Cyanobacterial nitrogen fixation in arid soils of Central Australia

Abstract The distribution of nitrogen-fixing cyanobacteria was studied in two regions of Central Australia, one near Yulara and the other in the Ti-Tree Basin. Soil and other samples were tested in situ for nitrogen fixation by the acethylene reduction assay. They were then enriched in low light in suitable growth medium containing no fixed carbon or nitrogen over a period of months. The cyanobacterial components were identified and their nitrogen-fixing capacity measured again both in the light and in darkness. It is concluded that a variety of genera of cyanobacteria survive in the arid region of Central Australia and can be assumed to contribute fixed nitrogen to the soils of the region.     

Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.     

Cyanobacterial inoculation and nitrogen fertilization in rice

During three rice-growing seasons in Uruguay, field experiments were conducted to study the contribution of cyanobacterial inoculation and chemical N fertilization to rice production. Neither grain yield nor fertilizer recovery by the plant were affected by inoculation with native cyanobacterial isolates. A low fertilizer use efficiency (around 20%) was observed when labelled (NH₄)₂SO₄ was applied at sowing. Recovery of applied ¹⁵N by the soil–plant system was 50%. Inoculation did not modify ¹⁵N uptake by the plant when the fertilizer was three-split applied either. The total N-fertilizer recovery was higher when the fertilizer was split than when applied in a single dose. Plant N-fertilizer uptake was higher when the fertilizer was applied at tillering. Uptake of ¹⁵N from cyanobacteria by rice was studied in a greenhouse pots experiment without chemical nitrogen addition. Recovery of ¹⁵N from labelled cyanobacteria by rice in greenhouse growth conditions was similar to that of partial recovery of (NH₄)₂SO₄ applied at sowing in the field. Cyanobacterial N mineralization under controlled conditions was fast as cyanobacterial N was detected in plants after 25 days. Moreover 40 days after inoculation non-planted and inoculated soil had more inorganic N than the non-inoculated one.     

Effect of nitrogen- and potassium-based fertilizers on nitrogen fixation in the winged bean,Psophocarpus tetragonolobus

Summary Five rates of urea application (0 to 40 kg N/ha) and three rates of K⁺ (0 to 65 kg/ha) on the nitrogen-fixing activity of two varieties (SLS-40 and SLS-44) of winged bean were examined in the field. Specific activity of nitrogenase was inhibited with increasing amounts of urea up to 75 days after sowing (d.a.s.), but this was not so apparent at 90 d.a.s. Nitrogenase activity increased in proportion to K⁺ fertilization especially during the early stages of growth (30 to 60 d.a.s.) and continued even with added N fertilizer. Nitrogenase activities correlated better with nodule dry weight than with nodule number. Nodule number and nodule dry weight were significantly decreased by added N. K⁺ had a significant effect on nodule number and nodule dry weight. Forty-five day-old plants gave high nodule number but the highest nodule dry weight was recorded on 75 day-old plants. K⁺ fertilizer stimulated early root growth (30 to 45 d.a.s.) but the overall root biomass at the end of the experiment was similar under all K⁺ treatments. The shoot weight increased with N but not with K⁺.

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Effet d'une fertilisation basée sur l'azote et le potassium sur la fixation de l'azote chez la fève ailée (Psophocarpus tetragonolobus)

Résumé On examine sur le terrain l'effet de cinq doses d'application d'urée (de 0 à 40 kg d'azote par ha) et de trois doses d'application de K⁺ (de 0 à 65 kg/ha) sur la fève ailée. Les activités spécifiques de nitrogénase sont inhibées par des doses croissantes d'urée jusqu'à 75 jours après le semis (j.a.s.) mais ceci n'est pas aussi apparent 90 j.a.s. Les activités de nitrogénase augmentent proportionnellement aux doses de fertilisation par le K⁺, surtout pendant les premiers temps de la croissance (de 30 à 60 j.a.s.) et se maintiennent même en cas d'ajout de fertilisant azoté. Les activités de nitrogénase sont mieux correlées avec les poids secs des nodules qu'avec les nombres de nodules. Tant les nombres de nodules que les poids secs de nodules diminuent de manière significative par ajout d'azote. K⁺ a un effet significatif tant sur les nombres de nodules que sur les poids secs de nodules. Des plants agés de 45 jours démontrent des nombres élevés de nodules mais les poids secs les plus élevés de nodules sont enregistrés chez des plants agés de 75 jours. La fertilisation par K⁺ stimule la croissance précoce des racines (de 30 à 45 j.a.s.) mais les biomasses radiculaires totales à la fin de l'expérimentation sont semblables quelque soit le traitement potassique. Les poids de pousses augmentent avec les doses d'azote mais pas avec les doses de K⁺.

     

15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

Plant and Soil - To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for...     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Consequences of banding nitrogen fertilizers in soil

Summary An account is given of the growth of wheat roots in the environment produced by banded urea and ammonium sulphate in both sterile (γ-irradiated) and non-sterile soil. In the non-sterile soil, (i) roots were absent from the fertilizer-affected zone (diameter about 10 cm) for both fertilizers at 2 weeks, presumably owing to unfavourable osmotic suctions there, (ii) roots proliferated enormously in the zone at both 4 and 8 weeks with ammonium sulphate, and (iii) roots were absent from the zone at both 4 and 8 weeks with urea (presumably owing to the large amounts of nitrite there), but they proliferated at the edge of the zone and completely encased it by eight weeks. In the sterile soil there was no marked proliferation of roots with ammonium sulphate, and with urea there was a region of intense proliferation surrounding a small zone (diameter 2 cm) which was devoid of roots. There were no universal correlations between the proliferations and the distributions of nitrate, nitrite, ammonium, pH, or (presumed) activity of nitrifiers. Nevertheless, ammonium seemed to be the main stimulus for the roots; it only failed to correlate with the root distributions if the concentration of nitrite was high or if the pH was below 4.     

Possible causes of the physiological decline in soybean nitrogen fixation in the presence of nitrate

Nodulated soybean plants (Glycine max (L.) Merr. cv. Clarke)were supplied with 10 mol m^-3 nitrate at the vegetative stage.This treatment caused a rapid decline in nitrogen fixation (acetylenereduction) activity and a consequent decline in ureides in thexylem sap. However, there was virtually no effect on the nitrogenasecomplex, according to Western blots against components 1 and2. The effect on nitrogen fixation was matched by a decreasein nitrogenase-linked respiration and increases in nodule oxygendiffusion resistance and the carbon cost of nitrogen fixation.The addition of nitrate had little effect on protein contentfrom either nodule plant or bacteroid fractions. Activitiesof nitrate reductase (NR) and nitrite reductase (NiR) from eitherthe plant fraction or the bacteroids were affected in differentways during 8 d of supply. Nodule plant NR and bacteroid NiR were not affected. However,nodule plant NiR increased 5-fold within 2 d of supplying Bacteroid NR only increased after6 d. These results could be interpreted in terms of a restrictednitrate access into the infected region of nodules. However,denitrification was detected within 2 d of nitrate supply insoybean nodules. The results are discussed in relation to possiblecauses of the nitrate-induced decline in nitrogenase activity. Key words: Glycine max, nitrate, nitrogen fixation, nodules     

Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4.

The metabolic fate of gaseous nitrogen (15N2) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N2-fixation system was followed. A majority of the fixed 15N2 was found to be exported into the cell supernatant. For example, as much as 94% of the 15N2 fixed by Rhizobium japonicum (soybean symbiont) was recovered as 15NH+4 from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH+4 from L-histidine. Evidence is presented that overproduction and export of NH+4 by free-living Rhizobia may be closely linked to the control of several key enzymes of NH+4 assimilation. For instance, NH+4 was found to repress glutamine synthetase whereas L-glutamate repressed glutamate synthase. Assimilation of NH+4 as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH+4 production by root nodule bacteria is discussed.     

长效氮肥施用对黑土水旱田CH4和N2O排放的影响

通过在黑土玉米地与水稻田施用长效氮肥后发现 ,长效碳酸氢铵 (长碳 )与长效尿素(长尿 )能显著减少黑土玉米地N2 O的排放。与施用普通尿素相比 ,其排放量分别减少了5 9 2 %和 73 3%。长碳和长尿还能促进黑土玉米地对CH4的吸收作用。黑土水稻田施用长尿后 ,N2 O的排放减少了 6 1%。而CH4的排放却略有增加     

Alternate nitrogen amendments for organic fertilizers

The use of compost or manure in agriculture as an organic source of nutrients is common in many tropical, developing countries like Nigeria. One of the drawbacks of such materials is their low nitrogen (N) content (=1% N). Farmers commonly use chemical N fertilizers such as urea, calcium ammonium nitrate (CAN), and NPK formulations to obtain better crop growth and yield. These chemical supplements may have a negative impact on the environment through nitrate leaching into water, leading to eutrophication of surface waters that can affect public health. Gliricidia sepium, a fast-growing, tropical, perennial hedge plant was tested as a source of N in organo-mineral fertilizer formulations. Average nutrient content of Gliricidia is 3.8% N, 0.32% P, 1.8% K, 0.8% Ca, and 0.2% Mg. Using a sand culture and Amaranthus caudatus as a test crop, it was shown that amending commercial composts with 30% Gliricidia prunings would benefit many small-scale farmers and control environmental pollution.     

Role of N2 fixation in the soybean N credit in maize production

Many studies have shown that maize (Zea mays L.) requires less fertilizer N for optimum yield when grown in rotation with soybean [Glycine max (L.) Merr] than when grown in monoculture, which is referred to as the `soybean N credit' in the maize growing areas of the United States. Because the specific source of this soybean N credit is unclear, our objective was to determine the role of nodules and N₂ fixation as a contributing source of the soybean N credit. Our research approach was designed to separate the effect of symbiotic N₂ fixation from other rotational effects, as the treatments included: maize grown after nodulated (N₂ fixing) soybean and maize grown after non-nodulated (non N₂ fixing) soybean. A separate experiment examined maize grown after maize. For each previous crop, maize was grown the following year with varying rates of fertilizer applied N. In both years, the yield differences between nodulated and non-nodulated soybean as the previous crop were much smaller than the apparent yield decrease associated with continuous maize. Although small in magnitude, maize following non-nodulated soybean accumulated less total N, was paler in leaf color, and yielded less than maize following nodulated soybean in the more favorable year of 1999, while most of these differences were not observed in 2000. These findings indicate that soybean nodules and N₂ fixation, while having a certain role, are not the major determinants of the soybean N credit.     

Effect of nitrogen fertilization and cropping system of the reference crop on estimation of N2 fixation by vetch using15N methodology

This study was conducted to examine the effects of varying N rates and cropping systems (mixedversus pure stand) on the suitability of oats (Avena sativa L.) for estimating N₂ fixed in sequentially harvested vetch (Vicia sativa L.) over two growing seasons (1984–85 and 1985–86). The N rates were, 20 and 100 kg N ha^–1 in 1984–85 and 15 and 60 kg N ha^–1 in 1985–86. In the 1984–85 season, vetch at maturity derived 76 and 63% N from fixation at the high and low N rates respectively. The corresponding values for the second season were 66 and 42%. Except in the 1985–86 season when some significantly higher values of % N₂ fixed were estimated by using the reference crop grown at the higher (A-value approach) than at the lower N rate (isotope-dilution approach), both approaches resulted in similar measurements of N₂ fixed. In the 1984–85 season, similar values of N₂ fixed were obtained using either the pure or mixed stand oats reference crops. Although in the 1985–86 season, the mixed reference crop occasionally estimated lower % N₂ fixed than pure oats, total N₂ fixed estimates were always similar (P<0.05). Thus, in general, N fertilization and cropping system of the reference crop did not significantly influence estimates of N₂ fixation.     

Biological nitrogen fixation

And he gave it for his opinion, that whoever could make two ears of corn or two blades of grass to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country than the whole race of politicians put together. {Jonathan Swift, ‘Gulliver's Travels’, Voyage to Brobdingnag, Ch. 7.)     

Interactions between white clover and ryegrass under contrasting nitrogen availability: N2 fixation,N fertilizer recovery,N transfer and water use efficiency

Seasonal variation in N₂ fixation, N transfer from clover to ryegrass, and soil N absorption in white clover–ryegrass swards were investigated under field conditions over three consecutive years. The plots were established with different seeding ratios of clover and ryegrass and contrasting fertilizer N ranging from 3 to 72 kg ha^-1 year^-1.An initially poor clover population needed at least one growing season to reach the same yield output as an initially well established clover population. The clover content of the sward decreased by the annual application of 72 kg N ha^-1 but not by smaller N dressings.The total amount of atmospherically derived N in clover growing in mixture with ryegrass was, on average over the three years equal to 83, 71, 68 and 60 kg N ha^-1 for the treatments of 3, 24, 48 and 72 kg N ha^-1, respectively. The proportion of atmospherically derived N declined with increasing N application, but never became smaller than 80% of total clover N. The proportion of atmospherically derived N in a pure stand white clover amounted to 60–80% of the total N content, equivalent to 109, 110, 103 and 90 kg N ha^-1 for the treatments of 3, 24, 48 and 72 kg N ha^-1, respectively.Only small amounts of atmospherically derived N was transferred to the associated ryegrass during the first production year, while in each of the following years up to 21 kg ha^-1 was transferred. The average amount of N transferred from clover to ryegrass was equivalent to 3, 16 and 31% of the N accumulated in ryegrass in the first, second and third production year, respectively. Expressed relative to the total amount of fixed N₂ in the clover–ryegrass mixture, the transfer amounted to 3, 17 and 22% in the first, second and third production year, respectively. Thus transfer of atmospherically derived N from clover contributed significantly to the N economy of the associated ryegrass.The clover–ryegrass mixture absorbed constantly higher amount of soil derived N than the pure stands of the two species. Only 11% of the total accumulated fertilizer N and soil derived N in the mixture was contained within the clover component. Lower water use efficiencies for the plants grown in mixture compared to pure stands were mainly related to the increased N uptake in the mixture, with the subsequent increase in growth compared to the pure stands.It is concluded that positive interactions between clover and ryegrass growing in mixture ensure a more efficient fixation of atmospheric N₂ and absorption of fertilizer N and soil derived N than pure stands of the same species.