Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants

Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N₂-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc. A heavy inoculum of S. lipoferum grown on NH₄⁺ was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N₂. Conversion to N₂ fixation was prompt upon exhaustion of fixed nitrogen; growth on N₂ was most rapid at a pO₂ of 0.005 to 0.007 atm. Numbers of S. lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium. Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C₂H₂ are presumptive evidence that they are S. lipoferum. With most-probable-number tables the observations can be converted to numbers of S. lipoferum in the samples. The most-probable-number method indicated that numbers of S. lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30°C at a pO₂ initially adjusted to 0.01 atm.     

Nitrogen Stress and Apparent Photosynthesis in Symbiotically Grown Pisum sativum L

Pea plants (Pisum sativum L. cv. Alaska) were inoculated individually with one of 15 Rhizobium leguminosarum strains and grown under uniform environmental conditions in the absence of combined N. Differences in effectiveness of the Rhizobium strains produced plants with differing rates of whole plant apparent N₂ fixation and total N content at the same morphological stage of development. Plants were analyzed to determine interactions between N₂ fixation, N allocation, apparent photosynthesis, and growth. Total leaf N increased linearly with total N₂ fixation (R² = 0.994). The proportion of total N allocated to leaves, the per cent N content of individual leaves, and the photosynthetic efficiency of individual leaves showed a curvilinear response with increasing plant N content. Differences in allocation patterns of leaf N between plants with low and high N content resulted in differences in the relationship between total N content and plant dry weight. Results from this study show that N₂ fixation interacts with leaf photosynthetic efficiency and plant growth in a manner that is dependent upon the allocation of symbiotically fixed N.     

Carbon and nitrogen assimilation and partitioning in soybeans exposed to low root temperatures

Low root temperature effects on vegetative growth of soybean (Harosoy 63 × Rhizobium japonicum USDA 16) were examined in 35 day old plants exposed to temperatures of 15°C (shoots at 25°C) for an 11 day period. Duing this period various aspects of C and N assimilation and partitioning were monitored including shoot night and nodulated root respiration, C and N partitioning to six plant parts, C₂H₂ reduction, H₂ evolution, leaf area, transpiration, net photosynthesis, and N₂ fixation. The low temperature treatment resulted in a decrease in the net rate of N₂ fixation but nitrogenase relative efficiency increased. In response, the plant retained N in the tissues of the nodulated root and decreased N partitioning to young shoot tissues, thereby inducing the remobilization of N from older leaves, and reducing leaf area development. The leaf area specific rate of net photosynthesis was not affected over the study period; however, shoot and nodulated root respiration declined. Consequently, C accumulated in mature leaves and stems, partly in the form of increased starch reserves. Three possibilities were considered for increasing low temperature tolerance in nodulated soybeans: (a) decrease in temperature optima for nitrogenase, (b) increased development of nodules and N₂ fixation capacity at low temperature, and (c) alterations in the pattern of C and N partitioning in response to low temperature conditions.     

Incorporation of Different N Sources and Light Response Curves of Nitrogenase and Photosynthesis by Cyanobacterial Blooms from Rice Fields

In this work, we estimate the contributions of the different sources of N incorporated by two N₂-fixing cyanobacterial blooms (Anabaena sp. and Microchaete sp.) in the rice fields of Valencia (Spain) during the crop cycles of 1999 and 2000, and evaluate the response of nitrogenase and C assimilation activities to changing irradiances. Our results show that, far from the generally assumed idea that the largest part of the N incorporated by N₂-fixing cyanobacterial blooms in rice fields comes from N₂ fixation, both cyanobacterial blooms incorporated about three times more N from dissolved combined compounds than from N₂ fixation (only about 33–41% of the N incorporated came from N₂ fixation). Our results on the photodependence of C and N₂ fixation indicate that in both cyanobacterial blooms, N₂ fixation showed a steeper initial slope (α) and was saturated with less irradiance than C fixation, suggesting that N₂ fixation was more efficient than photosynthesis under conditions of light limitation. At saturating light, N₂ fixation and C fixation differed depending on the bloom and on the environmental conditions created by rice plant growth. Carbon assimilation but not nitrogenase activity appeared photoinhibited in the Anabaena but not in the Microchaete bloom in August 1999, when the plants were tall and the canopy was important, and there was no limitation of dissolved inorganic carbon. The opposite was found in the Microchaete bloom of June 2000, when plants were small and produced little shade, and dissolved inorganic carbon was very low.     

The effect of combined and separate inoculation of alfalfa plants with <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> on denitrification and nitrogen-fixing activities

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N₂O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N₂O with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C₂H₄ nmol/flask · 24 h) and low denitrification activity (1.8 μmol N₂O/flask · 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass

The effect of oxygen on N₂-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 μM O₂, some of them being carbon limited. All strains needed a minimum amount of oxygen for N₂-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O₂ concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N₂ fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N₂ fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O₂ concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.     

Effect of Light Intensity on Efficiency of Carbon Dioxide and Nitrogen Reduction in Pisum sativum L

Photosynthetic efficiency, primary productivity, and N₂ reduction were determined in peas (Pisum sativum L. var. Alaska) grown at light intensities ranging from severely limiting to saturating. Plants grown under higher light intensities showed greater carboxylation and light capture potential and higher rates of net C exchange. Uptake of N₂, computed from measured C₂H₂ reduction and H₂ evolution rates, also increased with growth light intensity, while the previously proposed relative efficiency of N₂ fixation, based on these same parameters, declined. The plot of N/C ratios (total nitrogen content/plant dry weight) increased hyperbolically with light intensity, and the plot of N₂/CO₂ uptake ratios (N₂ uptake rate/net CO₂ uptake rate) increased linearly. Both plots extrapolated to the light compensation point. The data indicate that the relative efficiency of N₂ fixation is not necessarily correlated with maximum plant productivity and that evaluation of a plant's capacity to reduce N₂ is related directly to concurrent CO₂ reduction. A measure of whole plant N₂ fixation efficiency based on the N₂/CO₂ uptake ratio is proposed.     

Evaluation of the Relative Ureide Content of Xylem Sap as an Indicator of N(2) Fixation in Soybeans: GREENHOUSE STUDIES

The use of the relative ureide content of xylem sap [(ureide-N/total N) × 100] as an indicator of N₂ fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N₂ fixation were imposed upon the root systems of plants totally dependent upon N₂ fixation as their source of N and of plants dependent upon both N₂ fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N₂ fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N₂ fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. `Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N₂ fixation. The relative ureide content of xylem sap was shown to indicate reliably the N₂ fixation of these plants during vegetative growth using a ¹⁵N method to measure N₂ fixation activity. The use of the relative ureide content of xylem sap for quantification of N₂ fixation in soybeans should be evaluated further.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Bryophyte-Cyanobacteria Associations during Primary Succession in Recently Deglaciated Areas of Tierra del Fuego (Chile)

Bryophyte establishment represents a positive feedback process that enhances soil development in newly exposed terrain. Further, biological nitrogen (N) fixation by cyanobacteria in association with mosses can be an important supply of N to terrestrial ecosystems, however the role of these associations during post-glacial primary succession is not yet fully understood. Here, we analyzed chronosequences in front of two receding glaciers with contrasting climatic conditions (wetter vs drier) at Cordillera Darwin (Tierra del Fuego) and found that most mosses had the capacity to support an epiphytic flora of cyanobacteria and exhibited high rates of N₂ fixation. Pioneer moss-cyanobacteria associations showed the highest N₂ fixation rates (4.60 and 4.96 µg N g⁻¹ bryo. d⁻¹) very early after glacier retreat (4 and 7 years) which may help accelerate soil development under wetter conditions. In drier climate, N₂ fixation on bryophyte-cyanobacteria associations was also high (0.94 and 1.42 µg N g⁻¹ bryo. d⁻¹) but peaked at intermediate-aged sites (26 and 66 years). N₂ fixation capacity on bryophytes was primarily driven by epiphytic cyanobacteria abundance rather than community composition. Most liverworts showed low colonization and N₂ fixation rates, and mosses did not exhibit consistent differences across life forms and habitat (saxicolous vs terricolous). We also found a clear relationship between cyanobacteria genera and the stages of ecological succession, but no relationship was found with host species identity. Glacier forelands in Tierra del Fuego show fast rates of soil transformation which imply large quantities of N inputs. Our results highlight the potential contribution of bryophyte-cyanobacteria associations to N accumulation during post-glacial primary succession and further describe the factors that drive N₂-fixation rates in post-glacial areas with very low N deposition.     

Nodulation and N2 fixation of guar at high root temperature

Guar (Cyamopsis tetregonoloba (L.) may be grown when soil temperatures are potentially high enough at the time of planting to inhibit nodulation and N₂ fixation. An experiment was conducted using controlled conditions to determine the influence of high root temperature on growth and N₂ fixation of guar. The experiment included two strains of rhizobia, two varieties of guar, two mineral N treatments, and root temperatures of 34, 37, and 40°C. Plants were grown for 44 days. The root temperature of 40°C reduced N fixation by at least 80% and nodule weight by more than 50%. Significant interactions occurred between most factors in influencing nodulation, N₂ fixation and dry matter production. Guar, nodulated by rhizobial strain GAR022-1 and fully dependent on N₂ fixation or provided with starter mineral N (25 mg pot^–1), was not influenced by the root temperature of 37°C as compared to 34°C. Nodulation and N₂ fixation by strain 32H1 was reduced by at least 40% when no starter mineral N was provided and the root temperature was 37°C. Providing starter mineral N to one variety of guar doubled the quantity of N₂ fixed by strain 32H1 at both 34 and 37°C but N₂ fixation was lower at the higher root temperature. It appears that root temperatures between 37° and 40°C bracketed the critical root temperature for N₂ fixation by nodulated guar and that the critical root temperature for guar dependent on mineral N was above 40°C.     

Functional significance of dinitrogen fixation in sustaining coral productivity under oligotrophic conditions

Functional traits define species by their ecological role in the ecosystem. Animals themselves are host–microbe ecosystems (holobionts), and the application of ecophysiological approaches can help to understand their functioning. In hard coral holobionts, communities of dinitrogen (N₂)-fixing prokaryotes (diazotrophs) may contribute a functional trait by providing bioavailable nitrogen (N) that could sustain coral productivity under oligotrophic conditions. This study quantified N₂ fixation by diazotrophs associated with four genera of hermatypic corals on a northern Red Sea fringing reef exposed to high seasonality. We found N₂ fixation activity to be 5- to 10-fold higher in summer, when inorganic nutrient concentrations were lowest and water temperature and light availability highest. Concurrently, coral gross primary productivity remained stable despite lower Symbiodinium densities and tissue chlorophyll a contents. In contrast, chlorophyll a content per Symbiodinium cell increased from spring to summer, suggesting that algal cells overcame limitation of N, an essential element for chlorophyll synthesis. In fact, N₂ fixation was positively correlated with coral productivity in summer, when its contribution was estimated to meet 11% of the Symbiodinium N requirements. These results provide evidence of an important functional role of diazotrophs in sustaining coral productivity when alternative external N sources are scarce.     

Denitrification and nitrogen fixation by Azospirillum

A model system is described where Azospirillum and germinated wheat seeds were grown in association for a week and then assayed for nitrogen fixation (C₂H₂-reduction) and denitrification (N₂O-formation) activities. The association performed C₂H₂-reduction and N₂O-formation under microaerobic conditions. Both activities were measurable after already 3–5 h of incubation with substantial rates and were strictly dependent on the presence of both plants and bacteria. During the week of the growth of the association, the bacteria had lived exclusively from the carbon compounds supplied by the roots of the plants. C₂H₂-reduction activity by the association was more or less the same with all the Azospirillum brasilense strains, but lower with A. lipoferum and with the A. amazonense strains tested. Two nitrogenase negative mutants of Azospirillum brasilense showed virtually no activity in the association. C₂H₂-reduction activity was strongly dependent on the growth temperature of the association. Denitrification (N₂O-formation) was high also at higher temperatures and at pH-values in the medium around 7.8 but not at neutrality and was strictly dependent on nitrate. The Azospirillum strain used strongly determined the rate of the N₂O-formation in the association. It is suggested that Azospirillum may be beneficial to crops particularly under tropical conditions.Dedicated to Professor Dr. Gerhart Drews, Freiburg, on the occasion of his 60th birthday     

Low Root Temperature Retardation of the Mineral Nitrogen Induced Decline in N2Fixation by a Northern Ecotype of White Clover

Nodulated white clover plants (Trifolium repensL.) of a Norwegian ecotype from Pasvik (70°N) were grown in flowing solution culture. Root temperature was 17°C until 51d after sowing, when it was lowered decrementally over 5d to 7°C in four of the eight plant culture units. After a further 24h, mineral N was supplied automatically at 20μMNH₄NO₃in three culture units at each root temperature (7 and 17°C) over 17d. The remaining two units provided control plants solely dependent on N₂fixation at 7 and 17°C.The supply of NH₄NO₃greatly reduced the nodule biomass per plant at 17°C over 17d compared with control plants, but had little effect at 7°C. The nodule decline at 17°C accompanied an acute and progressive decrease in specific rate of N₂fixation, from 9mmolN d^-1g^-1nodule d.wt on day 0 to zero by day 10. Whilst initial rates of N₂fixation were lower at 7°C, the mineral N-induced decrease in fixation rates was also less severe than at 17°C and specific fixation rates recovered after reaching a minimum on day 11. N₂fixation accounted for 36% of the total uptake of N by +min.N plants during the treatment period at 7°C as opposed to only 13% at 17°C. The total N₂fixed at 7°C was 86% of that fixed at 17°C, although the specific growth rate (d.wt) at 7°C was only 55% of that at 17°C. Addition of NH₄NO₃at 7°C had little effect on the gross amount of N₂fixed subsequently. In contrast, total N₂fixation by +min.N plants at 17°C was only 24% of that fixed by the corresponding controls. The possible mechanisms by which mineral N affects N₂fixation are discussed.     

Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O₂ (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.     

Quantification of N2-fixation and survival of inoculated diazotrophs associated with roots of Kallar grass

White clover plants were grown for 97 days under two temperature regimes (20/15°C and 8/5°C day/night temperatures) and were supplied with either small amounts (a total of 80 mg N pot^–1) of ammonium (NH ₄ ⁺ ) or nitrate (NO ₃ ^– ) nitrogen, or received no mineral N and relied on N₂ fixation. Greatest growth and total leaf area of clover plants occurred in N₂ fixing and NO ₃ ^– -fed plants grown at 20/15°C and poorest growth occurred in NH ₄ ⁺ -fed plants grown at 8/5°C. Nodule mass per plant was greater at 8/5°C due to increased nodule numbers rather than increased dry weight per nodule. This compensated to some extent for the reduced N₂-fixing activity per unit dry weight of nodule tissue found at the low growth temperature up to 116 d after sowing, but thereafter both activity per nodule dry weight and activity per plant were greater at the low temperature. Highest nitrate reductase activity (NRA) per g fresh weight and total activity per leaf, petiole or root occurred in NO ₃ ^– -fed plants at 8/5°C. Low growth temperature resulted in a greater partitioning of total plant NRA to the roots of NO ₃ ^– -fed plants. The results are considered in relation to the use of N fertiliser in the spring under field conditions.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

N2 Fixation in Feather Mosses is a Sensitive Indicator of N Deposition in Boreal Forests

Nitrogen (N) fixation in the feather moss–cyanobacteria association represents a major N source in boreal forests which experience low levels of N deposition; however, little is known about the effects of anthropogenic N inputs on the rate of fixation of atmospheric N₂ in mosses and the succeeding effects on soil nutrient concentrations and microbial community composition. We collected soil samples and moss shoots of Pleurozium schreberi at six distances along busy and remote roads in northern Sweden to assess the influence of road-derived N inputs on N₂ fixation in moss, soil nutrient concentrations and microbial communities. Soil nutrients were similar between busy and remote roads; N₂ fixation was higher in mosses along the remote roads than along the busy roads and increased with increasing distance from busy roads up to rates of N₂ fixation similar to remote roads. Throughfall N was higher in sites adjacent to the busy roads but showed no distance effect. Soil microbial phospholipid fatty acid (PLFA) composition exhibited a weak pattern regarding road type. Concentrations of bacterial and total PLFAs decreased with increasing distance from busy roads, whereas fungal PLFAs showed no distance effect. Our results show that N₂ fixation in feather mosses is highly affected by N deposition, here derived from roads in northern Sweden. Moreover, as other measured factors showed only weak differences between the road types, atmospheric N₂ fixation in feather mosses represents a highly sensitive indicator for increased N loads to natural systems.     

Effect of inoculation ofAzospirillum lipoferum on nitrogen fixation in rhizosphere soil,their association with root,yield and nitrogen uptake by mustard (Brassica juncea)

Summary A microplot field experiment was conducted in the presence or absence of P and N application to evaluate the influence of the seed inoculation of mustard (cv. Baruna T₅₉) withAzospirillum lipoferum on N₂-fixation in rhizosphere, association of the bacteria with the roots and grain yield and N uptake. Inoculation significantly increased the N content in rhizosphere soil particularly at early stage (40 days) of plant growth, which was accompanied by the increased association of the bacteria (A. lipoferum) in rhizosphere soil, root surface washing and surface-sterilized macerated root. A significant increase in grain yield and N uptake was also observed due to inoculation. Application of P particularly at the 20 kg. ha^–1 level further enhanced the beneficial effect ofAzospirillum lipoferum inoculation, while N addition markedly reduced such an effect.     

Growth and nitrogen accretion of dinitrogen-fixing Alnus glutinosa (L.) Gaertn. under elevated carbon dioxide

Short-term studies of tree growth at elevated CO₂ suggest that forest productivity may increase as atmospheric CO₂ concentrations rise, although low soil N availability may limit the magnitude of this response. There have been few studies of growth and N₂ fixation by symbiotic N₂-fixing woody species under elevated CO₂ and the N inputs these plants could provide to forest ecosystems in the future. We investigated the effect of twice ambient CO₂ on growth, tissue N accretion, and N₂ fixation of nodulated Alnus glutinosa (L.) Gaertn. grown under low soil N conditions for 160 d. Root, nodule, stem, and leaf dry weight (DW) and N accretion increased significantly in response to elevated CO₂. Whole-plant biomass and N accretion increased 54% and 40%, respectively. Delta-¹⁵N analysis of leaf tissue indicated that plants from both treatments derived similar proportions of their total N from symbiotic fixation suggesting that elevated CO₂ grown plants fixed approximately 40% more N than did ambient CO₂ grown plants. Leaves from both CO₂ treatments showed similar relative declines in leaf N content prior to autumnal leaf abscission, but total N in leaf litter increased 24% in elevated compared to ambient CO₂ grown plants. These results suggest that with rising atmospheric CO₂ N₂-fixing woody species will accumulate greater amounts of biomass N through N₂ fixation and may enhance soil N levels by increased litter N inputs.