Plant Growth–Promoting Bacteria (PGPB): Isolation and Screening of PGP Activities

Adriana Ambrosini1, Luciane M. P. Passaglia1

1 Genetics Department, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/pb.20054
Online Posting Date:  September, 2017
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Abstract

Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Plant growth–promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco‐friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that are able to improve plant development and productivity. In this unit, we present protocols to isolate bacteria from soil and plant roots (“putative” diazotrophic and endospore‐forming bacteria), as well to evaluate some of their beneficial characteristics for the promotion of plant growth (e.g., nitrogen fixation, production of indolic compounds and siderophores, phosphate solubilization, and 1‐aminocyclopropane‐1‐carboxylate deaminase activity). © 2017 by John Wiley & Sons, Inc.

Keywords: diazotrophs; endospore‐forming; PGPB; roots; soil

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing Plant Roots for the Isolation of “Putative” Endophytic Bacteria
  • Basic Protocol 2: Preparing the Root Rhizospheric Soil for Bacterial Isolation
  • Basic Protocol 3: Isolation of “Putative” Diazotrophic Bacteria
  • Basic Protocol 4: Isolation of Endospore‐Forming “Putative” Diazotrophic Bacteria
  • Basic Protocol 5: Acetylene Reduction Assay (ARA)
  • Basic Protocol 6: Indolic Compound Production Assay
  • Basic Protocol 7: Tricalcium Phosphate Solubilization Assay
  • Basic Protocol 8: Siderophore Production Assay
  • Basic Protocol 9: ACC Deaminase Activity Indirect Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing Plant Roots for the Isolation of “Putative” Endophytic Bacteria

  Materials
  • Plants of interest
  • 70% ethanol
  • Household bleach (sodium hypochlorite) diluted in sterile water (1:1)
  • King B solid medium (see recipe)
  • 0.85% NaCl solution (saline)
  • Paper towels
  • Scalpel
  • 1‐liter glass beaker
  • Glass stir rod
  • Filter paper
  • Glass petri dishes
  • Laminar flow hood
  • 28°C to 30°C incubator
  • 500‐ml Erlenmeyer flask
  • 4°C incubator with shaking at 160 to 180 rpm
  • Glass test tubes (20 mm diameter × 150 mm length)
NOTE: This protocol should be performed under axenic conditions and using sterile technique in a disinfected laminar flow hood.

Basic Protocol 2: Preparing the Root Rhizospheric Soil for Bacterial Isolation

  Materials
  • Bacterial suspension of interest (see Basic Protocols protocol 11 and protocol 22)
  • NFb, LGI, or LGI‐P semi‐solid medium (see recipe)
  • Agar plates containing NFb, LGI, or LGI‐P solid medium (see recipe)
  • King B medium (see recipe)
  • Glycerol
  • 10‐ml Bijou or penicillin vials with cotton or foam plugs
  • 28°C to 30°C incubator with shaking capabilities
  • Platinum loop
  • Glass test tubes (20 mm diameter × 150 mm length)
  • Centrifuge
  • 2‐ml cryogenic tubes with screw cap
  • Additional reagents and equipment for Gram staining and verification of culture purity (see Moyes, Reynolds, & Breakwell, )
NOTE: All materials should be sterile, and the protocol should be performed in a laminar flow hood.

Basic Protocol 3: Isolation of “Putative” Diazotrophic Bacteria

  Materials
  • Bacterial isolates to be tested (see Basic Protocols protocol 33 and protocol 44)
  • Positive control strain (nitrogen‐fixing bacterium; e.g., strains belonging to Azospirillum brasilense or Gluconacetobacter diazotrophicus species)
  • NFb, LGI, LGI‐P, or nitrogen‐free thiamine‐biotin semi‐solid medium (see recipe)
  • King B medium (see recipe)
  • Ultra‐pure acetylene gas
  • 0.2 N NaOH
  • Bradford's reagent (Bradford, )
  • Bovine serum albumin (BSA) standards
  • Glass test tubes (20 mm diameter × 150 mm length)
  • Spectrophotometer
  • 10‐ml Bijou or penicillin vials
  • Cotton plugs
  • Rubber stoppers
  • 28°C to 30°C incubator
  • Gas chromatograph equipped with a flame ionization detector (FID; 110°C) and Col‐Elite‐Alumina (50 m × 0.53 mm internal diameter × 10 µm) or Porapak N (100 to 200 mesh; 2 m × 1 mm diameter) column connected to a chromatography data computer system
  • 1‐ml sterile plastic syringe
  • 10‐µl to 500‐µl gas‐tight syringe
  • Water bath
  • 96‐well plate and plate reader

Basic Protocol 4: Isolation of Endospore‐Forming “Putative” Diazotrophic Bacteria

  Materials
  • Bacterial isolates to be tested (see Basic Protocols protocol 33 and protocol 44)
  • King B medium (see recipe) supplemented with 2.5 mM tryptophan
  • Salkowski's reagent (see recipe)
  • 3‐indoleacetic acid (IAA; 99%) standards
  • Glass test‐tubes (20 mm diameter × 150 mm length)
  • 28°C to 30°C incubator with shaking
  • Spectrophotometer
  • Cuvettes or 96‐well plates
  • 1.5‐ml and 2.0‐ml microcentrifuge tubes
  • Microcentrifuge
CAUTION: Use care in preparing and handling culture medium supplemented with 2.5 mM tryptophan and Salkowski's reagent.

Basic Protocol 5: Acetylene Reduction Assay (ARA)

  Materials
  • Bacterial isolates to be tested (see Basic Protocols protocol 33 and protocol 44)
  • King B medium (see recipe)
  • Agar plates containing GY/tricalcium phosphate solid medium (see recipe)
  • Glass test tubes (20 mm diameter × 150 mm length)
  • 28°C to 30°C incubator with shaking
CAUTION: Use care in preparing and handling GY/tricalcium phosphate agar plates.

Basic Protocol 6: Indolic Compound Production Assay

  Materials
  • Bacterial isolates to be tested (see Basic Protocols protocol 33 and protocol 44)
  • King B medium (see recipe)
  • Agar plates containing King B five‐fold diluted/CAS solid medium (see recipe)
  • Glass test tubes (20 mm diameter × 150 mm length)
  • 28°C to 30°C incubator with shaking
CAUTION: Use care in preparing and handling King B five‐fold diluted/CAS agar plates.

Basic Protocol 7: Tricalcium Phosphate Solubilization Assay

  Materials
  • Bacterial isolates to be tested (see Basic Protocols protocol 33 and protocol 44)
  • King B medium (see recipe)
  • 0.85% NaCl (saline)
  • Agar plates containing DF salts solid medium with and without ACC (see recipe)
  • Glass test tubes (20 mm diameter × 150 mm length)
  • 28°C to 30°C incubator with variable shaking
  • 1.5‐ml or 2.0‐ml microcentrifuge tubes
  • Microcentrifuge
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Figures

Videos

Literature Cited

Literature Cited
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  Penrose, D. M., & Glick, B. R. (2003). Methods for isolating and characterizing ACC deaminase‐containing plant growth‐promoting rhizobacteria. Physiologia Plantarum, 118, 10–15. doi: 10.1034/j.1399‐3054.2003.00086.x.
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