Workshop: Methods for quantifying AMF in roots

WORKSHOP: METHODS FOR QUANTIFYING AMF IN ROOTS

Taraneh Emam Grad Group in Ecology UC Davis M a r c h 8 t h , 2 01 3

OUTLINE       

Intro Sample collection and preservation Staining methods Preserving slides Staining Questions? Quantifying AMF Quantifying AMF Questions?

BREAK: reconvene in soil lab, 1149 PES  Microscope demonstration

INTRODUCTION

When you are examining…  Nutrients, water, plant stress  Soil food webs  Agricultural practices  Soil structure or erosion  Restoration ecology  Effects of non-native plants

 When might it not be interesting to examine AMF?  Plant taxa do not form associations with AMF  Plants grown in sterilized greenhouse soil  Plants grown only in high-nutrient soils

M. Brundrett, mycorrhizas.info

 When might it be interesting to examine arbuscular mycorrhizal fungi (AMF)? A few examples:

INTRODUCTION  Many factors af fect colonization levels of AMF:       

Plant species/morphology AMF species/morphology Plant nutrient status Plant phenology/plant age Root age and length Abiotic conditions AMF propagule availability

 Percent colonization ≠ biomass of AMF in the soil  Despite this, colonization level “is a reliable indicator of benefits to host plants” in terms of plant biomass and P content (Treseder 2012)

SAMPLE COLLECTION AND PRESERVATION    

In field, collect from 0–10 or 15cm layer of soil Store in refrigeration up to ~10 days Wash thoroughly as soon as possible Handle roots gently so as not to crush plant cells and AMF structures

Long term storage:  Place in 70% ethanol (or ethyl rubbing alcohol) in fridge centrifuge tubes 

SAMPLE CONTAINERS Histology cassettes (and inserts) • May pop open if not properly closed Centrifuge tubes: • Use for long-term root storage • Can poke holes and submerge in chemicals for cold staining (but not hot)

CellSafe insert

• Label samples with a nonspecific code – so that later, observer won’t know which treatment a given sample received • Separate by plant species as different species may require different methods

STAINING METHODS: OPTIONS HOT METHOD  Quicker (1-2 hours per batch)  More dangerous for roots (and you)  Not recommended for delicate roots COLD METHOD  Takes ≤ I week  Harder to mess up, gentler for roots  Easier to process large quantities STAIN OPTIONS  Ink and Vinegar (not recommended for cold staining), Chlorazol Black E (CBE), Trypan Blue, Aniline Blue, Acid Fuchsin

STAINING METHODS: MY PROTOCOL  I currently use a hot/cold method combination  Developed for native CA grass roots; adjustments may be needed SUPPLIES:  Hotplate or Bunsen burner  Large beakers (~3)  Sieve  Histology cassettes or other sample container  Safety gear and fume hood  Timer REAGENTS:  KOH: 10% (hot) or 20% (cold) and 0.5-1% w/v  Household vinegar  Quink Blue-Black ink  Mounting media

STAINING METHODS: MY PROTOCOL  Check roots frequently during clearing to determine appropriate clearing time

1-3 days 20% KOH Check!

STAINING METHODS: MY PROTOCOL  Drain KOH into new beaker, catching samples in sieve  Can reuse KOH 2-3 times or until it becomes tea colored Then rinse well with DI water

DI water 20% KOH

STAINING METHODS: MY PROTOCOL  Soak in household vinegar for several hours to acidify roots (do not rinse afterwards) Vinegar

   

5% ink in vinegar

Heat ink/vinegar mixture Remove samples from vinegar and drain excess vinegar Place samples in hot ink for 3-5 minutes Rinse well in DI water

STAINING METHODS: MY PROTOCOL  Place roots in cold 0.5 - 1% KOH beaker, swirl for 3 seconds  IMMEDIATELY drain roots and rinse well

QUICK!

DI water 0.5-1% KOH

PREPARING SLIDES Common mountants:  Polyvinyl Lacto-Glycerol (PVLG)  100mL lactic acid, 100mL DI water, 10mL glycerol and 16g polyvinyl alcohol powder  Dissolve together in a hot water bath (70-80 deg. C) for ~4hrs.  I’ve found that the stain begins to fade after 1-2 years in PVLG

 Corn syrup  Preserves roots well, but is sticky  Non-toxic, don’t need a chem lab  Can remove excess with rubbing alcohol

 Plain (50-100%?) glycerol  Haven’t tried this yet, will report back.

And it smells like vanilla!

PREPARING SLIDES SUPPLIES  Mountant  Slides  Long coverslips (22x50mm)  Forceps  Toothpick or other applicator  Rubbing alcohol  Weights for unruly roots  I use heavy ball bearings in centrifuge tubes

 Patience!

PREPARING SLIDES  Line up root segments on slides using forcepts  attempt parallel & non-overlapping

    

Let dry out for a few minutes Apply many small dots of mountant Gently place coverslip and press out bubbles For larger roots, may need to weight coverslip while drying Air dry and clean of f excess mountant

NOW THE FUN PART… SUPPLIES  Compound light microscope  10 or 20x, and 40x objectives recommended  Eyepiece cross-hatch reticle (or pointer)  Multichannel counter

 Can first view roots with 10 or 20x  But often necessary to use 40x to identify structures Make sure to take eye breaks.

CHARACTERISTICS OF AMF  Aseptate: no cell walls dividing hypal cells  “Blobbiness”: AMF tend to look much thicker and less uniform than other root fungi, and can appear knobby  Branching of hyphae tends to occur at angles of about 60 degrees  Arbuscules are always a sign of mycorrhizal fungi, but are often hard to see clearly

M. Brundrett, mycorrhizas.info

WHAT’S THAT IN MY MICROSCOPE? AMF STRUCTURES absorptive hyphae

*These two are good images of AMF

spores

arbuscule

hyphae All images photographed at 400x, except for top image this page (at 200x).

WHAT’S THAT IN MY MICROSCOPE? AMF STRUCTURES vesicles (Acaulospora?) hyphae

vesicles (Glomus?)

~2 year old slide with corn syrup with PVLG

vesicle

WHAT’S THAT IN MY MICROSCOPE? AMF STRUCTURES Fine hyphae 

hyphae

vesicles

WHAT’S THAT IN MY MICROSCOPE? FIELD ROOT MYSTERIES Things that are NOT mycorrhizal:

Polymyxa? Note “grape cluster” shape

??? 

Chytrid sporangia and zoospores. Note “cat eye” and hexagon shapes.

WHAT’S THAT IN MY MICROSCOPE? Things that are NOT mycorrhizal:

Dark septate endophytes: Note the septae, and dark staining Blue is AMF hyphae (PVLG mountant)

Empty root with no fungi 

Other things that may stain blue: • Root vascular tissue • Root meristem tissue • Uncleared plant organelles

QUANTIFYING AMF     

Magnified intersections method (McGonigle 1990) Use cross-hatch reticle in eyepiece of microscope Move back and forth across slide Each time cross-hatch hits a root = intersection Evaluate intercept: AMF structures present?

Turn eyepiece so that one line is perpendicular to the root

QUANTIFYING AMF

This shows intersection of hyphae

This shows intersection of a vesicle (includes hyphae)

No AMF structures intercepted

QUANTIFYING AMF  Each intersection gets only one designation: No AMF Hyphae only Vesicles + Hyphae Arbuscules + Hyphae Vesicles + Arbuscules + Hyphae

 Consistency is key!  More intersections = higher precision  At least 100 intersections recommended

x x xx

x x x x x

x x xx x x

x x x x xx

x x x x x x

x x x x x x

Move slide back and forth to cover roots evenly until you reach 100 intersections

% arbuscular colonization

    

x

McGonigle et al. 1990

THANKS TO

Shannon Schechter, Christine Hawkes, and Alison Berry for advice and training related to AMF

methods.

REFERENCES Azul A.M., Ramos V., Pato A., Azenha M. & Freitas H. 2008. Mycorrhizal types in the Mediterranean Basin: safety teaching and training. J. Biol. Educ., 42, 130-137. Brundrett MC. 2008. Mycorrhizal Associations: The Web Resource. McGonigle, T. P., M. H. Miller, D. G. Evans, G. L. Fairchild, and J. A. Swan. 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115:495–501. Robertson, G. P., Coleman, D. C., Bledsoe, C. S., & Sollins, P. (Eds.). 1999. Standard soil methods for longterm ecological research (Vol. 2). Oxford University Press, USA. Smith S.E. & Read D.J. 2008. Mycorrhizal symbiosis. Academic Pr. Treseder, K. K. 2012. Percent mycorrhizal root length is a reliable indicator of benefits to host plants: Results f r o m a m e t a - a n a l y s i s . M e e t i n g A b s t r a c t s , 9 7 th A n n u a l M e e t i n g o f t h e E c o l o g i c a l S o c i e t y o f A m e r i c a . Vierheilig, H., A. Coughlan, U. Wyss, and Y. Piche. 1998. Ink and vinegar, a simple staining technique for arbuscular-mycorrhizal fungi. Applied and environmental microbiology 64:5004–7. Vierheilig, H., Schweiger, P., & Brundrett, M. 2005. An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiologia Plantarum 125: 393-404. Wang B. & Qiu Y.L. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza, 16, 299-363.