Cyanobacterial N2 fixation in presence of nitrogen fertilizers

Anabaena oryzae ARM 570 was examined for its growth (chlorophyll and protein), heterocyst frequency, nitrogenase (acetylene reduction) activity, ammonia excretion, and glutamine synthetase and nitrate reductase in response to two levels of urea-N vis-à-vis N2-N. Growth of cyanobacterium increased with duration of incubation. Reduction in heterocyst frequency (40%) was observed at 30 ppm of urea-N, whereas at 60 ppm of urea-N, filaments were completely devoid of heterocysts and no nitrogenase activity was observed. Maximum excretion of ammonia occurred at 30 ppm of urea-N, which was concomitant with minimum glutamine synthetase activity. These results suggested that A. oryzae could be effectively utilized in cyanobacterial biofertilizer programme even in the presence of combined nitrogen, for improving N-budget in rice cultivation.     

15N natural abundance in plants of the Amazon River floodplain and potential atmospheric N2 fixation

Summary The¹⁵N natural abundance values of various Amazon floodplain (várzea) plants was investigated. Samples of young leaf tissues were collected during three different periods of the river hydrography (low water, mid rising water and high water) and during one period in the Madeira River (high water). A large variation of¹⁵N abundance was observed, both among the different plant types and between the different flood stages. This variation probably, reflected, in part, the highly variable nature of the floodplain, sometimes dry and oxygenated and at other times inundated and anaerobic and, in part, changes in plant nitrogen metabolism. Comparison of the nitrogen isotopic composition of leguminous plants with that of non-leguminous plants showed that, on average, the¹⁵N abundance was lower in the legumes than non-legumes, suggesting active N-fixation. Also, the¹⁵N natural abundance in aquatic grasses of the generaPaspalum, was in general, lower than the¹⁵N abundance of aquatic grasses of the generaEchinochloa. As both of these grasses grow in the same general habitat, it appears thatPaspalum grasses may also be nitrogen fixers.     

Enhancing biological nitrogen fixation: An appraisal of current and alternative technologies for N input into plants

Biological nitrogen fixation (BNF) involves a highly specialized and intricately evolved interactions between soil microorganisms and higher plants for harnessing the atmospheric elemental nitrogen (N). This process has been researched for almost a century for efficient N input into plants. The basic mechanism and biochemical steps involved in BNF have been unraveled. It has become abundantly clear that the host plant (legumes) dominates in regulating the BNF process. Environmental factors as well influence this process. Perturbation or any manipulation of the interactions between the bacteria and the legumes seems to offset the critical balance, usually to the detriment of N fixation efficiency. Not much success has been obtained in either enhancing BNF in legumes or transferring important BNF traits to non-nitrogen fixing organisms. An appraisal is given for the lack of success in making the BNF process a popular and efficient agronomic practice. Alternative physiological approaches are presented for improving mobilization, redistribution and utilization of stored N reserves within the host plant.     

Evidence against associative N2 fixation as a significant N source in long-term wheat plots

In monocropped cereal systems, annual N inputs from non-fertilizer sources may be more than 30 kg ha^-1. We examined the possibility that these inputs are due to biological N₂ fixation (BNF) associated with roots or decomposing residues. Wheat was grown under greenhouse conditions in pots (34 cm long by 10 cm diameter) containing soil from a plot cropped to spring wheat since 1911 without fertilization. The roots and soil were sealed from the atmosphere and exposed to a¹⁵N₂-enriched atmosphere for three to four weeks during vegetative, reproductive or post-reproductive stages. This technique permitted detection of as little as 1 μg fixed N plant^-1 in plant material and 40 μg fixed N plant^-1 in soil. No fixation of¹⁵N₂ occurred during either of the first two labelling periods. In the final labelling period, straw returned to the soil was significantly enriched in¹⁵N, especially in a pot with a higher soil moisture content. Total BNF in this pot was 13 μg N plant^-1, or about 30 g N ha^-1. In a separate experiment with soil from the same plot, we detected BNF only when soil was amended with glucose at a high soil moisture content. Measured associative BNF was insufficient to account for observed N gains under field conditions. Lethbridge Research Centre contribution no. 3879488. Lethbridge Research Centre contribution no. 3879488.     

Molecular-marker-facilitated investigation on the ability to stimulate N2 fixation in the rhizosphere by irrigated rice plants

An F₂ population, consisting of 231 individuals derived from a cross between rice cultivars with a similar growing duration, Palawan and IR42, was utilized to investigate the genetic nature of rice varietal ability to stimulate N₂ fixation in the rice rhizosphere. To assess rhizospheric N₂ fixation, an isotope-enriched ¹⁵N dilution technique was employed, using ¹⁵N-stabilized soil in pots. IR42, an indica variety, had 23% higher N derived from fixation (Ndfa) than Palawan, a javanica genotype. Normal segregation of atom% ¹⁵N excess was obtained in the F₂ population, with an average of 0.218 with 8% of plants below IR42 (0.188) and 10% of plants above Palawan (0.248). One-hundred-and-four RFLP markers mapped on 12 chromosomes were tested for linkage to the putative QTLs. Significant (P<0.01) associations between markers and segregation of atom% ¹⁵N excess were observed for seven marker loci located on chromosomes 1, 3, 6 and 11. Four QTLs defined by the detected marker loci were identified by interval-mapping analysis. Additive gene action was found to be predominant, but for at least one locus, dominance and partial dominance effects were observed. Significant (P<0.01) epistatic effects were also identified. Individual marker loci detected between 8 and 16% of the total phenotypic variation. All four putative QTLs showed recessive gene action, and no phenotypic effects associated with heterozygosity of marker loci were observed. The results of this study suggest that rice genetic factors can be identified which affect levels of atom% ¹⁵N excess in the soil by interacting with diazotrophs in the rice rhizosphere.     

N2 fixation in Thai soybeans: Effect of tillage and inoculation on15N-determined N2 fixation in recommended cultivars and advanced breeding lines

The practice of seeding soybeans following paddy rice in Thailand has encountered difficulties in seedling germination, nodulation and crop establishment. This research project evaluated the choice of a non-fixing control to quantify N₂ fixation by¹⁵N isotope dilution, and the effect of tillage regime, soybean cultivar, strain ofBradyrhizobium japonicum and P fertilization on yield and N₂ fixation after paddy rice in northern and central Thailand.Japanese non-nodulating lines Tol-0 and A62-2 were the most appropriatecontrol plants for¹⁵N isotope dilution for Thai soybeans in these soils which contained indigenous rhizobia. Cereals such as maize, sorghum and barley were also appropriate controls at some sites. The choice of the appropriate non-fixing control plant for the¹⁵N isotope dilution technique remains a dilemma and no alternative exists other than to use several possible controls with each experiment. Acetylene reduction assay (ARA) proved of little value for screening varieties on their N₂ fixing capacity.The recommended Thai soybean cultivars (SJ1, 2, 4, 5) and an advanced line 16–4 differed little in their ability to support N₂ fixation or yield, possibly due to similar breeding ancestry. The ten AVRDC (ASET) lines showed considerable genotypic control in their ability to utilize their three available N sources (soil, fertilizer, atmosphere) and to translate them into yields. None of these lines were consistently superior to Thai cultivars SJ4 or SJ5 although ASET lines 129, 209 and 217 showed considerable promise.Neither recommended Thai or ASET cultivars were affected by tillage regime. Zero tillage resulted in superior N₂ fixation and yield at two sites but conventional tillage was superior at another site. Soybean cultivars grown in Thailand were well adapted to zero tillage. Levels of N₂ fixation were similar to world figures, averaging more than 100 kg N ha^–1 and supplying over 50% of the plant's N yield. However, seed yields seldom exceeded 2 t ha^–1, well below yields for temperately-grown soybeans. It is not clear why Thai soybeans support N₂ fixation, but do not translate this into higher seed yields.     

Nitrogenase activity and N2 fixation are stimulated by elevated CO2 in a tropical N2-fixing tree

 Seeds of Gliricidia sepium, a fast-growing woody legume native to seasonal tropical forests of Central America, were inoculated with N₂-fixing Rhizobium bacteria and grown in environmentally controlled glasshouses for 67–71 days under ambient CO₂ (35 Pa) and elevated CO₂ (70 Pa) conditions. Seedlings were watered with an N-free, but otherwise complete, nutrient solution such that bacterial N₂ fixation was the only source of N available to the plant. The primary objective of our study was to quantify the effect of CO₂ enrichment on the kinetics of photosynthate transport to nodules and determine its subsequent effect on N₂ fixation. Photosynthetic rates and carbon storage in leaves were higher in elevated CO₂ plants indicating that more carbon was available for transport to nodules. A ¹⁴CO₂ pulse-chase experiment demonstrated that photosynthetically fixed carbon was supplied by leaves to nodules at a faster rate when plants were grown in elevated CO₂. Greater rates of carbon supply to nodules did not affect nodule mass per plant, but did increase specific nitrogenase activity (SNA) and total nitrogenase activity (TNA) resulting in greater N₂ fixation. In fact, a 23% increase in the rate of carbon supplied to nodules coincided with a 23% increase in SNA for plants grown in elevated CO₂, suggesting a direct correlation between carbon supply and nitrogenase activity. The improvement in plant N status produced much larger plants when grown in elevated CO₂. These results suggest that Gliricidia, and possibly other N₂-fixing trees, may show an early and positive growth response to elevated CO₂, even in severely N-deficient soils, due to increased nitrogenase activity. Received: 27 February 1996 / Accepted: 19 June 1996     

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.     

Interactions between nitrate uptake and N2 fixation in white clover

Summary White clover (Trifolium repens L.) plants grown in pots and supplied with the same concentration x days of¹⁵N labelled nitrate, but in contrasting patterns and doses had similar N concentrations but differed in the proportions devived from N₂ fixation and nitrate. N₂-fixation and nodule dry weight responded rapidly (2–3 days) to changes in nitrate availability. Plants exposed frequently to small doses of nitrate took up more nitrate (and hence relied less on N₂-fixation) and had greater dry weights and shoot: root ratios than those exposed to larger doses less often. In mixed ryegrass (Lolium perenne L.)/clover communities clover's ability to either successfully compete for nitrate or fix N₂ gave it consistently higher N concentrations than grass whether they were given high or low nitrate nutrient. This higher N concentration was accompanied by greater dry weights than grass in the low nitrate swards but not where high levels of nitrate were applied.     

14CO2 fixation and glycolate metabolism in the dark in isolated maize (Zea mays L.) bundle sheath strands

Bundle sheath strands capable of assimilating up to 68 μmoles CO₂ per mg chlorophyll per hr in the dark have been isolated from fully expanded leaves of Zea mays L. This dark CO₂-fixing system is dependent on exogenous ribose-5-phosphate, ADP or ATP, and Mg²⁺ for maximum activity. The principal product of dark fixation in this system is 3-phosphoglycerate, indicating that the CO₂-fixing reaction is mediated by ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39). The rate of dark CO₂ uptake in the strands in the presence of saturating levels of ribose-5-phosphate plus ADP is inhibited by oxygen. The inhibitory effect of oxygen is rapidly and completely reversible, and is relieved by increased levels of CO₂. Glycolate is synthesized in this dark system in the presence of [U-¹⁴C]ribose-5-phosphate, ADP, oxygen, and an inhibitor of glycolate oxidase (EC 1.1.3.1). Glycolate formation is completely abolished by heating the strands, and the rate of glycolate synthesis is markedly reduced by either lowering the oxygen tension or increasing the level of CO₂.These results, obtained with intact cells in the absence of light, indicate that the direct inhibitory effect of oxygen on photosynthesis is associated with photosynthetic carbon metabolism, probably at the level of ribulose-1,5-bisphosphate carboxylase, and not with photophosphorylation or photosynthetic electron transport. Furthermore, the findings indicate that the synthesis of glycolate from exogenous substrate can readily occur in the absence of photosynthetic electron transport, an observation consistent with the ribulose-1, 5-bisphosphate “oxygenase” scheme for glycolate formation during photosynthesis.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops

The effect of mixed intercropping of field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.), compared to monocrop cultivation, on the yield and crop-N dynamics was studied in a 4-yr field experiment using ¹⁵N-isotope dilution technique. Crops were grown with or without the supply of 5 g ¹⁵N-labeled N m^-2. The effect of intercropping on the dry matter and N yields, competition for inorganic N among the intercrop components, symbiotic fixation in pea and N transfer from pea to barley were determined. As an average of four years the grain yields were similar in monocropped pea, monocropped and fertilized barley and the intercrop without N fertilizer supply. Nitrogen fertilization did not influence the intercrop yield, but decreased the proportion of pea in the yield. Relative yield totals (RYT) showed that the environmental sources for plant growth were used from 12 to 31% more efficiently by the intercrop than by the monocrops, and N fertilization decreased RYT-values. Intercrop yields were less stable than monocrop barley yields, but more stable than the yield of monocropped pea. Barley competed strongly for soil and fertilizer N in the intercrop, and was up to 30 times more competitive than pea for inorganic N. Consequently, barley obtained a more than proportionate share of the inorganic N in the intercrop. At maturity the total recovery of fertilizer N was not significantly different between crops, averaging 65% of the supplied N. The fertilizer N recovered in pea constituted only 9% of total fertilizer-N recovery in the intercrop. The amount of symbiotic N₂ fixation in the intercrop was less than expected from its composition and the fixation in monocrop. This indicates that the competition from barley had a negative effect on the fixation, perhaps via shading. At maturity, the average amount of N₂ fixation was 17.7 g N m^-2 in the monocrop and 5.1 g N m^-2 in the intercropped pea. A higher proportion of total N in pea was derived from N₂ fixation in the intercrop than in the monocrop, on average 82% and 62%, respectively. The ¹⁵N enrichment of intercropped barley tended to be slightly lower than of monocropped barley, although not significantly. Consequently, there was no evidence for pea N being transferred to barley. The intercropping advantage in the pea-barley intercrop is mainly due to the complimentary use of soil inorganic and atmospheric N sources by the intercrop components, resulting in reduced competition for inorganic N, rather than a facilitative effect, in which symbiotically fixed N₂ is made available to barley.Abbreviations MC monocrop - IC intercrop - PMC pea monocrop - BMC barley monocrop - PIC pea in intercrop - BIC barley in intercrop     

Measurements of N2 fixation by common bean in Central Brazil as affected by different reference crops

An experiment was conducted at EMBRAPA/CNPAF, Goiânia, Goias, Brazil, on a typic haplustox soil to evaluate growth and N₂ fixation-related parameters of Phaseolus vulgaris L. Bean lines, which had been selected for N₂ fixation at CNPAF, including production cultivars, germplasm bank entries, and parents and progenies of a cross made to improve this characteristic. Wheat (Triticum aestivum L.) and dwarf sorghum (Sorghum bicolor (L.) Moench) were evaluated as non-N₂-fixing reference crops for difference method (DM) and ¹⁵N isotope dilution technique (IDT) estimates of N₂ fixation. IDT estimates ranges from 4 to 18 kg N₂ fixed ha^-1. High variability associated with low levels of N₂ fixation precluded definitive identification of the best N₂ fixing bean lines. Due to differences in growth cycle and in patterns and amounts of soil N uptake during the season, neither of the reference crops tested appears to be an adequate control for either DM or IDT estimates of N₂ fixation. However, ranking of lines for effectiveness in N₂ fixation could be performed without the use of any reference crops.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

Estimation of N2 fixation based on differences in the natural abundance of 15N among freshwater N2-fixing and non-N2-fixing algae

The hypothesis that small mammal burrows can increase the amount of water infiltrating into the soil profile was tested. The amount of water added to the soil profile from spring recharge in areas adjacent to ground squirrel (Spermophilus townsendii and S. elegans) burrows was compared to nearby areas without burrows. Recharge amounts in burrow areas were significantly higher than nonburrow areas. An average of 21% more of the winter precipitation infiltrated into the soil near burrows. The amount of recharge was also found to be positively related to burrow density. Burrows also affected the distribution of the recharge by adding significantly more water to the deeper portions (>50 cm) of the soil profile.     

Manganese transport is essential for N2‐fixation by Rhizobium leguminosarum in bacteroids from galegoid but not phaseoloid nodules

Rhizobium leguminosarum has two high‐affinity Mn²⁺ transport systems encoded by sitABCD and mntH. In symbiosis, sitABCD and mntH were expressed throughout nodules and also strongly induced in Mn²⁺‐limited cultures of free‐living cells. Growth of a sitA mntH double mutant was severely reduced under Mn²⁺ limitation and sitA and mntH single mutants were more sensitive to oxidative stress. The double sitA mntH mutant of R. leguminosarum was unable to fix nitrogen (Fix^‐) with legumes belonging to the galegoid clade (Pisum sativum, Vicia faba and Vicia hirsuta). The presence of infection thread‐like structures and sparsely‐packed plant cells in nodules suggest that bacteroid development was blocked, either at a late stage of infection thread progression or during bacteroid‐release. In contrast, a double sitA mntH mutant was Fix⁺ on common bean (Phaseoli vulgaris), a member of the phaseoloid clade of legumes, indicating a host‐specific symbiotic requirement for Mn²⁺ transport.