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 sampling 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 below for overall sampling. In total, you’ll have 9 samples from the same plot in the plastic bag. The grid can be resized to for studies sampling smaller locations that cannot fit within a 30m x 30m grid, although the prefered size is 30m x 30m.
  2. Fill in the metadata sheet.
    A. Note country of sampling, name of expedition leader, and collaborators in sampling.
    B. Classify the landuse of the site (e.g. small-farm, non-agricultural, urban, burned).
    C. Record GPS coordinates (WGS1984) using 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 know 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 GPS device.
  5. Collect the first sample from this central point (5 cm diam. to 10 cm depth) into the large plastic bag. Remove large roots and rocks by hand if present. Write the name of the sampling location GPS coordinates, Date, and Collector in marker on each bag. Take a photo of the bag.
  6. In each cardinal direction from the central point, measure 15 m and collect other 4 samples after removing the loose litter. From the projected corners of the four samples, collect other 4 samples after removing the loose litter.
  7. Rub the bag outside 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. Take a subsample of 10-20 grams into the smaller bag or plastic tube and store (e.g. put in backpack). Add a clean tea bag with 10-20 gr of silica gel to the soil bag. If possible keep refrigerated with ice or cold packs until you can air dry it. Do not explicitly freeze as we want to avoid the impact of freezing and thawing multiple times.
  9. Navigate to your next sampling site and repeat steps 1-2. At new location confirm you are at least 1 km from your previous sampling location, you can check this with your GPS.
  10. Perform a soil wash 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. Send samples to -80C storage as soon as all samples have been collected across all sites.