How does mycorrhizal symbiosis affect plant growth?

I tested this question by growing plants under different soil conditions.

Hypothesis:

If I add mycorrhizal fungi to the soil, this will result in colonization of plant roots and formation of mycorrhizal symbiosis. Plants colonized with mycorrhizal fungi (Myc+) will grow better than non-colonized plants (Myc-).

Mycorrhizal Symbiosis (Latin) = Mushroom-Roots Living Together

“The roots of most plants are colonized by symbiotic fungi to form mycorrhiza, which play a critical role in the capture of nutrients from the soil and therefore in plant nutrition.” Smith, S.E., & Read, D.J. (2008).

“Arbuscular mycorrhizae (AM) are a type of mycorrhiza in which the symbiont fungus (Arbuscular mycorrhizal fungi, or AMF) penetrates the cortical cells of the roots of a vascular plant forming arbuscules. Both paleobiological and molecular evidence indicate that AMF are an ancient symbiosis that originated at least 450 million years ago. Arbuscular mycorrhizae are believed to form associations with 85% of all terrestrial plants.“ Willis, A., Rodrigues, B. F., & Harris, P. J. C. (2013).

How it works:

“The mycorrhizosphere plays an important role in plant nutrition, stress tolerance (drought, metal toxicity) and soil microbiology. There is a cost for maintaining this symbiotic relationship. Plants exchange sugars (glycogen) with the fungus for nutrients and water. There is a trade off between plant growth and development of the mycorrhizal symbiosis.“ Audet, P. (2012).

Study Design:

Barley: Barley (Hordeum vulgare) is an important agricultural species in the grass family (Poaceae). It is fast growing and commonly grown across Canada. Barley, and other members of the grass family, are known to be colonized and benefit from arbuscular mycorrhizal fungi.

Rhizophagus irregularis & Funneliformis mosseae Rhizophagus irregularis (previously known as Glomus intraradices) and Funneliformis mosseae (previously known as Glomus mosseae) are arbuscular mycorrhizal fungi most commonly used as a soil inoculant in agriculture and horticulture. These AMF species are also the most commonly studied research species; the fungal inoculum is even available commercially at garden stores.

Project Experimental Design: The goal of this project is to explore the effects of mycorrhizal symbiosis on plant growth. Barley and Rhizophagus irregularis & Funneliformis mosseae were selected as a study species because (1) they are fast growing, (2) easily available, and (3) easily grown indoors under greenhouse conditions. Also, other people have studied mycorrhizal symbiosis using barley and oRhizophagus irregularis & Funneliformis mosseae. So there is available information for comparison of results.

Manipulated Types Soil: Sand vs. Potting Soil) With or Without Mycorrhizal Fungus: Myc+ vs. Myc-

Responding Plant Height Plant Biomass (Dry Weight) Root Colonization

Controlled Amount of Soil in the pots Amount of Water per week Amount of Sunlight Growing Period

Planting Procedure

1. Mix Soil Substrates in a Bassin

2. Sand (100% Sand)

3. Myc- (50% Sand, 50% Potting Soil without Mycorrhizal Fungus)

4. Myc+ (50% Sand, 50 Potting Soil with Mycorrhizal Fungus)

5. Label all pots clearly.

6. Sand 4x Pots

7. Myc- 4x Pots
8. Myc+ 4x Pots
9. Add soil to pots (300ml soil per pot)
10. Water all pots (250ml per pot)
11. Poke 3x holes (1cm depth) per pot (in a triangle shape)
12. Add 3x seeds per hole (9x seeds per pot)
13. Take lots of pictures
14. Grow plants in greenhouse for 5x weeks

Harvesting

1. Take plants carefully out of pots.
2. Gently remove soil from roots.
3. Separate roots and shoots.
4. Take a sample of roots by cutting off a portion of the outer roots and placing in a water filled container.
5. Place in bags and label them the same as what you labeled your pots.
6. Dry plants by placing them in the oven and heating for 40 minutes at 350 degrees.

Root staining

1. Collect fresh root subsamples (approx. 2cm) from plants grown in the Myc- and Myc+ treatments
2. Rinse clean all samples and combine all subsamples into two separate containers (Myc- and Myc+)
3. Add hydrogen peroxide to both containers; close containers and warm solution in a bain-marie
4. Make staining solution by combining vinegar and ink in a separate container; warm solution to help combine these substances
5. Carefully rinse roots from the hydrogen peroxide, and add staining solution to root sample container; allow up to 1 day to soak in solution
6. Carefully rinse roots from the staining solution using vinegar and then fresh water
7. Add glycerin to the stained roots to preserve until root microscope slide preparation.

Measuring Plant Growth

1. gather dried plants
2. separate roots from shoots
3. weigh roots by placing on the scale and recording on data sheet
4. lay shoots flat and measure height by using a tape measure
5. weigh shoots
6. record measurements

Preparing Microscope Slides

1. Label all microscope slides with soil treatments (Myc- or Myc+)
2. Transfer stained root samples to Petri dish
3. Use dissecting tools to pull apart the roots and cut into smaller pieces (up to 5-10mm long)
4. Place 8 root pieces onto each microscope slide
5. Using the pipette, add a small amount of the glycerin solution on to the roots
6. Cover the root pieces with the slide covers
7. Seal around the slide covers with clear nail polish; allow to dry and touch up if necessary.

For the Myc- treatment, 4x slides were prepared using a total of 32 root pieces For the Myc+ treatment, 10x slides were prepared using a total of 80 root pieces.

Results:

• Plant Growth
  • Average number of plants
  • Root biomass (dried)
  • Shoot biomass (dried)
  • Plant heights

• Root Colonization (Microspcope Analysis)
  • Myc+ Root fungal colonization
  • Myc- Root fungal colonization (Control)

11 fungal structure / 80 root segments were observed for Myc+ roots. This could mean approximately 10-15% root colonization frequency. Observed fungal vesicles (that look like balloons) and hyphae (that look wires).

0 fungal structure / 32 root segments were observed for Myc- roots. This means no root colonization (as expected).

The manipulated variables in this experiment were sand, potting soil without mycorrhiza (Myc-), and potting soil with mycorrhiza (Myc+). The responding variables were the plant height, dry weight, and mycelium root colonisation.

On the graphs you can see that Myc- surprisingly had the best growth over all. This is interesting because Myc+ had microbial colonisation. The mycorrhizal symbiosis was expected to help the plants grow. But there is a trade off, in which the plant gives nutrients to the mycorrhizal fungus and the mycelium acts as a root extension, probing new areas of soil to better the plants growth and nutrient intake, by absorbing larger pieces of nutrients found in the soil.

Hypothesis:

Plants colonized with mycorrhizal fungi (Myc+) will grow better than non-colonized plants (Myc-).

Conclusion:

This hypothesis was not supported by my data: My experiment was about growing plants in different soil conditions. I thought that Plants colonized with mycorrhizal fungi (Myc+) will grow better than non-colonized plants (Myc-).

Surprisingly Potting soil without mycelium (Myc-) grew the best overall. The potting soil with mycelium (Myc+) did not grow as well. This is interesting because plants with mycelium generally do better than plants without it. the reason plants with it do better is because of symbiosis, the mycelium in the soil bonds with the plant and acts as a root extension, this is a trade off as the plant gives the mycelium nutrients, and the mycelium breaks down bigger pieces of nutrients in the soil, which the plant absorbs and the cycle continues.

My experiment was to explore the effects of mycorrhizal symbiosis on plant growth. In the real world, we use chemical fertilisers to grow plants. But chemical fertiliser is expensive, and using too much is not good for the environment. A more eco-friendly solution would be to use mycorrhizal symbiosis to help plant growth and produce more food.

This technique has become so common that you can even buy mycorrhizal fungi in garden stores. More farmers using mycorrhiza means less chemical fertilizer which would be more eco-friendly. But what about other natural fertilisers like cow or animal manure? This can have the same effect, but you have to reapply it every year. Mycorrhizal networks only have to establish themselves once and will help plants grow better over the long term.

Potential sources of error in this study include:

1. Plants were grown for 5x weeks. Maybe they needed more time to grow. If plants were grown for longer, maybe the mycelium could have better helped the plants. 
2. Only 4x pots per treatment were grown. Maybe more pots (replicates) would be better.
3. The root staining could have been better. Differentiating the fungal structure was difficult.
4. The microscope that was used was basic. I did my best, but it was difficult to focus the image at higher magnification.

Audet, P. (2012). Arbuscular Mycorrhizal Symbiosis and Other Plant–Soil Interactions in Relation to Environmental Stress. In: Ahmad, P., Prasad, M. (eds) Environmental Adaptations and Stress Tolerance of Plants in the Era of Climate Change. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-0815-4_11

Willis, A., Rodrigues, B. F., & Harris, P. J. C. (2013). The Ecology of Arbuscular Mycorrhizal Fungi. Critical Reviews in Plant Sciences, 32(1), 1–20. https://doi.org/10.1080/07352689.2012.683375

Smith, S.E., & Read, D.J. (2008). Mycorrhizal Symbiosis (3rd ed.). Academic Press. 

Vierheilig H, Coughlan AP, Wyss U, Piché Y. (1998). Ink and Vinegar: a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi. Appl Environ Microbiol 64 (12). https://journals.asm.org/doi/epub/10.1128/aem.64.12.5004-5007.1998

I worked on this project with my dad who studied arbuscular mycorrhizal symbiosis from 2006-2013.  We worked together on the background research, study design, data collection and analysis.