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Factors affecting N fixation in legumes and their measurement using 15N natural abundance

Nitrogen fixation may be assessed using a variety of procedures. These include balance methods, direct labelling and indirect methods in pot and field trials. Each of these procedures has advantages and disadvantages. Frequently a combination of procedures is used.

The objective of this experiment was to investigate the factors affecting N fixation in legumes and their measurement using the 15N natural abundance method.


We will investigate how the application of inoculum and N affects N fixation of two summer legumes (cowpea and soybean).  Each treatment will be replicated 4 times. 1700 g of air dry soil was added to each pot in a plastic bag.  Soil was freshly collected, and had no history of growing either summer legume.

Preparation of solutions

Basal nutrients were applied in the form of pre-made 10 mL additions of liquid.  There were two solutions.  One containing everything except sulfates, and another containing just sulfates.  10 mL of each solution was added to each pot.  In half of the pots 10 mL of an N solution supplying 100 mg/kg of N (as urea) was added to each pot.


2 species (cowpea, soybean)

1 non-N fixing control (maize)

1 inoculated legume grown in sand (control for N fixation only)

4 replications of the design below.



No nitrogen

100 mg N/kg

5 seeds were planted evenly spaced in each pot to a depth of 1 cm using a rod or finger to press the seed into the soil.  In half of the pots, seed was soaked in the correct rhizobium inoculum slurry.  Pots were maintained at 19% field capacity by weight, taking into account an air-dry moisture content of 3%.

Plants were harvested after 5 weeks growth, rapidly dried to 60oC then weighed. All samples were finely ground for N analysis using the IRMS.  The total tissue N concentration and the 15N concentration were recorded for both legumes and the non-N fixing control plants grown in the same soil at the same time. Nodules were scored on three replicates on a 1-5 scale, with 1 being very few sparsely located nodules and 5 being extensive and complete nodulation on most of the root system.

The write-up should include Introduction, Methods, Results, Discussion and References sections.  You will need to calculate the %Ndfa for all treatments and the effect of inoculation and N addition. Statistical analysis of treatment effects and species differences is to be performed.  The Discussion should include comparison of your results with other values in the literature for those species and discussion of the effect of inoculation and N addition on N fixation.


Presence of higher acidity negatively impacts the growth of symbiosis, mostly involving the exchange pattern of molecular signals on the basic legume plant and microsymbiont. Limiting is necessary during overcoming of soil acidity and toxic aluminium mineral. However, in some regions such as Brazil soils are usually limited to pH levels which are in a neutral state. The selection of rhizobia inoculant strains level that is in a stable state under harsh soil conditions is effective but the challenging part is having minimal skills and knowledge regarding microsymbiont tolerance (Huang et al, 2014). Optimal results can be obtained by natural selection which occurs in rhizobia from acidic soils that are affected by water content and presence of extremely high temperatures. In some regions, the rise of grain production of beans has resulted from inoculation with a high level of tolerance. Necessary soil management operations are significant such as not till approaches lower high temperatures and maintain moisture. This is aimed at increasing the nitrogen fixation in leguminous plants. However, there exist several relationships that relate among nitrogen-rich plants, the biological nitrogen fixation and their immediate ability to respond to soil and prevailing environmental conditions which has been covered in various literary sources.

Continuous fluctuations in pH level, the presence of nutrients, high temperature, water availability and presence of other particular prevailing factors massively influence the growth and survival of metabolic natural operations on nitrogen-fixing bacteria. Low calcium with pH influence affects rhizobia attachment to plant root hairs section (Beversdorf, Miller ,McMahon, 2013).

The experiement started with the following methods which all directed us on how to compare how different plants absorb nitrogen content from soil.

The method involved comparing how the application of inoculum and the N fixation in two legumes that is the cowpea and the soybean which were used to carry the experiment (Biswas & Gresshoff, 2014).All treatment obtained were presented in 4 times .Each pot containing the two legumes contained a 1700 g of air dry soil and the soil was pure that no legumes which have aver planted before.

A 10 ml liquid was added in each pot to provide adequate nutrients needed (Biswas & Gresshoff, 2014).The method had two solutions whereby one of the solution had every nutrient except that it lacked the sulphate content and the other solution too contained all contents including the sulphate content. At half level of pots 10ml of an N solution with the 100mg/kg of N which contained the urea was added.

The method was carried out using the following;

  1. Legumes plants cowpea and the soybean.
  2. One plant that is maize (non N fixing plant) acting as control experiment.
  3. (Control for N fixation only) that is inoculated legume plant grown in sand soil.

In every pot 5 seeds were planted whereby they were all planted in depth of 1cm by use of metal rod and pressing the seeds in the soil by using fingers (Dwivedi, Sahrawat,Upadhyaya,Mengoni,Galardini,Bazzicalupo, Ortiz,2015).Seeds were socked and  then rhizobium inoculum added in each pot. The surroundings were maintained at 3% free from moisture.

After a period of 5 weeks, the 5 plants were harvested, dried at a temperature of 60oC and then weighted in a weighing balance. The 5 samples were finely grounded for the nitrogen analysis by the use of the IRMS.The N concentration in plants and the total 15N content was recorded in all the two legumes and also the same procedure done on the case of the non-nitrogen fixing control experiment (Dwivedi et al, 2015).

Treatment of the plants

The following results were recorded and the calculation done .In the experiment the %Ndfa in all samples was determined and the effects of nitrogen supply in conjunction with the inoculation concentration was identified. All the data analysis in the treatments were identified and the species differences identified (Hungria & Mendes, 2015).

The four species were recorded in the table as shown below.

(a).Legume plant (cowpeas) (Hungria & Mendes, 2015).

Non inoculated


No nitrogen






(b). Legume plant (soybean) (Sinharoy et al, 2018).

Non inoculated


No nitrogen






(c).Non N fixing control plant (maize) (Walker et al, 2015).

Non inoculated


No nitrogen






(d).Inoculated legume plant grown in sand acting as the control for the N fixation (Biswas & Gresshoff, 2014).

Non inoculated


No nitrogen






From the table above the experimental calculation of the %Ndfa was done by following the principle of δ15N (15N natural abundance) technique to determine biological N2 fixation (Mendonça, Lima, Guimarães, Moura, Andrade, 2017). The calculation was based on the data obtained during the experiment and the calculation done on each plant. After drying and grounding into finely measured particles the %N was determined whereby each plant gave different results.

The calculation was based on the delta units of the %Ndfa as compared to the normal expression of the % nitrogen atom in the soil .This expresses the natural variations in the composition of the 15N abundance which is relevant to the international standard atmospheric nitrogen absorption content from the atmosphere. The % standard nitrogen content o in atmosphere was identified that is 0.36630 (Mendonça et al, 2017).The %Ndfa was calculated in each table as shown below.

The basic formulae was;

                   %Ndfa = (sample atom%15N – 0.3663)/0.3663 x 1000 

  From the table (a) the %Ndfa is given by ;

                    %Ndfa = (0.36859-0.3663)/0.3663 *1000


From the table (b) the %Ndfa is given by;

                     %Ndfa = (0.36750-0.3663)/0.3663 *1000


From the table (c) the %Ndfa is given by;

                       %Ndfa = (0.36932-0.3663)/0.3663 *1000


From the table (d) the %Ndfa is given by;

                         %Ndfa = (0.41234-0.3663)/0.3663 *1000


Plants used gave different parameters.

Soybean and cowpeas growth parameters.

In the inoculated plants the height was larger compared to the non-inoculated plants (Mus, Crook, Garcia, Costas, Geddes, Kouri, Udvardi, 2016). In the experiment the inoculation of the legume plant with the Rhizobium species provided adequate nutrient that facilitated the growth of the plant at high rate as compared to the non-inoculated (Mus et al, 2016).

Preparing solutions

The highest nodule formation was high in the legumes planted in soil enriched with the N concentration as compared with the one planted in the sand (Sachs et al,2018).The control experiment that is from the maize gave no nodules. The %Ndfa in cowpeas and the soybean was high as compared to the results obtained in table (d) .The nodule content in inoculated seeds was significant as compared to the non I inoculated seeds (Walker,Agapakis, Watkin, Hirsch,2015).

Nitrogen fixation is an effective source of nitrogen mineral which contributes to land remediation. It has played a huge role in existing symbiotic systems of leguminous plants and rhizobia. This is due to the fact that these mutual associations have large quantitate effect on the nitrogen cycle. The deficiency in nitrogen mineral constraints leguminous plant growth. Symbiotic relationships have resulted from plants and several N2 fixing microorganisms in the soil. The symbiotically fixed N2 by the interrelationships of Rhizobium species and those particular plants involve a reliable source of Nitrogen mineral that is largely required in the agricultural sector (Rosenberg & Zilber, 2011). This aspect forms the advantages of Rhizobium and legume plant symbioses association which contributes highly to biological nitrogen fixation ability.N2-fixing and the ability of plants to make their own food as an energy source are the major contributors on the constant changing soil environment conditions brought about by micro and micro-organisms present in the stressed soil range. The following affect the growth, survival and metabolic operations of N2 fixing plant bacteria's in the soil (Taub & Wang, 2008).

  • Change in pH
  • Presence of required nutrients in their correct amount
  • Water
  • High temperature
  • Stress factors in soil

Despite these approaches, microbes, leguminous plants and soil constituents have increasingly adapted to the changing environment forming a basis for productive and successional ecospheres. Evidently, they have developed distinct mechanisms to deal with the existing stress factors.

It significant to know how microorganism and symbionts relate at various molecular points.In some cases, environmental stress usually affects the modulation aspect and N2-fixation process. Induction and limiting bacterial nodulation genes are regulated as a key factor the impact of host specificity and focuses based on the existing environmental conditions variables. The nodulation gene together with flavonoid signal long chain molecules contributes to the activation of reduced nod gene (Gruber & Galloway, 2008). The inducers are certain for a specific legume-Rhizobium association and their prevailing productive interactions which is largely affected by soil variables such as fertility levels, pH and plant nod factors. The available repressor proteins and salt availability influence the permeability levels of inner nodule cortex cells affecting the development on particular sensitivity of symbiotic N2-fixing.In addition, researchers argue that affecting oxygen gas diffusions and accumulation contribute to salinity modifications and inhibits the continuous growth of nodule formation as it leads to a higher number of development of small nodules.

The water content in soil affects the growth of small organism through various natural processes including diffusion, mass flow rate, and high ratio concentration of required nutrients. Availability of water directly affects the growth of rhizobia organism, by reducing water content activity way below required tolerance points and alters the growth of plants and root exudations. Low-level nodulation of plants in semi-arid areas is because of a reduced number of rhizobia levels especially in the dry parts due to lack of soil water. Rhizobia have adapted several strategic mechanisms to deal with osmotic stress mainly through the use of intracellular maintenance of both organic and inorganic solute levels (Beneduzi et al, 2008).


As expected, availability of required nutrients in soils has effects on symbiosis and their development and survival techniques. Nitrogen fixation, thus, reduces tremendously with legume plant age due to rise in soil Nitrogen to maximal levels. Fertilizers which are nutrients rich affect the N2 fixation such as soil pH and drought.

The influence of pH levels during nodulation activity has been extensively researched. Low soil pH is seen as a major inhibitor to soil properties limit leguminous growth. They decrease nodulation and nitrogen fixation. In that case, rhizobia have distinct limiting critical tolerance to acidity level tolerance.

This has contributed to the influence on growth and survival of rhizobia strains in soil environment as they are soil strenuous and dependent. Soil temperature usually affects the competition for nodulation. This influence may arise because of temperature-induced delay in nodulation restriction. Nodule dry weight of plants is greatly influenced by temperatures hence affecting the overall nitrogen fixation. Relative root temperature has an influence on N2 fixing capability and development of legumes. They affect a given strain and cultivar associations. In this instance, combining legume plant and Rhizobium has maximum temperature conditions relationship which is approximately 30-40 0C (Campo, Araujo , Hungria, 2009)

In many cases, inoculation with endophytes can limit the growth of the leguminous plans

The interaction between mycorrhizal fungi and rhizobium bacteria produces nodular structures which are dependent on the formation of mycorrhizal. It, therefore, contributes to the N2 fixation ability of legumes.


The treatment of the seeds with the different components influenced the differing results as discussed above (Walker et al, 2015).The increased supply of the nitrogen component in the non-inoculated plants insignificantly decreased the nodule formation in the seeds and also the dry weight .The increased supply of the nitrogen rich content in inoculated plant gave adequate results whereby the legumes had favourable features such as increased height and increased dry weight.


Beneduzi, A., Peres, D., Vargas, L. K., Bodanese-Zanettini, M. H., & Passaglia, L. M. P. (2008). Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Applied Soil Ecology, 39(3), 311-320.

Beversdorf, L. J., Miller, T. R., & McMahon, K. D. (2013). The role of nitrogen fixation in cyanobacterial bloom toxicity in a temperate, eutrophic lake. PLoS One, 8(2), e56103.

Biswas, B., & Gresshoff, P. M. (2014). The role of symbiotic nitrogen fixation in sustainable production of biofuels. International journal of molecular sciences, 15(5), 7380-7397.

Campo, R. J., Araujo, R. S., & Hungria, M. (2009). Nitrogen fixation with the soybean crop in Brazil: Compatibility between seed treatment with fungicides and bradyrhizobial inoculants. Symbiosis, 48(1-3), 154-163.

Dwivedi, S. L., Sahrawat, K. L., Upadhyaya, H. D., Mengoni, A., Galardini, M., Bazzicalupo, M., ... & Ortiz, R. (2015). Advances in host plant and rhizobium genomics to enhance symbiotic nitrogen fixation in grain legumes. In Advances in Agronomy (Vol. 129, pp. 1-116). Academic Press.

Gruber, N., & Galloway, J. N. (2008). An Earth-system perspective of the global nitrogen cycle. Nature, 451(7176), 293.

Haynes, R. (2012). Mineral nitrogen in the plant-soil system. Elsevier.

Huang, X., Liu, S., Wang, H., Hu, Z., Li, Z., & You, Y. (2014). Changes of soil microbial biomass carbon and community composition through mixing nitrogen-fixing species with Eucalyptus urophylla in subtropical China. Soil Biology and Biochemistry, 73, 42-48.

Hungria, M., & Mendes, I. C. (2015). Nitrogen fixation with soybean: the perfect symbiosis? 10.

Mendonça, E. D. S., Lima, P. C. D., Guimarães, G. P., Moura, W. D. M., & Andrade, F. V. (2017). Biological nitrogen fixation by legumes and N uptake by coffee plants. Revista Brasileira de Ciência do Solo, 41.

Mus, F., Crook, M. B., Garcia, K., Costas, A. G., Geddes, B. A., Kouri, E. D., ... & Udvardi, M. K. (2016). Symbiotic nitrogen fixation and challenges to extending it to non-legumes. Applied and environmental microbiology, AEM-01055.

Rosenberg, E., & Zilber?Rosenberg, I. (2011). Symbiosis and development: the hologenome concept. Birth Defects Research Part C: Embryo Today: Reviews, 93(1), 56-66.

Sachs, J. L., Quides, K. W., & Wendlandt, C. E. (2018). Legumes versus rhizobia: a model for ongoing conflict in symbiosis. New Phytologist.

Sinharoy, S., Torres-Jerez, I., González-Guerrero, M., Routray, P., Benedito, V. A., Roberts, D. M., ... & Finney, L. A. (2018). An iron-activated citrate transporter, MtMATE67, is required for symbiotic nitrogen fixation.

Taub, D. R., & Wang, X. (2008). Why are nitrogen concentrations in plant tissues lower under elevated CO2? A critical examination of the hypotheses. Journal of Integrative Plant Biology, 50(11), 1365-1374.

Walker, R., Agapakis, C., Watkin, E., & Hirsch, A. (2015). Symbiotic nitrogen fixation in legumes: perspectives on the diversity and evolution of nodulation by Rhizobium and Burkholderia species. Biological nitrogen fixation, 2, 913-923.

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