Wheat root exudate harvesting

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Wheat root exudates and the recruitment of root microbes

Plant roots are nutrient rich environments for soil microbes which form root-associated microbial communities. These communities make up the root microbiome consisting of the endosphere (the space inside of the root), the rhizosphere (the soil that is directly influenced by the root) and the bulk soil (any soil exterior to the root rhizosphere).

16S rRNA gene sequencing has shown us that there is lower bacterial species diversity in the endosphere of roots compared to the rhizosphere and bulk soil (Lundberg et al, 2012; Prudence et al, 2012). This demonstrates that some bacteria have developed adaptations that allows them to acquire this niche; it is hypothesised that this is either through outcompeting other bacteria or a mechanism of protrusion. Some initial studies have found that plants grown side by side in the same environment recruit different microbes which suggest that plants can select for specific microbes which benefit them (Smalla, et al. 2001; Reinhold-Hurek et al., 2015). One factor which is being explored is the role of plant exudates on this selection.

Exudates are metabolites excreted by plants into the rhizosphere which allows plants to support healthy growth, for example to break down nutrients in a more readily accessible form and to signal PGP soil microbes (Vives-Peris, 2019). This protocol outlines how to extract root exudates.

You will need:

Preparation of growth tubes

  • 50ml falcon/centrifuge tubes (1 tube = 1 plant)
  • Electric hand drill
  • 10mm drill bit
  • 2ml microcentrifuge tubes
  • Dog nail cutters
  • Vermiculite (washed and autoclaved)

Growing of wheat plants

  • Wheat seeds
  • 70% ethanol
  • 3% bleach
  • dH2O
  • Sterile universal tube
  • Sterile tweezers
  • Water agar plates
  • 1/2 MS0 liquid media

Exudate harvesting

  • 2 1L duran bottles
  • 0.22 micron sterile Büchner filter/bottletop vaccum filter funnel
  • Vacuum pump
  • 50ml falcon/centrifuge tubes
  • Syringe needle or sharp pointed item
  • -80°C freezer
  • Freeze drier
  • dH2O
  • 4ml HPLC vile

Protocol:

Week 1, Day 1

Preparation of growth tubes

  1. Prepare as many tubes as plants required (1 batch of 50 tubes ~ 500 mg final exudate).
  2. Drill a 10mm hole in each tube and rinse with water to remove any plastic debris.
  3. Using the dog nail cutters, cut the end of a 2ml microcentrifuge tube to create a ~0.5cm opening. Loosely fill the tube with the prepared vermiculite and close the lid.
  4. Push the microcentrifuge tube into the hole of the 50ml falcon.
  5. Autoclave.

Growing of wheat plants

  1. Sterilise the wheat seeds.
  2. Spreadingly put the seeds onto a water agar plate and place into the fridge.

Week 1, Day 3

Growing of wheat plants

  1. Remove the water agar plates from the fridge and leave on the bench.

Week 1, Day 4

Growing of wheat plants

  1. Fill the falcon tubes with ~45ml 1/2 MS0 media.
  2. When the seeds have sprouted two roots of ~0.5-1cm long, transfer the seed to the vermiculite tube so that the roots are pointing downwards into the vermiculite.
  3. Keep the growth tubes in a controlled environment room (temp 24C, light 18h)

Week 2-4

Growing of wheat plants

  1. Keep the growth tubes topped up with 1/2 MS0 media.

Week 4, day 4

Growing of wheat plants

  1. Pour away the 1/2 MS0 and rinse the roots and tube with water (to remove any remaining salts).
  2. Fill the growth tubes with ~45ml fresh water (this will be the media which the exudates will be extracted from).
  3. Keep the growth tubes in a controlled environment room.

Week 5

Growing of wheat plants

  1. Keep the growth tubes topped up with sterile water.

Week 5, day 4

Exudate harvesting

  1. Pour the water from the growth tubes into a clean Duran bottle.
  2. Attach a sterile 0.22 micron Büchner filter to another sterile Duran bottle and fix to a vacuum pump.
  3. Pour the exudate solution into the Büchner filter and turn on the vacuum pump.
  4. Separate the filtered exudate solution into clean 50ml falcon tubes (about 25-30ml each) and freeze at -80°C.

Week 5, day 5

Exudate harvesting

  1. Pierce a few holes in the lids of the frozen 50ml falcon tubes.
  2. Place the tubes into a freeze drier and leave over the weekend.

Week 6, day 1

Exudate harvesting

  1. The water should have evaporated and be left with some fluffy/web-like exudate.
  2. Resuspend the exudate in minimal sterile water so that it fits into a pre weighed (without lid) 4ml HPLC vile.
  3. Dry down your sample again (on a freeze drier or alternatively a Genevac) and weigh.
  4. Your sample can be resuspended at a known concentration. It is now ready for use.

References:

Lundberg, D., Lebeis, S., Paredes, S., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., Rio, T., Edgar, R., Eickhorst, T., Ley, R., Hugenholtz, P., Tringe, S. and Dangl, J., 2012. Defining the core Arabidopsis thaliana root microbiome. Nature, 488(7409), pp.86-90.

Prudence, S., Newitt†, J., Worsley, S., Macey, M., Murrell, J., Lehtovirta-Morley, L. and Hutchings, M., 2021. Soil, senescence and exudate utilisation: characterisation of the Paragon var. spring bread wheat root microbiome. Environmental Microbiome, 16(1).

Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.

References Lundberg, D., Lebeis, S., Paredes, S., Yourstone, S., Gehring, J., Malfatti, S., Tremblay, J., Engelbrektson, A., Kunin, V., Rio, T., Edgar, R., Eickhorst, T., Ley, R., Hugenholtz, P., Tringe, S. and Dangl, J., 2012. Defining the core Arabidopsis thaliana root microbiome. Nature, 488(7409), pp.86-90.

Prudence, S., Newitt†, J., Worsley, S., Macey, M., Murrell, J., Lehtovirta-Morley, L. and Hutchings, M., 2021. Soil, senescence and exudate utilisation: characterisation of the Paragon var. spring bread wheat root microbiome. Environmental Microbiome, 16(1).

Reinhold-Hurek, B., Bünger, W., Burbano, C., Sabale, M. and Hurek, T., 2015. Roots Shaping Their Microbiome: Global Hotspots for Microbial Activity. Annual Review of Phytopathology, 53(1), pp.403-424.

Smalla, K., Wieland, G., Buchner, A., Zock, A., Parzy, J., Kaiser, S., Roskot, N., Heuer, H. and Berg, G., 2001. Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed. Applied and Environmental Microbiology, 67(10), pp.4742-4751.

Vives-Peris, V., de Ollas, C., Gómez-Cadenas, A. and Pérez-Clemente, R., 2019. Root exudates: from plant to rhizosphere and beyond. Plant Cell Reports, 39(1), pp.3-17.