Specific Acetylene Reduction Activity Research Articles

Phylogeny of nodulation genes and symbiotic diversity of Acacia senegal (L.) Willd. and A. seyal (Del.) Mesorhizobium strains from different regions of Senegal.

Acacia senegal and Acacia seyal are small, deciduous legume trees, most highly valued for nitrogen fixation and for the production of gum arabic, a commodity of international trade since ancient times. Symbiotic nitrogen fixation by legumes represents the main natural input of atmospheric N2 into ecosystems which may ultimately benefit all organisms. We analyzed the nod and nif symbiotic genes and symbiotic properties of root-nodulating bacteria isolated from A. senegal and A. seyal in Senegal. The symbiotic genes of rhizobial strains from the two Acacia species were closed to those of Mesorhizobium plurifarium and grouped separately in the phylogenetic trees. Phylogeny of rhizobial nitrogen fixation gene nifH was similar to those of nodulation genes (nodA and nodC). All A. senegal rhizobial strains showed identical nodA, nodC, and nifH gene sequences. By contrast, A. seyal rhizobial strains exhibited different symbiotic gene sequences. Efficiency tests demonstrated that inoculation of both Acacia species significantly affected nodulation, total dry weight, acetylene reduction activity (ARA), and specific acetylene reduction activity (SARA) of plants. However, these cross-inoculation tests did not show any specificity of Mesorhizobium strains toward a given Acacia host species in terms of infectivity and efficiency as stated by principal component analysis (PCA). This study demonstrates that large-scale inoculation of A. senegal and A. seyal in the framework of reafforestation programs requires a preliminary step of rhizobial strain selection for both Acacia species.

Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil : 2: Nitrogen accumulation in the stands and biological N 2 fixation

The sustainability of plantation forests is closely dependent on soil nitrogen availability in short-rotation forests established on low-fertility soils. Planting an understorey of nitrogen-fixing trees might be an attractive option for maintaining the N fertility of soils. The development of mono-specific stands of Acacia mangium (100A:0E) and Eucalyptus grandis (0A:100E) was compared with mixed-species plantations, where A. mangium was planted in a mixture at a density of 50% of that of E. grandis (50A:100E). N 2 fixation by A. mangium was quantified in 100A:0E and 50A:100E at age 18 and 30 months by the 15N natural abundance method and in 50A:100E at age 30 months by the 15N dilution method. The consistency of results obtained by isotopic methods was checked against observations of nodulation, Specific Acetylene Reduction Activity (SARA), as well as the dynamics of N accumulation within both species. The different tree components (leaves, branches, stems, stumps, coarse roots, medium-sized roots and fine roots) were sampled on 5–10 trees per species for each age. Litter fall was assessed up to 30 months after planting and used to estimate fine root mortality. Higher N concentrations in A. mangium tree components than in E. grandis might be a result of N 2 fixation. However, no evidence of N transfer from A. mangium to E. grandis was found. SARA values were not significantly different in 100A:0E and 50A:100E but the biomass of nodules was 20–30 times higher in 100A:0E than in 50A:100E. At age 18 months, higher δ 15N values found in A. mangium tree components than in E. grandis components prevented reliable estimations of the percentage of N derived from atmospheric fixation (%Ndfa). At age 30 months, %Ndfa estimated by natural abundance and by 15N dilution amounted to 10–20 and 60%, respectively. The amount of N derived from N 2 fixation in the standing biomass was estimated at 62 kg N ha −1 in 100A:0E and 3 kg N ha −1 in 50A:100E by the 15N natural abundance method, and 16 kg N ha −1 in 50A:100E by the 15N dilution method. The total amount of atmospheric N 2 fixed since planting (including fine root mortality and litter fall) was estimated at 66 kg N ha −1 in 100A:0E and 7 kg N ha −1 in 50A:100E by the 15N natural abundance method, and 31 kg N ha −1 in 50A:100E by the 15N dilution method. The most reliable estimation of N 2 fixation was likely to be achieved using the 15N dilution method and sampling the whole plant.

Seedling treatments and phosphorus solution concentrations affect nodulation and nodule functions in soybean (Glycine max L.)

The effect of three seedling treatments: T<sub>0</sub>, normal germination; T<sub>1</sub>, cotyledons removed; T<sub>2</sub>, cotyledons removed 5 days earlier than in T<sub>1</sub>; and two phosphorus levels (P<sub>0</sub> and P<sub>30</sub>) on nodulation and nodule function in soybean [<i>Glycine max</i> (L.) Merr.] were investigated in nutrient solution culture. The number of nodules formed at P<sub>0</sub> was in the order T<sub>2</sub> > T<sub>0</sub> > T<sub>1</sub>, but it was T<sub>0</sub> > T<sub>2</sub> > T<sub>1</sub> at P<sub>30</sub>. Nodule dry weight per plant had the same tendency as the nodule number. Nodule size (dry weight per nodule) in seedlings ranged from 0.601 to 1.089 mg in the order T<sub>0</sub> > T<sub>1</sub> > T<sub>2</sub>, regardless of P level. For example, nodule size in T<sub>0</sub> was larger by 86% and 52% than T<sub>2</sub> at P<sub>0</sub> and P<sub>30</sub>, respectively. Furthermore, regardless of P level, a specific acetylene reduction activity (ARA, µM C<sub>2</sub>H<sub>4</sub>/h/g nodule) increased with P content in seedlings, but no significant difference was found (<i>P</i> < 0.05). Leghemoglobin (Lb) content was not significantly affected by P level; however, seedlings (T<sub>0</sub> and T<sub>1</sub>) significantly affected the Lb content per unit plant biomass (<i>P</i> < 0.05). All these results suggest that seedling P content plays a key role in nodulation and nodule function of soybean.

LIMITATIONS TO SYMBIOTIC NITROGEN FIXATION IN PRIMARY SUCCESSION ON THE TANANA RIVER FLOODPLAIN

Constraints on nitrogen fixation are the ultimate causes of N limitation of primary production, but hypotheses concerning limitations to N2 fixation remain largely untested in natural terrestrial ecosystems. We examined limitations to N2 fixation by thinleaf alder (Alnus tenuifolia) in two stages of primary forest succession on the Tanana River floodplain (interior Alaska, USA) and focused on the hypothesis that N, fixation was limited by low soil P availability. Paired control and P fertilized plots were established at four replicate early successional alder stands and four later successional poplar (Populus bal- sanzifera) stands (dense alder understories with mature poplar overstories) and N2 fixation was estimated with an acetylene reduction assay. In alder stands, P fertilization increased total nodule dry biomass and increased total ecosystem N inputs, but it had little effect on nitrogenase activity per unit nodule dry mass (specific acetylene reduction activity, ARA). Specific ARA increased only in late July when soil temperature and ARA were at their maximum values. In contrast, fertilization had no effect on these measures in poplar stands where reduced soil moisture may have superseded limitation by P. We detected no differ- ences in specific ARA, total nodule biomass, or N inputs, between alder and poplar stands but all of these measures were highly variable. Leaf area of the alder canopy emerged as the best predictor of ecosystem inputs of fixed N among control plots. Alders resorbed high amounts of P but little N (consistent with low P availability and a high P demand and a high N availability in alder), and P fertilization reduced P resorption but had no effect on N resorption. The timing of N, fixation and N resorption indicate that late-season increases in leaf N, following a midseason reduction in leaf N, were driven by N2 fixation in excess of plant N demands as nodules continued fixing N while alder leaves senesced. These results have shown that P limits N2 fixation in alder stands in this nitrogen-limited sere, but that factors limiting N2 fixation can change over short successional time scales.

Phosphorus Deficiency Impairs Early Nodule Functioning and Enhances Proton Release in Roots of Medicago truncatula L.

The effect of phosphorus supply (1–15μM) on proton release and the role of P in symbiotic nitrogen fixation in medic (Medicago truncatula L. ‘Jemalong’) was investigated. As P concentration in the nutrient solution increased, shoot and root growth increased by 19 and 15%, respectively by day 35, with maximal growth at 4μM P. A P concentration of 15μM appeared to be toxic to plants. Phosphorus supply had no influence on nodule formation by day 12 but increased nodule number by day 35. Nitrogenase activity was estimated by in situ measurement of acetylene reduction activity (ARA) in an open-flow system. During the assay, a C2H2-induced decline of ARA was observed under all P concentrations except 4μM. Specific ARA (per unit nodule weight) doubled when P supply was increased from 1 to 8μM. This effect of P was much greater than the effects of P on nodulation and host plant growth. Concentrations of excess cations in plants decreased with increasing P concentration in the nutrient solution. Phosphorus deficiency stimulated uptake of excess cations over anions by the plants and hence enhanced proton release. The results suggest that P plays a direct role in nodule functioning in medic and that P deficiency increases acidification which may facilitate P acquisition.

The effects of aluminium on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana Miq.

In order to investigate the effects of Al on nodule formation and function in the Casuarina-Frankia symbiosis, inoculated plants were grown in sand culture at five nominal Al concentrations (0-880 μM Al) at pH 4.0. There was an Al-free control at pH 6.0 to assess the effects of pH 4.0 treatments. Mean N concentration of nodules was significantly less at pH 4.0 (1.83%) than at pH 6.0 (2.01%). There were nodulated plants at all Al levels, though there were fewer nodulated plants at 440 and 880 μM Al. Dry weights of nodules, shoots and roots were not reduced by Al concentrations at or below 220 μM Al, but were decreased by Al concentrations at or above 440 μM Al. Nodule weight expressed as a percentage of total weight did not differ significantly with respect to an Al-free control at pH 4. N concentrations of shoots and whole plants were significantly reduced at 440 μM Al. Nodular specific acetylene reduction activity (ARA) did not differ significantly among Al treatments. However, N2-fixation efficiency was decreased from 0.20 to 0.10 mg N fixed mg nodule dry weight−1 at 880 μM Al.

Comparative growth and symbiotic performance of four Acacia mangium provenances from Papua New Guinea in response to the supply of phosphorus at various concentrations

Plant growth performance, the P content in root and nodule tissues, and nodulation and N2-fixing ability were studied in four provenances of Acacia mangium from Papua New Guinea following different levels of P fertilizer application. A. mangium did not seem to need high levels of P for growth and N2 fixation. The response by this leguminous tree to the P supply varied significantly according to provenance and to P concentrations in the culture solution. The provenances of A. mangium were classified into three types according to their P response: (1) Growth performance, nodulation, and N2 fixation of plants were stimulated as concentrations of P increased (provenance PH 482); (2) the maximal effect of P on plant growth was found only at P concentrations higher than 500 μM (provenance PH 484); and (3) the plant response to P fertilization was low, even with nutrient solutions containing P concentratins higher than 500 μM (provenances PH 483 and PH 485). Provenance PH 483 was distinguished by its low nodulating ability. However, this provenance grew well, probably because of its high N2 fixation efficiency as expressed by specific acetylene reduction activity and its high P content in nodule tissues. Therefore, in certain cases, these two parameters may be useful criteria in selecting leguminous plants for field use. Statistical analyses of the study results showed that the effect of the factor P supply on N2 fixation efficiency and nodule development was only significant at P concentrations lower than 250 μM whereas the effect of the factor plant provenance was significant regardless of the P concentration used. This observation emphasizes the value of provenance screening in the identification of plants for use in a wide range of soil types.

Comparison of twoPisum sativum nodulation mutants with their parental cultivar

In comparison with the parental cv. Finale the ‘RisfixC’ supernodulator exhibited higher, continuously increasing nodule number and fresh mass accumulation, but substantially lower individual nodule fresh mass, leghemoglobin concentration, and specific acetylene reduction activity of nodule tissue. There were no substantial differences between Finale and ‘RisfixC’ in total acetylene reduction, nodule leghemoglobin accumulation per nodulated root, total and specific CO2 evolution from nodulated roots and gross CO2 respiratory costs of acetylene reduction. The ‘RisfixC’ also exhibited a substantially lower plant dry mass production (by 30%), but nitrogen concentration in shoots and carotenoid concentration in leaf tissue were significantly higher by 33 and 14%, and the chlorophylla+b content insignficantly higher than in the parental cultivar. In contrast, the nodulation mutant ‘Risnod29’, exhibited a somewhat higher nodule fresh mass accumulation (by 21%) and individual nodule fresh mass (by 23%), total and specific acetylene reduction (by 49 and 19%) and a somewhat more rapid plant dry mass accumulation compared with the cv. Finale.

Adverse effects of nitrate on stem nodules of Sesbania rostrata Brem.

When the stems of Sesbania rostrata, growing vigorously in nutrient solution, are inoculated with Azorhizobium caulinodans ORS 571 and deprived of nitrate, hundreds of nodules with active N2 fixation (acetylene reduction) develop on each stem within 10 d. The numbers of nodules that develop are not affected by increasing the nitrate concentration in the medium from 0 to 9 mM. However, as the nitrate concentration is raised above about 2 mM the growth of the nodules is strongly inhibited and all aspects of acetylene reduction activity (ARA) are progressively curtailed as the nitrate concentration is increased from zero. In 6 mM nitrate, effects are seen at the earliest possible time of measurement (6 d after inoculation) with maximum inhibition of growth and ARA (about 80%) at 10 d. In plants with stem nodules that have developed in the absence of nitrate since inoculation, the addition of 9 mM nitrate leads to inhibition of further nodule growth and to prompt decrease in specific ARA. The 'growth' and 'activity' responses of the stem nodules of Sesbania are similar in degree and sensitivity to those of root nodules of other legumes and thus they can no longer be considered as resistant to fixed N.

Effect of low rhizosphere oxygen on growth, nitrogen fixation and nodule morphology in lucerne

Oxygen shortage in soils can occur following a wide range of natural circumstances, affecting the plant's physiology. In this paper the performance of nodulated lucerne plants under severe hypoxia is examined and the mechanisms involved to achieve this adaptation are discussed. Nodulated lucerne plants (Medicago sativa L.) were grown with their rooting medium exposed to 1 or 21 kPa oxygen. Final yield, as expressed on a shoot dry weight basis, was unaffected but root and nodule dry weights were reduced by 50%. Water content in roots and nodules was higher at 1 kPa as a result of the formation of aerenchyma. Specific acetylene reduction activity was higher in hypoxic nodules as a consequence of modified nodule structure, although they were more sensitive to the presence of acetylene or nitrate. Root respiration was insensitive to changes in external oxygen supply, therefore providing adequate support for mineral uptake. Nodule respiration rates were 5 times higher in control plants when measured as CO2 evolution, whereas no differences were observed in O2 uptake. It is suggested that adaptation of nodulated lucerne to low oxygen concentrations involves changes in photosynthate allocation and nodule morphology, which provide a more efficient nitrogen fixation.

Growth of Sesbania rostrata (Brem) with stem nodules under controlled conditions

ABSTRACTA series of experiments was carried out in an attempt to produce nodulated plants of Sesbania rostrata with qualities more closely resembling those in the wild than has been achieved to date. When groups of five plants were grown in a controlled climate chamber in pipes containing ∼12dm3 modified Jensen's medium with 6mol m−3 nitrate, the daily growth in height reached 5 cm and at 30 d the plants were ∼40cm high. At this time, the stems were inoculated with Azorhizobium caulinodans ORS 571 and the medium replaced with Jensen's medium without nitrate. In the subsequent 19‐d period ∼300 nodules (representing >50% of the potential infection sites) developed on each stem. The nodules increased linearly in size over this time to ∼15mg. Specific acetylene reduction activity, ARA ((μmol C2H4 mg−1 h−1) rose to 45 between days 5 and 10 after inoculation and plateaued; total ARA rose to ∼200 μmol C2H4 plant−1 h−1. Under the conditions described the plants grew vigorously, and reproducibly uniform yields of nodules with high ARA activities were obtained. As outlined, the procedure offers a standard system in which, within a 2‐week period after inoculation, individual strains of bacteria can be quantitatively compared in their ability to induce nodulation and N2‐fixation. Physiological and biochemical aspects of the nodulated system can be much more readily approached than with plants producing only root nodules. The inhibitory effects of stem nodules induced by wild type and two mutant strains of Azorhizobium on the development and activity of root nodules are described.

Variation in Nitrate Tolerance of Nitrogen Fixation in the Pea/Rhizobium Symbiosis

AbstractThe short‐term effects of nitrate on symbiotic nitrogen fixation were studied in six cultivars of pea (Pisum sativum L.) using two strains of Rhizobium leguminosarum. Plants grown symbiotically for three weeks, in order to eliminate the effect of NO3 on nodule formation, were exposed to nil, 5 or 15 mM NO3 for a period of seven days. Nodule mass, nitrogenase activity by acetylene reduction and the NO2 and NO3 concentrations in nodules were determined.Exposure to nitrate reduced the total acetylene reduction activity (ARA) in all cultivars, and a significant cultivar × nitrate treatment interaction was found. Nitrate influenced the components of N2 fixation, nodule mass and specific ARA (ARA/mg nodule), differently within and among cultivars. Nodule mass was reduced in all cultivars, but a variation among cultivars in the degree of reduction was found. Nitrate also reduced the specific ARA to different extents in the cultivars‐ The specific ARA of nodules from NO3‐treated plants, expressed as per cent of the specific ARA of nodules from plants grown without NOT3 was negatively correlated with the concentration of NO3 in nodules. No relationship was found between the reduction of N2 fixation and the nitrite concentration in nodules.The results indicate that it is possible to improve the nitrate tolerance of symbiotic N2 fixation of the established pea/Rhizobium symbiosis by selection of suitable host plants.

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N(2) than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N(2) than did the USDA 110 during the 20-day period. The superior N(2) fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N(2)-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N(2)-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N(2) in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N(2)-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N(2) fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N(2)-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.

Nitrate Inhibition of Legume Nodule Growth and Activity

The synthesis and accumulation of nitrite has been suggested as a causative factor in the inhibition of legume nodules supplied with nitrate. Plants were grown in sand culture with a moderate level of nitrate (2.1 to 6.4 millimolar) supplied continuously from seed germination to 30 to 50 days after planting. In a comparison of nitrate treatments, a highly significant negative correlation between nitrite concentration in soybean (Glycine max [L.] Merr.) nodules and nodule fresh weight per shoot dry weight was found even when bacteroids lacked nitrate reductase (NR). However, in a comparison of two Rhizobium japonicum strains, there was only 12% as much nitrite in nodules formed by NR(-)R. japonicum as in nodules formed by NR(+)R. japonicum, and growth and acetylene reduction activity of both types of nodules was about equally inhibited. In a comparison of eight other NR(+) and NR(-)R. japonicum strains, and a comparison of G. max, Phaseolus vulgaris, and Pisum sativum, the concentration of nitrite in nodules was unrelated to nodule weight per plant or to specific acetylene reduction activity. The very small concentration of nitrite found in P. vulgaris nodules (0.05 micrograms NO(2) (-)-N per gram fresh weight) was probably below that required for the inhibition of nitrogenase based on published in vitro experiments, and yet the specific acetylene reduction activity was inhibited 83% by nitrate. The overall results do not support the idea that nitrite plays a role in the inhibition of nodule growth and nitrogenase activity by nitrate.