Sampling and preserving mycorrhizal roots

Camille Truong, Guillermo Bueno and Adriana Corrales, August 2022.

This protocol described how to sample plant roots for mycorrhizal studies, such as % colonization and DNA profiling of root-associated fungi.

Interested in collecting roots? Please reach out.

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Field sampling

• Sample 3-5 individuals of the species of interest in each population.

• Record the date, location, canopy plants (putative mycorrhizal hosts) and basic habitat information (exposure, slope).

  If you can, record as much information as possible about the type of vegetation and any soil characteristic of the site, such as soil depth, pH, moisture, etc.

• Using a hand rack, excavate two lateral roots from the trunk until fine roots that are clearly connected to the tree are found.

  In the case of seedlings or smaller plants, carefully excavate the entire root system without damaging the fine roots.

• Store 2-3 root branches in a Ziplock bag and annotate on which horizon the roots were growing (organic or mineral soil).

• Keep samples refrigerated with icepacks in a cooler until processing.

Excavation of fine mycorrhizal roots:

Processing in the lab, field station or camp

• Clean roots with tap water and put them in a Petri dish.

• Cut fine roots into >20 pieces of ca. 1-2 cm long and check for ectomycorrhizal colonization (under a stereomicroscope if available).

• For DNA, rinse >10 pieces with distilled water, choosing the pieces that look more colonized, and store in an Eppendorf tube with a DNA buffer (such as CTAB); store at -20°C until DNA extraction.

  In DNA buffer, the samples will keep well at room temperature for several days/weeks.

Cleaning and sorting out roots for DNA in the field:

Terminal root colonized by ectomycorrhizal fungi (ECM root tip):

• To preserve roots for staining and anatomical observations you have two options:

  • If possible, save >10 root pieces in 95% ethanol in a Falcon tube and store at 5ºC.

    Room temperature is ok for several days/weeks but do not freeze.

  • Alternatively, dry root pieces in small envelopes or tea bags with silica gel in a sealed Tupperware, in a dehydrator (below 50°), or even air-drying them in the sun.

Preservation of dry roots for anatomical observations: