Quantification of Grass Colonization by Associative Bacteria

Eduardo Balsanelli1, Fábio de Oliveira Pedrosa1, Emanuel Maltempi de Souza1

1 Department of Biochemistry and Molecular Biology, Federal University of Paraná, Curitiba
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20047
Online Posting Date:  June, 2017
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Abstract

There is a growing interest in the use of plant growth–promoting rhizobacteria to improve crop productivity as a partial substitute for nitrogen fertilizer. Bacteria‐colonizing plants may be epiphytic or endophytic. This article describes reproducible protocols to quantify the level of colonization in each plant compartment. The protocols were developed using several cereal crops such as maize, rice, sorghum, and wheat. © 2017 by John Wiley & Sons, Inc.

Keywords: endophytic bacteria quantification; epiphytic bacteria quantification; hydroponic system; Poaceae‐bacteria interaction

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Quantification of Grass Colonization by Associative Bacteria
  • Basic Protocol 2: Plant Colonization Competition Assay
  • Basic Protocol 3: Effect of Carbohydrates on Bacterial Root Attachment
  • Basic Protocol 4: Effect of Lectins on Bacterial Root Attachment
  • Basic Protocol 5: Effect of Protease on Bacterial Root Attachment: Bacteria Treatment
  • Basic Protocol 6: Effect of Protease on Bacterial Root Attachment: Root Treatment
  • Basic Protocol 7: Plant Growth Promotion
  • Alternate Protocol 1: Quantification of Bacteria by Flow Cytometry
  • Support Protocol 1: Re‐Isolation of Inoculated Bacteria
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantification of Grass Colonization by Associative Bacteria

  Materials
  • Zea mays, Oryza sativa, Triticum aestivum, or Sorghum bicolor seeds
  • 70 % (v/v) ethanol
  • Acidified hypochlorite/Tween solution (see recipe)
  • Hypochlorite solution (see recipe)
  • 30% (v/v) hydrogen peroxide
  • 0.8% (w/v) water‐agar plates
  • Bacterial strains of interest
  • Growth plate containing solid medium appropriate for bacteria of interest
  • Liquid medium appropriate for bacteria of interest
  • Phosphate‐buffered saline (PBS), sterile (see recipe)
  • Plant medium, carbon‐free (see Egener, Hurek, & Reinhold‐Hurek, )
  • Fixing solution: 4% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde
  • Fluoromount (e.g., Sigma‐Aldrich)
  • 500‐ml beaker, sterile
  • Tweezers, sterile
  • Plant growth chamber, 28°C/16 hr light (150 µmol/seg) and 25°C/8 hr dark, 70% humidity (e.g., Conviron MTR30)
  • Bacterial incubator with variable temperature and shaking
  • Stereomicroscope (10× to 20× magnification)
  • Hydroponic system including:
    • 100‐ml glass tubes with foam caps
    • Polypropylene beads (2 to 3 mm in diameter)
    • Scalpel, sterile
    • Filter paper, sterile
    • 1.5‐ml microcentrifuge tubes, sterile
    • Vortex (e.g., Vortex‐Genie 2)
    • Mortar and pestle, sterile
    • Glass slides and coverslips
    • Fluorescence or confocal microscope

Basic Protocol 2: Plant Colonization Competition Assay

  Materials
  • Root‐attached bacteria (see protocol 1, Quantification assay 1)
  • Epiphytic bacteria (see protocol 1, Quantification assay 2)
  • Endophytic bacteria (see protocol 1, Quantification assay 3)
  • Flow cytometry marker buffer: 50% ethanol in PBS, 1× SYBR green (e.g., Thermo Fisher Scientific, cat. no. S7563)
  • 5‐ml syringe, sterile
  • Glass microfiber filter (e.g., Whatman, cat. no. 1823‐025)
  • Centrifuge
  • Flow cytometer (e.g., BD Accuri C5)

Basic Protocol 3: Effect of Carbohydrates on Bacterial Root Attachment

  Materials
  • Bacterial colonies from experimental plants (see protocol 1)
  • Solid medium containing selective antibiotics
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Figures

Videos

Literature Cited

Literature Cited
  Balsanelli, E., Baura, V. A., Pedrosa, F. O., Souza, E. M., & Monteiro, R. A. (2014). Exopolysaccharide biosynthesis enables mature biofilm formation on abiotic surfaces by Herbaspirillum seropedicae. PLoS One, 9, e110392. doi: 10.1371/journal.pone.0110392
  Balsanelli, E., Tuleski, T. R., de Baura, V. A., Yates, M. G., Chubatsu, L. S., de Olivera Pedrosa, F., … Monteiro, R. A. (2013). Maize root lectins mediate the interaction with Herbaspirillum seropedicae via N‐Acetyl glucosamine residues of lipopolysaccharides. PLoS One, 8, e77001. doi: 10.1371/journal.pone.0077001
  DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. doi: 10.1021/ac60111a017
  Egener, T., Hurek, T., & Reinhold‐Hurek, B. (1999). Endophytic expression of nif genes of Azoarcus sp. strain BH72 in rice roots. Molecular Plant‐Microbe Interactions, 12, 813–819. doi: 10.1094/MPMI.1999.12.9.813
  Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A., & Olsen, G. J. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology, 74, 2461–2470. doi: 10.1128/AEM.02272‐07
  Klassen, G., Pedrosa, F. O., Souza, E. M., Funayama, S., & Rigo, L. U. (1997). Effect of nitrogen compounds on nitrogenase activity in Herbaspirillum seropedicae SmR1. Canadian Journal of Microbiology, 43, 887–891. doi: 10.1139/m97‐129
  Kloepper, J. W., & Beauchamp, C. J. (1992). A review of issues related to measuring colonization of plant roots by bacteria. Canadian Journal of Microbiology, 38, 1219–1232. doi: 10.1139/m92‐202
  Valdameri, G., Kokot, T. B., Pedrosa, F. O., & Souza, E. M. (2015). Rapid quantification of rice root‐associated bacteria by flow cytometry. Letters in Applied Microbiology, 60, 237–241. doi: 10.1111/lam.12351
  Westphal, O., & Jann, K. (1965). Bacterial lipopolysaccharides: Extraction with phenol‐water and further applications of the procedure. Methodology Carbohydrate Chemistry, 5, 83–91.
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