Interactive effects of exogenous combined nitrogen and phosphorus on growth and nitrogen fixation by azolla

Effects of N source and media-N and P levels were examined on growth, N uptake, and N₂ fixation ofAzolla pinnata withAnabaena azollae association (azolla) at two inoculum-P concentrations. Each expeiment was conducted for 7 days in a growth chamber using azolla at a predetermined inoculum-P concentration and the growth media containing a combination of four levels of P (0, 15, 75, and 200 M) and three levels (0, 1, and 5 mM) of either¹⁵N-enriched NH ₄ ⁺ as ammonium sulfate or¹⁵N-enriched NO ₃ ^– as potassium nitrate. Nitrogen uptake and N₂ fixation were measured by¹⁵N isotopic dilution method. Tissue P and N, N uptake, and N₂-fixation increased with increasing P concentration in the media regardless of the inoculum-P level of azolla. Increasing P concentration in the media increased growth of azolla at low inoculum P, but the effect on high inoculum-P azolla was either small or absen. High inoculum-P concentration resulted in increased growth, tissue-N and P concentrations, N uptake, and N₂ fixation by azolla. Ammonium in the growth media caused larger increase in tissue-N and greater repression of N₂ fixation than equimolar concentration of NO ₃ ^– . In the presence of NH ₄ ⁺ or NO ₃ ^– , in the growth media, N uptake by azolla exceeded the corresponding decrease in N₂ fixation, resulting in an overall increase in tissue-N concentration. Phosphorus in the media tended to negate the inhibitory effect of NH ₄ ⁺ or NO ₃ ^– on N₂ fixation. A multiple regression model showed that the effect of tissue-N on N₂ fixation was negative while that of tissue-P was positive. Therefore, a relative change in tissue-N and P appeared to regulate N₂ fixation. Tissue-N and P had similar effects on relative growth rate of azolla also. Inoculum-P level of azolla was important in determining the response to media-P.This research was supported by a grant from USAID under Indo-US Science and Technology Initiative.     

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

Effects of nitrogen and phosphorus supply,and ofRhizobium and VAM fungus inoculants on dinitrogen fixation in soybean

The effect of inoculation of N₂ fixation by soybean plants, grown in sandy soil was studied in pot experiments.Bradyrhizobium japonicum (Rh) and/or mycorrhizæ, in the presence of basic application of a P fertilizer (super or rock P), and two levels of¹⁵N-labelled ammonium sulfate (20 and 100 mg N per kg soil), were used. Highest N₂ fixation was observed after a dual inoculation (Rh+VAM), followed by single inoculation (Rh) and by mycorrhizal infection. Higher doses of N fertilizer depressed the capacity of the plant nodules and the inoculants for N₂ fixation.     

The effect of phosphorus deficiency on nutrient uptake,nitrogen fixation and photosynthetic rate in mashbean,mungbean and soybean

Phosphorous (P) fertilization is the major mineral nutrient yield determinant among legume crops. However, legume crops vary widely in the ability to take up and use P during deficiency. The aim here was to compare P uptake and translocation, biological nitrogen fixing ability and photosynthetic rate among mashbean (Vigna aconitifolia cv. ‘Mash-88’), mungbean (Vigna radiata cv. ‘Moong-6601’) and soybean (Glycine max L. cv. ‘Tamahomare’) during deficiency in hydroponics. Two treatments, the withdrawal of P from the solution (P-deprivation) and continued P at 160 μM (P sufficient) were effected at the pod initiation stage. Plants were grown for 20 days. Short-term labeling with ³²P showed the uptake and distribution of P into plant parts. Withdrawal of P from the solution reduced biomass, photosynthetic activity, and nitrogen fixing ability in mungbean, and mashbean more than in soybean. P deprivation decreased P accumulation more than N accumulation. The decrease was more severe in mungbean and mashbean than soybean. More P was translocated and distributed into leaves in soybean than in mungbean and mashbean. Leaf P amount was more correlated to leaf area than to photosynthetic rate per unit leaf area among all three legume species. The results indicate that selection for increased efficiency of P utilization and leaf area may be used to improve leguminous crops.     

Effects of drought on nitrogen fixation in soybean root nodules

Soybean plants [Glycine max (L.) Merr.] were grown in silica sand and were drought stressed for a 4 week period during reproductive development and without any mineral N supply in order to maximize demand for fixed nitrogen. A strain of Bradyrhizobium japonicum that forms large quantities of polysaccharide in nodules was used to determine whether or not the supply of reduced carbon to bacteroids limits nitrogenase activity. A depression of 30–40% in nitrogen content in leaves and pods of stressed plants indicated a marked decline in nitrogen fixation activity during the drought period. A 50% increase in the accumulation of bacterial polysaccharide in nodules accompanied this major decrease in nitrogen fixation activity and this result indicates that the negative impact of drought on nodules was not due to a depression of carbon supply to bacteroids. The drought treatment resulted in a statistically significant increase in N concentration in leaves and pods. Because N concentration and chlorophyll concentration in leaves were not depressed, there was no evidence of nitrogen deficiency in drought‐stressed plants, and this result indicates that the negative impact of drought on nodule function was not the cause of the depression of shoot growth. At the end of the drought period, the concentration of carbohydrates, amino nitrogen, and ureides was significantly increased in nodules on drought‐stressed plants. The overall results support the view that, under drought conditions, nitrogen fixation activity in nodules was depressed because demand for fixed N to support growth was lower.     

低磷和干旱胁迫对大豆植株干物质积累及磷效率的影响

土壤缺磷和季节性干旱已经成为南方酸性红壤地区大豆生产的主要限制因素之一。选取2个大豆品种巴西10号(磷高效)和本地2号(磷低效),研究其在不同磷素(0,15, 30 mg/kg P)和水分处理(分别在开花期和结荚期进行干旱胁迫)下的反应,从植株生物量、叶绿素含量、磷效率指标等方面研究不同基因型大豆对水磷耦合胁迫的适应机制。研究结果表明,随着土壤磷素水平的增加,两个品种的生物量和叶片叶绿素含量显著增加,根冠比则显著下降。在同一磷素水平处理下,干旱胁迫则导致较高的根冠比,对叶片叶绿素含量影响不大,两个品种表现一致。两个基因型大豆受到干旱胁迫后,其产量均显著低于正常水分处理。中等施磷能显著提高两个大豆品种的产量,但高磷处理对产量的增加幅度有限,甚至高磷处理还造成本地2号减产。巴西10号的产量随土壤中磷素的增加而增加,而本地2号的产量则为中磷>高磷>低磷,不管是磷处理还是水分处理,巴西10号的产量均高于本地2号。无论是花期干旱还是结荚期干旱,巴西10号和本地2号的根磷效率比、磷吸收效率及磷转移效率均随土壤磷浓度的增加而增加,磷利用效率则降低。总体上来讲,巴西10号的磷吸收效率和利用效率高于本地2号,而根磷效率比、磷转移效率则小于本地2号。     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Fructose uptake and influence on growth of and nitrogen fixation by Anabaena variabilis

Fructose is specifically taken up by nitrogen-fixing cultures of Anabaena variabilis in the light and lowers the doubling time from 24 to 8 h. The kinetics for both fructose-dependent growth and fructose uptake are exponential. The apparent Km for fructose uptake in N2-fixing cultures is 160 microM for cells not previously exposed to fructose and 50 microM in cells adapted to fructose. Picomolar amounts of [14C]fructose are scavenged from the medium and accumulate in filaments. Heterocysts of fructose-adapted filaments accumulate 14C from fructose within 20 min. Short-term experiments with fructose-starved cultures provide evidence that nitrogenase activity, protein, and chlorophyll content change within one generation time upon addition of fructose. In long-term experiments, the amount of fructose initially present in the medium determines heterocyst number and packed-cell volume. Photosynthetic oxygen evolution and amounts of chlorophyll decrease with exogenous fructose concentrations greater than 20 mM.     

Nodulation and nitrogen fixation on vegetative soybean plants as affected by photoperiod and gibberellic acid

Long-day photoperiods and applications of gibberellic acid promoted shoot growth by stimulating leaf enlargement as a result of increasing avaliable photosynthates, which was also reflected in the higher leaf, stem and nodule plant dry weights. Short-day plants had more nodules, but they were smaller (by weight) than those of long-day plants. Gibberellic acid at 1.5×10^–6 m enhanced nodule growth without preventing nodule formation. Factors other than just gibberellic acid are concluded to be involved in the responses of nodulation and nitrogen fixation to day length.

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Résumé Des photopériodes de longue journée et l'application d'acide gibbérellique a promu la croissance des pousses en stimulant l'élargissement des feuilles. Celle-ci résuitalent d'un accroissement de photosynthates disponibles. Cet effet s'est aussi réflecté dans un accroissement de la masse de feullies, de tiges et de nodules chez la plante. Des plants soumis à des photopériodes de courte journée présentaient plus de nodules mais ceux-ci étaient plus petits et de masse moindre que ceux de plants soumis à des photopériode de longue journée. L'acide gibbérellique à la dose de 1.5 10^–6 m a accru la croissance des nodules sans empêcher la formation des nodules. On a conclu que des facteurs autres que le seul acide gibbérellique sont mêlés aux réponses de la nodulation et de la fixation d'azote à la longueur de la journée.

     

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha^-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the cowpea cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.     

Effects of nitrogen: phosphorus supply ratios on nitrogen fixation in agricultural and pastoral ecosystems

An analysis of data compiled from the literature confirms a strong inverse relationship between annual rates of nitrogen fixation and the soil nitrogen content in agricultural and pastoral ecosystems. However, this inverse relationship is strongly modified by the rate of application of phosphorus fertilizer, which strongly influences the activities of both symbiotic and non-symbiotic nitrogen fixing organisms. In the case of symbiotic legumes, the response of N-fixation to N and P is in part a result of changes in legume dominance within the plant community. These results, as well as supporting data presented from a review of experiments on nitrogen fixation in a variety of other terrestrial and aquatic ecosystems, provide important support for the hypothesis that phosphorus availability is a key regulator of nitrogen biogeochemistry. Published as Paper No. 9950, Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE, USA.     

The influence of soybean planting density on dinitrogen fixation and yield

We investigated the effect of planting density on soybean (Glycine max (L.) Merr.) yield in glasshouse and field experiments. Because net canopy photosynthesis increases with increasing plant density, we hypothesized that increasing planting density would result in increasing rates of dinitrogen fixation in soybeans and higher yields per unit land area.In glasshouse studies, Wayne variety soybeans were planted in 10-cm diameter pots, 1 plant pot^-1 in matrices of 10-, 15-, 20-, 25-, or 30-cm equidistant intervals. Bradyrhizobium japonicum inoculum was added to half of the plants in each treatment. Replicate measurements of total stem height, internode lengths, leaf mass, stem mass, root mass, nodule number, nodule mass, and nitrogenase activity were obtained at 3, 6, and 9 weeks post-emergence. Fruits were harvested and counted at week 14. As planting density increased there were (1) altered morphology and growth rates, (2) increased apparent specific nodule activity (SNA), (3) decreased nodule number and mass, and (4) nearly constant fruit and seed production/plant. Expressed on a unit area basis, nitrogen influx and yield increased geometrically as planting density increased, with maximum values observed for 10-cm plantings.Field studies of Wayne, Stein, Williams, and Gold Harvest soybean varieties were made in 1985. Plots were established containing 100 plants spaced at 10-, 20-, and 30-cm distances. Measurements made during the growing season and at harvest established the same relative trends identified from the glasshouse studies. Increasing plant densities resulted in higher yields per unit land. Varietal differences were almost significant.     

Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods

We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using ¹⁵N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar ¹⁵N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum-extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), α-amino-N, and nitrate, and of dry matter for N and ¹⁵N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap (100 [ureide-N]/[ureide-N+ α-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.     

Interactive effect of warming,nitrogen and phosphorus limitation on phytoplankton cell size

Cell size is one of the ecologically most important traits of phytoplankton. The cell size variation is frequently related to temperature and nutrient limitation. In order to disentangle the role of both factors, an experiment was conducted to determine the possible interactions of these factors. Baltic Sea water containing the natural plankton community was used. We performed a factorial combined experiment of temperature, type of nutrient limitation (N vs. P), and strength of nutrient limitation. The type of nutrient limitation was manipulated by altering the N:P ratio of the medium (balanced, N and P limitation) and strength by the dilution rate (0% and 50%) of the semicontinuous cultures. The negative effect of temperature on cell size was strongest under N limitation, intermediate under P limitation, and weakest when N and P were supplied at balanced ratios. However, temperature also influenced the intensity of nutrient imitation, because at higher temperature there was a tendency for dissolved nutrient concentrations to be lower, while the C:N or C:P ratio being higher…higher at identical dilution rates and medium composition. Analyzing the response of cell size to C:N ratios (as index of N limitation) and C:P ratios (as index of P limitation) indicated a clear dominance of the nutrient effect over the direct temperature effect, although the temperature effect was also significant.     

Translocation of iron citrate and phosphorus in xylem exudate of soybean

Soybean plants, Glycine max (L.) Merrill, in standard solution received 2.5 μm ferric ethylenediamine di(o-hydroxyphenylacetate (FeEDDHA) and 0 to 128 μm phosphorus. Their stem exudates contained: 32 to 52 μm Fe, 120 to 5000 μm P, and 120 to 165 μm citrate. Electrophoresis of exudates with high P caused Fe trailing that precluded identification of any major form of Fe. Exudate with low P gave an anodic band of Fe citrate as the major Fe compound. Phosphate added to exudate in vitro depressed the Fe citrate peak and cause Fe trailing. EDDHA added to exudate in vitro pulled Fe from Fe citrate; citrate then migrated as a slower form and Fe migrated as FeEDDHA. A modified preculture system, involving 2-day renewals of 0.2 μm FeEDDHA with 3.2, 9.6, or 16 μm P and low levels of other ions, controlled pH depression and produced considerable change in citrate and P levels. The exudates contained: 45 to 57 μm Fe, 200 to 925 μm P, and 340 to 1025 μm citrate. The high citrate was from plants grown with low P. The major form of Fe in the exudates was Fe citrate. This is probably the form translocated in the plants.     

Effect of irradiance on development of apparent nitrogen fixation and photosynthesis in soybean

Soybeans grown with 2 millimolar NO₃⁻, which optimized apparent N₂ fixation by Rhizobium symbionts, showed significantly different rates of apparent photosynthesis and C₂H₂ reduction during seedling development at two irradiances. Those physiological processes were lower for several weeks in plants grown at 1,500 microeinsteins per meter² per second than in those exposed to 700 microeinsteins per meter² per second. The irradiance-induced retardation was evident in short-term rates of apparent photosynthesis and N₂ fixation, as well as in measures of dry matter and total N accumulation. In spite of their previously inhibited development, plants grown at 1,500 microeinsteins per meter² per second were indistinguishable by day 28 from those exposed to 700 microeinsteins per meter² per second in terms of whole-shoot CO₂-exchange rate; by day 35 they were identical in terms of whole-plant C₂H₂-reduction rate. On day 38 there was no significant difference in dry weight or N content between treatments. Shifting plants between irradiance treatments on day 21 showed that the higher irradiance also had a short-term inhibitory effect on C₂H₂ reduction. The fact that 16 millimolar NO₃⁻ prevented the continuous exposure to 1,500 microeinsteins per meter² per second from inhibiting apparent photosynthesis suggested that seedlings grown on 2 millimolar NO₃⁻ with Rhizobium were N-limited. Although rates of apparent photosynthesis were similar by day 28, the additional week required to produce equal rates of apparent N₂ fixation between irradiance treatments showed that physiological adaptations of shoots, as well as photosynthesis per se, can affect root nodule activity.