N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

New type of N2-fixing unicellular cyanobacterium (blue-green alga)

Abstract A new N₂-fixing unicellular cyanobacterium identified as a Synechococcus sp. was isolated and purified as an axenic culture. It fixed N₂ aerobically either under continuous illumination or in alternating light-dark cycle. The N₂-fixing properties of the new isolate and Gloeocapsa are discussed.     

Effects of moisture,nitrogen, grass competition and simulated browsing on the survival and growth of Acacia karroo seedlings

The effects of irrigation, nitrogen fertilization, grass competition and clipping were investigated for one growing season at the research farm of the University of Fort Hare in the Eastern Cape Province of South Africa. The aim of the experiment was to assess the short‐term performance of Acacia karroo seedlings under different environmental conditions and the implications of such factors on the long‐term recruitment of plant species in savanna rangelands. There were no significant treatment effects on the survival of A. karroo seedlings. Using stem length and basal diameter as growth parameters, it was observed that irrigation enhanced both variables, while nitrogen fertilization did not have any significant effects. Clipping, grass competition and their interaction greatly suppressed the growth of the seedlings. Clipping increased the mean stem length when they were irrigated and fertilized. Control and fertilized plants had the highest stem length in the absence of grass competition, while grass competition combined with clipping resulted in the lowest stem length in both irrigated and nonirrigated plants. It was concluded that in the presence of grass competition, controlled browsing could be a viable solution to the problem of bush encroachment in savanna rangelands.     

Modification of NO−3 metabolism in heterocysts of the N2-fixing cyanobacterium Anabaena 7120 (ATCC27893)

Abstract The utilization of NO⁻₃, NO⁻₂ and NH⁺₄ was studied in whole filaments and isolated heterocysts of Anabaena 7120 (ATCC27893). NO⁻₃- and NO⁻₂-uptake were detectable in whole filaments but not in heterocysts, whereas NH⁺₄-uptake was detectable in both. Activity of NO⁻₃-reductase was present in cell-free extracts of whole filaments but not of heterocysts, whereas activities of NO⁻₂-reductase and glutamine synthetase were present in both. NO⁻₃-uptake and reductase activities could not be induced in heterocysts even after prolonged incubation in NO⁻₃ medium. It is suggested that NO⁻₃-metabolism in heterocysts is impaired due to a selective and irreversible loss of NO⁻₃-uptake and reductase systems resulting in the abolition of competition for molybdenum cofactor (Mo-Co) and reductant between nitrogenase and NO⁻₃-reductase, and an increase in glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase levels.     

Herbivory effects on saplings are influenced by nutrients and grass competition in a humid South African savanna

Woody plant encroachment is a common consequence of disturbance in savannas. Grazers and browsers interfere with sapling establishment dynamics by direct consumption of plant tissue, changing soil nutrient status (through fertilization and trampling) and grass competition. Studies evaluating the effects of herbivory on sapling establishment have mostly been extrapolated from single species. In a controlled field experiment, we studied the effects of clipping (simulating grazing and browsing), nutrients, grass competition, and their interactive effects on sapling survival and growth of four dominant humid and four dominant mesic savanna species. We conducted this experiment in a humid South African savanna. We found no effects on sapling survival by the treatments provided. However, clipped saplings of all species increased their investment in relative growth rate of stem length (RGR_L). Clipping had a greater negative impact on relative growth rate of more humid than mesic species in terms of stem diameter (RGR_D), total dry biomass and proportion of leaf biomass. Nutrients had a positive effect on the RGR_L and sapling biomass of three mesic species. Positive effects of nutrients on RGR_L of one humid and two mesic species were observed in their clipped saplings only. Grass competition had a strong negative impact on all growth parameters measured. Clipped saplings of one humid and two mesic species had lower RGR_L with grass competition whereas intact saplings showed no significant response. After clipping, humid savanna species were more vulnerable to grass competition than mesic species, with reduced ability to use nutrients. In conclusion, herbivory increases sapling vulnerability to grass competition, with humid species being more susceptible than mesic species, indicating that woody-plant control strategies are more likely to be effective in humid savannas.     

Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment

The combined effects of carbon dioxide (CO₂) enrichment and water deficits on nodulation and N₂ fixation were analysed in soybean [Glycine max (L.) Merr.]. Two short-term experiments were conducted in greenhouses with plants subjected to soil drying, while exposed to CO₂ atmospheres of either 360 or 700 μmol CO₂ mol^–1. Under drought-stressed conditions, elevated [CO₂] resulted in a delay in the decrease in N₂ fixation rates associated with drying of the soil used in these experiments. The elevated [CO₂] also allowed the plants under drought to sustain significant increases in nodule number and mass relative to those under ambient [CO₂]. The total non-structural carbohydrate (TNC) concentration was lower in the shoots of the plants exposed to drought; however, plants under elevated CO₂ had much higher TNC levels than those under ambient CO₂. For both [CO₂] treatments, drought stress induced a substantial accumulation of TNC in the nodules that paralleled N₂ fixation decline, which indicates that nodule activity under drought may not be carbon limited. Under drought stress, ureide concentration increased in all plant tissues. However, exposure to elevated [CO₂] resulted in substantially less drought-induced ureide accumulation in leaf and petiole tissues. A strong negative correlation was found between ureide accumulation and TNC levels in the leaves. This relationship, together with the large effect of elevated [CO₂] on the decrease of ureide accumulation in the leaves, indicated the importance of ureide breakdown in the response of N₂ fixation to drought and of feedback inhibition by ureides on nodule activity. It is concluded that an important effect of CO₂ enrichment on soybean under drought conditions is an enhancement of photoassimilation, an increased partitioning of carbon to nodules and a decrease of leaf ureide levels, which is associated with sustained nodule growth and N₂ rates under soil water deficits. We suggest that future [CO₂] increases are likely to benefit soybean production by increasing the drought tolerance of N₂ fixation.     

Differences in nitrogen metabolism of Faidherbia albida and other N2-fixing tropical woody acacias reflect habitat water availability

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida , a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO₃⁻ and NH₄⁺ preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO₃⁻-free solution or placed under N₂-fixing conditions, and also in nodules of N₂-fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N₂-fixing Sahelian acacias ( Acacia seyal , A. nilotica and A. tortilis ), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water ( A. nilotica ), F. albida can be distinguished from acacias growing strictly in arid zones ( A. seyal and A. tortilis ) in terms of initial growth, water and nitrate management.     

Distribution and N2-fixing activity of Frankia strains in relation to soil depth

The qualitative and quantitative distribution of Frankia strains infective on Elaeagnus angustifolia L. from different depths in the same soil (10–20 cm; 30–40 cm; 50–60 cm) were compared. The soil samples collected from a natural stand devoid of Elaeagnaceae were planted with Elaeagnus angustifolia L. seedlings. All plants became nodulated, demonstrating that Frankia strains were present at least down to 60 cm in the soil. The decreased density of Frankia strains was correlated to the decline of soil organic matter content with soil depth. DNA extracted from nodules produced on Elaeagnus angustifolia L. seedlings were polymerase chain reaction (PCR) characterized by amplifying the nif D-K intergenic spacer (IGS), using the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. This showed the presence of seven nif-Hae III profiles within this Frankia community. Diversity of Frankia strains was maintained throughout the soil column, but the relative distribution of strains varied. Some nif-Hae III profiles were only found in the deeper soil, suggesting different selective advantages to withstand the constraints of soil depth. ¹⁵N₂ experiments indicated that all the strains tested had N₂-fixing activity. However, efficiency was not significantly different among nodules of different nif-Hae III profiles. Therefore, N₂-fixing activity does not seem to be the main factor responsible for the different distribution of Frankia strains at different soil depths.     

Relationship between boron and calcium in the N2-fixing legume–rhizobia symbiosis

Because boron (B) and calcium (Ca²⁺) seem to have a strong effect on legume nodulation and nitrogen fixation, rhizobial symbiosis with leguminous plants, grown under varying concentrations of both nutrients, was investigated. The study of early pre‐infection events included the capacity of root exudates to induce nod genes, and the degree of adsorption of bacteria to the root surface. Both phenomena were inhibited by B deficiency, and increased by addition of Ca²⁺, resulting in an increase of the number of nodules. The infection and invasion steps were investigated by fluorescence microscopy in pea nodules harbouring a Rhizobium leguminosarum strain that constitutively expresses green fluorescent protein. High Ca²⁺ enhanced cell and tissue invasion by Rhizobium, which was highly inhibited after B deficiency. This was combined with an increased B concentration in nodules of plants grown on B‐free medium and supplemented with high Ca²⁺ concentrations, and that can be attributed to an increased B import to the nodules. Histological examination of indeterminate (pea) and determinate (bean) nodules showed an altered nodule anatomy at low B content of the tissue. The moderate increase in nodular B due to additional Ca²⁺ was not sufficient to prevent the abnormal cell wall structure and the aberrant distribution of pectin polysaccharides in B‐deficient treatments. Overall results indicate that the development of the symbiosis depends of the concentration of B and Ca²⁺, and that both nutrients are essential for nodule structure and function.     

Isolation and identification of a N2-fixing zoogloea-forming bacterium from kallar grass histoplane

Semi-solid medium was used to isolate an aerobic, N₂-fixing (C₂H₂-reducing), H₂-utilizing bacterium from the roots of kallar grass ( Leptochloa fusca ). The organism was identified by morphological, cultural and biochemical characteristics. The N₂-fixing, zoogloeal floc-forming isolate described here is a new species.     

Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean

The effects of increasing rhizosphere pO₂on nitrogenase activity and nodule resistance to O₂diffusion were investigated in soybean plants [Glycine max (L.) Merr. cv. Harosoy 63] in which nitrogenase (EC 1.7.99.2) activities were inhibited by (a) removal of the phloem tissue at the base of the stem (stem girdling), (b) exposure of roots to 10 mM NO₃over 5 days (NO₃-treated), or (c) partial inactivation of nitrogenase activity by an exposure of nodulated roots to 100 kPa O₂(O₂-inhibitcd). In control plants and in plants which had been treated with 100 kPa O₂, increasing rhizosphere O₂concentrations in 10 kPa increments from 20 to 70 kPa did not alter the steady-state nitrogenase activity. In contrast, in plants in which nitrogenase activities were depressed by stem girdling or by exposure to NO₃, increasing rhizosphere pO₂resulted in a recovery of 57 or 67%, respectively, of the initial, depressed rates of nitrogenase activity. This suggests that the nitrogenase activity of stem-girdled and NO₃-treated soybeans was O₂-limited. For each treatment, theoretical resistance values for O₂diffusion into nodules were estimated from measured rates of CO₂exchange, assuming a respiratory quotient of 1.1 and 0 kPa of O₂in the infected cells. At an external partial pressure of 20 kPa O₂, the stem-girdled and NO₃^--treated plants displayed resistance values which were 4 to 8.6 times higher than those in the nodules of the control plants. In control and O₂-inhibited plants, increases in pO₂from 20 to 70 kPa in 10 kPa increments resulted in a 2.5- to 3.9-fold increase in diffusion resistance to O₂, and had little effect on either respiration or nitrogenase activity. In contrast, in stem-girdled and NO₃^--treated plants, increases in external pO₂had little effect on diffusion resistance to O₂, but resulted in a 2.3- to 3.2-fold increase in nodule respiration and nitrogenase activity. These results are consistent with stem-girdling and NO₃^--inhibition treatments limiting phloem supply to nodules causing an increase in diffusion resistance to O₂at 20 kPa and an apparent insensitivity of diffusion resistance to increases in external pO₂.     

Physiological and growth responses of two African species, Acacia karroo and Themeda triandra, to combined increases in CO2 and UV-B radiation

The interactive effects of increased carbon dioxide (CO₂) concentration and ultraviolet-B (UV-B, 280–320 nm) radiation on Acacia karroo Hayne, a C₃ tree, and Themeda triandra Forsk., a C₄ grass, were investigated. We tested the hypothesis that A. karroo would show greater CO₂-induced growth stimulation than T. triandra, which would partially explain current encroachment of A. karroo into C₄ grasslands, but that increased UV-B could mitigate this advantage. Seedlings were grown in open-top chambers in a greenhouse in ambient (360 μmol mol^-1) and elevated (650 μmol mol^-1) CO₂, combined with ambient (1.56 to 8.66 kJ m^-2 day^-1) or increased (2.22 to 11.93 kJ m^-2 day^-1) biologically effective (weighted) UV-B irradiances. After 30 weeks, elevated CO₂ had no effect on biomass of A. karroo, despite increased net CO₂ assimilation rates. Interaction between UV-B and CO₂ on stomatal conductance was found, with conductances decreasing only where elevated CO₂ and UV-B were supplied separately. Increases in water use efficiencies, foliar starch concentrations, root nodule numbers and total nodule mass were measured in elevated CO₂. Elevated UV-B caused only an increase in foliar carbon concentrations. In T. triandra, net CO₂ assimilation rates were unaffected in elevated CO₂, but stomatal conductances and foliar nitrogen concentrations decreased, and water use efficiencies increased. Biomass of all vegetative fractions, particularly leaf sheaths, was increased in elevated CO₂. and was accompanied by increased leaf blade lengths and individual leaf and leaf sheath masses. However, tiller numbers were reduced in elevated CO₂. Significantly moderating effects of elevated UV-B were apparent only in individual masses of leaf blades and sheaths, and in total sheath and shoot biomass. The direct CO₂-induced growth responses of the species therefore do not support the hypothesis of CO₂-driven woody encroachment of C₄ grasslands. Rather, differential changes in resource use efficiency between grass and woody species, or morphological responses of grass species, could alter the competitive balance. Increased UV-B radiation is unlikely to substantially alter the CO₂ response of these species.     

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm⁻) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an 'in vitro' assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm⁻ mutant class in Rhizobium spp.     

The Glycine-Glomus-Bradyhizobium symbiosis. VIII. Phosphorususe efficiency of CO2 and N2 fixation in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants nodulated by Bradyhizobium japonicum strain USDA 110 were grown in pot cultures in severely P- and N-deficient soil and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or fertilized with a high (HP) or low (LP) level of KH₂PO₄ (0.6 or 0.3 m M , respectively), After 7 weeks of growth, nodule and chloroplast activities (C₂H₂ reduction and CO₂ exchange rate) were determined. Photosynthetic P-use efficiency of CO₂ fixation was significantly higher in VAM than in HP plants, while that of nitrogenase activity was lower. The LP plants were intermediate in both respects. The ratio of nodule to chloroplast activity [mol C₂H₂ reduced (mol CO₂ fixed)⁻¹] was highest in HP and lowest in VAM plants. Root colonization by the VAM fungus significantly increased nodule number and dry weight and reduced nodule specific mass and activity in comparison to HP plants. In spite of lower nodule activity, VAM plants were significantly larger and had higher N concentrations than the HP plants. The results suggest nonnutritional. VAM-elicited and host-mediated effects on the symbiotic functions of the legume association.