Effect of different levels of mineral nitrogen on nodulation and N2 fixation of two cultivars of common bean (Phaseolus vulgaris L.)

An experiment was conducted under greenhouse conditions to evaluate the effect of mineral nitrogen on N₂ fixation of two cultivars of Phaseolus vulgaris L., Puebla 152 and Negro Argel. Nitrogen application was 0, 2.5, 12.5 and 25 mg N Kg^–1 of a vermiculite-sand-mixture at planting time. Shoot and root growth were elevated by nitrogen application at all growth stages. During vegetative growth (V 5) nodule dry weight and nitrogenase activity (acetylene reducing activity) per plant were reduced by nitrogen supply in both cultivars, but less in Negro Argel than in Puebla 152. At later stages nodulation in nitrogen-treated Puebla 152 did not differ from that in non-treated plants, whereas increased nodule number was found in Negro Argel at high nitrogen levels. The influence of mineral N on the total amount of nitrogen fixed in the two bean cultivars was only slightly different.     

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

Summary A field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha^–1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha^–1 of¹⁵N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N₂ fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N₂ fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha^–1 respectively. These two cultivars produced 991 and 883 kg ha^–1 of grain, respectively, when inoculated and 663 and 620 kg ha^–1 with the addition of 100 kg N ha^–1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.     

Selection for increased nodule number in common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) plants were grown for 21–28 days in plastic container-modified Leonard jar assemblies and placed in a controlled-environment room. The nodules on each plant were removed, counted; selected plants were repotted, grown and intercrossed to produce progenies for the next cycle of recurrent selection. Among the ten parent lines, Puebla 152 and WBR 22–34 produced the most nodules and Rio Tibagi and Negro Argel the fewest, when averaged over five experiments. An analysis of number of nodules on F₁ plants resulting from crosses made in a partial diallel design among the ten parents revealed highly significants variation for general combining ability (GCA) but not for specific combining ability (SCA). After three cycles of recurrent selection for nodule number per plant, the mean nodule number was 211% of the mean for the 10 parents control. Total nodule weight on selected plants also increased, but individual nodule weight decreased. Nineteen C₁ and 18 C₂ lines resulting from the individual plants selected for greater nodule number, along with the ten parents and two non-nodulating soybean lines included as non-fixing check plants were grown in a single experiment in a low-N field. C₂ lines on average accumulated significantly more N per plant than either the parents or C₁ lines, providing evidence for increased N₂ fixation measured by the N difference method. These data show that more nodules, possibly resulting from greater susceptibility to nodulation, are an important, heritable component of symbiosis and that selection for increased nodule number resulted in lines capable of fixing more atmospheric N₂.     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.     

The influence of nitrate supply on nitrogen fixation during growth of the field bean Vicia faba in sand

Field bean (Vicia faba L.) cv. Maris Bead seeds were inoculated with Rhizobium Catalogue No. 1001, supplied by Rothamsted Experimental Station and grown in sand culture supplied with ¹⁵N-labelled nitrate at two concentrations. Plants were sampled at intervals throughout their growth for ¹⁵N and total N analysis. The rate of nitrate uptake was almost uniform up to pod-fill and was proportional to the nitrate concentration. Nodule weight was slightly depressed by the larger nitrate concentration at all samplings, and there was a corresponding reduction in the amount of atmospheric nitrogen fixed. However, at harvest the bean seeds from plants given most nitrate contained slightly more total N, as the enhanced nitrate uptake outweighed the reduction in fixation.     

Differential effects of mineral and organic N sources,and of ectomycorrhizal infection by Hebeloma cylindrosporum,on growth and N utilization in Pinus pinaster

The effect of ectomycorrhizal association of Pinus pinaster with Hebeloma cylindrosporum was investigated in relation to the nitrogen source supplied as mineral (NH₄⁺ or NO₃^?) or organic N (L ‐glutamate) and at 5 mol m^?3. Plants were grown for 14 and 16 weeks with mineral and organic N, respectively, and samples were collected during the last 6 weeks of culture. Total fungal biomass was estimated using glucosamine amount and its viability was assessed using the glucosamine to ergosterol ratio. Non‐mycorrhizal plants grew better with NH₄⁺ than with NO₃^? and grew very slowly when supplied with L ‐glutamate. The presence of the fungus decreased the growth of the host plant with mineral N whereas it increased it with L ‐glutamate. Whatever the N source, most of the living fungal biomass was associated with the roots, whereas the main part of the total biomass was assayed outside the root. The form of mineral N did not significantly affect N accumulation rates over the 42 d in control plants. In mycorrhizal plants grown on either N source, the fungal tissues developing outside of the root were always the main N sink. The ectomycorrhizal association did not change ¹⁵NH₄⁺ uptake rate by roots, suggesting that the growth decrease of the host‐plant was related to the carbon cost for fungal growth and N assimilation rather than to a direct effect on NH₄⁺ acquisition. In contrast, in NO₃^?‐grown plants, in addition to draining carbon for NO₃^? reduction the fungus competed with the root for NO₃^? uptake. With NH₄⁺ or NO₃^? feeding, although mycorrhizal association improved N accumulation in shoots, we concluded that it was unlikely that the fungus had supplied the plant with N. In L ‐glutamate‐grown plants, the presence of the fungus increased the proportion of glutamine in the xylem sap and improved both N nutrition and the growth rate of the host plant.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

The use of different soil nitrogen sources by young Norway spruce plants

 Three-year-old Norway spruce trees were planted into a low-nitrogen mineral forest soil and supplied either with two different levels of mineral nitrogen (NH₄NO₃) or with a slow-release form of organic nitrogen (keratin). Supply of mineral nitrogen increased the concentrations of ammonium and nitrate in the soil solution and in CaCl₂-extracts of the rhizosphere and bulk soil. In the soil solution, in all treatments nitrate concentrations were higher than ammonium concentrations, while in the soil extracts ammonium concentrations were often higher than nitrate concentrations. After 7 months of growth, ¹⁵N labelled ammonium or nitrate was added to the soil. Plants were harvested 2 weeks later. Keratin supply to the soil did not affect growth and nitrogen accumulation of the trees. In contrast, supply of mineral nitrogen increased shoot growth and increased the ratio of above-ground to below-ground growth. The proportion of needle biomass to total above-ground biomass was not increased by mineral N supply. The atom-% ¹⁵N was higher in younger needles than in older needles, and in younger needles higher in plants supplied with ¹⁵N-nitrate than in plants supplied with ¹⁵N-ammonium. The present data show that young Norway spruce plants take up nitrate even under conditions of high plant internal N levels. Received: 1 April 1998 / Accepted: 9 October 1998     

Nitrogen accumulation and distribution in Danthonia richardsonii swards in response to CO2 and nitrogen supply over four years of growth

Nitrogen‐stressed microcosms of the C3 grass Danthonia richardsonii gained nitrogen from the environment when grown under ambient or enriched (359, ‘amb’ or 719 μL L^{? 1}‘enr’, respectively) atmospheric CO₂ concentrations over a 4‐y period. This gain was apparent at all rates of supplied mineral N (2.2, 6.7 or 19.8 g N m^{? 2} y^{? 1}– low‐N, mid‐N or high‐N), although it was small at high‐N. Small losses of N occurred from the microcosm as leachate, while gaseous losses of N were estimated to be between 10% and 25% of applied mineral N. Losses of applied mineral N were slightly lower under CO₂ enrichment only at the highest rate of mineral N supply. Levels of ¹⁵N natural abundance in green leaf (δ¹⁵Ν) of ? 2‰ (amb low‐N) and of below ? 4‰ (enr low‐ & mid‐N) suggest that absorption of atmospheric NH₃ may have been a source of some of the extra N in the low and mid‐N treatments. Biological N₂ fixation, of up to 2 g m^{? 2} y^{? 1} was hypothesized to form the remainder of the environmental N source. Microcosm C:N ratio was higher under CO₂ enrichment. Nitrogen productivity of microcosm carbon gain (g C accumulated g^{? 1} leaf N day^{? 1}) was increased (up to 100%) by CO₂ enrichment at all rates of mineral N supply. Green leaf %N was reduced by CO₂ enrichment, and there was less nitrogen in the green leaf pool under CO₂ enrichment. Less, or the same amount of nitrogen was present in senesced leaf, surface litter and root under CO₂ enrichment while more nitrogen was present in the soil in organic forms, and as NH₄ + at the highest rate of mineral N supply.     

Changes in photosynthesis and antioxidant defenses of Picea asperata seedlings to enhanced ultraviolet-B and to nitrogen supply

The paper mainly studied the effects of ultraviolet‐B (UV‐B) radiation, nitrogen, and their combination on photosynthesis and antioxidant defenses of Picea asperata seedlings. The experimental design included two levels of UV‐B treatments (ambient UV‐B, 11.02 KJ m⁻² day⁻¹; enhanced UV‐B, 14.33 KJ m⁻² day⁻¹) and two nitrogen levels (0; 20 g m⁻² a⁻¹ N) – to determine whether the adverse effects of UV‐B are eased by supplemental nitrogen. Enhanced UV‐B significantly inhibited plant growth, net photosynthetic rate (A), stomatal conductance to water vapor (Gs), transpiration rate and photosynthetic pigment, and increased intercellular CO₂ concentration, UV‐B absorbing compounds, proline content, malondialdehyde (MDA) content, and activity of antioxidant enzymes (peroxidase (POD), superoxide dimutase, and glutathione reductase). Enhanced UV‐B also reduced needle DW and increased hydrogen peroxide (H₂O₂) content and the rate of superoxide radical (O₂⁻) production only under supplemental nitrogen. On the other hand, supplemental nitrogen increased plant growth, A, Gs, chlorophyll content and activity of antioxidant enzymes (POD, ascorbate peroxidase, and catalase), and reduced MDA content, H₂O₂ content, and the rate of O₂⁻ production only under ambient UV‐B, whereas supplemental nitrogen reduced activity of antioxidant enzymes under enhanced UV‐B. Carotenoids content, proline content, and UV‐B absorbing compounds increased under supplemental nitrogen. Moreover, significant UV‐B × nitrogen interaction was found on plant height, basal diameter, A, chlorophyll a, activity of antioxidant enzymes, H₂O₂, MDA, and proline content. These results implied that supplemental nitrogen was favorable for photosynthesis and antioxidant defenses of P.asperata seedlings under ambient UV‐B. However, supplemental nitrogen made the plants more sensitive to enhanced UV‐B, although some antioxidant indexes increased.     

The uptake and distribution of 15N2 into the various organs of Typha latifolia L.

Direct evidence of heterotrophic dinitrogen fixation associated with the emergent aquatic angiosperm, Typha latifolia L., was obtained through the exposure of actively growing plants to ¹⁵N₂ gas for 7 days in a gas-tight exposure vessel. Highest enrichments of ¹⁵N were found in roots/rhizomes and leaf bases. Slight enrichments were also found in the leaves due to translocation from the roots, rhizomes and leaf bases. Total fixed ¹⁵N values were 71.8 μg for the plant and 49.1 μg for the soil.Plants growing in silica sand, which received a nutrient solution containing combined nitrogen, exhibited higher enrichments and fixed 86% more ¹⁵N after exposure to ¹⁵N₂ gas than plants which received a nutrient solution lacking combined nitrogen. It is hypothesized that the concentration of combined nitrogen added was insufficient to repress nitrogen fixation and resulted in an increase in nitrogen fixation by associated microorganisms.Propane was used to trace the loss and movement of gases from the ¹⁵N₂ vessel and between the upper leaf chamber and the lower root chamber. Gas was rapidly exchanged between the upper and lower chambers through the leaves and roots of T. latifolia. Further investigations showed that propane moved at a rate of 1223 μmol day⁻¹ from the leaves to the roots and 2652 μmol day⁻¹ from the roots to the leaves. These data demonstrated that gases diffuse rapidly through the plant body of T. latifolia.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Growth and nutrient allocation in Phaseolus vulgaris L. colonized with endomycorrhizae or Rhizobium

Two cultivars of Phaseolus vulgaris L., one responsive to colonization with microsymbionts (Mexico 309) and one less-responsive (Rio Tibagi) were grown in Leonard jars containing sand/vermiculite under greenhouse conditions. Bean plants were either left non-inoculated (controls) or were inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus etunicatum or a strain of Rhizobium leguminosarum bv. phaseoli (UMR-1899). Plants from the Mexico 309 cultivar maintained a higher growth rate, supported proportionately more nodules and mycorrhizae, and assimilated relatively more N or P when colonized by Rhizobium or Glomus, respectively, than did plants of the Rio Tibagi cultivar. Estimated specific nodule activity for Mexico 309 beans was more than twice that of Rio Tibagi, whereas the specific phosphorus uptake rate (SPUR) was 35% greater in the non-inoculated roots of Rio Tibagi compared to Mexico 309. Colonization by G. etunicatum more than doubled the SPUR for each cultivar compared to control roots. New acid phosphatase isozymes appeared in VAM-colonized roots of both cultivars compared to controls. Acid and alkaline phosphatase activities were significantly higher in G. etunicatum-colonized Mexico 309 roots, but not in Rio Tibagi mycorrhizae, compared to uninfected roots. Polyphosphate hydrolase activity was elevated in mycorrhizae of both cultivars compared to control roots. These results indicate that the dependence of a host on a specific endophyte increases when there are limitations to the supply of a nutrient that the endophyte can provide. The greater the increase in absorption or utilization capacity following colonization by the microsymbiont, the greater the dependence by the host. More importantly, identification of enzymatic activities that influence these plant-microbe associations opens the possibility that the specific genes that code for these enzymes could be targeted for future manipulation.     

The response of Vallisneria spinulosa (Hydrocharitaceae) to different loadings of ammonia and nitrate at moderate phosphorus concentration: a mesocosm approach

1. While phosphorus (P) is often considered the most important growth limiting factor for plants in lakes, recent studies of shallow lakes indicate that nitrogen (N) may be of greater importance than realized hitherto and that submerged macrophytes may be lost when the N concentration exceeds a certain threshold, as long as the concentration of P is sufficiently high. 2. We studied the effects of different loadings of NH₄‐N and NO₃‐N on chlorophyll a and on a macrophyte tolerant of eutrophication, Vallisneria spinulosa (Hydrocharitaceae). In outdoor mesocosms we used water from a pond as control and created four levels of NH₄‐N and NO₃‐N (approximately 2.5, 5, 7.5 and 10 mg L⁻¹) by dosing with NH₄Cl and NaNO₃, respectively. After the experiment, the plants were transferred back to a holding pond to study their recovery. In contrast to previous research, we used a low background concentration of phosphorus (TP 0.024 ± 0.003 mg L⁻¹) so we could judge whether any effects of N were apparent when P is in short supply. 3. Chlorophyll a increased significantly with N dosing for both forms of N, but the increase was highest in the NH₄‐N dosed mesocosms (maximum 58 μg L⁻¹ versus 42 μg L⁻¹ in the NO₃‐N mesocosms), probably due to a higher total inorganic N concentration (part of the added ammonia was converted to nitrate during the experiment). 4. Although the number of ramets of V. spinulosa was not affected by the N treatment, the biomass increased up to concentrations of 7.5 mg L⁻¹, while biomass at 10 mg L⁻¹ remained at the control level for both N ions treatments. A similar pattern was apparent for the content of N and soluble sugar of the plant, while there were no differences in the plant P content among treatments. Five months after transplantation back to the pond no difference was found in the number of ramets or in biomass, except that the biomass of plants grown at 10 mg N L⁻¹ during the experiment was greater than that in the control, while the N and P contents of plants were similar to those of the controls. 5. Nitrogen concentration had little influence on the growth of the eutrophication tolerant submerged macrophyte at moderately low concentrations of phosphorus. Moreover, the two N ions showed no toxic effects, suggesting that loss of macrophytes observed in other studies, run at higher phosphorus concentrations, was probably related to enhanced shading by periphyton and/or phytoplankton rather than to any toxic effects of N.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Interaction between atmospheric and pedospheric nitrogen nutrition in spruce (Picea abies L. Karst) seedlings

The effect of NO₂ fumigation on root N uptake and metabolism was investigated in 3-month-old spruce (Picea abics L. Karst) seedlings. In a first experiment, the contribution of NO₂ to the plant N budget was measured during a 48 h fumigation with 100mm³m^?3 NO₂. Plants were pre-treated with various nutrient solutions containing NO₂ and NH₄⁺, NO₃^? only or no nitrogen source for 1 week prior to the beginning of fumigation. Absence of NH₄⁺ in the solution for 6d led to an increased capacity for NO₃^? uptake, whereas the absence of both ions caused a decrease in the plant N concentration, with no change in NO₃^? uptake. In fumigated plants, NO₂ uptake accounted for 20–40% of NO₃^? uptake. Root NO₃^? uptake in plants supplied with NH₄⁺plus NO₃^? solutions was decreased by NO₂ fumigation, whereas it was not significantly altered in the other treatments. In a second experiment, spruce seedlings were grown on a solution containing both NO₂ and NH₄⁺ and were fumigated or not with 100mm³m^?3 NO₂ for 7 weeks. Fumigated plants accumulated less dry matter, especially in the roots. Fluxes of the two N species were estimated from their accumulations in shoots and roots, xylem exudate analysis and ¹⁵N labelling. Root NH₄⁺ uptake was approximately three times higher than NO₃^? uptake. Nitrogen dioxide uptake represented 10–15% of the total N budget of the plants. In control plants, N assimilation occurred mainly in the roots and organic nitrogen was the main form of N transported to the shoot. Phloem transport of organic nitrogen accounted for 17% of its xylem transport. In fumigated plants, neither NO₃^? nor NH₄⁺ accumulated in the shoot, showing that all the absorbed NO₂ was assimilated. Root NO₃^? reduction was reduced whereas organic nitrogen transport in the phloem increased by a factor of 3 in NO₂-fimugated as compared with control plants. The significance of the results for the regulation of whole-plant N utilization is discussed.     

H2-Recycling system in mungbean Rhizobium in relation to N2-fixation

Thirty isolates of mungbean Rhizobium were tested for the presence of H₂-recycling system. All the isolates were preliminary screened for detecting H₂-recycling system in free culture using triphenyltetrazolium chloride reduction as screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle hydrogen both in vivo as well as in vitro. Nitrogen fixing efficiency of hydrogenase positive, hydrogenase negative isolates and Hup^– mutants was compared by green house experiments. There was 13–56% increase in dry matter and 21–46% increase in total nitrogen of the plants inoculated with H₂-recycling isolates over the plants inoculated with non-recycling isolates. There was reduction in dry matter and total nitrogen content of the plants inoculated with Hup^– mutants as compared to plants inoculated with wild type strain. The per cent decrease due to inoculation with Hup^– mutants over wild type strain was 19–22 and 20–26 of dry weight and total nitrogen in plants, respectively.Abbreviations TTC triphenyltetrazolium chloride     

The Role of Plant Size and Nutrient Concentrations in Associations between Medicago, and Rhizobium and/or Glomus

The aim of this research was to carry out a critical study of the method of obtaining size equivalence between non-symbiotic alfalfa and alfalfa associated with Glomus and/or Rhizobium by applying fixed addition rates of nutrients to the non-symbiotic controls. The experimental design included three nutrient response curves in which the levels of added phosphorus and/or nitrogen were constant during the whole plant growth process: 1) a phosphorus response curve, in order to compare the growth of double symbiotic plants with that of only-Rhizobium inoculated ones; 2) a nitrogen response curve, that consisted of a comparison between the growth of double symbiotic alfalfa and four treatments associated only with Glomus; 3) a phosphorus and nitrogen response curve, to compare the growth of non-inoculated alfalfa with that of double symbiotic plants. Although similar size was achieved among some treatments at harvest, shoot growth over time and nutrient concentrations in tissues differed, indicating that growth equivalence did not mean functional equivalence. A second experimental design was performed taking into account the establishment of microsymbionts for determining the adequate moment to add supplemental phosphorus and/or nitrogen. It included four treatments: a) double symbiotic plants (MR); b) plants inoculated with Rhizobium only (R); c) plants inoculated with Glomus only (M), and d) non-inoculated plants (N). Great similarity in terms of plant growth and nutrient contents in tissues were obtained. Moreover, symbiotic plants were able to produce similar dry matter than non-symbiotic ones under P and N limitations.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

Cultivar and Rhizobium strain effect on nitrogen fixation and transport inPhaseolus vulgaris L.

The effects of Rhizobium strain and its interaction with plant cultivar were examined in glasshouse-grownPhaseolus vulgaris in two experiments where the physiological attributes defining the symbiotic efficiency were determined. Strains of Rhizobium significantly affected nodulation, rates of N accumulation, partitioning of N within the mature shoot and remobilizaton of the N stored in the vegetative organs to the seeds. The most efficient symbiosis (strain CO5 with Negro Argel), in comparison with the least efficient symbiosis (strain 127 K-17 with Venezuela-350) showed higher rates of C₂H₂ reduction from flowering to mid pod fill stage, evolved less hydrogen from nodules and showed higher rates of N transport as well as higher percentages of ureide-N in the xylem sap. At maturity, the best cultivar/strain association exceeded the total N accumulated in the seed and the harvest index of the poorest symbiosis in 88% and 20%, respectively. The other symbiotic combinations were intermediate in all characteristics. Nitrogen accumulation in plant shoot showed highly significant correlation with acetylene reduction rates, nodule relative efficiency, total N transport in the xylem sap and percentage of N transported as ureides.