How to Test Soil for Mycorrhizal Activity

Mycorrhizal fungi form symbiotic relationships with the roots of most terrestrial plants, playing a vital role in soil health and plant growth. These fungi enhance nutrient uptake, improve soil structure, and increase plant resilience against pathogens and environmental stresses. Understanding and measuring mycorrhizal activity in soil is crucial for gardeners, farmers, ecologists, and restoration specialists aiming to optimize plant productivity and ecosystem sustainability. This article provides a comprehensive guide on how to test soil for mycorrhizal activity, including methods, tools, and interpretation of results.

Understanding Mycorrhizal Fungi and Their Importance

Mycorrhizae refer to the mutualistic association between fungi and plant roots. The two main types are arbuscular mycorrhizal fungi (AMF) and ectomycorrhizal fungi (EMF). AMF typically associate with herbaceous plants and many crops, while EMF are more common with woody plants such as trees.

The benefits of mycorrhizal fungi include:

• Increased nutrient acquisition (especially phosphorus)
• Enhanced water absorption
• Improved soil aggregation
• Protection against root pathogens
• Increased tolerance to salinity and drought

Given these advantages, assessing mycorrhizal activity in soil can inform management practices such as inoculation, crop selection, or organic amendments.

Signs of Active Mycorrhizal Colonization

Before conducting tests, it is useful to identify visual or indirect indicators of mycorrhizal activity:

• Healthy root systems with fine branching and root hairs
• Presence of external fungal structures like hyphae or fungal mats around roots
• Improved plant vigor without excessive fertilizer use
• Dark or orange staining in some roots (typical of EMF colonization)

However, these observations are subjective. Therefore, reliable testing methods are necessary to quantify mycorrhizal presence and activity.

Methods for Testing Soil for Mycorrhizal Activity

There are several approaches to assess mycorrhizal activity in soil ranging from simple bioassays to advanced molecular techniques. The choice depends on the purpose, budget, and available resources.

1. Root Staining and Microscopic Examination

Overview

This classical method involves collecting root samples from plants growing in the soil under study, then staining the roots to visualize fungal structures under a microscope.

Procedure

1. Sample Collection: Collect fine roots from representative plants in the area.
2. Cleaning: Gently wash roots with water to remove adhering soil.
3. Clearing: Soak roots in a potassium hydroxide (KOH) solution (typically 10%) at 90°C for 30–60 minutes to clear cellular contents.
4. Acidification: Rinse roots and soak briefly in an acidic solution (e.g., hydrochloric acid) to prepare for staining.
5. Staining: Immerse roots in a stain such as trypan blue, ink-vinegar solution, or chlorazol black E for 15–30 minutes.
6. Destaining: Rinse roots briefly in acidified glycerol or water to remove excess stain.
7. Microscopy: Mount stained root segments on slides and examine under a compound microscope at 100x–400x magnification.

What to Look For

• Arbuscules (branched fungal structures inside root cells)
• Vesicles (storage structures)
• Hyphal coils or networks inside cortical cells

Quantification

Estimate percent root length colonized by mycorrhizae by examining multiple fields of view per sample using gridline intersect or point count methods.

Advantages

• Direct visualization confirms colonization
• Relatively inexpensive materials
• Established protocols available

Limitations

• Labor-intensive
• Requires microscopy skills
• Does not directly measure soil fungal abundance but root colonization status

2. Soil Bioassays Using Trap Plants

Overview

This method grows host plants known to form associations with mycorrhizal fungi in collected soil samples under controlled conditions. After a set growth period, roots are examined for colonization levels.

Procedure

1. Collect representative soil samples from the area.
2. Fill pots or containers with the test soil.
3. Plant seeds or seedlings of a suitable host species (e.g., clover, corn for AMF).
4. Grow plants under controlled conditions (greenhouse or growth chamber) without fertilization that may suppress colonization.
5. After 6–12 weeks, harvest plants and assess root colonization by staining and microscopy (see method 1).

Advantages

• Measures presence of infective mycorrhizal propagules capable of colonizing roots
• Reflects biological activity rather than just spore counts or DNA presence

Limitations

• Time-consuming (weeks to months)
• Requires space and care for growing plants
• May not reflect natural field conditions perfectly if environmental factors differ substantially

3. Spore Extraction and Counting

Overview

This method extracts spores from soil using wet sieving and centrifugation techniques followed by microscopic counting.

Procedure

1. Place a known quantity of soil in water.
2. Shake/agitate vigorously to dislodge spores.
3. Pass suspension through a series of sieves with decreasing mesh sizes (e.g., 500 μm down to 25 μm).
4. Centrifuge samples using sucrose or other density gradients to separate spores.
5. Collect spores from centrifuge tubes onto filter paper.
6. Count spores under a dissecting microscope.

Advantages

• Provides information about spore density in soil
• Useful for assessing inoculum potential

Limitations

• Does not indicate colonization or active hyphal presence
• Some spores may be dormant or nonviable
• Requires specialized equipment like centrifuge and sieves

4. Molecular Techniques: DNA-Based Assays

Advances in molecular biology have enabled direct detection and quantification of mycorrhizal fungi DNA in soils.

Common Methods

• qPCR (Quantitative Polymerase Chain Reaction): Targets specific fungal DNA sequences; quantifies fungal abundance relative to soil mass.

• Metagenomic Sequencing: High-throughput sequencing identifies diverse fungal taxa present simultaneously.

Procedure Overview for qPCR:

1. Extract total DNA from soil samples using commercial kits optimized for soil.
2. Use primers specific for mycorrhizal groups (e.g., AMF-specific small subunit rRNA genes).
3. Run qPCR reactions with standards for quantification.
4. Analyze data as gene copy number per gram of soil.

Advantages

• Highly sensitive and specific
• Detects fungi regardless of spore stage or viability
• Enables community composition analysis via sequencing

Limitations

• Requires molecular biology lab facilities and expertise
• More expensive compared to microscopy methods
• DNA presence does not always correlate with active colonization or functional activity

5. Enzymatic Activity Assays

Certain enzymes secreted by mycorrhizal fungi contribute to nutrient cycling; measuring their activity can indirectly indicate fungal function.

Examples include phosphatases involved in phosphorus mineralization.

Though not specific solely to mycorrhizae, enzyme assays provide complementary data on microbial activity related to nutrient transformations.

Interpreting Results from Mycorrhizal Tests

Understanding what the results mean is critical:

| Test Method | What It Measures | Interpretation |
|————————–|————————————-|————————————-|
| Root staining | Actual colonization level | Higher % means more active symbiosis|
| Bioassay | Infective propagule presence | Successful colonization indicates viable inoculum|
| Spore counts | Spore abundance | Indicative of potential but may overestimate active fungi|
| Molecular assays | Fungal DNA abundance & diversity | Shows presence; need functional correlation|
| Enzyme assays | Microbial extracellular enzyme activity| Related to nutrient cycling; indirect measure|

High mycorrhizal activity generally correlates with improved plant growth conditions but must be balanced with other soil health indicators.

Factors Affecting Mycorrhizal Activity Testing Accuracy

Certain considerations impact testing outcomes:

• Soil Moisture & Sampling Time: Dry soils may reduce fungal activity temporarily; seasonal variations affect results.

• Host Plant Species: Some plants form stronger associations influencing colonization levels observed.

• Soil Disturbance & Fertilizers: Tillage and high phosphorus fertilizers can reduce natural colonization rates.

• Sample Handling: Avoid prolonged storage; keep samples cool to preserve fungal viability.

Practical Applications of Soil Mycorrhizal Testing

Testing helps guide practical decisions such as:

• Selecting appropriate crop rotations maximizing beneficial symbioses
• Determining need for commercial mycorrhizal inoculants in degraded soils
• Monitoring restoration success by tracking recovery of native fungi communities
• Optimizing fertilization regimes reducing chemical inputs while maintaining yields
• Researching effects of land management on belowground biodiversity

Summary: Best Practices for Testing Soil Mycorrhizal Activity

1. Define your objective clearly—quantify colonization, detect inoculum potential, analyze community structure?
2. Choose a method suited to your resources: root staining for basic studies; molecular methods for detailed analysis.
3. Collect representative samples carefully avoiding contamination.
4. Combine multiple approaches when possible for robust assessments.
5. Interpret results within ecological context considering plant species, land use history, and environmental factors.

By accurately testing soils for mycorrhizal activity, growers and scientists can harness these ancient symbionts’ power to foster healthier plants, more productive soils, and sustainable ecosystems.

References available upon request.