sampling protocol

Sampling of soil for molecular analysis of AMF and EMF DNA sequences modified from the Silva Nova/SoilBon protocol.

in field: necessary equipment
  1. GPS device
  2. Notebook with pens
  3. Transect tape
  4. Mini-grip or ZipLock plastic bags (larger and small size; gallon and quart)
  5. Metal or PVC soil corer 5 cm diameter x 10 cm length (or similar)
  6. Rubber mallet or hammer
  7. Plastic or nylon gloves
  8. Permanent marker
  9. Plastic tubes or bags (50ml)
  10. Silica gel
  11. Camera
  1. Navigate to the sampling site and place two transects at right angles to outline a 900m2 (30m x 30m) area for sampling. 9 subsamples (5 cm diameter to 10 cm depth) will be collected. Please see the figure for overall sampling scheme. In total, you will have combine  9 samples from the same plot in the plastic bag. The grid can be resized for studies sampling smaller locations that cannot fit within a 30m x 30m grid, although the preferred size is 30m x 30m.
  2. Fill in the metadata sheet.
    A. Note the country of sampling, name of the expedition leader, and collaborators.
    B. Classify the land use of the site (e.g. small farm, non-agricultural, urban, burned).
    C. Record GPS coordinates (WGS1984) using a GPS device and ideally in decimal format (e.g. -39.5818, -71.51917).
    D. Roughly estimate the number of plant species present and % coverage of trees vs grass/smaller plants. Important to document ectomycorrhizal and ericoid (Ericaceae) mycorrhizal plants. If plant names are known, write them down.
    E: Final notes: record anything else of importance not listed above.
  3. Put on gloves. Remove the litter layer (loose material) from the central point.
  4. At the first central point, record elevation and GPS coordinates using a GPS device.
  5. Collect the first sample from this central point (5 cm diam. to 10 cm depth) using the soil corer and place soil into the large plastic bag. Remove large roots and rocks by hand (or using a sieve, if preferred). Write the name of the sampling location GPS coordinates, Date, and Collector in marker on each bag. Take a photo of each bag.
  6. In each cardinal direction from the central point, measure 15 m and collect additional  samples 4 after removing the loose litter. Then complete a full square to collect the remaining 4 samples (see figure). There should be 9 subsamples in total.
  7. Rub the outside of the bag with your hands, mix the samples thoroughly but gently so that the soil becomes a homogenous mixture. Mixing should be done for at least 2 minutes to secure sufficient homogenisation.
  8. Process soil samples depending on field conditions (wet vs dry):

    For dry soils
    :  Take a subsample of 10-20 grams into the smaller bag (or tube) and add a clean tea bag with 10-20 gr of silica gel to the soil bag/tube. If preferred, keep samples refrigerated with ice or cold packs in a field cooler. If you are on a collecting trip lasting several days and don’t have access to refrigeration, you can air dry the samples or continue to replace silica gel when necessary.  Do not freeze samples in the field as we want to avoid the impact of freezing and thawing multiple times during transportation.

    For wet soils: Take a subsample of 10-20 grams into the smaller bag (or tube), keep on ice or cold packs in the field. Refrigerate at the end of the day (if possible) until you reach the final storage facility. Do not freeze samples in the field as we want to avoid the impact of freezing and thawing multiple times. If refrigeration is not possible, then use the same protocol suggested for dry soil.
  9. Navigate to your next sampling site and repeat steps 1-2. At the new location, confirm you are at least 1,2 km (suggested distance) from your previous sampling location.Verify this using your GPS device.
  10. Perform a ‘soil wash’ on the soil core to remove soil from the previous location. You do this by collecting a soil core within the sampling area and dispose of it on the ground.
  11. Repeat until all sites have been collected.
  12. As soon as possible, samples should be moved to -20 ºC or -80 ºC for storage until DNA can be extracted.