Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1986 Oct;52(4):847–851. doi: 10.1128/aem.52.4.847-851.1986

Rapid Colored-Nodule Assay for Assessing Root Exudate-Enhanced Competitiveness of Bradyrhizobium japonicum

Abateni Ayanaba 1,†, Richard A Haugland 1,‡, Michael J Sadowsky 1,§,*, Robert G Upchurch 1,∥, Karen D Weiland 1, Robert M Zablotowicz 1,
PMCID: PMC239125  PMID: 16347177

Abstract

The effects of root exudate (RE) treatment on nodule occupancy by Bradyrhizobium japonicum were investigated by a rapid colored-nodule assay, which is based on the observation that B. japonicum L-110 and its antibiotically marked derivatives form dark-red nodules on certain soybean (Glycine max) cultivars, whereas other strains form beige nodules. The efficacy of the assay was confirmed by direct immunofluorescence and by antibiotic platings of nodule bacteria. Both logarithmic- and stationary-phase cultures of the reference strain, L-110Nal, were used in paired-competition studies with RE-treated or untreated cells of seven challenge strains. On the basis of field and greenhouse competition studies, these strains were placed into three competitiveness groups: high (AN-11, AN-16aStrRif, and AN-6), intermediate (AN-3 and 122SR), and low (I-110ARS and AN-18). Seedlings of G. max cv. Centennial were inoculated with two ratios of challenge to reference strain, 1:1 and 1:9, and nodule occupancy was determined after the V4 to V5 stage of ontogeny. Two of the strains showed increased occupancy in response to RE treatment at the 1:1 inoculation ratio. Logarithmic- and stationary-phase cultures of AN-6 showed increased occupancy, from 22 to 38% (P < 0.10) and from 23 59 39% (P < 0.05), respectively. While the maximum increase for stationary-phase cultures of AN-16aStrRif was from 34 to 47% (P < 0.05), logarithmic-phase cultures failed to respond to RE treatment. The results of these studies indicate that RE treatment increases the nodule occupancy of some, but not all, B. japonicum strains and that the colored-nodule assay could be rapidly and reliably used to determine the competitive ability of B. japonicum.

Full text

PDF
848

Images in this article

849Image
on p.849

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bhagwat A. A., Thomas J. Legume-Rhizobium interactions: cowpea root exudate elicits faster nodulation response by Rhizobium species. Appl Environ Microbiol. 1982 Apr;43(4):800–805. doi: 10.1128/aem.43.4.800-805.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhuvaneswari T. V., Bauer W. D. Role of Lectins in Plant-Microorganism Interactions: III. Influence of Rhizosphere/Rhizoplane Culture Conditions on the Soybean Lectin-binding Properties of Rhizobia. Plant Physiol. 1978 Jul;62(1):71–74. doi: 10.1104/pp.62.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop P. E., Guevara J. G., Engelke J. A., Evans H. J. Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max. Plant Physiol. 1976 Apr;57(4):542–546. doi: 10.1104/pp.57.4.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohlool B. B., Schmidt E. L. Nonspecific staining: its control in immunofluorescence examination of soil. Science. 1968 Nov 29;162(3857):1012–1014. doi: 10.1126/science.162.3857.1012. [DOI] [PubMed] [Google Scholar]
  5. Currier W. W., Strobel G. A. Chemotaxis of Rhizobium spp. to Plant Root Exudates. Plant Physiol. 1976 May;57(5):820–823. doi: 10.1104/pp.57.5.820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Currier W. W., Strobel G. A. Chemotaxis of Rhizobium spp. to a Glycoprotein Produced by Birdsfoot Trefoil Roots. Science. 1977 Apr 22;196(4288):434–436. doi: 10.1126/science.196.4288.434. [DOI] [PubMed] [Google Scholar]
  7. Eaglesham A. R., Ahmad M. H., Hassouna S., Goldman B. J. Cowpea rhizobia producing dark nodules: use in competition studies. Appl Environ Microbiol. 1982 Sep;44(3):611–618. doi: 10.1128/aem.44.3.611-618.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Halverson L. J., Stacey G. Host recognition in the Rhizobium-soybean symbiosis: detection of a protein factor in soybean root exudate which is involved in the nodulation process. Plant Physiol. 1984 Jan;74(1):84–89. doi: 10.1104/pp.74.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kosslak R. M., Bohlool B. B., Dowdle S., Sadowsky M. J. Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation. Appl Environ Microbiol. 1983 Oct;46(4):870–873. doi: 10.1128/aem.46.4.870-873.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kuykendall L. D., Elkan G. H. Rhizobium japonicum derivatives differing in nitrogen-fixing efficiency and carbohydrate utilization. Appl Environ Microbiol. 1976 Oct;32(4):511–519. doi: 10.1128/aem.32.4.511-519.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Leonard L. T. A Simple Assembly for Use in the Testing of Cultures of Rhizobia. J Bacteriol. 1943 Jun;45(6):523–527. doi: 10.1128/jb.45.6.523-527.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. MEANS U. M., JOHNSON H. W., DATE R. A. QUICK SEROLOGICAL METHOD OF CLASSIFYING STRAINS OF RHIZOBIUM JAPONICUM IN NODULES. J Bacteriol. 1964 Mar;87:547–553. doi: 10.1128/jb.87.3.547-553.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. May S. N., Bohlool B. B. Competition Among Rhizobium leguminosarum Strains for Nodulation of Lentils (Lens esculenta). Appl Environ Microbiol. 1983 Mar;45(3):960–965. doi: 10.1128/aem.45.3.960-965.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Meade J., Higgins P., O'gara F. Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia. Appl Environ Microbiol. 1985 Apr;49(4):899–903. doi: 10.1128/aem.49.4.899-903.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moawad H. A., Ellis W. R., Schmidt E. L. Rhizosphere Response as a Factor in Competition Among Three Serogroups of Indigenous Rhizobium japonicum for Nodulation of Field-Grown Soybeans. Appl Environ Microbiol. 1984 Apr;47(4):607–612. doi: 10.1128/aem.47.4.607-612.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mulligan J. T., Long S. R. Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6609–6613. doi: 10.1073/pnas.82.19.6609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schmidt E. L., Bakole R. O., Bohlool B. B. Fluorescent-antibody approach to study of rhizobia in soil. J Bacteriol. 1968 Jun;95(6):1987–1992. doi: 10.1128/jb.95.6.1987-1992.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES