Suppression of nitrification and nitrous oxide emission by the tropical grass Brachiaria humidicola

Nitrification by soil nitrifiers may result in substantial losses of applied nitrogen through NO₃ ^– leaching and N₂O emission. The biological inhibition of nitrification by crop plants or pasture species is not well known. This study was conducted to evaluate the ability of three pasture species, Brachiaria humidicola, B. decumbens and Melinis minutiflora to inhibit nitrification. Plants were grown in a growth chamber for sixty days, fertilized with (NH₄)₂SO₄. After harvesting, the soil was incubated with (NH₄)₂SO₄ for 24 days. Ammonium oxidizing bacteria (AOB), NH₄-N levels, and N₂O emission were monitored at 4 d intervals. Among the species studied, B. humidicola inhibited nitrification and maintained NH₄-N in soil to a much greater extent than the other two species. This nitrification inhibition lasted for 12 days after initiation of soil incubation study (i.e. from 60 DAS when the plants were harvested). The AOB populations and N₂O emission from the soil were significantly lower in the soils where B. humidicola has been grown compared to the other two species. Root exudates and soil extracts of B. humidicola suppressed AOB populations, whereas those of B. decumbens and M. minutiflora did not. The results are in consistence with the hypothesis that B. humidicola suppressed nitrification and N₂O emissions through an inhibitory effect on the AOB population.     

Dinitrogen fixation (Acetylene reduction) and nitrogen accumulation in peas cultivated under the standard growth conditions

InPisum sativum cultivated under standard growth conditions the extent of N₂ fixation with time estimated by the acetylene reduction assay (PN₂F) and rates of the actual nitrogen accumulation of plant biomass (ANA) were calculated from six independent growth experiments. In the plants inoculated with indigenous soilRhizobium populations and cultivated on 0.63 mmol/L nitrate level the percentage PN₂F:ANA ratios ranged from 25.7 to 61.5%. In peas inoculated with the inoculant strain the PN₂F:ANA ratios were markedly higher, ranging from 59.8 to 65.1%. The plants cultivated on N-free nutrient solutions showed both PN₂F:ANA and C₂H₄N₂ ratios to be somewhat higher compared with the 0.63 mmol/L nitrate cultivated plants.     

The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation

All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N₂ for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N₂ in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N₂‐fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species‐dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N₂, RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N₂‐fixing ability of the rhizobia strains.     

No evidence for adaptation to local rhizobial mutualists in the legume Medicago lupulina

Local adaptation is a common but not ubiquitous feature of species interactions, and understanding the circumstances under which it evolves illuminates the factors that influence adaptive population divergence. Antagonistic species interactions dominate the local adaptation literature relative to mutualistic ones, preventing an overall assessment of adaptation within interspecific interactions. Here, we tested whether the legume Medicago lupulina is adapted to the locally abundant species of mutualistic nitrogen‐fixing rhizobial bacteria that vary in frequency across its eastern North American range. We reciprocally inoculated northern and southern M. lupulina genotypes with the northern (Ensifer medicae) or southern bacterium (E. meliloti) in a greenhouse experiment. Despite producing different numbers of root nodules (the structures in which the plants house the bacteria), neither northern nor southern plants produced more seeds, flowered earlier, or were more likely to flower when inoculated with their local rhizobia. We then used a pre‐existing dataset to perform a genome scan for loci that showed elevated differentiation between field‐collected plants that hosted different bacteria. None of the loci we identified belonged to the well‐characterized suite of legume–rhizobia symbiosis genes, suggesting that the rhizobia do not drive genetic divergence between M. lupulina populations. Our results demonstrate that symbiont local adaptation has not evolved in this mutualism despite large‐scale geographic variation in the identity of the interacting species.     

Multi‐symbiotic systems: functional implications of the coexistence of grass–endophyte and legume–rhizobia symbioses

The coexistence of symbionts with different functional roles in co‐occurring plants is highly probable in terrestrial ecosystems. Analyses of how plants and microbes interact above‐ and belowground in multi‐symbiotic systems are key to understand community structure and ecosystem functioning. We performed an outdoor experiment in mesocosms to investigate the consequences of the interaction of a provider belowground symbiont of legumes (nitrogen‐fixing bacteria) and a protector aerial fungal symbiont of grasses (Epichloё endophyte) on nitrogen dynamics and aboveground net primary productivity. Four plants of Trifolium repens (Trifolium, a perennial legume) either inoculated or not with Rhizobium leguminosarum, grew surrounded by 16 plants of Lolium multiflorum (Lolium, an annual grass), with either low or high levels of the endophyte Neotyphodium occultans. After five months, we quantified the number of nodules in Trifolium roots, shoot biomass of both plant species, and the contribution of atmospheric nitrogen fixation vs. soil nitrogen uptake to above ground nitrogen in each plant species. The endophyte increased grass biomass production (+ 16%), and nitrogen uptake from the soil – the main source for the grass. Further, it reduced the nodulation of neighbour Trifolium plants (?50%). Notably, due to a compensatory increase in nitrogen fixation per nodule, this reduced neither its atmospheric nitrogen fixation – the main source of nitrogen for the legume – nor its biomass production, both of which were doubled by rhizobial inoculation. In consequence, the total amount of nitrogen in aboveground biomass and aboveground productivity were greatest in mesocosms with both symbionts (i.e. high rhizobia + high endophyte). These results show that, in spite of the deleterious effect of the endophyte on the establishment of the rhizobia–legume symbiosis, the coexistence of these symbionts, leading to additive effects on nitrogen capture and aboveground productivity, can generate complementarity on the functioning of multi‐symbiotic systems.     

Effective nodulation of rhamnaceous actinorhizal plants induced by air dry soils

Air dry soil samples stored at room temperature for more than one and a half years, were used as inocula for actinorhizal plants. Seedlings of Colletia paradoxa, Discaria americana, D. articulata and D. trinervis (Rhamnaceae) cultivated in nitrogen-free nutrient solution, were inoculated by adding dry soil to the solution. All the soil samples tested were able to induce nodulation, showing the presence of infective propagules of Frankia. Healthy growth of nodulated plants suggested the occurrence of nitrogen fixation.     

Effects of inoculation ofPoa pratensis andTriticum aestivum with root-associated,N2-fixing Klebsiella,Enterobacter and Azospirillum

Strains ofKlebsiella pneumoniae, Klebsiella terrigena, Enterobacter agglomerans andAzospirillum lipoferum were compared as diazotrophic inoculants in association withPoa pratensis andTriticum aestivum. Each strain colonized both plants in numbers ranging from 10⁴ to 10⁷ bacteria per root, and electron microscopy and immunofluorescence staining of inoculated roots revealed bacteria mainly on root hairs. Indirect immunofluorescence with specific antifimbriae antibodies showed that the enteric bacteria expressed their fimbria in both associations. All associations were positive in an acetylene reduction test but only in half of them was atmospheric nitrogen transferred to the plant. In the inoculated plants, variable effects in the dry matter and N yields in both hosts were observed and no correlation was found between dry matter, nitrogen content or the amount of fixed nitrogen. In infected plants, the number of root hairs and lateral roots increased and the length of the zone of elongation decreased. The changes in root morphology were more evident in associations with enteric bacteria than with Azospirillum. The results give further evidence on the importance of bacterial adhesion in associative N₂ fixation and suggest that bacteria-induced physiological changes in plant roots may be more important than the amount of nitrogen transferred to the plant.     

Arbuscular Mycorrhizal Fungi and Arsenate Uptake by Brachiaria Grass (Brachiaria decumbens)

ABSTRACT Using a compartmentalized treatment technique, the role of arbuscular mycorrhizal fungi (AMF; Acaulospora scrobiculata) on arsenic (As) uptake and translocation in Brachiaria decumbens. Treatments consisted of a factorial arrangement of three As doses (0, 50, and 100 mg kg^?1) and the presence/absence of AMF inoculates. In the absence of AMF, B. decumbens did not show As accumulation, indicating the probable presence of tolerance mechanism via As exclusion by the roots. B. decumbens plants showed high AMF colonization levels, especially in the arsenic treatments, with AMF improving shoot and root growth independent of As concentrations. Arsenic accumulation occurred only with AMF inoculation. Phosphorous uptake was reduced in B. decumbens roots in the presence of arsenic with and without inoculation of AMF. Results suggest that B. decumbens can be used in phytoremediation procedures when inoculated with A. scrobiculata, although pasture formation should be strictly avoided in contaminated sites.     

Aluminum tolerance measured by root growth and mucilage protection in Urochloa brizantha and Urochloa decumbens

Aluminum toxicity on root systems was analyzed through comparing root growth and evaluating the protective function of mucilage in Urochloa decumbens and Urochloa brizantha. Seedlings were grown in a solution with different concentrations of AlCl₃ and with mucilage removed or present. The root elongation rate, total length, number of roots and presence of aluminum at the root apex were measured. Root development was inhibited by aluminum and the elongation rate was maintained without any difference between the two species. A significant reduction in root length was found in U. brizantha. Aluminum did not influence root branching in either species and accumulated mostly in the rhizosphere of U. brizantha, where the mucilage has less of a protective function. The greatest aluminum tolerance was found in U. decumbens, observed through maintenance of total root system growth from lower to higher aluminum toxicity.     

Inoculation of hybrid poplar with the endophytic bacterium Enterobacter sp. 638 increases biomass but does not impact leaf level physiology

Endophytic bacteria have been shown to provide several advantages to their host, including enhanced growth. Inoculating biofuel species with endophytic bacteria is therefore an attractive option to increase the productivity of biofuel feedstocks. Here, we investigated the effect of inoculating hard wood cuttings of Populus deltoides Bartr. × Populus. nigra L. clone OP367 with Enterobacter sp. 638. After 17 weeks, plants inoculated with Enterobacter sp. 638 had 55% greater total biomass than un‐inoculated control plants. Study of gas exchange and fluorescence in developing and mature leaves over a diurnal cycle and over a 5 week measurement campaign revealed no effects of inoculation on photosynthesis, stomatal conductance, photosynthetic water use efficiency or the maximum and operating efficiency of photosystem II. However, plants inoculated with Enterobacter sp. 638 had a canopy that was 39% larger than control plants indicating that the enhanced growth was fueled by increased leaf area, not by improved physiology. Leaf nitrogen content was determined at two stages over the 5 week measurement period. No effect of Enterobacter sp. 638 on leaf nitrogen content was found indicating that the larger plants were acquiring sufficient nitrogen. Enterobacter sp. 638 lacks the genes for N₂ fixation, therefore the increased availability of nitrogen likely resulted from enhanced nitrogen acquisition by the 84% larger root system. These data show that Enterobacter sp. 638 has the potential to dramatically increase productivity in poplar. If fully realized in the production environment, these results indicate that an increase in the environmental and economic viability of poplar as a biofuel feedstock is possible when inoculated with endophytic bacteria like Enterobacter sp. 638.     

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a Threatened Species Native to the Brazilian Cerrado

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N₂-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N₂ fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N₂-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.     

Acetobacter diazotrophicus,an indoleacetic acid producing bacterium isolated from sugarcane cultivars of México

Thirteen cane cultivars grown on fields in México were sampled to assess the occurrence of Acetobacter diazotrophicus, a recently identified N₂-fixing bacterium. Results showed that the isolation frequencies extended over a broad range (1.1 to 67%), likely to be related to the nitrogen fertilization level. The lowest isolation frequencies (1.1 to 2.5%) were obtained from plants growing at high nitrogen doses (275–300 kg ha^-1) and the highest values (10–67%) from plants cultivated with 120 kg N ha^-1. All eighteen strains of A. diazotrophicus produced indoleacetic acid (IAA) in defined culture medium. Estimates obtained from HPLC analyses revealed that A. diazotrophicus strains produced from 0.14 to 2.42 g IAA mL^-1 in culture medium. Considering that A. diazotrophicus is found within the plant tissue, the biosynthesis of IAA suggests that the bacteria could promote rooting and improve sugarcane growth by direct effects on metabolic processes, in addition to their role in N₂ fixation.     

Competition for alfalfa nodulation under metal stress by the metal‐tolerant strain Ochrobactrum cytisi Azn6.2

Legume plants, in association with rhizobia, are gaining increasing interest for heavy metal rhizoremediation. This symbiotic interaction combines the advantages of rhizoremediation and soil nitrogen enrichment. In metal polluted soils, Ochrobactrum cytisi can elicit non‐fixing nodules on legumes, including Medicago sativa. Nodulation kinetics was much slower when M. sativa plants were inoculated with O. cytisi Azn6.2 compared with the natural symbiont Ensifer meliloti 1021 and nodules were ineffective in nitrogen fixation. A competition experiment was performed using alfalfa grown on heavy metals, and co‐inoculated with equal amounts of the metal‐sensitive E. meliloti 1021 and the metal‐resistant O. cytisi Azn6.2. When plants were inoculated in non‐polluted substrates, all nodules were formed by E. meliloti 1021. Nevertheless, under increasing metal concentrations, the number of nodules occupied by O. cytisi grew. At the highest metal concentration, all nodules were elicited by O. cytisi, suggesting that the resistant species can take the place of the natural symbiont. This fact has important ecological and environmental implications when proposing legume–rhizobia symbioses for rhizoremediation and highlights the need of selecting highly resistant rhizobia in order to be competitive in polluted soils.     

Tomato Growth in Soil Amended with Sugar Mill By-Products Compost

Sugar mill by-products compost may be a good soil amendment to promote tomato (Lycopersicon esculentum L.) growth. In addition, the compost may further promote plant growth by inoculation with N₂-fixing bacteria. Compost from sugar-mill waste was prepared with and without the N₂-fixing bacteria, Azotobacter vinelandii, Beijerinckia derxii and Azospirillum sp. and incubated for 50 days. Each compost type was added to 10 kg of soil in pots at rates of 0, 15, and 45 g with and without fertilizer N at rates of 0, 0.75, and 1.54 g. A blanket application of P and K was applied to all pots. Shoot and root dry weights and N content of the whole plant was measured at 55 days. Dry weight of tomato shoots was increased by 40% by addition of fertilizer N and root weight was increased by 66%. Without fertilizer N the high rate of inoculated compost increased shoot growth 180% and uninoculated compost increased shoot growth 112%. For most treatments with and without fertilizer N, inoculated compost enhanced shoot growth and nitrogen content more than uninoculated compost. Root weights were nearly doubled by addition of either compost in comparison to the 0 N treatment. At the low rate of compost addition without fertilizer N, root weight was the same for uninoculated and inoculated compost but at the high rate of compost addition root weight was 32% higher for inoculated compost. The N₂-fixing bacteria colonized roots when inoculated compost was used. Sugar mill by-products compost proved to be an effective soil amendment for promoting the growth of tomato plants.     

Control of symbiotic nitrogen fixation in Rhizobia regulation of NH4 assimilation

The metabolic fate of gaseous nitrogen (¹⁵N₂) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N₂-fixation system was followed. A majority of the fixed ¹⁵N₂ was found to be exported into the cell supernatant. For example, as much as 94% of the ¹⁵N₂ fixed by Rhizobium japonicum (soybean symbiont) was recovered as ¹⁵NH₄⁺ from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH₄⁺ from l-histidine. Evidence is presented that overproduction and export of NH₄⁺ by free-living Rhizobia may be closely linked to the control of several key enzymes of NH₄⁺ assimilation. For instance, NH₄⁺ was found to repress glutamine synthetase whereas l-glutamate repressed glutamate synthase. Assimilation of NH₄⁺ as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH₄⁺ production by root nodule bacteria is discussed.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Rhizobial-induced increase in internode length and identification of endogenous GAs of cowpea (Vigna unguiculata [L.] Walp) stems and nodules

Inoculation with Bradyrhizobium sp. strain 127E14 has been shown to cause a dramatic increase in the internode length of lima bean (Phaseolus lunatus L.), when compared to control plants inoculated with strain 127E15. This rhizobial-induced growth also occurs in cowpea (Vigna unguiculata [L.] Walp), an alternate host for the symbiont. Cowpea plants inoculated with strain 127E14 were 23% taller than those inoculated with strain 127E14 after 6 weeks of growth. Petiole length was found to be significantly greater in plants inoculated with strain 127E14. Cowpea plants treated at the apex with exogenous GA₃ or GA_4/7 responded by increasing internode length when compared to controls. As in lima beans, the rhizobial-induced growth response observed in cowpeas may be in response to an imbalance in the levels of GA-like substances within the plants. Gibberellins A₁, A₃, A₈, A₁₉, A₂₀, A₂₉, and A₄₄have been identified by GC-MS analysis in stems of cowpea, whereas the gibberellins A₁, A₁₉, A₂₀, A₂₉, and A₄₄ were found to be present in nodule tissue formed by strain 127E14. The presence of these GAs indicates that the early 13-hydroxylation biosynthetic pathway is operative in cowpea. GAs identified in cowpea nodules are similar to those found in lima bean nodules formed by the same rhizobia. The finding that rhizobial strain 127E14 induces GA-dependent growth responses in two host legumes further supports the hypothesis that the presence of this bacteria alters the GA balance within the plant.     

Differential growth costs and nitrogen fixation in Cytisus multiflorus (L′Hér.) Sweet and Cytisus scoparius (L.) Link are mediated by sources of inorganic N

• Shrubby legumes in Mediterranean‐type ecosystems face strong nutrient limitations that worsen in summer, when water is absent. Nitrogen‐fixing legumes are likely to be able to switch between soil N and atmospheric N (N₂) sources to adjust the C costs of N acquisition in different seasons.
• We investigated the utilisation of different inorganic N sources by two indigenous shrubby legumes (Cytisus multiflorus and Cytisus scoparius). Plant performance in terms of photosynthesis and biomass production was also analysed. Plants were cultivated in sterile river sand supplied with Hoagland nutrient solution, grown in N‐free nutrient solution and inoculated with effective rhizobial strains from nodules of adult plants of the same species. A second treatment consisted of plants given 500 μm NH₄NO₃ added into the nutrient solution. In a third treatment, plants were watered with another source of N (500 μm NH₄NO₃) as well as being inoculated with effective rhizobial strains.
• The application of NH₄NO₃ to the legumes resulted in a larger increase in plant dry matter. Carbon construction costs were higher in plants supplied with mineral and symbiotic N sources and always higher in the endemic C. multiflorus. Differences in photosynthesis rates were only observed between species, regardless of the N source. Non‐fertilised inoculated plants had more effective root nodules and a clear dependence on N₂ fixation.
• We propose that the ability of C. scoparius to change N source makes it a plastic species, which would account for its broader distribution in nature.

     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

Nitrogenase Inhibition in Nodules from Pea Plants Grown Under Salt Stress Occurs at the Physiological Level and can be Alleviated by B and Ca

In order to shed new light on the mechanisms of salt-mediated symbiotic N₂-fixation inhibition, the effect of salt stress (75 mM) on N₂-fixation in pea root nodules induced by R. leguminosarum was studied at the gene expression, protein production and enzymatic activity levels. Acetylene reduction assays for nitrogenase activity showed no activity in salt-stressed plants. To know whether salt inhibits N₂-fixing activity at a molecular or at a physiological level, expression of the nifH gene, encoding the nitrogenase reductase component of the nitrogenase enzyme was analyzed by RT-PCR analysis of total RNA extracted from nodulated roots. The nifH messenger RNA was present both in plants grown in the presence and absence of salt, although a reduction was observed in salt-stressed plants. Similar results were obtained for the immunodetection of the nitrogenase reductase protein in Western-blot assays, indicating that nitrogen fixation failed mainly at physiological level. Given that nutrient imbalance is a typical effect of salt stress in plants and that Fe is a prosthetic component of nitrogenase reductase and other proteins required by symbiotic N₂-fixation, as leghemoglobin, plants were analyzed for Fe contents by atomic absorption and the results confirmed that Fe levels were severely reduced in nodules developed in salt-stressed plants. In a previous papers (El-Hamdaoui et al., 2003b), we have shown that supplementing inoculated legumes with boron (B) and calcium (Ca) prevents nitrogen fixation decline under saline conditions stress. Analysis of salt-stressed nodules fed with extra B and Ca indicated that Fe content and nitrogenase activity was similar to that of non-stressed plants. These results indicate a linkage between Fe deprivation and salt-mediated failure of nitrogen fixation, which is prevented by B and Ca leading to increase of salt tolerance.