Critical evaluation of the acetylene reduction test for estimating the activity of nitrogen-fixing bacteria associated with the roots of wheat and barley

The reliability of the C₂H₂ reduction test for estimating the activity of N₂-fixing bacteria associated with the roots of cereals has been evaluated in Scottish soils. Six wheat cultivars, including two chromosome substitution lines, and five barley cultivars were grown in a glasshouse in nine soils from the North East of Scotland. All the soils exhibited C₂H₄ oxidase activity which was completely inhibited by 0.0001–0.1 atm C₂H₂. Over-estimation of C₂H₂ reduction, resulting from the accumulation of endogenous C₂H₄, could, therefore, occur in assays of undisturbed plants, with the real possibility of deducing the existence of N₂-fixation where none existed. However, radiolabelled C₂H₂ reduction tests on undisturbed plants producing 2.4–18.0 μmol C₂H₄ day^?1, showed that all the C₂H₄ had been derived from the C₂H₂. With less active plants, the source of the C₂H₄ could not be accurately determined by this tracer method. These low rates of C₂H₄ production (< 2.4 μmol C₂H₄ day^?1), referred to as apparent C₂H₂ reduction, should, therefore, not be considered proof of N₂-fixation.The highest C₂H₂ reduction activities were observed in soils at maximum water holding capacity (MWHC). Roots removed from these soils reduced C₂H₂ immediately, if the initial partial pressure of O₂ (pO₂) was < 0.1 atm. Roots washed free of soil did not oxidize C₂H₄ during the 8 h assay. The C₂H₂ reduction activities of these excised roots could not be related to the activity of plants in soil for three reasons. (1) Development of C₂H₂ reduction was dependent on protein synthesis (inhibited by chloramphenicol), indicating re-establishment of activity destroyed by exposure to atmospheric pO₂, rather than continuation of the activity of undisturbed roots. (2) A lag period, dependent on the volume of the incubation vessel, was observed, indicating the involvement of root respiration in the assay. (3) Growth of N₂-fixing bacteria on compounds released from the roots (reducing sugars, amino acids and α-keto acids) occurred during the assay.Even with the possibility of over-estimation of N₂-fixation, the C₂H₂ reduction activities measured were considered to be too low to contribute significantly to the nitrogen requirement of the cereals grown under field conditions in Scotland.Some guidelines for screening programmes of N₂-fixation associated with the roots of grasses are suggested.