Table 1 Estimating the contribution of biological nitrogen fixation

Determining the rate of nitrogen fixation is a difficult task, especially in field conditions. Five categories of techniques have been used, and all of them have their pitfalls.

(1) The acetylene reduction assay (ARA) is a sensitive and accurate method of assessing nitrogenase activity, via the indirect measure of reduction from acetylene to ethylene by nitrogenase. However, different types of nitrogenases reduce acetylene differently, leading to discrepancies with other methods, and this method is challenging in field conditions due to the flammable acetylene gas and difficulties in tightly enclosing the plant. Most importantly, this technique cannot evaluate how much of the fixed nitrogen is assimilated by the plant.

(2) The ¹⁵N natural abundance technique relies on the higher abundance of this naturally occurring and stable nitrogen isotope in most soils [92]. A diazotroph acquiring its nitrogen from the air and its host will, therefore, have a lower ¹⁵N abundance than plants only obtaining their nitrogen from the soil. Variations in isotope ratios are reported as ∂-values, commonly expressed in parts per mil (‰). These variations are measured using isotope-ratio mass spectrometry. This technique is high throughput and can be performed in fields. The stable nature of ¹⁵N isotopes allows storing and shipping samples efficiently. Unfortunately, variations in ¹⁵N abundance across the experimental field or from a geographical location to another and soil horizons can lead to artifacts, and the use of abundant controls, including soil samples, is required.

(3) ¹⁵N isotope dilution is a variant of the previous technique where the soil is enriched with a ¹⁵N-enriched nitrogen source to increase the differential between the ground and the air and limits the natural variations in ¹⁵N abundance. However, the cost of ¹⁵N-enriched nitrogen restricts the scale of these experiments. ¹⁵N-enriched sources can also move vertically or horizontally during the growing season, which mandates frequent soil sampling for controls [92, 93].

(4) Another ¹⁵N-based technique, called ¹⁵N gas enrichment, is conceptually the reverse of the previous ones. In this case, dinitrogen from the air is labeled with ¹⁵N and the incorporation of ¹⁵N in bacteria and its host plant indicates that they acquired some of their nitrogen from the air. This technique is one of the best pieces of evidence to prove that plants obtained nitrogen through nitrogen fixation. However, bacterial contaminations must always be considered as another source of N reaching the host. Sensitivity can be enhanced using radioactive nitrogen isotopes, such as ¹³N, but these are challenging to use given their short half-life [94]. Determining if ¹⁵N was incorporated in the host tissues is best achieved by mass spectrometry imaging or by extracting plant-specific metabolites such as chlorophyll [95, 96].

(5) Nitrogen-balance experiments evaluate the amount of nitrogen acquired by the plant from the soil and the total amount of nitrogen in the plant. The difference between the two measurements gives the amount of nitrogen from the air. However, evaluating soil nitrogen is difficult, introducing a significant level of uncertainty in these evaluations.