah-MP) is unique in that it is the next-generation land surface model for the Weather Research and Forecasting meteorological model and for the operational weather/climate models in the National Centers for Environmental Prediction. In this study, we add a capability to Noah-MP to simulate nitrogen dynamics by coupling the Fixation and Uptake of Nitrogen (FUN) plant model and the Soil and Water Assessment Tool (SWAT) soil nitrogen dynamics. This model development incorporates FUN's state-of-the-art concept of carbon cost theory and SWAT's strength in representing the impacts of agricultural management on the nitrogen cycle. Parameterizations for direct root and mycorrhizal-associated nitrogen uptake, leaf retranslocation, and symbiotic biological nitrogen fixation are employed from FUN, while parameterizations for nitrogen mineralization, nitrification, immobilization, volatilization, atmospheric deposition, and leaching are based on SWAT. The coupled model is then evaluated at the Kellogg Biological Station – a Long Term Ecological Research site within the US Corn Belt. Results show that the model performs well in capturing the major nitrogen state/flux variables (e.g., soil nitrate and nitrate leaching). Furthermore, the addition of nitrogen dynamics improves the modeling of net primary productivity and evapotranspiration. The model improvement is expected to advance the capability of Noah-MP to simultaneously predict weather and water quality in fully coupled Earth system models.

Regional constraints to biological nitrogen fixation in post‐fire forest communities

Biological nitrogen fixation (BNF) is a key ecological process that can restore nitrogen (N) lost in wildfire and shape the pace and pattern of post-fire forest recovery. To date, there is limited information on how climate and soil fertility interact to influence different pathways of BNF in early forest succession. We studied asymbiotic (forest floor and soil) and symbiotic (the shrub Ceanothus integerrimus) BNF rates across six sites in the Klamath National Forest, California, USA. We used combined gradient and experimental phosphorus (P) fertilization studies to explore cross-site variation in BNF rates and then related these rates to abiotic and biotic variables. We estimate that our measured BNF rates 22 years after wildfire (6.1-12.1 kg N x ha(-1) x yr(-1)) are unlikely to fully replace wildfire N losses. We found that asymbiotic BNF is P limited, although this is not the case for symbiotic BNF in Ceanothus. In contrast, Ceanothus BNF is largely driven by competition from other vegetation: in high-productivity sites with high potential evapotranspiration (Et), shrub biomass is suppressed as tree biomass increases. Because shrub biomass governed cross-site variation in Ceanothus BNF, this competitive interaction led to lower BNF in sites with high productivity and Et. Overall, these results suggest that the effects of nutrients play a larger role in driving asymbiotic than symbiotic fixation across our post-fire sites. However, because symbiotic BNF is 8-90x greater than asymbiotic BNF, it is interspecific plant competition that governs overall BNF inputs in these forests.

Four allantoinase genes are expressed in nitrogen-fixing soybean

Soybean (Glycine max L. [Merr]) plants export nitrogen from the nodules as ureides during symbiotic biological nitrogen fixation. Ureides also play a role as nitrogen storage compounds in the seeds and are broken down in germinating seedlings. In this work we identified four soybean genes encoding allantoinase (E.C. 3.5.2.5), an enzyme involved in both ureide production in nodules and ureide catabolism in leaves and other sink tissues. We examined ureide content, allantoinase enzyme activity and expression of these genes, which we term GmALN1 through GmALN4, in germinating seedlings and in vegetative tissues from 45 day old soybean plants. GmALN1 and GmALN2 transcripts were measured in all tissues, but similar levels of expression of GmALN3 and GmALN4 was only observed in nodules. The soybean allantoinase genes seem to have arisen through tandem gene duplication followed by a whole genome duplication. We looked for evidence of the tandem duplication in common bean (Phaseolus vulgaris L.) and present evidence that it occured sometime in the bean lineage before these two species diverged, but before soybean became a tetraploid.

Genome Sequence of Rhizobium etli CNPAF512, a Nitrogen-Fixing Symbiont Isolated from Bean Root Nodules in Brazil

Rhizobium etli is a Gram-negative soil-dwelling alphaproteobacterium that carries out symbiotic biological nitrogen fixation in close association with legume hosts. R. etli strains exhibit high sequence divergence and are geographically structured, with a potentially dramatic influence on the outcome of symbiosis. Here, we present the genome sequence of R. etli CNPAF512, a Brazilian isolate from bean nodules. We anticipate that the availability of genome sequences of R. etli strains from distinctly different areas will provide valuable new insights into the geographic mosaic of the R. etli pangenome and the evolutionary dynamics that shape it.

The genome of the versatile nitrogen fixer Azorhizobium caulinodans ORS571

BackgroundBiological nitrogen fixation is a prokaryotic process that plays an essential role in the global nitrogen cycle. Azorhizobium caulinodans ORS571 has the dual capacity to fix nitrogen both as free-living organism and in a symbiotic interaction with Sesbania rostrata. The host is a fast-growing, submergence-tolerant tropical legume on which A. caulinodans can efficiently induce nodule formation on the root system and on adventitious rootlets located on the stem.ResultsThe 5.37-Mb genome consists of a single circular chromosome with an overall average GC of 67% and numerous islands with varying GC contents. Most nodulation functions as well as a putative type-IV secretion system are found in a distinct symbiosis region. The genome contains a plethora of regulatory and transporter genes and many functions possibly involved in contacting a host. It potentially encodes 4717 proteins of which 96.3% have homologs and 3.7% are unique for A. caulinodans. Phylogenetic analyses show that the diazotroph Xanthobacter autotrophicus is the closest relative among the sequenced genomes, but the synteny between both genomes is very poor.ConclusionThe genome analysis reveals that A. caulinodans is a diazotroph that acquired the capacity to nodulate most probably through horizontal gene transfer of a complex symbiosis island. The genome contains numerous genes that reflect a strong adaptive and metabolic potential. These combined features and the availability of the annotated genome make A. caulinodans an attractive organism to explore symbiotic biological nitrogen fixation beyond leguminous plants.

Observed and modelled soil carbon and nitrogen changes after planting a Pinus radiata stand onto former pasture

After reforesting pasture land, it is often observed that soil carbon stocks decrease. The present work reports findings from a site near Canberra, Australia, where a pine forest ( Pinus radiata) was planted onto a former unimproved pasture site. We report a number of detailed observations seeking to understand the basis of the decline in soil C stocks. This is supported by simulations using the whole-ecosystem carbon and nitrogen cycling model CenW 3.1. The model indicated that over the first 18 years after forest establishment, the site lost about 5.5 t C ha −1 and 588 kgN ha −1 from the soil. The C:N ratio of soil organic matter did not change in a systematic manner over the observational period. Carbon and nitrogen stocks contained in the biomass of the 18-year old pine stand exceeded that of the pasture by 88 t C ha −1 and 393 kgN ha −1. An additional 6.1 t C ha −1 and 110 kgN ha −1 accumulated in above-ground litter. These changes, together with the vertical distribution of carbon and nitrogen in the soil, agreed well with the observation at the site. It was assumed that over 18 years, there was also a loss of 86 kgN ha −1 from the ecosystem because of normal gaseous losses during nitrogen turn-over and a small amount of nitrogen leaching. Those losses could not be replenished in the pine system without symbiotic biological nitrogen fixation, and there were no fertiliser additions. A simple mass balance approach indicated that the amount of nitrogen accumulating in plant biomass and the litter layer plus the assumed nitrogen loss from the site matched the amount of nitrogen lost from the soil organic nitrogen pool. This reduction in soil nitrogen, together with an unchanged C:N ratio, provided a simple and internally consistent explanation for the observed reduction of soil carbon after reforestation. It supports the general notion that trends in soil carbon upon land-use change can often be controlled by the possible fates of available soil nitrogen.

Enzimas hidrolíticas extracelulares de isolados de rizóbia nativos da Amazônia Central, Amazonas, Brasil

A associação rizóbia x leguminosa contribui para enriquecer o solo com nitrogênio por meio da fixação biológica. Entretanto, pouco se conhece a respeito do perfil enzimático desses microrganismos. Nesse contexto, a presente investigação propõe avaliar a produção de enzimas hidrolíticas extracelulares por isolados de rizóbia nativos da Amazônia Central. Essa triagem constitui o primeiro passo na seleção de microrganismos nativos que são potencialmente exploráveis como produtores de enzimas. Foram testados 67 isolados nativos de rizóbia para as atividades amilolítica, celulolítica, lactolítica, lipolítica, pectinolítica e proteolítica, em meio YMA modificado. A atividade ureolítica foi detectada em meio ágar-uréia. As bactérias isoladas dos nódulos de feijão caupi mostraram maior capacidade em produzir enzimas do que os isolados bacterianos de soja. De todas as enzimas hidrolíticas avaliadas, apenas a pectinase não foi detectada neste estudo. Amilase (32,8%), protease (28,4%), urease (20,9%) e carboximetilcelulase (9,0%) foram as enzimas mais freqüentes produzidas pelos isolados. Neste trabalho, apenas as enzimas amilase e protease variaram significativamente entre os isolados de rizóbia. Os isolados INPA R-926 e INPA R-915 exibiram os maiores índices amilolíticos (IE = 3,1) e proteolíticos (IE = 6,6), respectivamente. Este estudo revelou alguns isolados de rizóbia nativos da Amazônia Central como fontes promissoras de enzimas de importância industrial para uso biotecnológico.

Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil.

A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.