Sustainable agriculture in the semi-arid tropics through biological nitrogen fixation in grain legumes

Sustainable agriculture relies greatly on renewable resources like biologically fixed nitrogen. Biological nitrogen fixation plays an important role in maintaining soil fertility. However, as BNF is dependent upon physical, environmental, nutritional and biological factors, mere inclusion of any N₂-fixing plant system does not guarantee increased contributions to the soil N pool. In the SAT where plant stover is also removed to feed animals, most legumes might be expected to deplete soil N. Yet beneficial legume effects in terms of increased yields in succeeding cereal crops have been reported. Such benefits are partly due to N contribution from legumes through BNF and soil N saving effect. In addition, other non-N rotational benefits, for example, improved nutrient availability, improved soil structure, reduced pests and diseases, hormonal effects are also responsible. In this paper we have reviewed the research on the contribution of grain legumes in cropping systems and the factors affecting BNF. Based on the information available, we have suggested ways for exploiting BNF for developing sustainable agriculture in the semi-arid tropics (SAT). A holistic approach involving host-plant, bacteria, environment and proper management practices including need based inoculation for enhancing BNF in the cropping systems in the SAT is suggested.     

Enhancing legume N2 fixation through plant and soil management

Atmospheric N₂ fixed symbiotically by associations between Rhizobium spp. and legumes represents a renewable source of N for agriculture. Contribution of legume N₂ fixation to the N-economy of any ecosystem is mediated by: (i) legume reliance upon N₂ fixation for growth, and (ii) the total amount of legume-N accumulated. Strategies that change the numbers of effective rhizobia present in soil, reduce the inhibitory effects of soil nitrate, or influence legume biomass all have potential to alter net inputs of fixed N. A range of management options can be applied to legumes growing in farming systems to manipulate N₂ fixation and improve the N benefits to agriculture and agroforestry.     

Outdoor mass culture ofAzolla spp.: yields and efficiencies of nitrogen fixation

Summary The productivity of three species of Azolla (A. pinnata, A. filiculoides andA. caroliniana) in outdoor culture has been evaluated at different planting densities. The highest yields were obtained with biomass concentration ranging from 40 to 70g d.w. m^–2. The mean productivity over a 90 days period (from May 10th to August 10th) ranged from 10g d.w. m^–2 day^–1 forA. filiculoides up to 11.5 g d.w. m^–2 day^–1 forA. caroliniana. The nitrogen content of the dried biomasses was 48.3 mg (g d.w.)^–1 forA. pinnata, 51.5mg (g d.w.)^–1 forA. filiculoides and 52.3 mg (g d.w.)^–1 forA. caroliniana. Very little variations of the nitrogen content of the ferns during the experimental period were observed.The nitrogen-fixing efficiency of the Azolla-Anabaena azollae symbiosis grown in outdoor conditions was evaluated both by direct measurement of the amount of N₂ fixed by the culture and by the C₂H₂-reduction and H₂-evolution tests in an air atmosphere. These tests were performed outdoor under the same environmental conditions as the growing cultures. For all the species the ratios of C₂H₂-reduced to N₂-fixed were unexpectedly low, ranging from 2.04 (A. pinnata) to 1.50 (A. caroliniana).The results suggest that the reliability of the C₂H₂-reduction assay, particularly when applied to complex biological N₂-fixing systems, must be re-examined.     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

Nitrogen fixation by non-legumes in tropical agriculture with special reference to wetland rice

Summary Of the 143 million hectares of cultivated rice land in the world, 75% are planted to wetland rice. Wet or flooded conditions favour biological nitrogen fixation by providing (1) photic-oxic floodwater and surface soil for phototrophic, free-living or symbiotic blue-green algae (BGA), and (2) aphotic-anoxic soil for anaerobic or microaerobic, heterotrophic bacteria. TheAzolla-Anabaena symbiosis can accumulate as much as 200 kg N ha^–1 in biomass. In tropical flooded fields, biomass production from a singleAzolla crop is about 15 t fresh weight ha^–1 or 35 kg N ha^–1. Low tolerance for high temperature, insect damage, phosphorus requirement, and maintenance of inoculum, limit application in the tropics. Basic work on taxonomy, sporulation, and breeding ofAzolla is needed. Although there are many reports of the positive effect of BGA inoculation on rice yield, the mechanisms of yield increase are not known. Efficient ways to increase N₂-fixation by field-grown BGA are not well exploited. Studies on the ecology of floodwater communities are needed to understand the principles of manipulating BGA. Bacteria associated with rice roots and the basal portion of the shoot also fix nitrogen. The system is known as a rhizocoenosis. N₂-fixation in rhizocoenosis in wetland rice is lower than that ofAzolla or BGA. Ways of manipulating this process are not known. Screening rice varieties that greatly stimulate N₂-fixation may be the most efficient way of manipulating the rhizocoenosis. Stimulation of N₂-fixation by bacterial inoculation needs to be quantified.     

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.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Biological N2 Fixation in wetland rice fields: Estimation and contribution to nitrogen balance

This paper 1) reviews improvements and new approaches in methodologies for estimating biological N₂ fixation (BNF) in wetland soils, 2) summarizes earlier quantitative estimates and recent data, and 3) discusses the contribution of BNF to N balance in wetland-rice culture.Measuring acetylene reducing activity (ARA) is still the most popular method for assessing BNF in rice fields. Recent studies confirm that ARA measurements present a number of problems that may render quantitative extrapolations questionable. On the other hand, few comparative measures show ARA's potential as a quantitative estimate. Methods for measuring photodependent and associative ARA in field studies have been standardized, and major progress has been made in sampling procedures. Standardized ARA measurements have shown significant differences in associative N₂ fixation among rice varieties.The ¹⁵N dilution method is suitable for measuring the percentage of N derived from the atmosphere (% Ndfa) in legumes and rice. In particular, the ¹⁵N dilution technique, using available soil N as control, appears to be a promising method for screening rice varieties for ability to utilize biologically fixed N. Attempts to adapt the ¹⁵N dilution method to aquatic N₂ fixers (Azolla and blue-green algae [BGA]) encountered difficulties due to the rapid change in ¹⁵N enrichment of the water.Differences in natural ¹⁵N abundance have been used to show differences among plant organs and species or varieties in rice and Azolla, and to estimate Ndfa by Azolla, but the method appears to be semi-quantitative.Recent pot experiments using stabilized ¹⁵N-labelled soil or balances in pots covered with black cloth indicate a contribution of 10–30 kg N ha^-1 crop^-1 by heterotrophic BNF in flooded planted soil with no or little N fertilizer used.Associative BNF extrapolated from ARA and ¹⁵N incorporation range from 1 to 7 kg N ha^-1 crop^-1. Straw application increases heterotrophic and photodependent BNF. Pot experiments show N gains of 2–4 mg N g^-1 straw added at 10 tons ha^-1.N₂ fixation by BGA has been almost exclusively estimated by ARA and biomass measurements. Estimates by ARA range from a few to 80 kg N ha^-1 crop^-1 (average 27 kg). Recent extensive measurements show extrapolated values of about 20 kg N ha^-1 crop^-1 in no-N plots, 8 kg in plots with broadcast urea, and 12 kg in plots with deep-placed urea.Most information on N₂ fixed by Azolla and legume green manure comes from N accumulation measurements and determination of % Ndfa. Recent trials in an international network show standing crops of Azolla averaging 30–40 kg N ha^-1 and the accumulation of 50–90 kg N ha^-1 for two crops of Azolla grown before and after transplanting rice. Estimates of % Ndfa in Azolla by ¹⁵N dilution and delta ¹⁵N methods range from 51 to 99%. Assuming 50–80% Ndfa in legume green manures, one crop can provide 50–100 kg N ha^-1 in 50 days. Few balance studies in microplots or pots report extrapolated N gains of 150–250 kg N ha^-1 crop^-1.N balances in long-term fertility experiments range from 19 to 98 kg N ha^-1 crop^-1 (average 50 kg N) in fields with no N fertilizer applied. The problems encountered with ARA and ¹⁵N methods have revived interest in N balance studies in pots. Balances are usually highest in flooded planted pots exposed to light and receiving no N fertilizer; extrapolated values range from 16 to 70 kg N ha^-1 crop^-1 (average 38 kg N). A compilation of balance experiments with rice soil shows an average balance of about 30 kg N ha^-1 crop^-1 in soils where no inorganic fertilizer N was applied.Biological N₂ fixation by individual systems can be estimated more or less accurately, but total BNF in a rice field has not yet been estimated by measuring simultaneously the activities of the various components in situ. As a result, it is not clear if the activities of the different N₂-fixing systems are independent or related. A method to estimate in situ the contribution of N₂ fixed to rice nutrition is still not available. Dynamics of BNF during the crop cycle is known for indigenous agents but the pattern of fixed N availability to rice is known only for a few green manure crops.     

New techniques for studying competition by Rhizobia and for assessing nitrogen fixation in the field

One of the key factors limiting the proper assessment and use of rhizobial strains in the field is the lack of suitable methodology to screen the success of individual isolates in competing for nodule occupancy with different cultivars of legumes and in different soil and agronomic conditions. The use of marker genes enables individual rhizobial strains to be identified by a simple colour assay, thus enabling a dramatic increase in throughput of strain screening. One such marker system for rhizobial ecology, the GUS system, is already in use to facilitate rapid screening of rhizobial isolates. Other markers, which will allow the competitive behaviour of several strains to be studied at once, are under development.Likewise, breeding of the host legume for a high efficiency of nitrogen fixation is hampered by the difficulty in assessing this property. The method which currently gives the highest throughput of analysis, and has been successfully used in soybean breeding programs, is the ureide technique. However, it remains somewhat laborious for use in routine breeding programs. In this paper we discuss the potential use of reporter genes to provide information on the relative levels of ureides and other nitrogenous compounds in plants growing in the field. This would greatly increase the rate at which this trait could be scored, and would thus enable routine assays for increased symbiotic nitrogen fixation for breeding or management purposes in legume crops such as soybean (Glycine max) and common bean (Phaseolus vulgaris).     

Azolla and other plant-cyanobacteria symbioses: Aspects of form and function

Nostoc, a genus of filamentous, heterocystous, cyanobacteria, is widely distributed in the free-living state. It is also the most common phycobiont in N₂-fixing lichens and occurs as the N₂-fixing symbiont in a small and diverse group of green plants. These include several bryophyte genera (e.g. Anthoceros and Blasia), a pteridophyte genus (Azolla; while the symbiont is referred to asAnabaena azollae, it may be aNostoc spp.), a division of gymnosperms (the 10 cycad genera) and one angiosperm genus (Gunnera). In Gunnera the Nostoc apparently penetrates into the cells of the host. In the other associations Nostoc is extracellular but specific morphological modifications and/or structures of the host plant organs create an environment which fosters interaction and metabolite interchange.The individual group of Nostoc-green plant symbioses other than Azolla are summarized in regard to the current understanding of their establishment, perpetuation, and host-symbiont interaction. This includes available information on recognition and specificity, mode(s) of infection if applicable, and a synopsis of morphological modifications of the partners. The symbiosis withAzolla is then addressed separately with a more indepth account of the foregoing areas. In addition, the concept ofAzolla harboring a dominant, obiligately symbiotic Nostoc which has not been cultured as well as minor symbionts capable of free-living growth, the distinction between re-constituting and simply re-establishing the symbiosis, and current approaches to improving the symbiosis and to authenticating the establishment of new associations are considered.     

Nitrogen fixation in several grain legume species with contrasting sensitivities to copper nutrition

The nitrogen fixation response to copper nutrition in faba bean, yellow lupin and soybean was studied. Copper nutrition significantly increased the pod yields of all tested grain legumes but faba bean gave the greatest Cu-use efficiency for pod and grain production. The accumulation of dry matter in vegetative parts, nodules, N and leghemoglobin concentration in nodules and nitrogen accumulation in the whole plants were increased by copper supply in faba bean and yellow lupin in contrast with soybean. Cu nutrition significantly increased the Cu concentrations in nodules of all cultivated plants. The differential sensitivity of N₂ fixation in tested grain legume species to copper nutrition could be connected with the level of phenols in nodules and depended on both the host plants and strains of rhizobia, which differ in their ability to produce catechol-like siderophores. Copper requirements by symbiotic N₂ fixation could also depend on the nature of phenols in nodules (presence of o-dihydroxyphenols or number of hydroxyls in molecule).     

Trends in biological nitrogen fixation research and application

In the world each year 17.2×10⁷ tons of N are biologically fixed. Biological nitrogen fixation (BNF) contributes to plant production in arable lands and in natural ecosystems. Research to improve BNF is progressing through the breeding of efficient N-fixing organisms and host plants, selection of the best combinations of host plant and microsymbiont, and by the improvement of inoculation techniques and field management. Biotechnology is useful for the creation of promising N₂-fixing organisms. However, to increase plant production through enhanced BNF the constraints in establishing effective N₂-fixing systems in the field should be understood and eliminated.     

Nitrogen cycling in low input legume-based agriculture,with emphasis on legume/grass pastures

Low input legume-based agriculture exists in a continuum between subsistence farming and intensive arable and pastoral systems. This review covers this range, but with most emphasis on temperate legume/grass pastures under grazing by livestock. Key determinants of nitrogen (N) flows in grazed legume/grass pastures are: inputs of N from symbiotic N₂ fixation which are constrained through self-regulation via grass/legume interactions; large quantities of N cycling through grazing animals with localised return in excreta; low direct conversion of pasture N into produce (typically 5–20%) but with N recycling under intensive grazing the farm efficiency of product N: fixed N can be up to 50%; and regulation of N flows by mineralisation/immobilisation reactions. Pastoral systems reliant solely on fixed N are capable of moderate-high production with modest N losses e.g. average denitrification and leaching losses from grazed pastures of 6 and 23 kg N ha^–1 yr^–1. Methods for improving efficiency of N cycling in legume-based cropping and legume/grass pasture systems are discussed. In legume/arable rotations, the utilisation of fixed N by crops is influenced greatly by the timing of management practices for synchrony of N supply via mineralisation and crop N uptake. In legume/grass pastures, the spatial return of excreta and the uptake of excreta N by pastures can potentially be improved through dietary manipulation and management strategies. Plant species selection and plant constituent modification also offer the potential to increase N efficiency through greater conversion into animal produce, improved N uptake from soil and manipulation of mineralisation/immobilisation/nitrification reactions.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

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.     

Effect of varying periods of inoculation ofAzolla pinnata R. Brown on its growth,nitrogen fixation and response to rice crop

Summary Inoculation of water fernAzolla pinnata R. Brown (Bangkok isolate) at the rate of 500kg fresh weight ha⁻¹ in rice fields at weekly intervals after planting in addition to 30 kg N ha⁻¹ as urea showed a decrease in its growth and N₂-fixation with delay in application. Use of Azolla up to 3 weeks after planting (WAP) during wet and 4 WAP during dry season produced significantly more grain yield than 30 kg N ha⁻¹, whereas its application upto one WAP produced more grain yield than 60 kg N ha⁻¹. Grain yield with Azolla applied at the time of planting was similar to that of 60 kg N treatment during the wet season. Higher grain yields in zero and one WAP Azolla treatments resulted due to increase in both number of panicles m⁻² and number of grains/panicle while the subsequent Azolla inoculations increased grain yield mainly by producing more number of grains/panicle. Dry matter and total N yields at maturity of rice crop were more with Azolla application upto 3 WAP during wet and 2 WAP during dry season while the reduction in sterility (%) was observed upto one WAP over 30 kg N ha⁻¹ during both seasons. Number of tillers m⁻² and dry matter production at maximum tillering and flowering were more than 30 kg N ha⁻¹ with the use of Azolla upto one WAP. Increased grain N yield was observed with the use of Azolla upto 4 WAP during two seasons whereas straw N yield increased upto one WAP during wet and 2 WAP during dry season.     

Biological N2 fixation by heterotrophic and phototrophic bacteria in association with straw

Much of the crop residues, including cereal straw, that are produced worldwide are lost by burning. Plant residues, and in particular straw, contain large amounts of carbon (cellulose and hemicellulose) which can serve as substrates for the production of microbial biomass and for biological N₂ fixation by a range of free-living, diazotrophic bacteria. Microorganisms with the dual ability to utilise cellulose and fix N₂ are rate, but some strains that utilize hemicellulose and fix N₂ have been found. Generally, cellulolysis and diazotrophy are carried out by a mixed microbial community in which N₂-fixing bacteria utilise cellobiose and glucose produced from straw by cellulolytic microorganisms. N₂-fixing bacteria include heterotrophic and phototrophic organisms and the latter are apparently more prominent in flooded soils such as rice paddies than in dryland soils. The relative contributions of N₂ fixed by heterotrophic diazotrophic bacteria compared with cyanobacteria and other phototrophic bacteria depend on the availability of substrates from straw decomposition and on environmental pressures. Measurements of asymbiotic N₂ fixation are limited and variable but, in rice paddy systems, rates of 25 kg N ha^-1 over 30 days have been found, whereas in dryland systems with wheat straw, in situ measurements have indicated up to 12 kg N ha^-1 over 22 days. Straw-associated N₂ fixation is directly affected by environmental factors such as temperature, moisture, oxygen concentration, soil pH and clay content as well as farm management practices. Modification of managements and use of inoculants offer ways of improving asymbiotic N₂ fixation.In laboratory culture systems, inoculation of straws with cellulolytic and diazotrophic microorganisms has resulted in significant increases in N₂ fixation in comparison to uninoculated controls and gains of N of up to 72 mg N fixed g^-1 straw consumed have been obtained, indicating the potential of inoculation to improve N gains in composts that can then be used as biofertilisers. Soils, on the other hand, contain established, indigenous microbial populations which tend to exclude inoculant microorganisms by competition. As a consequence, improvements in straw-associated N₂ fixation in soils have been achieved mostly by specific straw-management practices which encourage microbial activity by straw-decomposing and N₂-fixing microorganisms.Further research is needed to quantify more accurately the contribution of asymbiotic N₂ fixation to cropping systems. New strains of inoculants, including those capable of both cellulolytic and N₂-fixing activity, are needed to improve the N content of biofertilisers produced from composts. Developments of management practices in farming systems may result in further improvements in N₂ fixation in the field.     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

Improving nitrogen-fixing systems and integrating them into sustainable rice farming

This paper summarizes recent achievements in exploiting new biological nitrogen fixation (BNF) systems in rice fields, improving their management, and integrating them into rice farming systems. The inoculation of cyanobacteria has been long recommended, but its effect is erratic and unpredictable. Azolla has a long history of use as a green manure, but a number of biological constraints limited its use in tropical Asia. To overcome these constraints, the Azolla-Anabaena system as well as the growing methods were improved. Hybrids between A. microphylla and A. filiculoides (male) produced higher annual biomass than either parent. When Anabaena from high temperature-tolerant A. microphylla was transferred to Anabaena-free A. filiculoides, A. filiculoides became tolerant of high temperature. Azolla can have multiple purposes in addition to being a N source. An integrated Azolla-fish-rice system developed in Fujian, China, could increase farmers' income, reduce expenses, and increase ecological stability. A study using Azolla labeled with ¹⁵N showed the reduction of N losses by fish uptake of N. The Azolla mat could also reduce losses of urea N by lowering floodwater-pH and storing a part of applied N in Azolla. Agronomically useful aquatic legumes have been explored within Sesbania and Aeschynomene. S. rostrata can accumulate more than 100kg N ha^-1 in 45 d. Its N₂ fixation by stem nodules is more tolerant of mineral N than that by root nodules, but the flowering of S. rostrata is sensitive to photoperiod. Aquatic legumes can be used in rainfed rice fields as N scavengers and N₂ fixers. The general principle of integrated uses of BNF in rice-farming systems is shown.