Asymbiotic biological nitrogen fixation in a temperate grassland as affected by management practices

Estimates of asymbiotic biological N fixation (BNF) in temperate grasslands are few with large variations. In the past six decades, European grasslands have been subjected to intensive management practices and presently it is not known how asymbiotic BNF is influenced by these practices. Our objective was to assess the impact of fertilizer application and mowing frequency on asymbiotic BNF in a Central European grassland. In 2008, we established a three-factorial experiment with two fertilizer treatments (no fertilizer application and combined nitrogen (N), phosphorus (P) and potassium (K) fertilization at 180–30–100 kg ha⁻¹ yr⁻¹), two mowing frequencies (cut once and thrice per year) and three sward compositions through the application of herbicides (control, monocot- and dicot-enhanced swards). Three years after the initial sward manipulation, there was no more difference in functional group composition. Between June 2011 and May 2012, we measured in-situ asymbiotic BNF using the acetylene reduction assay, calibrated with ¹⁵N₂-fixation method. Across treatments, asymbiotic BNF rates in the 0–5-cm soil depth ranged from 1.7 (±0.2 SE) kg ha⁻¹ yr⁻¹ for fertilized plots cut once a year to 5.7 (±2.3 SE) kg ha⁻¹ yr⁻¹ for unfertilized plots cut thrice a year. Fertilization decreased asymbiotic BNF, suggesting that the potential positive effect of increased soil P levels might have been overruled by the negative effect of increased soil mineral N levels. Intensive mowing stimulated asymbiotic BNF, which was probably due to an increase in rhizodeposition. Our calibration of the acetylene reduction assay with the ¹⁵N₂-fixation method resulted in a conversion factor of 0.61, which largely deviates from the theoretical conversion factor of 3. Furthermore, laboratory incubations under increased soil moisture and temperature conditions overestimated BNF rates compared to in-situ measurements. Thus, laboratory measurements with altered soil moisture, temperature or disturbed soil may lead to strong biases in estimates of asymbiotic BNF. Our results suggest that input of N through BNF may be considerable in temperate grasslands. We conclude that BNF studies should be conducted in-situ and that the acetylene reduction assay should be calibrated against ¹⁵N₂-fixation calibration for reliable estimates.     

Variation in nitrogen fixation among populations ofFrankia sp. andCeanothus sp. in actinorhizal association

Summary Wildland shrub improvement is needed for sound range and disturbed land revegetation practice. The possibility of selecting superior N₂-fixingFrankia-Ceanothus spp. actinorhizal associations was examined. Greenhouse tests were used to expose various soil-borne microsymbiont andCeanothus sp. population accessions in reciprocal combination. The acetylene reduction rate was used as a measure of N₂-fixation capacity. There was no significant interaction between host and microsymbiont regardless of source for all variables measured. The acetylene reduction rate, nodule number and mass, plant biomass, and root: shoot ratio were significantly different among soil sources. The acetylene reduction rate was not significantly different amongCeanothus sp. accessions. Neither was it strongly correlated with other variables. It was concluded that the N₂-fixation rate is more a function ofFrankia sp. than the hostCeanothus sp. in actinorhizal associations. It appears possible to select soil sources with superior N₂-fixing microsymbiont populations.The use of trade or firm names in this paper is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.This article was produced by government employees and is in the public domain and not subject to copyright. It may be freely reprinted with customary crediting of the source.     

Influence of phosphorus or phosphorus-potassium fertilization on biomass and dinitrogen fixation of the stem-nodulating green-manure legume Sesbania rostrata in different marginally productive wetland rice soils

The performance of Sesbania rostrata varies widely from site to site. This makes it difficult to predict the N yield and biomass of this plant in marginally productive soils, and to arouse the interest of farmers in green manure technology. Three consecutive pot experiments were conducted in a greenhouse at the International Rice Research Institute (IRRI) to evaluate growth, nodulation, N₂ fixation (C₂H₂ reduction assay and ¹⁵N dilution method), and N yield of 6-week-old S. rostrata on 13 physicochemically different wetland rice soils of the Philippines and on three artificial substrates. The performance of S. rostrata on the unfertilized controls was compared with two fertilizer treatments containing either P (100 mg P kg^-1 dry soil) or P+K (100 mg P kg^-1 and 200 mg K kg^-1 dry soil). In the control soils and substrates, the N yield of S. rostrata varied between 20 and 470 mg N per pot, with the N rate from N₂ fixation ranging between 0 and 95%. In three of the nutritionally poor soils even Mn toxicity symptoms apparently occurred with S. rostrata. P application alleviated these symptoms and increased the overall N yield considerably, mainly through increased biological N₂ fixation. An additional increase in N yield was obtained by the PK treatment. Multiple regression analysis between soil characteristics and the N yield of S. rostrata showed that the original level of P (Olsen-extracted) and Mn in the soil accounted for 73% of the variance in biomass production by S. rostrata among the unfertilized soils and substrates.     

Seasonal changes in periphyton nitrogen fixation in a protected tropical wetland

Cyanobacteria are important for global nitrogen cycle and often form complex associations referred to as cyanobacterial mats or periphyton that are common in tropical, limestone-based wetlands. The objective of this study was to monitor the nitrogen fixation rate using the acetylene reduction assay of these cyanobacterial mats in a tropical, unfertilized, and protected wetland. To account for temporal and spatial variation of nitrogenase activity, we were interested in seasons in a hydrological cycle (dry, rains, and end of rains), sites with different vascular vegetation, and rates of nitrogenase activity in a 24-h cycle. The annual average of nitrogenase activity was 22 nmol C₂H₄ cm⁻² h⁻¹, with a range of <6 to 35 nmol C₂H₄ cm⁻² h⁻¹, and the annual nitrogen fixation rate of our study site (9.0 g N m⁻² year⁻¹) is higher than similar estimates from other freshwater wetlands. There was a clear temporal pattern in nitrogenase activity with a maximum rate occurring during the rainy season (August) and a maximum nitrogenase activity occurring between 0600 and 1200 hours. We found spatial differences in nitrogenase activity among the four sites that could be attributed to variations in species composition within the periphyton.     

Conditions for successful Rhizobium-legume symbiosis in saline environments

Summary The Rhizobium-legume symbiosis in arid ecosystems is particularly important for locations where the area of saline soils is increasing and becoming a threat to plant productivity. Legumes, which are usually present in arid ecosystems, may be adapted to fix more N₂ under saline conditions than legumes grown in other habitats.Legumes are known to be either sensitive or moderately resistant to salinity. The salt sensitivity can be attributed to toxic ion accumulations in different plant tissues, which disturb some enzyme activities.Among the basic selection criteria for salt-tolerant legumes and rhizobia are genetic variability within species with respect to salt tolerance, correlation between accumulations of organic solutes (e. g., glycine betaine, proline betaine, and proline) and salt tolerance, and good relationships between ion distribution and compartmentation, and structural adaptations in the legumes.Salt stress reduces the nodulation of legumes by inhibiting the very early symbiotic events. Levels of salinity that inhibit the symbiosis between legumes and rhizobia are different from those that inhibit the growth of the individual symbionts. The poor symbiotic performance of some legumes under saline conditions is not due to salt limitations on the growth of rhizobia.Prerequisites for a successful Rhizobium-legume symbiosis in saline environments include rhizobial colonization and invasion of the rhizosphere, root-hair infection, and the formation of effective salt-tolerant nodules.The possibility of exploring the Rhizobium-legume symbiosis to improve the productivity of saline soils is reviewed in this paper.     

Azospirillum — plant root associations: A review

Summary Bacteria of the genus Azospirillum are extensively studied for their plant-growth promoting effect following inoculation. Physiological and biochemical studies of these diazotrophic bacteria are now benefiting from recent breakthroughs in the development of genetic tools for Azospirilum. Moreover, the identification and cloning of Azospirillum genes involved in N₂ fixation, plant interaction, and phytohormone production have given new life to many research projects on Azospirillum. The finding that Azospirillum genes can complement specific mutations in other intensively studied rhizosphere bacteria like Rhizobia will certainly trigger the exploration of new areas in rhizosphere biology. Therefore a review of the Azospirillum-plant interactions is particularly timely.     

Effect of high temperature on growth,nitrogen fixation,and chlorophyll content of five species of Azolla Anabaena symbiosis

Summary Cultures of Azolla sp. ST-SI, A. microphylla BR-GI, A. mexicana BR-GL, A. caroliniana WT-V, and A. filiculoides BR -H were grown in N-free International Rice Research Institute growth medium in the glasshouse at 38±1 °C (day) and 25±1 °C (night) under a light intensity of 350 Em²s^–1 for 27 days. Biomass, chlorophyll contents and nitrogenase activity (acetylene reduction assay) were recorded on the 19th and 27th day. For comparison the same parameters were studied in Azolla spp. under normal growth conditions at 26±1 °C (day) and 19±1 °C (night). Azolla sp. STSI and A. microphylla BR-GI had produced a larger biomass by the 19th and the 27th day of incubation than A. caroliniana WTV and A. filiculoides which showed poor growth. Under normal growth conditions A. caroliniana WTV and A. filiculoides BRH produced less biomass than the other Azolla spp. cultures tested. A. mexicana BR-GL had a higher total chlorophyll content in both incubation periods than A. caroliniana WT-V and A. filiculoides BR-H. The N content was high in Azolla sp. ST-SI, A. microphylla BR-GI, and A. mexicana BR-GL compared with the low N content of A. filiculoides BR-H and A. caroliniana WT-V. At the higher temperature (38±1 °C/25±1 °C) Azolla sp. ST-SI and A. microphylla BR-GI consistently showed a higher growth rate than A. filiculoides BR-H and A. caroliniana WTV, while the growth rate of A. mexicana BR-GL was intermediate.The study was carried out at C.F. Kettering Research Laboratory, Yellowsprings, OH - 45387, USA     

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT

Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics.     

Nitrogen fixation,biomass production,and nutrient uptake by annual Sesbania species in an alkaline soil

Summary In three field trials conducted during the summer season of 1986, 1987 and 1989 in an alkaline soil, 17 accessions of annual Sesbania spp. were evaluated for nodulation, N₂ fixation (acetylene reduction assay), dry weight of roots and shoots, woody biomass production, and nutrient uptake. At 50 days after sowing all the accessions were effectively nodulated (average 36.4 root nodules plant^-1) with a high nodule score (3.4). There was a lot of variation in nodule volume and mass and in acetylene reduction activity but not in N content (5.2%). N uptake in shoots, roots and nodules averaged 639, 31, and 13 mg plant^-1, respectively, and much of the fixed N remained in shoots. Accessions of S. cannabina complex performed better than others. S. rostrata had poor root nodulation but exhibited excellent stem nodulation (300 nodules plant^-1) even though not inoculated with Azorhizobium sp. Average concentrations of N, P, K, S, Ca, and Mg in the shoots were high, at 3.2, 0.28, 1.5, 0.28, 1.5, and 0.4% respectively, and Na was low (0.15%), reflecting the usefulness of Sesbania spp. as an integrated biofertilizer source. Green matter production was 26.0 Mg ha^-1 (5.9 Mg dry matter) and N uptake was 158 kg ha^-1, 54 days after sowing. Average woody biomass of six accessions at maturity, 200 days after sowing, was high (19.9 Mg ha^-1), showing its potential for shortterm firewood production. Total nutrient uptake for production of woody biomass (200 days of growth) was no more demanding than growing the plant to the green-manuring stage of 50–60 days' growth.     

Nitrogen-fixing stem nodules on Aeschynomene afraspera

Summary Aeschynomene afraspera is a wild annual legume growing in periodically waterlogged soils in western Africa. This legume is characterized by a profuse stem nodulation. Nodules are formed on the stem at the emergence of lateral root primordia, called nodulation sites. These sites are irregularly distributed on vertical rows all along the stem and branches. Stem nodules are hemispherically shaped. Their outside is dark green and they contain a red-pigmented central zone. Stem nodules exhibit a high nitrogen-fixing potential. Acetylene reduction assays result in stem nodule activity of 309 mol C₂H₄ g^–1 dry nodule h^–1. Field-grown stem nodulated Aeschynomene accumulated more N (51 g N m^–2 in 10 weeks) than the root nodulated one. Because of this nitrogenfixing potential and its ability to grow in waterlogged conditions, A. afraspera could probably be introduced into tropical rice cropping systems.     

Influence of methyl isocyanate on the growth,ATP levels,and nitrogen fixation of Azotobacter chroococcum

Summary Growth, ATP levels, and N₂-fixing activity of Azotobacter chroococcum culture exposed to 500 g ml^-1 of methyl isocyanate were investigated. In methyl isocyanate-exposed cells, the lag phase was extended up to 16 h but the control cells started to multiply immediately. ATP levels and N₂-fixing activity as determined by acetylene reduction assay were markedly reduced in the treated cells.     

Short- and long-term effects of ammonium on photodependent nitrogen fixation in wetland rice fields of Spain

Short- and long-term experiments were conducted in the rice fields of Valencia, Spain, to determine the ecological significance of ammonium on nitrogen fixation. A significant inhibition of nitrogenase activity by ammonium, at concentrations higher than 0.5mM, was observed after 8h of incubation in short-term experiments done with a bloom of the N₂-fixing cyanobacterium Anabaena sp. In a second set of short-term experiments for in situ assays of nitrogenase activity in the field, a significant correlation between nitrogenase activity and the number of N₂-fixing cyanobacteria in soil was found. No significant inhibition of nitrogenase activity by ammonium at concentrations up to 2mM was observed in these assays after 24h of incubation. This lack of inhibition was probably due to the rapid decrease in ammonium content in the flood water. Only 5% of the ammonium initially added remained in the water 24h later. In the long-term experiments, nitrogenase activity was assayed in plots fertilized with 0, 70 and 140kgNha^–1, over the cultivation cycle, for 5 years. A partial inhibition of nitrogenase activity by deep-placed N fertilizers was observed. Differences were only significant in 2 years. Mean results from 5 years only showed significant differences between plots fertilized with 0 and 140kgNha^–1. The partial inhibition of nitrogenase activity by ammonium increased over the cultivation cycle. Inhibition was only significant in September, at the end of the cultivation cycle. Received: 28 January 1996     

Quantitative effects of field water deficits on N2(C2H2) fixation in selected legumes grown in the Indian desert

Summary The nitrogenase activity of irrigated and rainfed plants of mung bean, cluster bean and moth bean was studied throughout the growth period in order to estimate the reduction in the potential nitrogen fixation (C₂H₂ reduction) rate due to field water deficits. Nitrogenase activity followed a similar trend in all crops and was dependent on both plant ontogeny and soil moisture levels. The loss of activity due to water deficits varied from 13% to 100% at different growth stages and was related to the plant water potential. The specific activity was directly correlated with the plant water potential under both the treatments. The average loss of nitrogen fixation rate during the season did not differ markedly among crops. There was an accumulation of ureides in the nodules with increasing field moisture stress in mung bean and moth bean while no such effect was found in cluster bean. The significance of these results is discussed in the N-economy of these legumes grown in the drought-prone areas of the Indian desert.     

Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Yield response of spring wheat cultivars (Triticum aestivum and T. turgidum) to inoculation with Azospirillum brasilense under field conditions

Summary Eight commercial Israeli spring wheat cultivars (six Triticum aestivum and two T. turgidum) grown with 40 and 120 kg N/ha were tested for responses to inoculation with Azospirillum brasilense. At the low level of N fertilization (40 kg/ha), five cultivars showed significant increases in plant dry weight measured at the milky ripe stage; however, by maturation only the cultivar Miriam showed a significant increase in grain yield. Two cultivars, which had shown a positive inoculation effect at the earlier stages, had a significant decrease in grain yield. No significant effect of inoculation was found at the high N level. To confirm those results, four wheat (T. aestivum) cultivars were tested separately over 4 years in 4 different locations under varying N levels. Only Miriam showed a consistently positive effect of Azospirillum inoculation on grain yield. Inoculation increased the number of roots per plant on Miriam compared with uninoculated plants. This effect was found at all N levels. Nutrient (N, P and K) accumulation and number of fertile tillers per unit area were also enhanced by Azospirillum, but these parameters were greatly affected by the level of applied N. It is suggested that the positive response of the spring wheat cultivar Miriam to Azospirillum inoculation is due to its capacity to escape water stresses at the end of the growth season.     

Diurnal and seasonal patterns of nitrogenase activity of red alder in comparison with white clover in silvopastoral agroforestry systems

 Simultaneous measurements were made to assess the diurnal and seasonal patterns of nitrogenase activity of red alder (Alnus rubra Bong.) and white clover (Trifolium repens L.) growing together in a silvopastoral agroforestry system using the acetylene reduction assay. Diurnal measurements were made in the summer and autumn at 3-h intervals whereas seasonal nitrogenase activity was assessed based on observations made at midday in July, September and January to represent the summer, autumn and winter seasons, respectively. No obvious diurnal patterns of nitrogenase activity were found in either red alder or white clover in summer and no significant variations in nitrogenase activity were observed between day and night. However, in autumn, pronounced diurnal patterns were observed in both species. Significantly higher rates of nitrogenase activity per unit dry weigh (dwt) of nodules were detected at 1500 hours in red alder, whereas, in white clover, significantly higher rates were obtained at 2100 hours. There was no significant correlation between diurnal nitrogenase activity and air temperature, photosynthetically active radiation and soil temperature at 10 cm depth in either red alder or white clover. Seasonal rates of nitrogenase activity showed significantly higher activity in summer, which subsequently decreased in autumn, to reach very low levels in the winter. The rates of nitrogenase activity of white clover were consistently higher than those of red alder both diurnally and seasonally. In the three seasons sampled, the average nitrogenase activity for white clover was 66.42 μmol C₂H₄ g dwt^–1 h^–1, which was 3.5 times higher than the 18.67 μmol C₂H₄ g dwt^–1 h^–1 obtained for red alder. Received: 11 November 1997     

Nitrogenase activity and nitrogen-fixing bacteria associated with the roots ofAtriplex spp. growing in saline sodic soils of Pakistan

Summary TwoAtriplex spp. growing in low-fertility saline sodic soils were assayed for root-associated nitrogenase activity. The excised washed and unwashed root of the two species.A. lentiformis andA. amnicola, showed high root-associated nitrogenase activity. Acetylene-reducing activity seemed to be directly influenced by moisture. The highest number of diazotrophs, enumerated using a most probable number technique was observed on the root surface. Most of the isolated diazotrophs were identified asEnterobacter agglomerans. Root-associated nitrogenase activity inAtriplex spp. may explain the high protein and biomass content of these plants growing in low-fertility saline sodic soils.     

Estimation of associative nitrogen fixation in relation to water regime and plant nutrition in a long-term pot experiment with rice (Oryza sativa L.)

Summary The contribution of associative N₂ fixation to the N nutrition of lowland rice was estimated in a long-term pot experiment with ten consecutive crops of rice. The experiment comprised two N and two K levels with wet (WF) and dry fallow (DF) between the cropping seasons. Growth of N₂-fixing cyanobacteria was prevented. Greatest yields were obtained in the high NK fertilizer treatment, but with continuation of the experiment yields responsed more to DF than to WF. Nitrogenase activity, however, was favoured by WF. Higher K application increased and higher N application decreased nitrogenase activity. Under WF treatments the organic C and total N contents of the soil remained unchanged during the experimental period, but alternate drying and flooding in DF treatments caused a decline. Lower N fertilizer rates in the second five-crop period did not affect yields, but increased the ratio of N removed to N applied. For the ten-crop period the estimated N balance was positive in the low-N and negative in the high-N treatments. N balances were also established separately for both the first and the second halves of the ten-crop period. In the first period N losses were higher, and the N balance was mostly negative. In the second period only high-N combined with low-K fertilization resulted in a negative N balance. DF favoured N losses in the first but not in the second period. The highest N gain in the second period was found in the DF treatment with low-N and high-K application. In this treatment, nearly one-quarter of the N taken up by the above-ground parts of the plants could be ascribed to associative N₂ fixation. In the corresponding treatment with the higher N level and a 49% higher yield, the contribution of fixed N declined to less than 5%. When harvested straw contained more than 10 mg N g^–1, the N balance was mostly negative, while at N contents less than 10 mg N g^–1, the N balance was generally positive.