Differential responses ofAzolla microphylla Kaulf andAzolla filiculoides Lam. to lead nitrate

The aquatic fernAzolla is used as a green manure for rice production systems in warm temperate and tropical regions throughout the world. We used lead nitrate in nutrient media to assess the tolerance/response of twoAzolla microphylla andAzolla filiculoides to heavy metals. While both species showed negative responses in growth with an increase in lead concentrations, They were distinctly different in their tolerance for higher concentration of the heavy metal. This was apparent in their growth and biochemical characteristics. Amicrophylla was more tolerant of the higher concentrations of lead nitrate (25 and 50 ppm), whereasA. filiculoides had a higher rate of lead uptake.     

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.     

Adaptation of exotic Azolla to tropical environment of Thailand

There are seven known species of Azolla, two of which have been used in cultivated systems, the tropical speciesA. pinnata, and the temperate speciesA. filiculoides. OnlyA. pinnata is indigenous in Thailand. In this study the two exotic species,A. caroliniana andA. microphylla, were evaluated under the various tropical field conditions in Thailand. When compared with seven selected strains ofA. pinnata under three field conditions,A. caroliniana andA. microphylla were similar to the indigenous species, in terms of growth performance, N₂ fixation, and yield. This study suggests thatA. caroliniana andA. microphylla can be successfully cultivated as a nitrogen fixing green manure for rice production in the tropics.     

Biological nitrogen fixation for sustainable agriculture: A perspective

The economic and environmental costs of the heavy use of chemical N fertilizers in agriculture are a global concern. Sustainability considerations mandate that alternatives to N fertilizers must be urgently sought. Biological nitrogen fixation (BNF), a microbiological process which converts atmospheric nitrogen into a plant-usable form, offers this alternative. Nitrogen-fixing systems offer an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. Symbiotic systems such as that of legumes and Rhizobium can be a major source of N in most cropping systems and that of Azolla and Anabaena can be of particular value to flooded rice crop. Nitrogen fixation by associative and free-living microorganisms can also be important. However, scientific and socio-cultural constraints limit the utilization of BNF systems in agriculture. While several environmental factors that affect BNF have been studied, uncertainties still remain on how organisms respond to a given situation. In the case of legumes, ecological models that predict the likelihood and the magnitude of response to rhizobial inoculation are now becoming available. Molecular biology has made it possible to introduce choice attributes into nitrogen-fixing organisms but limited knowledge on how they interact with the environment makes it difficult to tailor organisms to order. The difficulty in detecting introduced organisms in the field is still a major obstacle to assessing the success or failure of inoculation. Production-level problems and socio-cultural factors also limit the integration of BNF systems into actual farming situations. Maximum benefit can be realized only through analysis and resolution of major constraints to BNF performance in the field and adoption and use of the technology by farmers.     

Differential response ofAzolla microphylla kaulf. andAzolla filiculoides Lam. to sodium fluoride

We assessed the differential response ofAzolla microphylla andAzolla filiculoides to fluoride stress by growing them in culture media containing 1 to 50 ppm sodium fluoride (NaF).A microphylla had a higher total chlorophyll content thanA. filiculoides. Both species showed gradual decreases in protein content as the concentration of NaF increased.A. microphylla accumulated less proline thanA. filiculoides when more NaF was added to the culture medium. For all concentrations tested, the amount of residual fluoride in the nutrient media that supportedA. microphylla was higher than in the nutrient media used for growingA. filiculoides.     

Managing native and legume-fixed nitrogen in lowland rice-based cropping systems

Lowlands comprise 87% of the 145 M ha of world rice area. Lowland rice-based cropping systems are characterized by soil flooding during most of the rice growing season. Rainfall distribution, availability of irrigation water and prevailing temperatures determine when rice or other crops are grown. Nitrogen is the most required nutrient in lowland rice-based cropping systems. Reducing fertilizer N use in these cropping systems, while maintaining or enhancing crop output, is desirable from both environmental and economic perspectives. This may be possible by producing N on the land through legume biological nitrogen fixation (BNF), minimizing soil N losses, and by improved recycling of N through plant residues. At the end of a flooded rice crop, organic- and NH₄-N dominate in the soil, with negligible amounts of NO₃. Subsequent drying of the soil favors aerobic N transformations. Organic N mineralizes to NH₄, which is rapidly nitrified into NO₃. As a result, NO₃ accumulates in soil during the aerobic phase. Recent evidence indicates that large amounts of accumulated soil NO₃ may be lost from rice lowlands upon the flooding of aerobic soil for rice production. Plant uptake during the aerobic phase can conserve soil NO₃ from potential loss. Legumes grown during the aerobic phase additionally capture atmospheric N through BNF. The length of the nonflooded season, water availability, soil properties, and prevailing temperatures determine when and where legumes are, or can be, grown. The amount of N derived by legumes through BNF depends on the interaction of microbial, plant, and environmental determinants. Suitable legumes for lowland rice soils are those that can deplete soil NO₃ while deriving large amounts of N through BNF. Reducing soil N supply to the legume by suitable soil and crop management can increase BNF. Much of the N in legume biomass might be removed from the land in an economic crop produce. As biomass is removed, the likelihood of obtaining a positive soil N balance diminishes. Nonetheless, use of legumes rather than non-legumes is likely to contribute higher quantities of N to a subsequent rice crop. A whole-system approach to N management will be necessary to capture and effectively use soil and atmospheric sources of N in the lowland rice ecosystem.IRRI-NifTAL-IFDC joint contribution.     

Effect of Sesbania,Azolla and rice straw incorporation on the kinetics of NH4, K,Fe, Mn,Zn and P in some flooded rice soils

In a greenhouse study, with and without rice plants, of five flooded Philippine rice soils whose organic C (OC) content varied from 0.5 to 3.6%, incorporation ofSesbania rostrata, Azolla microphylla and rice straw affected the kinetics of soil solution NH ₄ ⁺ −N, K⁺, Fe²⁺, Mn²⁺, Zn²⁺, and P. Sesbania and Azolla increased NH ₄ ⁺ −N concentration above the control treatment, whereas rice straw depressed it. In all soils Azolla released less NH ₄ ⁺ −N than Sesbania. The apparent net N release depended on the soil and ranged from 44–81% for Sesbania and 27–52% for Azolla. These effects persisted throughout the growth of IR36. Soil solution and exchangeable NH ₄ ⁺ −N increased initially but levelled off between 30 to 80 days and between 20 to 40 days after flooding (DF), respectively. With rice, soil solution NH ₄ ⁺ −N concentration, reached a peak at 15–40 DF and declined to very low levels (<4mg L⁻¹). In the 3 soils of low OC content nitrogen derived from green manure ranged from 34–53% and the apparent revovery of added green manure N varied from 29–67%. Almost all N released from both Azolla and Sesbania were recovered in the rice plant in all soils except Concepcion with only 77%. The concentration of K⁺, Fe²⁺, Mn²⁺ and P in the soil solution were higher with rice straw than Sesbania and Azolla in all soils except Hanggan which showed no change in Fe²⁺ and Mn²⁺ but increased K⁺ and P. In general, rice straw, Sesbania and Azolla decreased Zn²⁺ concentration in all soils.     

Amount of nitrogen fixed by forage,pasture and grain legumes in Cyprus,estimated by the A-value and a modified difference method

The increasing need for protein at low cost has created a need to evaluate the biological nitrogen fixing potential of legumes in Cyprus. In field studies which were conducted over the growing years of 1982–3 and 1983–4, legumes which are traditionally grown in the country were evaluated for dry matter and nitrogen yield and biological nitrogen fixation (BNF). The legumes studied were medic (Medicago truncatula Gearth), ochrus vetch (Lathyrus ochrus L.), bitter vetch (Vicia ervilia L.) and faba bean (Vicia faba L. var major) in the first year and in addition chickpea (Cicer arietinum L.), woollypod vetch (Vicia dasycarpa Ten.) and tickbean (Vicia faba L. var minor) in the second year. Using the A-value method with barley and oats as reference crops, nitrogen (N) fixed by the various legumes in the first year was 30–50% and from 55–67% of total N yield for the two reference crops, respectively. In the second year the estimates of N fixed ranged from 70 to 80% with similar results obtained for the two reference crops barley and ryegrass. However, in the second year chickpea, which had limited nodulation, fixed only 40% of its N yield. Estimates of nitrogen from the atmosphere (Ndfa) obtained by the difference method (DM) were 10 to 14% lower than those from the A-value method. These results were obtained after correcting for the amount of N derived from the applied fertilizer. The two methods were highly correlated (r=0.98) for estimates of amount of BNF. The rates of N₂ fixation of uninoculated legumes which are nodulated by the indigenous populations of Rhizobium in Cyprus are comparable to those of legumes inoculated with selected strains of Rhizobium in other countries. An exception was the amount of N fixed by chickpea. The appearance of the first nodules at late stages of growth may be the reason for the low BNF of this crop.     

Effect of NPK on growth and nitrogen fixation of Sesbania rostrata as a green manure for lowland rice (Oryza sativa L.)

The stem-nodulating tropical legume Sesbania rostrata is a promising green manure species for low input rice-farming systems in lowland areas. However, its success as biofertilizer depends on its biomass production and N₂ fixation. Nutrient imbalances and soils low in available nutrients can considerably affect biofertilizer production. Use of mineral N, P, and K fertilizers in growing S. rostrata as biofertilizer for lowland rice was therefore evaluated in pot experiments, and in the fields in Central Luzon, Philippines. Two soils low in Olsen P (3–7.3 mg kg^–1) and exchangeable K (0.05–0.08 meq 100g^-1) were used. Increasing amounts of N (0, 10, 20, 30, and 40 mg kg^-1), P (0, 50, and 100 mg kg^-1), and K (0, 100, 200, and 300 mg kg^-1) were applied to S. rostrata grown in the greenhouse, whereas small amounts of N, P, and K fertilizers (30, 15, and 33 kg ha^-1, respectively) were applied in the field.Mineral N application depressed nodulation and N₂ fixation in roots. It however, stimulated nodulation and N₂ fixation in stems. Applying 30 kg N ha^-1 as urea increased total N accumulation by S. rostrata and yield of the subsequent rice crop (IR64). Applied P and K both stimulated growth, nodulation, and N₂ fixation of S. rostrata. Nitrogen accumulation in P- and K-fertilized S. rostrata was about 40% higher than that in nonfertilized green manure. Thus integration of mineral N, P, and K fertilizers in a green manure-based rice-farming system can considerably improve biofertilizer production and increase rice grain yield.     

Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production?

A fundamental shift has taken place in agricultural research and world food production. In the past, the principal driving force was to increase the yield potential of food crops and to maximize productivity. Today, the drive for productivity is increasingly combined with a desire for sustainability. For farming systems to remain productive, and to be sustainable in the long-term, it will be necessary to replenish the reserves of nutrients which are removed or lost from the soil. In the case of nitrogen (N), inputs into agricultural systems may be in the form of N-fertilizer, or be derived from atmospheric N₂ via biological N₂ fixation (BNF).Although BNF has long been a component of many farming systems throughout the world, its importance as a primary source of N for agriculture has diminished in recent decades as increasing amounts of fertilizer-N are used for the production of food and cash crops. However, international emphasis on environmentally sustainable development with the use of renewable resources is likely to focus attention on the potential role of BNF in supplying N for agriculture. This paper documents inputs of N via symbiotic N₂ fixation measured in experimental plots and in farmers' fields in tropical and temperate regions. It considers contributions of fixed N from legumes (crop, pasture, green manures and trees), Casuarina, and Azolla, and compares the relative utilization of N derived from these sources with fertilizer N.     

Use of Azolla as a test organism in a growth chamber of simple design

Summary The construction and application of a new type of growth chamber, in which different growth conditionsi.e.: temperature, humidity, pH, light intensity, light colour, change in nutrient composition and gas exchange can easily be controlled, are presented. The method has previously been applied to twoAzolla speciesviz. Azolla filiculoides, which is cold tolerant andAzolla pinnata (distinguished in Vietnam as the form Xanh), which is heat tolerant. In the growth chamber natural growth conditions of the Azolla —Anabaena azollae symbiotic association were imitated as much as possible. For testing the system, methods discussed earlier^8,14 and some previously presented data, concerning photosynthetic activities, such as oxygen evolution and nitrogen fixation (acetylene reduction) of twoAzolla species³⁹, were partially used. Biomass ofA. filiculoides was measured and reactions to its environment at conditions when grown in the field and in the growth chamber, were studied. Growth and photosynthesis measurements were performed under special light conditions and with whole plants grown under laboratory conditions. Anthocyanin synthesis was studied in relation with humidity. Anthocyanin spectra were analyzed by means of a spectrum-deconvolution method. On leave from the Department of Plant Physiology of the University of Hanoi, Vietnam.     

Nitrogen fixation in rice systems: state of knowledge and future prospects

Rice is the most important cereal crop. In the next three decades, the world will need to produce about 60% more rice than today's global production to feed the extra billion people. Nitrogen is the major nutrient limiting rice production. Development of fertilizer-responsive varieties in the Green Revolution, coupled with the realization by farmers of the importance of nitrogen, has led to high rates of N fertilizer use on rice. Increased future demand for rice will entail increased application of fertilizer N. Awareness is growing, however, that such an increase in agricultural production needs to be achieved without endangering the environment. To achieve food security through sustainable agriculture, the requirement for fixed nitrogen must increasingly met by biological nitrogen fixation (BNF) rather than by using nitrogen fixed industrially. It is thus imperative to improve existing BNF systems and develop N₂-fixing non-leguminous crops such as rice. Here we review the potentials and constraints of conventional BNF systems in rice agriculture, as well as the prospects of achieving in planta nitrogen fixation in rice.     

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.     

Amino acid composition of azolla as affected by strains and population density

The total nitrogen and amino acid composition of seven Azolla strains were compared at four different growth phases. Total nitrogen content of the individual strains ranged from 2.6% to 5.7% of dry matter and was not significantly influenced by growth phase or population density. The concentration of the sixteen amino acids determined was maximal during the linear growth stage and specific differences occurred among Azolla strains. AnAzolla microphylla strain was the best source of amino acids and anA. filiculoides strain the poorest under the cultural conditions used. The chemical index score demonstrated the potential of some species, such as theA. microphylla strain, as contributor of protein for animals. Strains of other species, such asA. filiculoides, had several limiting amino acids and appear more suited for use solely as a green manure. All Azolla strains contained a similar proportion of essential (55%) and non-essential (45%) amino acids. Leucine, lysine, arginine and phenylanine+tyrosine were the predominant essential amino acids whereas the sulfur containing amino acids (methionine and cystine) were present in smaller amounts.     

Nitrogen uptake and recovery from urea and green manure in lowland rice measured by15N and non-isotope techniques

In the recent past considerable attention is paid to minimize dependence on purchased inputs such as inorganic nitrogen fertilizer. Green manure in the form of flood-tolerant, stem-nodulatingSesbania rostrata andAeschynomene afraspera is an alternative N source for rice, which may also increase N use efficiency. Therefore research was conducted to determine the fate of N applied to lowland rice (Oryza sativa L.) in the form ofSesbania rostrata andAeschynomene afraspera green manure and urea in two field experiments using¹⁵N labeled materials.¹⁵N in the soil and rice plant was determined, and¹⁵N balances established. Apparent N recoveries were determined by non-tracer method. ¹⁵N recoveries averaged 90 and 65% of N applied for green manure and urea treatments, respectively. High partial pressures of NH₃ in the floodwater, and high pH probably resulted from urea application and favoured losses of N from the urea treatment. Results show that green manure N can supply a substantial proportion of the N requirements of lowland rice. Nitrogen released fromSesbania rostrata andAeschynomene afraspera green manure was in synchrony with the demand of the rice plant. The effect of combined application of green manure and urea on N losses from urea fertilizer were also investigated. Green manure reduced the N losses from¹⁵N labeled urea possibly due to a reduction in pH of the floodwater. Positive added N interactions (ANIs) were observed. At harvest, an average of 45 and 25% of N applied remained in the soil for green manure and urea, respectively.Contribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, GermanyContribution from IRRI, Los Baños, Philippines and Justus-Liebig-University, Giessen, Germany     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Natural 15N abundance in shrub and tree legumes,Casuarina, and non N2 fixing plants in Thailand

The leaves and nodules from the shrub and tree legumes, particularly, Aeschynomene spp., Sesbania spp., Mimosa spp. and Leucaena spp., and Casuarina spp. and the leaves from neighbouring non-fixing plants were analyzed for their natural abundances of ¹⁵N ( ¹⁵N).The ¹⁵N in the leaves of non-fixing plants was +5.9% on average, whereas those from shrub legumes and Casuarina spp. were lower and close to the values of atmospheric N₂, suggesting the large contribution of N₂ fixation as the N source in these plants. The ¹⁵N values of the leaves from tree legumes except for Leucaena spp. were between the shrub legumes and non-fixing plants, which suggests that the fractional contribution of fixed N₂ in tree legumes may be smaller than that in the shrub legumes. Casuarina spp. was highly dependent on N₂ fixation. The ¹⁵N values of the nodules from most of the shrub legumes investigated were higher than those of the leaves.     

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 response ofAzolla pinnata R. Brown to the split application of phosphorus and the transfer of assimilated phosphorus to flooded rice plants

A field experiment was conducted at the Bangladesh Rice Research Institute, Joydebpur, Dhaka during the late wet season. Basal application of P at both 5 and 10 kg ha⁻¹ significantly increased total biomass production and nitrogen fixation byAzolla pinnata R. Brown (local strain). Addition of both 5 and 10 kg P ha⁻¹ in equal splits at inoculation and at six day intervals thereafter during growth periods of 12, 24 and 36 days increased biomass production and nitrogen fixation by Azolla over that attained with the basal application. Biomass and nitrogen fixation using a split application of 5 kg P ha⁻¹ exceeded that attained with basal application of 10 kg P ha⁻¹ and split application of 10 kg P ha⁻¹ resulted in 0.58, 11.2, and 18.3 t ha⁻¹ more biomass, and 0.47, 18.9, and 18.3 more kg fixed N ha⁻¹ at 12, 24 and 36 days, respectively, than the same amount applied as a basal application. Analyses indicated that the critical level of dry weight P in Azolla for sustained growth was in the range of 0.15–0.17%. Compared with the control, where no P was added, and additional 30 and 36 kg N ha⁻¹ were fixed after 24 and 36 days, respectively, when P was provided at 10 kg ha⁻¹ using a split application. A separate field study showed that flooded rice plants received P from incorporated Azolla with about 28% of the P present in the supplied Azolla being incorporated into the rice plants.     

Response of the floating aquatic fern <Emphasis Type="Italic">Azolla filiculoides</Emphasis> to elevated CO<Subscript>2</Subscript>, temperature,and phosphorus levels

Azolla filiculoides is a floating aquatic fern growing in tropical and temperate freshwater ecosystems. As A. filiculoides has symbiotic nitrogen-fixing cyanobacteria (Anabaena azollae) within its leaf cavities, it is cultivated in rice paddies to improve N availability and suppress other wetland weeds. To understand how C assimilation and N accumulation in A. filiculoides respond to elevated atmospheric carbon dioxide concentration (CO₂) in combination with P addition and higher temperatures, we conducted pot experiments during the summer of 2007 and 2008. In 2007, we grew A. filiculoides in pots at two treatment levels of added P fertilizer and at two levels of [CO₂] (380 ppm for ambient and 680 ppm for elevated [CO₂]) in controlled-environment chambers. In 2008, we grew A. filiculoides in four controlled-environment chambers at two [CO₂] levels and two temperature levels (34/26°C (day/night) and 29/21°C). We found that biomass and C assimilation by A. filiculoides were significantly increased by elevated [CO₂], temperature, and P level (all P < 0.01), with a significant interaction between elevated [CO₂] and added P (P < 0.01). Tissue N content was decreased by elevated [CO₂] and increased by higher temperature and P level (all P < 0.01). The acetylene reduction assay showed that the N-fixation activity of A. filiculoides was not significantly different under ambient and elevated [CO₂] but was significantly stimulated by P addition. N-fixation activity decreased at higher temperatures (34/26°C), indicating that 29/21°C was more suitable for A. azollae growth. Therefore, we conclude that the N accumulation potential of A. filiculoides under future climate warming depends primarily on the temperature change and P availability, and C assimilation should be increased by elevated [CO₂].