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It is expected that CO2 concentration will increase in the air, thereby stimulating the photosynthesis process and, hence, plant biomass production. In the case of legumes, increased biomass due to higher CO2 concentration can stimulate atmospheric nitrogen (N2) fixation in the nodules. However, N2 fixation is inhibited by external N supply. Thus, biomass production and N2 fixation were analysed in two legumes (Pisum sativum L. and Vicia faba L.) grown at two levels of CO2 and three N levels. P. sativum reduces fixation with high soil N (facultative), while V. faba maintains high fixation regardless of soil N levels (obligate). The N2 fixation and plant and nodule biomass of the two species were evaluated in a pot experiment under controlled conditions using growth chambers with artificial CO2 supply and N addition. The proportion of N derived from the air (%Ndfa) present in the plants' biomass was calculated from the natural abundance of 15N and the N concentration of plant tissues using nonlegumes reference plants. Additionally, N content data are presented for both species growing at two levels of air CO2. The data may be useful for plant physiologists, especially those working on biological N2 fixation with non-model legumes at elevated CO2.

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Intercropping legumes with cereals can lead to increased overall yield and optimize the utilization of resources such as water and nutrients, thus enhancing agricultural efficiency. Legumes possess the unique ability to acquire nitrogen (N) through both N2 fixation and from the available N in the soil. However, soil N can diminish the N2 fixation capacity of legumes. It is postulated that in intercropping, legumes uptake N mainly through N2 fixation, leaving more soil N available for cereals. The latter, in turn, has larger root systems, allowing it to explore greater soil volume and absorb more N, mitigating its adverse effects on N2 fixation in legumes. The goal of this study was to evaluate how the supply of N affects the intercropping of faba beans (Vicia faba L.) and peas (Pisum sativum L.) with wheat under varying plant densities and N levels. We measured photosynthetic traits, biomass production, the proportion of N derived from air (%Ndfa) in the shoot of the legumes, the N transferred to the wheat, and the land equivalent ratio (LER). The results revealed a positive correlation between soil N levels and the CO2 assimilation rate (An), chlorophyll content, and N balance index (NBI) in wheat. However, no significant effect was observed in legumes as soil N levels increased. Transpiration (E) increased in wheat intercropped with legumes, while stomatal conductance (gs) increased with N addition in all crops. Water use efficiency (WUE) decreased in faba beans intercropped with wheat as N increased, but it showed no significant change in wheat or peas. The shoot dry matter of wheat increased with the addition of N; however, the two legume species showed no significant changes. N addition reduced the %Ndfa of both legume species, especially in monoculture, with peas being more sensitive than faba beans. The intercropping of wheat alleviated N2 fixation inhibition, especially at high wheat density and increased N transfer to wheat, particularly with peas. The LER was higher in the intercropping treatments, especially under limited N conditions. It is concluded that in the intercropping of wheat with legumes, the N2 fixation inhibition caused by soil N is effectively reduced, as well as there being a significant N transfer from the legume to the wheat, with both process contributing to increase LER.

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Introduction and aims: In the context of increasing population and decreasing soil fertility, food security is one of humanity's greatest challenges. Large amounts of waste, such as sewage sludge, are produced annually, with their final disposal causing environmental pollution and hazards to human health. Sludge has high amounts of nitrogen (N), and, when safely recycled by applying it into the soil as composted sewage sludge (CSS), its residual effect may provide gradual N release to crops. A field study was conducted in the Brazilian Cerrado. The aims were to investigate the residual effect of successive applications of CSS as a source of N in the common bean (Phaseolus vulgaris L. cv. BRS Estilo)-palisade grass (Urochloa brizantha (A.Rich.) R.D. Webster)-soybean (Glycine max L.) rotation under no-tillage. Additionally, N cycling was monitored through changes in N metabolism; the efficiency of biological N2 fixation (BNF) and its implications for plant nutrition, development, and productivity, was also assessed. Methods: The experiment consisted of a randomized complete block design comparing four CSS rates (10, 15, 20, and 25 Mg ha-1, wet basis) to a control treatment (without adding mineral or organic fertilizer) over two crop years. Multiple plant and soil analyses (plant development and crop yield, Falker chlorophyll index (FCI), enzymatic, biochemical, 15N natural abundance, was evaluated, root and shoot N accumulation, etc.) were evaluated. Results and discussion: Results showed that CSS: i) maintained adequate N levels for all crops, increasing their productivity; ii) promoted efficient BNF, due to the stability of ureide metabolism in plants and increased protein content; iii) increased the nitrate content and the nitrate reductase activity in soybean; iv) affected urease activity and ammonium content due to changes in the plant's urea metabolism; v) increased N accumulation in the aerial part of palisade grass. Composted sewage sludge can be used as an alternative source to meet crops' N requirements, promoting productivity gains and N cycling through forage and improving N metabolism.

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Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

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Biological nitrogen fixation is a key process for the maintenance of natural ecosystems productivity. In tropical forests, the contribution of asymbiotic nitrogen fixation (ANF) to the nitrogen (N) input has been underestimated, even though few studies have shown that ANF may be as important as symbiotic nitrogen fixation in such environments. The inputs and abiotic modulators of ANF in the Amazon forest are not completely understood. Here, we determined ANF rates and estimated the N inputs from ANF in the phyllosphere, litter and rhizospheric soil of nine tree species in the Amazon forest over time, including an extreme drought period induced by the El Niño-Southern Oscillation. Our data showed that ANF rates in the phyllosphere were 2.8- and 17.6-fold higher than in the litter and rhizospheric soil, respectively, and was highly dependent on tree taxon. Sampling time was the major factor modulating ANF in all forest compartments. At the driest period, ANF rates were approximately 1.8-fold and 13.1-fold higher than at periods with higher rainfall, before and after the extreme drought period, respectively. Tree species was a key modulator of ANF in the phyllosphere, as well as N and Vanadium concentrations. Carbon, molybdenum and vanadium concentrations were significant modulators of ANF in the litter. Based on ANF rates at the three sampling times, we estimated that the N input in the Amazon forest through ANF in the phyllosphere, litter and rhizospheric soil, was between 0.459 and 0.714 kg N ha-1 yr-1. Our results highlight the importance of ANF in the phyllosphere for the N input in the Amazon forest, and suggest that changes in the patterns of ANF driven by large scale climatic events may impact total N inputs and likely alter forest productivity.

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The use of plant growth-promoting bacteria as agricultural inoculants of plants should be encouraged because of their prominent role in biological nitrogen fixation, the increase of nutrient uptake by roots, abiotic stress mitigation, and disease control. The complex mechanisms underlying the association between plant and beneficial bacteria have been increasingly studied, and proteomic tools can expand our perception regarding the fundamental molecular processes modulated by the interaction. In this study, we investigated the changes in protein expression in maize roots in response to treatment with the endophytic diazotrophic Herbaspirillum seropedicae and the activities of enzymes related to nitrogen metabolism. To identify maize proteins whose expression levels were altered in the presence of bacteria, a label-free quantitative proteomic approach was employed. Using this approach, we identified 123 differentially expressed proteins, of which 34 were upregulated enzymes, in maize roots cultivated with H. seropedicae. The maize root colonization of H. seropedicae modulated the differential expression of enzymes involved in the stress response, such as peroxidases, phenylalanine ammonia-lyase, and glutathione transferase. The differential protein profile obtained in the inoculated roots reflects the effect of colonization on plant growth and development compared with control plants.

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The objective was to evaluate milk production, N2-fixation and N transfer, forage yield and composition (under two cutting intervals) in a silvopastoral system (SPS) with Leucaena leucocephala-Megathyrsus maximus and M. maximus-monoculture (MMM) with crossbred cows in a completely randomized design. Forage yield in the SPS was 6490 and 6907 kg DM ha-1 for cutting intervals (CI) of 35 and 50 days. Forage yield for the MMM was 7284 and 10,843 kg DM ha-1, and forage crude protein (CP) was 29.0% and 26.1% for L. leucocephala, harvested at 35 and 50 days, respectively. CP for the associated M. maximus was 9.9% and 7.8% for CI 35 and 50 days, respectively, and for MMM was 7.4% and 8.4%, harvested at 35 and 50 days. Milk production was 4.7 kg cow-1 day-1 for cows grazing MMM and 7.4 kg cow-1 day-1 under SPS. Nitrogen fixation in L. leucocephala (%Ndfa) was estimated to be 89% and 95%, at 35 and 50 days, with an N2 transfer to the associated grass of 34.3% and 52.9%. SPS has the potential to fix and transfer important amounts of N2 to the associated grass, and increase forage CP content and milk production.

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Due to diverse human activities zinc (Zn) may reach phytotoxic levels in the soil. Here, we evaluated the differential sensibility of three Brazilian tree species from the Fabaceae to increasing soil Zn concentrations and its physiological response to cope with excess Zn. A greenhouse experiment was conducted with the species: Mimosa caesalpiniaefolia, Erythrina speciosa and Schizolobium parahyba, and the addition of 0, 200, 400 and 600 mg Zn kg-1 to the soil. Plants were harvested after three months of cultivation, and growth, root symbiosis, biochemical markers and elemental composition were analyzed. Soil Zn addition reduced seedling growth, irrespective of the species, with a strong reduction in M. caesalpiniaefolia. Regarding root symbiosis, in N2-fixing species, nitrogenase activity was reduced by the highest Zn concentrations. Zn addition caused plants nutritional imbalances, mainly in roots. The content of photosynthetic pigments in leaves decreased up to 40%, suggesting that high Zn contents interfered with its biosynthesis, and altered the content of foliar polyamines and free amino acids, depending on the species and the soil Zn concentration. Zn toxicity in M. caesalpiniaefolia plants was observed at available soil Zn concentrations greater than 100 mg kg-1 (DTPA-extractable), being the most sensitive species and E. speciosa was moderately sensitive. S. parahyba was a moderately tolerant species, which seems to be related to polyamines accumulation and to mycorrhizal association. This last species has the potential for revegetation of areas with moderately high soil Zn concentration and for phytostabilization purposes. Future research evaluating the tolerance to multiple metal stress under field conditions should confirm S. parayba suitability in Zn contaminated areas of tropical regions.

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Soybean is the most important oilseed in the world, cropped in 120-130 million hectares each year. The three most important soybean producers are Argentina, Brazil, and United States, where soybean crops are routinely inoculated with symbiotic N2-fixing Bradyrhizobium spp. This extended inoculation gave rise to soybean-nodulating allochthonous populations (SNAPs) that compete against new inoculant for nodulation, thus impairing yield responses. Competitiveness depends on intrinsic factors contributed by genotype, extrinsic ones determined by growth and environmental conditions, and strain persistence in the soil. To assess these factors in Argentinean SNAPs, we studied 58 isolates from five sites of the main soybean cropping area. BOX-A1R DNA fingerprint distributed these isolates in 10 clades that paralleled the pHs of their original soils. By contrast, reference Bradyrhizobium spp. strains, including those used as soybean-inoculants, were confined to a single clade. More detailed characterization of a subset of 11 SNAP-isolates revealed that five were Bradyrhizobium japonicum, two Bradyrhizobium elkanii, two Rhizobium radiobacter (formerly Agrobacterium tumefaciens), one Bradyrhizobium diazoefficiens, and one Paenibacillus glycanilyticus-which did not nodulate when inoculated alone, and therefore was excluded from further characterization. The remaining subset of 10 SNAP-isolates was used for deeper characterization. All SNAP-isolates were aluminum- and heat-tolerant, and most of them were glyphosate-tolerant. Meanwhile, inoculant strains tested were sensitive to aluminum and glyphosate. In addition, all SNAP-isolates were motile to different degrees. Only three SNAP-isolates were deficient for N2-fixation, and none was intrinsically more competitive than the inoculant strain. These results are in contrast to the general belief that rhizobia from soil populations evolved as intrinsically more competitive for nodulation and less N2-fixing effective than inoculants strains. Shoot:root ratios, both as dry biomass and as total N, were highly correlated with leaf ureide contents, and therefore may be easy indicators of N2-fixing performance, suggesting that highly effective N2-fixing and well-adapted strains may be readily selected from SNAPs. In addition, intrinsic competitiveness of the inoculants strains seems already optimized against SNAP strains, and therefore our efforts to improve nodules occupation by inoculated strains should focus on the optimization of extrinsic competitiveness factors, such as inoculant formulation and inoculation technology.

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Efficient N2-fixing Leguminosae nodulating bacteria resistant to As may facilitate plant growth on As-contaminated sites. In order to identify bacteria holding these features, 24 strains were isolated from nodules of the trap species Crotalaria spectabilis (12) and Stizolobium aterrimum (12) growing on an As-contaminated gold mine site. 16S rRNA gene sequencing revealed that most of the strains belonged to the group of α-Proteobacteria, being representatives of the genera Bradyrhizobium, Rhizobium, Inquilinus, Labrys, Bosea, Starkeya, and Methylobacterium. Strains of the first four genera showed symbiotic efficiency with their original host, and demonstrated in vitro specific plant-growth-promoting (PGP) traits (production of organic acids, indole-3-acetic-acid and siderophores, 1-aminocyclopropane-1-carboxylate deaminase activity, and Ca3(PO4)2 solubilization), and increased resistance to As, Zn, and Cd. In addition, these strains and some type and reference rhizobia strains exhibited a wide resistance spectrum to ß-lactam antibiotics. Both intrinsic PGP abilities and multi-element resistance of rhizobia are promising for exploiting the symbiosis with different legume plants on trace-element-contaminated soils.

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Plants on contaminated mining soils often show a reduced growth due to nutrient depletion as well as trace elements (TEs) toxicity. Since those conditions threat plant's survival, plant growth-promoting rhizobacteria (PGPRs), such as rhizobia, might be of crucial importance for plant colonization on TE-contaminated soils. Native rhizobia from mining soils are promising candidates for bioaugmented phytoremediation of those soils as they are adapted to the specific conditions. In this work, rhizobia from Zn- and Cd-contaminated mining soils were in vitro screened for their PGP features [organic acids, indole-3-acetic acid (IAA), and siderophore (SID) production; 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity; and Ca3(PO4)2 solubilization] and Zn and Cd tolerance. In addition, some type and reference rhizobia strains were included in the study as well. The in vitro screening indicated that rhizobia and other native genera have great potential for phytoremediation purposes, by exerting, besides biological N2 fixation, other plant growth-promoting traits. Leucaena leucocephala-Mesorhizobium sp. (UFLA 01-765) showed multielement tolerance and an efficient symbiosis on contaminated soil, decreasing the activities of antioxidative enzymes in shoots. This symbiosis is a promising combination for phytostabilization.

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We review waterlogging and submergence tolerances of forage (pasture) legumes. Growth reductions from waterlogging in perennial species ranged from >50% for Medicago sativa and Trifolium pratense to <25% for Lotus corniculatus, L. tenuis, and T. fragiferum. For annual species, waterlogging reduced Medicago truncatula by ~50%, whereas Melilotus siculus and T. michelianum were not reduced. Tolerant species have higher root porosity (gas-filled volume in tissues) owing to aerenchyma formation. Plant dry mass (waterlogged relative to control) had a positive (hyperbolic) relationship to root porosity across eight species. Metabolism in hypoxic roots was influenced by internal aeration. Sugars accumulate in M. sativa due to growth inhibition from limited respiration and low energy in roots of low porosity (i.e. 4.5%). In contrast, L. corniculatus, with higher root porosity (i.e. 17.2%) and O2 supply allowing respiration, maintained growth better and sugars did not accumulate. Tolerant legumes form nodules, and internal O2 diffusion along roots can sustain metabolism, including N2 fixation, in submerged nodules. Shoot physiology depends on species tolerance. In M. sativa, photosynthesis soon declines and in the longer term (>10 d) leaves suffer chlorophyll degradation, damage, and N, P, and K deficiencies. In tolerant L. corniculatus and L. tenuis, photosynthesis is maintained longer, shoot N is less affected, and shoot P can even increase during waterlogging. Species also differ in tolerance of partial and complete shoot submergence. Gaps in knowledge include anoxia tolerance of roots, N2 fixation during field waterlogging, and identification of traits conferring the ability to recover after water subsides.

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The bacterial genus Burkholderia comprises species occupying several habitats, including a group of symbionts of leguminous plants-also called beta-rhizobia-that has been recently ascribed to the new genus Paraburkholderia We used common bean (Phaseolus vulgaris L.) plants to trap rhizobia from an undisturbed soil of the Brazilian Cerrado under the vegetation type 'Cerradão'. Genetic characterization started with the analyses of 181 isolates by BOX-PCR, where the majority revealed unique profiles, indicating high inter- and intra-species diversity. Restriction fragment length polymorphism-PCR of the 16S rRNA of representative strains of the BOX-PCR groups indicated two main clusters, and gene-sequencing analysis identified the minority (27%) as Rhizobium and the majority (73%) as Paraburkholderia Phylogenetic analyses of the 16S rRNA and housekeeping (recA and gyrB) genes positioned all strains of the second cluster in the species P. nodosa, and the phylogeny of a symbiotic gene-nodC-was in agreement with the conserved genes. All isolates were stable vis-à-vis nodulating common bean, but, in general, with a low capacity for fixing N2, although some effective strains were identified. The predominance of P. nodosa might be associated with the edaphic properties of the Cerrado biome, and might represent an important role in terms of maintenance of the ecosystem, which is characterized by acid soils with high saturation of aluminum and low N2 content.

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Symbiotic nitrogen fixation is a process of considerable economic, ecological and scientific interest. The central enzyme nitrogenase reduces H(+) alongside N2 , and the evolving H2 allows a continuous and non-invasive in vivo measurement of nitrogenase activity. The objective of this study was to show that an elaborated set-up providing such measurements for periods as long as several weeks will produce specific insight into the nodule activity's dependence on environmental conditions and genotype features. A system was developed that allows the air-proof separation of a root/nodule and a shoot compartment. H2 evolution in the root/nodule compartment can be monitored continuously. Nutrient solution composition, temperature, CO2 concentration and humidity around the shoots can concomitantly be maintained and manipulated. Medicago truncatula plants showed vigorous growth in the system when relying on nitrogen fixation. The set-up was able to provide specific insights into nitrogen fixation. For example, nodule activity depended on the temperature in their surroundings, but not on temperature or light around shoots. Increased temperature around the nodules was able to induce higher nodule activity in darkness versus light around shoots for a period of as long as 8 h. Conditions that affected the N demand of the shoots (ammonium application, Mg or P depletion, super numeric nodules) induced consistent and complex daily rhythms in nodule activity. It was shown that long-term continuous measurements of nodule activity could be useful for revealing special features in mutants and could be of importance when synchronizing nodule harvests for complex analysis of their metabolic status.

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This study was carried out to establish comparative effects of drought and recovery on the nitrate assimilation and nodule activity related to N2 fixation in cowpea plants [Vigna unguiculata L. (Walp.)] previously inoculated with Bradyrhizobium spp. BR-3256 (CB-756) strain in the presence of 5 mol m-3 NO-3. Twenty-eight-day-old nodulated plants were submitted to water deprivation during 4 consecutive days and afterwards resupplied with nutrient solution during 2 days. The water deprivation caused a rapid increase in the nitrate content in root and a marked reduction in leaf nitrate reductase (NR) activity. In contrast nodule NR activity was slightly increased by water deprivation. Concomitantly, in nodules of water stressed plants, leghemoglobin and glutamine synthetase (GS) activity declined and a progressive reduction in ureide-N concentration in xylem sap was observed. Leaf-NR activity increased rapidly after rehydration while leaf nitrate content declined. In contrast both GS activity and soluble protein content in the nodule continued to decline in rewatered plants. In addition the concentration of leghemoglobin recovered well, while the xylem ureide-N content experienced a slight increase after rehydration. Despite the nitrate assimilation in leaves and the nodule activity had been both severely affected by water stress, the rapid recovery of nitrate reductase activity suggests that the nitrate assimilation process is less sensitive to drought/rehydration cycle when cowpea plants are nodulated in presence of moderate nitrate level.

Este estudo foi feito com a finalidade de estabelecer efeitos comparativos da seca e da reidratação na assimilação do nitrato e atividade do nódulo relacionado com a fixação de N2 em planta de caupi [Vigna unguiculata L. (Walp.)] previamente inoculada com Bradyrhizobium spp., estirpe BR-3256 (CB-756), na presença de NO-3 (5 mol m-3). Aos 28 dias após a emergência, as plantas noduladas foram submetidas à seca, durante 4 dias sucessivos e, depois, reidratadas com solução nutritiva durante 2 dias. A seca causou um aumento rápido no conteúdo de nitrato da raiz e uma acentuada redução na atividade da redutase do nitrato de folhas (NR). Em contraste, no nódulo esta atividade foi aumentada ligeiramente pelo déficit de água. Concomitantemente, nos nódulos das plantas estressadas, observou-se uma redução progressiva na concentração de leghemoglobina, atividade de glutamina sintetase (GS) e na concentração de ureídeos na seiva do xilema. A atividade da NR nas folhas aumentou rapidamente após a reidratação enquanto que o conteúdo de nitrato da mesma decresceu. Em contraste, a atividade de GS e a concentração de proteínas solúveis nos nódulos continuaram diminuindo nas plantas reidratadas. A concentração de leghemoglobina apresentou uma boa recuperação, enquanto que o conteúdo de ureídeos sofreu um leve aumento após a reidratação. Apesar da assimilação de nitrato em folhas e a atividade do nódulo terem sido severamente afetados pelo déficit de água, a rápida recuperação da atividade de redutase do nitrato nas folhas sugere que o primeiro processo seja menos susceptível ao ciclo de seca/reidratação quando plantas de caupi são noduladas em presença de nível moderado de nitrato.

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The suitability of the natural 15N abundance and of total N concentration of leaves as indicators of the type of plant N nutrition in a rain forest of French Guiana were tested. Leaf samples from primary legume species, non-legumes (pioneer species) and from the non-N2-fixing species Dicorynia guianensis were analyzed. Both δ15N and total leaf N varied widely (-1 ?δ15N (‰) ? 7 and 1 ? leaf N(%) ? 3.2) suggesting possible distinctions between diazotrophic and non-fixing plants. The δ15N also revealed two statistically distinct groups of non-N2-fixing species (δ15N = 5.14 ± 0.3 vs δ15N = 1.65 ± 0.17) related to the different ecological behaviors of these species in the successional processes. We conclude that the δ15N signature of plant leaves combined with their total N concentration may be relevant indicators for identifying functional groups within the community of non-N2-fixing species, as well as for detecting diazotrophy. Despite the variability in the δ15N of the non-N2-fixing species, N2-fixing groups can still be identified, provided that plants are simultaneously classified taxonomically, by their leaf δ15N and total N concentration and by the presence or absence of nodules. The variability in the δ15N of the non-fixing species is discussed.

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We report on the structure of N2 -fixing nodules formed on the stem of Discolobium pulchellum Benth., an aquatic legume in the subfamily Pupilionoideae, tribe Aeschynomeneae, from the Hooded areas in the 'Pantanal Matogrossense' region of Brazil. The stern (and root) nodules were obligately aquatic, requiring permanent submergence in water or flooded soil, and receive oxygen via profuse aerenchyma covering The lower stem. Of the 69 isolates of rhizobia isolated from stem and root nodules, 70% were fast-growing acid producers and 38% were slow growers. The rhizobia were not photosynthetic. Nodules were connected to the stem, and the vascular system from the stem branched throughout the nodule, penetrating the infected, tissue within finger-like ingrowths of cortex. In both stem and root nodules, infected tissue was aeschynomenoid or desmodioid, that is, without uninfected (interstitial) cells. The infected cells in stem nodules were vacuolate, with visible infection threads. The inner cortex was rich in amyloplasts and contained the components of an oxygen diffusion barrier (a boundary cell layer without intercellular spaces and glycoprotein occlusions of intercellular spaces in other cell layers). The mid-cortex, external to the boundary layer, consisted of loosely-packed cells and these were continuous with stem aerenchyma. The outer part of the nodules was made up of phellogen-derived cells forming a periderm, or 'corky' layer of cells. The periderm formed large lenticels above cortical vascular bundles. These lenticels also connected with the stem aerenchyma. Root nodules differed only in that infected cells were not vacuolate, bacteroids were larger and contained more poly-ß-hydroxybutyrate (PHB) and there was less aerenchyma/lenticellular tissue. Stem and root nodule structure is discussed in terms of adaptations to O2 constraints in an aquatic environment.