Skip to main content
Log in

Evaluation of effectiveness in mutant strains of rhizobium by acetylene reduction relative to other criteria of N2 fixation

  • Published:
Plant and Soil Aims and scope Submit manuscript

Summary

The feasibility of the acetylene reduction technique for evaluation of comparative effectiveness inRhizobium was tested inR. leguminosarum, R. trifolii, andR. meliloti with strains which were closely related but differed widely in effectiveness. Several variables in sampling and handling of nodules were found to introduce significant error into this sensitive assay. Freezing of nodules destroyed all reducing activity. Removal of nodules from the roots, storage of detached nodules for several hours before assay, and the dry-wet condition of nodules during assay contributed to lowered ethylene production. The time pattern of appearance, increase, and decline of acetylene reducing activity paralleled that of leghemoglobin content in effective pea nodules assayed at different stages of development. In a comparison of strains from all 3 species, there was generally good agreement between the rate of acetylene reduction (assayed at a stage of peak activity for effective nodules only) and plant dry weight, plant nitrogen, or visual ratings of effectiveness. Several exceptional mutants which were rated as partly effective on the basis of nodule type or leghemoglobin content showed little acetylene reduction or N2 fixation. Suitability of the acetylene assay for strain comparison and the question of units of comparison with other criteria of N2 fixation are considered.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Aprison, M. H., Magee, W. E., and Burris, R. H., Nitrogen fixation by excised soybean root nodules. J. Biol. Chem.208, 29–39 (1954).

    CAS  PubMed  Google Scholar 

  2. Bergersen, F. J., Haemoglobin content of legume root nodules. Biochim. Biophys. Acta50, 576–578 (1961).

    Article  CAS  Google Scholar 

  3. Bergersen, F. J. and Turner, G. L., Nitrogen fixation by the bacteroid fraction of breis of soybean root nodules. Biochim. Biophys. Acta141, 507–515 (1967).

    CAS  PubMed  Google Scholar 

  4. Chen, H. K. and Thornton, H. G., The strycture of ‘ineffective’ nodules and its influence on nitrogen fixation. Proc. Roy. Soc. (Lond.) B129, 208–229 (1940).

    Google Scholar 

  5. Chopra, C. L. and Subba-Rao, N. S., Mutual relationships among bacteroids, leghaemoglobin and nitrogen content of Egyptian clover (Trifolium alexandrinum) and gram (Cicer arietinum). Arch f. Mikrobiol.58, 71–76 (1967).

    Article  Google Scholar 

  6. Dilworth, M. J., Acetylene reduction by nitrogen-fixing preparations fromClostridium pasteurianum. Biochim. Biophys. Acta127, 285–294 (1966).

    CAS  PubMed  Google Scholar 

  7. Dobereiner, J., Evaluation of nitrogen fixation in legumes by the regression of total plant nitrogen with nodule weight. Nature (Lond.)210, 850–852 (1966).

    Google Scholar 

  8. Hardy, R. W. F. and Burns, R. C., Biological nitrogen fixation. Ann. Rev. Biochem.37, 331–358 (1968).

    CAS  Google Scholar 

  9. Hardy, R. W. F., Holsten, R. D., Jackson, E. K. and Burns, R. C., The acetylene-ethylene assay for N2 fixation: Laboratory and field evaluation. Plant Physiol.43, 1185–1207 (1968).

    CAS  Google Scholar 

  10. Keilin, D. and Hartree, E. F., Purification of horse-radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. Biochem. J.49, 88–104 (1951).

    CAS  PubMed  Google Scholar 

  11. Klucas, R. V., Koch, B., Russell, S. A. and Evans, H. J., Purification and some properties of the nitrogenase from soybean (Glycine max Merr.) nodules. Plant Physiol.43, 1906–1912 (1968).

    CAS  Google Scholar 

  12. Koch, B. and Evans, H. J., Reduction of acetylene to ethylene by soybean root nodules. Plant Physiol.41, 1748–1750 (1966).

    CAS  Google Scholar 

  13. Koch, B., Evans, H. J. and Russell, S., Reduction of acetylene and nitrogen gas by breis and cell-free extracts of soybean root nodules. Plant Physiol.42, 466–468 (1967).

    CAS  PubMed  Google Scholar 

  14. Methods of Analysis, Association of Official Agricultural Chemists, 6th Ed., pp. 26–27, College Offset Press, Philadelphia (1945).

  15. Moustafa, E. and Mortenson, L. E., Determination of azoferredoxin activity using cold-inactivated crude-extracts ofClostridium pasteurianum. Anal. Biochem.24, 226–231 (1968).

    Article  CAS  Google Scholar 

  16. Schöllhorn, R. and Burris, R. H., Study of intermediates in nitrogen fixation. Federation Proc.25, 710 (1966).

    Google Scholar 

  17. Schwinghamer, E. A., Studies on induced variation in the rhizobia. I. Defined media and nodulation test techniques. Applied Microbiol.8, 349–352 (1960).

    CAS  Google Scholar 

  18. Schwinghamer, E. A., Effectiveness ofRhizobium as modified by mutation for resistance to antibiotics. Antonie van Leeuwenhoek33, 121–136 (1967).

    Article  CAS  PubMed  Google Scholar 

  19. Schwinghamer, E. A., Loss of effectiveness and infectivity in mutants ofRhizobium resistant to metabolic inhibitors. Can. J. Microbiol.14, 355–367 (1968).

    CAS  PubMed  Google Scholar 

  20. Schwinghamer, E. A., Mutation to auxotrophy and prototrophy as related to symbiotic effectiveness inRhizobium leguminosarum andR. trifolii. Can. J. Microbiol.15, 611–622 (1969).

    CAS  PubMed  Google Scholar 

  21. Sloger, C. and Silver, W. S., Biological reductions catalyzed by symbiotic nitrogenfixing tissues. Bacteriol. Proc.1967, 112 (1967).

    Google Scholar 

  22. Stewart, W. D. P., Fitzgerald, G. P. and Burris, R. H., In situ studies on N2 fixation using the acetylene reduction technique. Proc. Natl. Acad. Sci. U.S.58, 2071–2078 (1967).

    CAS  Google Scholar 

  23. Virtanen, A. I., Erkama, J. and Linkola, H., On the relationship between nitrogen fixation and leghaemoglobin content of leguminous root nodules. II. Acta Chem. Scand.1, 861–870 (1947).

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

This investigation was supported in full by a Public Health Service research career development award (1-K3-GM-22, 594) and research grant (GM-12, 131) to the senior author, from the National Institutes of Health. Technical paper no.2648, Oregon Experimental Station.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schwinghamer, E.A., Evans, H.J. & Dawson, M.D. Evaluation of effectiveness in mutant strains of rhizobium by acetylene reduction relative to other criteria of N2 fixation. Plant Soil 33, 192–212 (1970). https://doi.org/10.1007/BF01378210

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01378210

Keywords

Navigation