Soil collection methods for fungal metabarcoding

By Emily McIntyre and Aviya Naccarella.

Materials

• PVC pipes
• Saw, e.g. tennon saw
• Mitre box
• Drill
• Sand paper
• Sharpie
• Sponge
• Dish soap
• Plastic gloves
• Small, medium, and large zip log bags
• Bleach
• Tub to sterilise soil cores
• Mallet
• Wooden block
• Spoon
• Peg
• Antiseptic wipes to sterilise spoon/peg in field

1. Making soil cores

These methods provide instructions to create soil cores that will collect a 10 cm x 5 cm cylinder of soil.

1. Purchase PVC pipe with appropriate dimensions, e.g. 50 mm x 1 m
2. Fig 1 provides a guide of how to cut up the pipe to make soil cores. Mark out where to cut. Use a saw, e.g. tennon saw, to cut the pipe. A mitre box can be helpful to make accurate cuts at the required angles.
3. Once the cores are cut to shape, use a drill to create holes in the sides of the top of each core (see Fig 1). During soil sampling, once core is pushed into the ground, a peg can be inserted into the holes and used to lift the core out of the ground if the core is stuck.
4. Use sand paper to sand down rough edges at the top, bottom, and drill holes. The smoother this surface is, the less dirt that sticks to it, and the easier it is to clean.
5. Use a sharpie to mark the top 10cm line (see Fig 1). This is useful to see how far the soil core needs to be pushed into the soil.

Figure 1: How to make soil cores out of PVC pipes.

2. Cleaning the soil cores

Sterilise all soil cores before use.

1. Scrub and wash soil cores (a heavy sponge/chux is helpful for persistent soil). Hot water and dishwashing liquid is helpful for cleaning thick/clay soils from the core.
2. Put plastic gloves on.
3. Mix up 10% bleach solution. For 1L of solution, use 131 mL of 4% bleach + 868 mL of water.
4. Fully immerse soil cores in the tub of bleach solution for 10 mins.
5. Rinse off residue.
6. Leave to air dry.
7. Seal individually in medium zip loc collection bags with 2x plastic gloves.

3. Soil sampling

The following provides information on collecting soil samples for DNA metabarcoding and soil chemistry analysis. Soil samples should be collected at every 10 m along a 60 m transect (0, 10, 20, 30, 40, 50 m), totalling to 7 samples per site. Each sample comprises 4 sub-samples (5 cm diameter x 10 cm deep) collected with a soil core (Fig 2). The 7 soil samples will be pooled at the site level for soil chemistry analysis. To collect each sample:

1. Use sharpie to label a large and small zip lock bag.
2. Put gloves on.
3. Brush leaf litter off the soil.
4. Open zip lock bag containing sterilised soil core.
5. Use a wooden block and mallet to knock the core into the ground until the 10 cm marker line is flush with the ground.
6. Remove the soil core, if necessary using a sterilised peg through the holes to lift it out of the ground.
7. Dump the soil into the plastic bag which previously held the soil core. If soil does not easily come out, use a sterilised spoon to push it or else knock it with the wooden block to loosen it.
8. Repeat steps 5-7 for each sub sample. Sub-samples should be taken within a 1m radius, if possible within 50cm above, below, and to the sides of the sampling location (Fig 2).
9. Brush leaf litter back onto uncovered soil once the samples have been collected.
10. Rub the bag containing 4 subsamples together to thoroughly mix the soil.
11. Remove ~2 tbsp of soil, using the spoon, and place in little zip lock bag. Refrigerate until freezing as soon as possible.

12. Repeat for each of the 7 samples.

13. Mix soil from the 7 large bags together in a single bag. Rub bag together to mix soil thoroughly. Empty approx. 300 g into a large paper bag to be used for soil chemistry analysis. Place in tub with lid with a layer of silica gel beads. Keep as dry as possible and heat in oven at 40 degrees until dry as soon as possible. Sieve to 2mm. Store paper bags in tub with silica beads.

Figure 2 . Soil sample made of 4 sub-samples (5 cm diameter x 10 cm deep) collected with a soil core.