Mycorrhizae Root Colonization - AM and EM Assessments

What are Mycorrhizae?

Root-tip Ectomycorrhizae of the Amanita type.

Mycorrhizae are fungus that form symbiotic or pathenogenic relationships with the roots of plants in the soil. They are responsible for decomposition and nutrient cycling processes, and produce and consume gases governing the global climate and they have many biotechnological applications (e.g. waste water treatment, bioremediation, biocontrol, antibiotic and other medicinal products, food, and alcohol).

Types of Mycorrhizae

Although thousands of different mycorrhizal species exist, there are 3 main types:

1. Arbuscular Mycorrhizae (AM) which are formed on the majority of herbaceous plants
2. Ericoid Mycorrhizae which are formed on ericaceous plants such as salal
3. Ectomycorrhizae (EM) which are formed on the majority of temperate tree species.

Why are Mycorrhizae Important?

Soils contain billions of bacteria and millions of fungal propagules per gram. Examples of beneficial symbiotic associations between plant roots and microorganisms include mycorrhizae which are a mutualistic or mildly pathenogenic association between fungi and plants. The fungus receives its carbon for growth from the host plant and in return supplies the plant with increased water and nutrient uptake capacity.

Another common and extremely important symbiosis is that between legumes and Rhizobium or Bradyrhizobium species of bacteria and that between alder trees and Frankia species of actinomycetes. Rhizobia and Frankia infect root hairs of the leguminous plants and produce nodules. The nodules become the home for the

An alder root nodule gall.

microorganisms where they obtain energy from the host plant and in return take free nitrogen (N) from the soil air and process it into combined N through biological nitrogen fixation (BNF). This is the process whereby atmospheric nitrogen (N₂) is reduced to ammonium in the presence of the nitrogenase enzyme. Nitrogenase is an

oxygen sensitive enzyme. The low oxygen tension condition is realized through synthesis of leghaemoglobin (in Rhizobium legume). Leghaemoglobin is a macromolecule synthesized by both symbiotic partners, the rhizobia and the host plant. Rhizobium synthesizes the heme portion, and the plant the globine. Like human haemoglobin, leghaemoglobin fixes oxygen (O₂). It is responsible for the red or brown colour of active (i.e., N₂-fixing) nodules. Non-N₂-fixing nodules have a white nodule content, or a green content when the globine has degenerated. Plants receive the fixed N from nodules and produce food and forage protein and can be used to enrich soils with N for following crops. N₂-fixing systems can thrive in soils poor in N, explaining the ability of alder to colonize highly disturbed ecosystems, such as abandoned logging roads and quarries.

Nodules are easy to identify on legume and alder roots. They are generally assessed by enumerating nodule number and mass and then nitrogenase activity using the acetylene reduction technique. Nitrogenase not only

A sectioned alder root nodule gall.

catalyzes the reduction of atmospheric N₂ to ammonium (NH₃), but can also reduce acetylene (C₂H₄). The acetylene reduction assay (ARA) is carried out on detached nodules, de-topped roots, or whole plants in a closed vessel containing 10% acetylene. A gas chromatograph is used to determine the amount of ethylene formed. Data

are usually expressed as nanomoles or micromoles of ethylene produced per hour per plant or per weight unit of nodules. The acetylene reduction assay provides an instant measure of nitrogenase activity (but not necessarily of N₂ fixed) under the experimental conditions. For the purposes of acting as a case study example provided here, rates of BNF per nodule will be provided and then you can calculate rates of BNF per plant for your sites.

Nitrogen fixing nodules in clover roots.

Rhizobium bacteria. The Rhizobium species of bacteria are nitrogen fixing bacteria that convert atmospheric nitrogen, into forms available for plant uptake. The Rhizobium species typically formed symbiotic nodules on the roots of agricultural legumes (e.g.. Clover, Lucerne, Beans) and non-agricultural legumes (such as Acacia, Cassia).

How are Mycorrhizal infection rates measured?

Though the advent of molecular methods to identify soil microorganisms has replaced many of the traditional techniques for studying these organisms, the isolation of these microorganisms from the complex mixed populations in soil is still essential for physiological studies. In addition, in many cases, rapid, relatively inexpensive methods are preferred for first-stage screening and assessment of the effects of, for example, different reclamation treatments, different cover crops, pollutant contamination, on these symbioses.

Methods for studying mycorrhizae are numerous, some first-stage assessments of mycorrhizae are:

1. Assessing % colonization of roots using root-clearing, staining and visual estimation of % infection for arbuscular mycorrhizae (as the majority of the fungus in AM is internal within the root cortical cells).
2. Morphotyping and visual estimation of % infection for ectomycorrhizae (as the majority of the fungus in EM is external, forming a mantle on the fine root tips) using microscopy and photographic keys. The morphotyping of ectomycorrhizae also enables an initial identification of the mycorrhizal fungus partner in the symbiosis as there are many different growth forms and colours characteristic of these fungi.
3. Count nodules on roots, dissect nodules to determine if nodules are active, weigh total nodule mass per plant. This information can be used to estimate the BNF rate per nodule (given) to calculate the overall rate of BNF per plant.

Cross section of a vascular plant's root that has arbuscular mycorrhizal fungi (AMF) present (visible as red stained structures in the root's cortex). *Actual lab procedure results may vary.

Materials

• Petri dishes
• Beakers
• Fine forceps
• Razor blades/scalpels
• 10% KOH
• Schaeffer's black ink
• White vinegar
• Bunsen burner
• Dissecting microscope
• Lamps
• Pipettes
• Filter paper
• Weighing balance
• Microscope camera (digital eyepiece)

Procedures

Sampling of soils and roots

a. Soils and root samples must first be obtained from field sites. General recommended guidelines for soil sampling can be found HERE.

b. For a root nodule assessment, whole plants and their roots are excavated from soil (for herbaceous plants –legumes and tree seedlings).

c. For larger trees, roots are excavated and representative roots and nodules removed.

1. Clearing and staining of herbaceous roots for AM assessment

a. Stain the roots of at least five replicate plants per treatment.

Root length is measured and then roots are cut into 1 cm segments and cleared of tannins by boiling in 10% (wt/vol) KOH for 3 minutes and then rinsed several times with tap water. To stain cleared roots are boiled for 3 min in a 5% Shaeffer black ink-vinegar solution with pure white household vinegar (5% acetic acid). Roots are destained by rinsing in tap water (acidified with a few drops of vinegar) for 3 minutes. The stained root pieces are then spread out evenly on a petri dish and observed using a dissecting microscope. By moving the dish and observing the whole sample an overall estimate of the % cortex infected can be made. Replication to find the SD of the method is obtained by rearranging the same root sample and making another visual estimate.

2. Visualizing EM on fine roots of trees

Visually compare roots that aren't infected by EM with roots that are. Make note of some of the differences. What do you notice?

3. Cleaning Procedure and ECM Extraction from Soil

Immerse the soil core in water and soak carefully in water until saturated. Wash the roots gently with pipettes to limit damage to the ectomycorrhizas then place cleaned roots (<1 mm diameter) in a Petri dish with filter paper soaked with water (Petri dishes with filter paper prevent color changes of ectomycorrhizas and hyphal growth). ECM tips can be stored in the refrigerator up to seven days after sampling.

4. ECM Morphology

Observe ECM root tips in water under a dissecting microscope (6×, 12×, 25×) using a black background and lamps of daylight quality. Isolate morphotypes by morphology, i.e., color, ramification type, systems, size and texture, presence of emanating hyphae, cystidia, rhizomorphs, and/or sclerotia (see B.C. ectomycorrhizal research website and Agerer and Rambold 2004–2007). (3) Take photos of mycorrhizal systems, maintaining the black background and lamps of daylight quality (Agerer 1991). Estimate % infection of roots by different mycorrhizal fungi. Estimate ECM abundance and richness.

5. Nodules

Wash roots gently with pipettes to minimize damage to nodules. Place nodules in petri dish and count nodule numbers. Calculate nodule mass by weighing nodules. Using a scalpel dissect nodules and determine the % active (brown/red) versus inactive (white/green) nodules. Using a BNF rate of 10 nmol per hour per nodule calculate the overall BNF per plant per day.

Calculations

$\%\ infection=\left({\frac {Number\ of\ roots\ with\ AM/EM\ infection}{Number\ of\ uninfected\ roots}}\right)(100\%)$

Steps for Lab Analysis

**Mycorrhizae**

1.     Calculate % infection of roots by AM fungi. Were there quantitative differences in % AM infection in your different plant samples? Explain why you think this is the case.

3.     Illustrate what you saw under the microscope with drawings/photos and labelling of key features.

4.     Calculate % infection of woody roots by ECM fungi. Note differences in color, ramification type, systems, size and texture, presence of emanating hyphae, cystidia, rhizomorphs, and/or sclerotia. Were there different ECM fungi on your root samples? Explain why you think this is the case.

6.     Illustrate what you saw under the microscope with drawings/photos.

7.     Did you manage to identify your main Ectomycorrhizae with the keys?

**Nodules**

1.     Count the total number of nodules for each plant.

2.     Calculate nodule mass by weighing nodules.

3.     Using a scalpel dissect nodules and determine the % active (brown/red) versus inactive (white/green) nodules.

4.     Using a biological nitrogen fixation (BNF) rate of 10 nmol per hour per nodule calculate the overall BNF per plant per day.

5.     Were there qualitative and quantitative differences in nodule numbers and mass in your different plant samples? Explain why you think this is the case.

6.     Did some samples contain more active nodules than others? Why might this be the case?

References

1. B.C. Ectomycorrhizal research atlas at: http://cfs.nrcan.gc.ca/projects/111?lang=en_CA
2. Fisher RF and Binkley D. 2000. Ecology and Management of Forest Soils. Third Edition. John Wiley & Sons Inc., Toronto.
3. Giovannetti M and Mosse B. 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84: 489-500.
4. Paul EA. 2007. Soil Microbiology, Ecology & Biochemistry.3^rd Edition. Elsevier Academic Press.
5. Pepper IL and Gerber CP 2004. Environmental Microbiology: A Laboratory Manual.2^nd Edition. Elsevier Academic Press.
6. Suz, LM, Azul AM, Morris MH, Bledsoe CS, & Martín MP. 2008. Morphotyping and Molecular Methods to Characterize Ectomycorrhizal Roots and Hyphae in Soil. In: C.S. Nautiyal, P. Dion (eds.) Molecular Mechanisms of Plant 437 and Microbe Coexistence. Soil Biology 15, DOI: 10.1007/978-3-540-75575-3. Springer-Verlag Berlin Heidelberg.
7. Vierheilig, H, Coughlan, AP, Wyss, U, & Piche Y. 1998. Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi. Applied and Environmental Microbiology 64: 5004–5007.