Plant Microbiome Identification and Characterization

Sarah L. Lebeis1

1 Department of Microbiology, University of Tennessee, Knoxville, Tennessee
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20048
Online Posting Date:  June, 2017
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Abstract

To fully exploit the potential of plant microbiome alterations to improve plant health, reliable methods must be used to prepare and characterize microbiome samples. The power of culture‐independent studies is that they allow the characterization of novel microbial community members, but only microbial members consistently represented between different research groups are likely to become broadly applicable treatments. The identification of plant microbiome members can be affected by several experimental stages, including design, sample preparation, nucleic acid extraction, sequencing, and analysis. The protocols described here therefore aim to highlight crucial steps that experimenters should consider before beginning a plant microbiome study. © 2017 by John Wiley & Sons, Inc.

Keywords: epiphyte; endophyte; plant microbiomes; phyllosphere; rhizosphere

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differential Harvest of Plant Microbiomes in the Laboratory
  • Basic Protocol 2: Isolation of Microbes from Surface Sterilized Plant Tissue
  • Basic Protocol 3: Tissue Preparation and Nucleic Acid Extraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Differential Harvest of Plant Microbiomes in the Laboratory

  Materials
  • Seeds
  • 70% (v/v) ethanol with 0.01% (v/v) Triton‐X‐100
  • 1% (v/v) bleach with 0.01% (v/v) Triton‐X‐100
  • Germinating plate medium (see recipe)
  • 70% and 95% ethanol
  • Natural soil from field site
  • Sterile sand
  • Phosphate buffer with 0.02% silwet (see recipe)
  • Plant growth chamber
  • Petri dishes
  • Autoclavable pots
  • Miracloth (Millipore)
  • Aluminum foil
  • Flats and domes
  • Flame resistant pan, mallet, and mesh kitchen strainer
  • Gloves
  • Metal spatula
  • Metal tweezers and scissors
  • 50‐ml conical tubes
  • 100‐μm basket cell strainers (Fisher)
  • Squeeze bottle containing distilled water
  • Centrifuge
  • Liquid nitrogen
  • Bath ultrasonicator (Branson) or Biotruptor (Diagenode)

Basic Protocol 2: Isolation of Microbes from Surface Sterilized Plant Tissue

  Materials
  • Plant tissue (see protocol 1, step 15)
  • 1% (v/v) bleach with 0.01% (v/v) Triton‐X100
  • 2.5% (w/v) sodium thiosulfate
  • Dilute bacterial agar media (e.g., PDA, R2A, 10% LB, 2% TSA, and 20% KB)
  • DNeasy UltraClean Microbial DNA Isolation Kit (Qiagen)
  • 40% to 80% (v/v) glycerol
  • Oligonucleotides (e.g., 27F and 1492R for bacteria or ITS9F and ITS4R for fungi)

Basic Protocol 3: Tissue Preparation and Nucleic Acid Extraction

  Materials
  • Plant tissue and pellets (from protocol 1)
  • Nucleic acid extraction kit, e.g., DNeasy PowerSoil Kit (Qiagen)
  • DNA quantification kit, e.g., Qubit or Pico Green (Thermo Fisher Scientific)
  • PCR primers (see Table 20.4.8000)
  • PCR Master Mix, e.g., HiFi HotStart Ready Mix (Kapa Biosystems)
  • Conical tubes (appropriate size for the volume of samples)
  • Dissecting probe, needle, or other flame‐resistant sharp object
  • Liquid nitrogen
  • Bell jar and lyophilizer/freeze drier
  • Beads of various sizes, glass, metal, ceramic, or garnet (Corning, Qiagen)
  • Mechanical disruptor, e.g., a grinding mill, Geno/Grinder (SPEX SamplePrep), or FastPrep24 (MP Biomedicals)
  • Vortex
  • Additional reagents and equipment for PCR (Kramer and Coen, ) and agarose gel electrophoresis (Voytas, )
Table 0.4.1   MaterialsPrimer Pairs for Phylogenetic Analysis of Bacterial Components of Plant Microbiomes

Primer Pair Variable region(s) Uses Strengths Weaknesses
27F‐1492R V1‐V9 Full length Sanger sequencing of isolates Accurate Bad for sample multiplexing
515F‐806R V4 454 and MiSeq (Caporaso et al., ; Lebeis, ; Lundberg et al., ; Tremblay et al., ) High microbial accuracy Few primer biases
779F‐1192R V5‐V7 454 and MiSeq (Agler et al., ; Bulgarelli et al., ) Good plant DNA exclusion Primer biases
926F‐1392R V6‐V8 454 and MiSeq (Tremblay et al., ) Captures most sequences Universal
1114F‐1392R V8‐V9 454 and MiSeq (Lundberg et al., ) Some plant DNA exclusion Primer biases against archaea

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Literature Cited

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