Azospirillum brasilense, a Beneficial Soil Bacterium: Isolation and Cultivation

Gladys Alexandre1

1 Biochemistry & Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3E.1
DOI:  10.1002/cpmc.40
Online Posting Date:  November, 2017
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Abstract

Bacteria of the genus Azospirillum comprise 15 species to date, with A. brasilense the best studied species in the genus. Azospirillum are soil bacteria able to promote the growth of plants from 113 species spanning 35 botanical families. These non‐pathogenic and beneficial bacteria are ubiquitous in soils and inhabit the roots of diverse plants. These bacteria are microaerophilic, able to fix nitrogen under free‐living conditions, motile, and able to navigate in gradients of various chemicals, including oxygen. These physiological traits are used to isolate these soil bacteria from soil and plant root samples, providing isolates that can be used for studying microbial physiology and plant growth promotion. © 2017 by John Wiley & Sons, Inc.

Keywords: Azospirillum; chemotaxis; motility; nitrogen‐fixation; plant roots

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of A. brasilense from Roots
  • Alternate Protocol 1: Isolation of Endophytic A. brasilense Strains
  • Basic Protocol 2: Isolation of A. brasilense from Soil Samples
  • Basic Protocol 3: Crypreservation of A. brasilense Isolates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of A. brasilense from Roots

  Materials
  • Root sample
  • Nfb medium (semisolid and solid; see recipe)
  • Cycloheximide (see recipe)
  • Congo Red (see recipe)
  • Sterile water
  • Tryptone yeast (TY) medium (solid; see recipe)
  • Sterile glass tubes (e.g., 18 mm diameter) containing semisolid Nfb medium supplemented with cycloheximide
  • Sterile Petri plates containing solid Nfb medium supplemented with Congo Red
  • Sterile glass tubes containing Nfb medium without cycloheximide
  • Sterile glass plates containing solid TY medium
  • Sterile loop
  • Bunsen burner
  • 1.5‐ml microcentrifuge tubes
  • Glass slide and coverslip
  • Light microscope with an objective of at least 40× but preferably, oil‐immersion 100×

Alternate Protocol 1: Isolation of Endophytic A. brasilense Strains

  Additional Materials (also see protocol 1)
  • Fresh root samples or intact roots removed from storage at cold temperatures.
  • Liquid commercial bleach containing 6% sodium hypochlorite

Basic Protocol 2: Isolation of A. brasilense from Soil Samples

  Additional Materials (also see protocol 1)
  • Soil sample
  • Sterile glass tubes (e.g., 18 mm diameter) with caps containing 9 ml of sterile H 2O
  • Beaker
  • Stir bar
  • Magnetic stirring plate

Basic Protocol 3: Crypreservation of A. brasilense Isolates

  Materials
  • A. brasilense isolate freshly grown on a solid Nfb medium plate supplemented with Congo Red
  • Tryptone yeast (TY) medium (liquid; see recipe)
  • Sterile glass tubes with caps containing 5 ml of liquid TY medium each
  • Sterile 50% glycerol stock solution
  • Bunsen burner
  • Sterile loop
  • 2‐ml sterile cryogenic vials
  • −80°C freezer
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Figures

Videos

Literature Cited

Literature Cited
  Assmus, B., Hutzler, P., Kirchhof, G., Amann, R., Lawrence, J. R., & Hartmann, A. (1995). In situ localization of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA‐targeted oligonucleotide probes and scanning confocal laser microscopy. Applied and Environmental Microbiology, 61, 1013–1019.
  Baldani, V. L. D., Alvarez, M. A.deB., Baldani, J. I., & Döbereiner, J. (1986). Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum. Plant and Soil, 90, 35–46. doi: 10.1007/BF02277385.
  Beijerinck, M. (1925). Über ein Spirillum, welches freien Stickstoff binden kann? Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Abteilung II, 63, 353–359.
  Boddey, R. M. (1987). Methods for quantification of nitrogen fixation associated with graminae. Critical Reviews in Plant Sciences, 6, 209–266. doi: 10.1080/07352688709382251.
  Cassán, F. D., & Diaz‐Zorita, M. (2016). Azospirillum sp. in current agriculture: From the laboratory to the field. Soil Biology & Biochemistry, 103, 117–130. doi: 10.1016/j.soilbio.2016.08.020.
  Döbereiner, J., & Day, J. M. (1976). Associative symbioses in tropical grasses: Characterization of microorganisms and dinitrogen‐fixing sites. In W. E. Newton, & C. J. Nyman (Eds.), Proceedings of the 1st International Symposium on N2 Fixation (pp. 518–538). Pullman, WA: Washington State University Press.
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  Reis, V. M., Baldani, V. L. D., & Baldani, J. I. (2015). Isolation, identification and biochemical characterization of Azospirillum spp. and other nitrogen‐fixing bacteria. In F. D. Cassán, Y. Okon, & C. M. Creus (Eds.), Handbook for Azospirillum (pp. 3‐26). Switzerland: Springer International Publishing. 06542‐7.
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