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

• Published in: Soil biology & biochemistry 1982, Vol.14 (1), p.27-35
• Main Authors: Lethbridge, G., Davidson, M.S., Sparling, G.P.
• Format: Article
• Language: English
• Subjects: Hordeum; Triticum
• ISSN: 0038-0717; 1879-3428
• DOI: 10.1016/0038-0717(82)90073-6

The reliability of the C 2H 2 reduction test for estimating the activity of N 2-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 C 2H 4 oxidase activity which was completely inhibited by 0.0001–0.1 atm C 2H 2. Over-estimation of C 2H 2 reduction, resulting from the accumulation of endogenous C 2H 4, could, therefore, occur in assays of undisturbed plants, with the real possibility of deducing the existence of N 2-fixation where none existed. However, radiolabelled C 2H 2 reduction tests on undisturbed plants producing 2.4–18.0 μmol C 2H 4 day −1, showed that all the C 2H 4 had been derived from the C 2H 2. With less active plants, the source of the C 2H 4 could not be accurately determined by this tracer method. These low rates of C 2H 4 production (< 2.4 μmol C 2H 4 day −1), referred to as apparent C 2H 2 reduction, should, therefore, not be considered proof of N 2-fixation. The highest C 2H 2 reduction activities were observed in soils at maximum water holding capacity (MWHC). Roots removed from these soils reduced C 2H 2 immediately, if the initial partial pressure of O 2 ( pO 2) was < 0.1 atm. Roots washed free of soil did not oxidize C 2H 4 during the 8 h assay. The C 2H 2 reduction activities of these excised roots could not be related to the activity of plants in soil for three reasons. (1) Development of C 2H 2 reduction was dependent on protein synthesis (inhibited by chloramphenicol), indicating re-establishment of activity destroyed by exposure to atmospheric pO 2, 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 N 2-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 N 2-fixation, the C 2H 2 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 N 2-fixation associated with the roots of grasses are suggested.