Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.     

Physical Factors Control Phytoplankton Production and Nitrogen Fixation in Eight Texas Reservoirs

We compared regression tree analyses and multiple linear regression models to explore the relative importance of physical factors, land use, and water quality in predicting phytoplankton production and N₂ fixation potentials at 85 locations along riverine to lacustrine gradients within eight southern reservoirs. The regression tree model (r ² = 0.73) revealed that differences in phytoplankton production were primarily a function of water depth. The highest rates of production (mg C m⁻³ h⁻¹) occurred at shallow sites (<0.9 m), where rates were also related to total phosphorus (TP) levels. At deeper sites, production rates were higher at sites with relative drainage area (RDA, ratio of drainage area to water surface area) below 45, potentially due to longer hydraulic residence times. In contrast, multiple linear regression selected TP, RDA, dissolved phosphorus, and percent developed land as significant model variables (r ² = 0.63). The regression tree model (r ² = 0.67) revealed that N₂ fixation potentials (mg N m⁻³ h⁻¹) were substantially higher at sites with relatively smaller drainage areas (RDA < 45). Within this subgroup, fixation rates were additionally related to TP values (threshold = 41 μg l⁻¹). The multiple linear regression model (r ² = 0.67) also selected RDA as the primary predictor of N₂ fixation. Regression tree models suggest that nutrient controls (phosphorus) were subordinate to physical factors such as depth and RDA. We concluded that regression tree analysis was well suited to revealing nonlinear trends in data (for example, depth), but yielded large uncertainty estimates when applied to linear data (for example, phosphorus).     

Biological nitrogen fixation: Investments,expectations and actual contributions to agriculture

Inputs of biologically fixed N into agricultural systems may be derived from symbiotic relationships involving legumes and Rhizobium spp., partnerships between plants and Frankia spp. or cyanobacteria, or from non-symbiotic associations between free-living diazotrophs and plant roots. It is assumed that these N₂-fixing systems will satisfy a large portion of their own N requirements from atmospheric N₂, and that additional fixed N will be contributed to soil reserves for the benefit of other crops or forage species. This paper reviews the actual levels of N₂ fixation attained by legume and non-legume associations and assesses their role as a source of N in tropical and sub-tropical agriculture. We discuss factors influencing N₂ fixation and identify possible strategies for improving the amount of N₂ fixed.     

Biological Nitrogen Fixation is not a Major Contributor to the Nitrogen Demand of a Commercially Grown South African Sugarcane Cultivar

It has previously been reported that endophytic diazotrophic bacteria contribute significantly to the nitrogen budgets of some graminaceous species. In this study the contribution of biological nitrogen fixation to the N-budget of a South African sugarcane cultivar was evaluated using ¹⁵N natural abundance, acetylene reduction and ¹⁵N incorporation. Plants were also screened for the presence of endophytic diazotrophic bacteria using acetylene reduction and nifH-gene targeted PCR with the pure bacterial strains. ¹⁵N natural abundance studies on field-grown sugarcane indicated that the plants did not rely extensively on biological nitrogen fixation. Furthermore, no evidence was found for significant N₂-fixation or nitrogenase activity in field-grown or glasshouse-grown plants using ¹⁵N incorporation measurements and acetylene reduction assays. Seven endophytic bacterial strains were isolated from glasshouse-grown and field-grown plants and cultured on N-free medium. The diazotrophic character of these seven strains could not be confirmed using acetylene reduction and PCR screening for nifH. Thus, although biological nitrogen fixation may occur in South African sugarcane varieties, the contribution of this N-source in the tested cultivar was not significant.     

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.     

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).     

The Improvement of Spinach Growth by Nano-anatase TiO<Subscript>2</Subscript> Treatment Is Related to Nitrogen Photoreduction

The improvement of spinach growth is proved to relate to N₂ fixation by nano-anatase TiO₂ in this study. The results show that all spinach leaves kept green by nano-anatase TiO₂ treatment and all old leaves of control turned yellow white under culture with N-deficient solution. And the fresh weight, dry weight, and contents of total nitrogen, , chlorophyll, and protein of spinach by nano-anatase TiO₂ treatment presented obvious enhancement compared with control. Whereas the improvements of yield of spinach were not as good as nano-anatase TiO₂ treatment under N-deficient condition, confirming that nano-anatase TiO₂ on exposure to sunlight could chemisorb N₂ directly or reduce N₂ to NH₃ in the spinach leaves, transforming into organic nitrogen and improving the growth of spinach. Bulk TiO₂ effect, however, was not as significant as nano-anatase TiO₂. A possible metabolism of the function of nano-anatase TiO₂ reducing N₂ to NH₃ was discussed.     

Nitrogen fixation in coniferous bark litter

Summary Non-symbiotic heterotrophic N₂ fixation in coniferous bark litter was investigated with the acetylene reduction assay under aerobic and anaerobic conditions. The litter studied was composed essentially of bark, of pH 5 and a C/N ratio of 101; the ratio of available C to available N, which governs N₂ fixation, was considerably higher. The rate of N₂ fixation was estimated as 2.5–4.4 g N. g^–1 dry wt. day^–1. Nitrogenase activity was still evident after seven months of incubation under aerobic conditions. The N₂-ase activity was O₂ dependent: under anaerobic conditions no N₂-ase activity was found unless a fermentable C source was added. The importance of N₂ fixation in N-poor litter for the maintenance of soil fertility is emphasized.     

A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane

During surveys of bacteria possibly responsible for N₂ fixation in sugarcane, root and stem samples were collected in four sugarcane-growing regions in Brazil. A new microaerobic N₂-fixing bacterium was isolated from most samples of washed roots and stems from all regions. Isolation procedures were based on semisolid diluted sugarcane juice medium followed by replication to N-free 10% sugar medium acidified with acetic acid to pH 4.5. The new bacterium is an aerobic rod, motile by 1 to 3 lateral flagella, fixes N₂ in semisolid media under air but not in liquid media except when a starter dose of N is added. It has no nitrate, reductase and N₂ fixation proceeds in the presence of 10mM NO ₃ ⁻ . Best growth occurs with high sucrose concentrations (10%). Growth occurs up to 30% sucrose but not at 35%. Acid is formed reaching a final pH of below 3.0. Growth and N₂ fixation proceed at this acidity. Ethanol is used for growth and is “overoxidised” (oxidized to CO₂ and H₂O). Acetic and lactic acids are also oxidized to CO₂ and H₂O. Acids produced from glucose are consumed with precipitation of CaCO₃. Dark brown colonies are formed on potato agar with 10% sugar and dark orange colonies on N poor agar (20 mg yeast extract per 1) containing bromothymol blue. In view of the distinct characteristics which do not allow identification within either Frateuria, Gluconobacter, Acetobacter or any known N₂-fixing bacterium a new genus and species are proposed and namedSaccharobacter nitrocaptans.     

Nitrogen Transfer Between Plants: A 15N Natural Abundance Study with Crop and Weed Species

An increasing amount of evidence indicates that N can be transferred between plants. Nonetheless, a number of fundamental questions remain. A series of experiments was initiated in the field to examine N transfer between N₂-fixing soybean (Glycine max [L.] Merr.) varieties and a non-nodulating soybean, and between N₂-fixing peanut (Arachis hypogaea L.) or soybean and neighboring weed species. The experiments were conducted in soils with low N fertilities and used differences in N accumulation and/or ¹⁵N natural abundance to estimate N transfer. Mixtures of N₂-fixing and non-nod soybean indicated that substantial inter-plant N transfer occurred. Amounts were variable, ranging from negligible levels to 48% of the N found in the non-nod at maturity. Transfer did not appear to strongly penalize the N₂-fixing donor plants. But, in cases where high amounts of N were transferred, N content of donors was noticeably lowered. Differences were evident in the amount of N transferred from different N₂-fixing donor genotypes. Results of experiments with N₂-fixing crops and the weed species prickly sida (Sida spinosa L.) and sicklepod (Senna obtusifolia [L.] Irwin & Barneby) also indicated substantial N transfer occurred over a 60-day period, with amounts accounting for 30–80% of the N present in the weeds. Transfer of N, however, was generally very low in weed species that are known to be non-hosts for arbuscular mycorrhizae (yellow nutsedge, Cyperus esculentus L. and Palmer amaranth, Amaranthus palmeri [S.] Watson). The results are consistent with the view that N transfer occurs primarily through mycorrhizal hyphal networks, and they reveal that N transfer may be a contributing factor to weed problems in N₂-fixing crops in low N fertility conditions.     

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).     

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.     

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.     

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.     

Leguminosae native nodulating bacteria from a gold mine As-contaminated soil: Multi-resistance to trace elements,and possible role in plant growth and mineral nutrition

Efficient N₂-fixing Leguminosae nodulating bacteria resistant to As may facilitate plant growth on As-contaminated sites. In order to identify bacteria holding these features, 24 strains were isolated from nodules of the trap species Crotalaria spectabilis (12) and Stizolobium aterrimum (12) growing on an As-contaminated gold mine site. 16S rRNA gene sequencing revealed that most of the strains belonged to the group of α-Proteobacteria, being representatives of the genera Bradyrhizobium, Rhizobium, Inquilinus, Labrys, Bosea, Starkeya, and Methylobacterium. Strains of the first four genera showed symbiotic efficiency with their original host, and demonstrated in vitro specific plant-growth-promoting (PGP) traits (production of organic acids, indole-3-acetic-acid and siderophores, 1-aminocyclopropane-1-carboxylate deaminase activity, and Ca₃(PO₄)₂ solubilization), and increased resistance to As, Zn, and Cd. In addition, these strains and some type and reference rhizobia strains exhibited a wide resistance spectrum to β-lactam antibiotics. Both intrinsic PGP abilities and multi-element resistance of rhizobia are promising for exploiting the symbiosis with different legume plants on trace-element-contaminated soils.     

Interspecific Competition for Soil N and its Interaction with N2 Fixation, Leaf Expansion and Crop Growth in Pea–Barley Intercrops

Field experiments were carried out during three successive years to study through a dynamic approach the competition for soil N and its interaction with N₂ fixation, leaf expansion and crop growth in pea–barley intercrops. The intensity of competition for soil N varied between experiments according to soil N supply and plant densities. This study demonstrates the key role of competition for soil N which occurs early in the crop cycle and greatly influences the subsequent growth and final performance of both species. Relative yield values for grain yield and N accumulation increased with the intensity of competition for soil N. Barley competed strongly for soil N in the intercrop. Its competitive ability increased steadily during the vegetative phase and remained constant after the beginning of pea flowering. The period of strong competition for soil N (500–800 degree-days after sowing) also corresponded to the period of rapid growth in leaf area for both species and therefore an increasing N demand. For each species, the leaf area per plant at the beginning of pea flowering was well correlated with crop nitrogen status. Barley may meet its N needs more easily in intercrops (IC) and has greater leaf area per plant than in sole crops (SC). Barley having a greater soil N supply results in an even higher crop N status and greater competitive ability relative to pea in intercrop. Competition by barley for soil N increased the proportion of pea N derived from fixation. The nitrogen nutrition index (NNI) values of pea were close to 1 whatever the soil N availability in contrast to barley. However N₂ fixation started later than soil N uptake of pea and barley and was low when barley was very competitive for soil N. Due to the time necessary for the progressive development and activity of nodules, N₂ fixation could not completely satisfy N demand at the beginning of the crop cycle. The amount of N₂ fixed per plant in intercrops was not only a response to soil N availability but was largely determined by pea growth and was greatly affected when barley was too competitive.     

Nitrogen and phosphorus cycling in relation to stand age of Eucalyptus regnans F. Muell

Lupins, canola, ryegrass and wheat fertilized with Na₂ ³⁵SO₄ and either ¹⁵NH₄Cl or K¹⁵NO₃(N:S=10:1), were grown in the field in unconfined microplots, and the sources of N and S (fertilizer, soil, atmosphere, seed) in plant tops during crop development were estimated. Modelled estimates of the proportion of lupin N derived from the atmosphere, which were obtained independently of reference plants, were used to calculate the proportion of lupin N derived from the soil. Total uptake of N and S and uptake of labelled N and S increased during crop development. Total uptake of S by canola was higher than lupins, but labelled S uptake by lupins exceeded uptake by canola. The form of N applied had no effect on uptake of labelled and unlabelled forms of N or S. Ratios of labelled to unlabelled S and ratios of labelled to unlabelled N derived from soil sources decreased during growth, and were less for S than for N for each crop at each sampling time. Although ratios of labelled to unlabelled soil-derived N were similar between crops at 155, 176 and 190 days after sowing, ratios of labelled to unlabelled S for lupins were higher than for the reference crops and declined during this period. The ratios of labelled to unlabelled S in lupins and the reference plants therefore bore no relationship either to ratios of labelled to unlabelled soil-derived N in the plants, or to total S uptake by the plants. Therefore the hypothesis that equal ratios of labelled N to unlabelled soil-derived N in legumes (Rleg) and reference plants (Rref) would be indicated by equal ratios of labelled to unlabelled S was not supported by the data. The results therefore show that the accuracy of reference plant-derived values of Rleg cannot be evaluated by labelling with ³⁵S.     

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.     

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.     

Nitrogen inputs and losses from New Zealand dairy farmlets, as affected by nitrogen fertilizer application: year one

Inputs and losses of nitrogen (N) were determined in dairy cow farmlets receiving 0, 225 or 360 kg N ha^-1 (in split applications as urea) in the first year of a large grazing experiment near Hamilton, New Zealand. Cows grazed perennial ryegrass/white clover pastures all year round on a free-draining soil. N₂ fixation was estimated (using ¹⁵N dilution) to be 212, 165 and 74 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. The intermediate N rate had little effect on clover growth during spring but favoured more total pasture cover in summer and autumn, thereby reducing overgrazing and resulting in 140% more clover growth during the latter period.Removal of N in milk was 76,89 and 92 kg N ha^-1 in the 0, 225 and 360 N treatments, respectively. Denitrification losses were low (7–14 kg N ha^-1 yr^-1), increased with N application, and occurred predominantly during winter. Ammonia volatilization was estimated by micrometeorological mass balance at 15, 45 and 63 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. Most of the increase in ammonia loss was attributed to direct loss after application of the urea fertilizer.Leaching of nitrate was estimated (using ceramic cup samplers at 1 m soil depth, in conjunction with lysimeters) to be 13, 18 and 31 kg N ha^-1 yr^-1 in a year of relatively low rainfall (990 mm yr^-1) and drainage (170–210 mm yr^-1). Drainage was lower in the N fertilized treatments and this was attributed to enhanced evapotranspiration associated with increased grass growth.Nitrate-N concentrations in leachates increased gradually over time to 30 mg L^-1 in the 360 N treatment whereas there was little temporal variation evident in the 0 (mean 6.4 mg L^-1) and 225 (mean 10.1 mg L^-1) N treatments. Thus, the 360 N treatment had a major effect by greatly reducing N₂ fixation and increasing N losses, whereas the 225 N treatment had little effect on N₂ fixation or on nitrate leaching. However, these results refer to the first year of the experiment and further measurements over time will determine the longer-term effects of these treatments on N inputs, transformations and losses.