Detection and Isolation of Epichloë Species, Fungal Endophytes of Grasses

Simona Florea1, Christopher L. Schardl1, Walter Hollin1

1 University of Kentucky, Department of Plant Pathology, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 19A.1
DOI:  10.1002/9780471729259.mc19a01s38
Online Posting Date:  August, 2015
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Abstract

Epichloë species (including former Neotyphodium species) are endophytic fungi that significantly affect fitness of cool‐season grass hosts, potentially by increasing nutrient uptake and resistance to drought, parasitism and herbivory. Epichloë species are obligately biotrophic, living in the intercellular spaces of their plant hosts, and spreading systemically throughout host aerial tissues. The reproduction of Epichloë species is versatile; some strains have both sexual and asexual modes of reproduction, but others are restricted to one or the other mode. The reproduction mode determines the dissemination mechanism, and the asexual species most important to agriculture are strictly seed‐borne, cause no signs or symptoms, and are undetectable except by specialized microscopic, molecular or antigenic procedures. These procedures can be used to identify endophytes in a variety of plant tissues. Similar protocols can be modified to detect less common symbionts, such as the penicillate “p‐endophytes,” when they occur by themselves or together with Epichloë species. © 2015 by John Wiley & Sons, Inc.

Keywords: symbiosis; isolation; detection; aniline‐blue; tissue staining; serology; multiplex PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Fungal Endophytes from Cool Season Grass Plants by using Aniline Blue‐Lactic Acid Solution
  • Alternate Protocol 1: Detection of Fungal Endophytes from Grass Tissue using Aniline Blue‐Lactic Acid Solution Without Peeling Epidermal Tissue
  • Alternate Protocol 2: Detection of Fungal Endophytes from Grass Seeds Using Aniline Blue‐Lactic Acid
  • Alternate Protocol 3: Detection of Fungal Endophytes from Grass Seeds with Rose Bengal Stain
  • Basic Protocol 2: Detection of Cool Season Grass Fungal Endophytes from Plant Tissue using Tissue Print‐Immunoblot (TPIB)
  • Support Protocol 1: Protein Extraction From Epichloë Coenophiala for Antiserum Production
  • Basic Protocol 3: Direct Detection of Epichloë Species in Plant Tissue by PCR
  • Alternate Protocol 4: Multiplex PCR for Detection of Epichloë Species in Plant Tissue
  • Basic Protocol 4: Isolation of Fungal Endophytes from Cool‐Season Grass Tissue
  • Alternate Protocol 5: Isolation of Fungal Endophytes from Cool‐Season Grass Seeds
  • Alternate Protocol 6: Ascospore Isolation of Epichloë Fungal Endophytes from Cool‐Season Grass
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of Fungal Endophytes from Cool Season Grass Plants by using Aniline Blue‐Lactic Acid Solution

  Materials
  • Grass tissue (tiller or leaf blade)
  • Aniline blue‐lactic acid solution (see recipe)
  • Purified water, optional
  • Scalpel
  • Tweezer
  • Glass slides
  • Alcohol or gas (Bunsen) burner
  • Coverslips
  • Tissue paper
  • Light or compound microscope

Alternate Protocol 1: Detection of Fungal Endophytes from Grass Tissue using Aniline Blue‐Lactic Acid Solution Without Peeling Epidermal Tissue

  Additional Materials (also see protocol 1)
  • 70% ethanol [mix 70 ml 200 Proof (100%) pure ethanol with 30 ml pure water]
  • Carnoy's solution (see recipe)
  • Aniline blue ethanol‐lactic acid solution (see recipe)
  • 25‐ml glass Corex tubes with screw caps (Corning)

Alternate Protocol 2: Detection of Fungal Endophytes from Grass Seeds Using Aniline Blue‐Lactic Acid

  Additional Materials (also see protocol 1)
  • Grass seeds
  • 5% NaOH
  • 50‐ml plastic Corning tubes with screw caps

Alternate Protocol 3: Detection of Fungal Endophytes from Grass Seeds with Rose Bengal Stain

  Additional Materials (also see protocol 1)
  • Standard 0.5 % Rose Bengal solution for fresh tissue (see recipe).
  • Alkaline solution: 2.5% NaOH for air‐dried samples and seeds (see recipe)

Basic Protocol 2: Detection of Cool Season Grass Fungal Endophytes from Plant Tissue using Tissue Print‐Immunoblot (TPIB)

  Materials
  • Samples
  • Blocking buffer (see recipe; prepared day before membrane development)
  • Rabbit antiserum
  • Anti‐rabbit IgG‐alkaline phosphatase conjugate (Sigma)
  • TPIB developing buffer (see recipe)
  • Nitrocellulose membrane (0.45 μm; Bio‐Rad Laboratories)
  • 10 ×10‐cm Pyrex container
  • Gloves
  • Black pen
  • Plastic/glass board
  • 3‐mm chromatography filter paper
  • Scalpel
  • Tweezers
  • Wax paper
  • Ceramic pestle (medium‐sized head)
  • Protective plastic sheets
  • Plastic bags
  • Shaker
  • Plastic wrap
  • Paper towels

Support Protocol 1: Protein Extraction From Epichloë Coenophiala for Antiserum Production

  Materials
  • Fungus, Epichloë coenophiala (see protocol 9)
  • Agar plates
  • PDB (Difco, cat. no. DF0549‐17‐9)
  • Phosphate‐buffered saline (PBS) minus K (see recipe)
  • Ice
  • Polyethylene glycol 8000 (PEG), 10% (w/v)
  • Sodium dodecyl sulfate (SDS)
  • Acetone
  • 300‐ml flasks
  • Orbital shaker
  • Vacuum filtration apparatus
  • Buchner funnel
  • Whatman filter paper
  • Polytron PT20 homogenizer (or similar)
  • 250‐ml beakers
  • Centrifuge
  • Spectrophotometer

Basic Protocol 3: Direct Detection of Epichloë Species in Plant Tissue by PCR

  Materials
  • Plant tissue
  • DNeasy 96 Plant kit (Qiagen)
  • ZR Fungal/Bacterial DNA MiniPrep (Epigenetics)
  • 1× PCR reaction buffer containing MgCl 2 (Applied Biosystems‐Life Technologies)
  • Primers (forward and reverse; see Table 19.1.1)
  • dATP, dCTP, dGTP and dTTP (100 mM each dNTP)
  • AmpliTaq Gold DNA Polymerase with buffer (Applied Biosystems)
  • Sterile purified water
  • 1% TBE (Tris‐borate‐EDTA)
  • Agarose
  • 1‐kb ladder DNA marker
  • Ethidium bromide or GelGreen for gel staining
  • Loading buffer (loading dye)
  • Scalpel
  • 1.5‐ml microcentrifuge tubes
  • PCR 96‐well plates
  • Gloves
  • Geno/Grinder 2000 or similar instrument
  • 3.2‐mm stainless steel beads
  • Pellet pestles
  • 30–50‐μl PCR tubes with cap
  • Thermal cycler
  • Electrophoresis chamber and power supply (Bio‐Rad) or similar
  • Gel casting trays and sample combs (Bio‐Rad) or similar
  • UV box: Gel Doc XR+ System, or similar

Alternate Protocol 4: Multiplex PCR for Detection of Epichloë Species in Plant Tissue

  Additional Materials (also see protocol 7)
  • MagAttract 96 DNA plant core kit (Qiagen)
  • GoTaq DNA Polymerase (Promega)

Basic Protocol 4: Isolation of Fungal Endophytes from Cool‐Season Grass Tissue

  Materials
  • Fresh plant material (pseudostems, leaf tissue)
  • PDA (Difco‐Fisher Scientific)
  • Penicillin G
  • Streptomycin sulfate
  • 95% Ethanol
  • 5.25% NaClO (sodium hypochlorite; Clorox bleach)
  • Sterile purified water (autoclaved)
  • 15‐ml Corning sterile screw‐cap plastic tubes
  • Beaker of 70% ethanol for sterilizing instruments
  • Alcohol or gas (Bunsen) burner
  • Biological containment/microbial transfer hood
  • 100 × 15‐mm petri dishes
  • Tweezers
  • Scalpel with blades
  • Parafilm (Bemis)
  • Incubator set to 22°C
NOTE: All the steps should be performed in a biological hood under sterile conditions. Scalpel and tweezers should be sterilized as needed, by immersion in 70% ethanol, followed by ignition of the ethanol with the burner flame to allow the ethanol to burn off.

Alternate Protocol 5: Isolation of Fungal Endophytes from Cool‐Season Grass Seeds

  Additional Materials (also see protocol 9)
  • 50% H 2SO 4
  • 50‐ml Corning sterile plastic tubes

Alternate Protocol 6: Ascospore Isolation of Epichloë Fungal Endophytes from Cool‐Season Grass

  Additional Materials (also see protocol 9)
  • Water agar plates (2% Bacto agar in water)
  • Double‐sided sticky tape (optional)
  • Microscope with standard 4×, 10× (and preferably 20×) objectives (these need to clear the stage plus a distance the thickness of the petri plate)
  • Wire transfer loop
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Figures

Videos

Literature Cited

Literature Cited
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