by Danielle Stevenson, D.I.Y. Fungi, This content was taught at workshop with Danielle for Healthy Soils, Healthy Trees program and part of tree drought research with TreeFolks. Thanks to Ecology Action and City of Austin Urban Forestry Grant for supporting this program.
There are several types of mycorrhizal fungi, but ectomycorrhizal (EMF) and arbuscular mycorrhizal fungi (AMF) are the most common. It is a good idea to look up what kind of mycorrhizal relationships the plant(s) you’re trying to cultivate form and cultivate those.
Watch part one of our workshop series with Danielle to learn more about mycorrhizal fungi.
Do you need to restore mycorrhizal populations? Yes in:
intensively managed agricultural lands (industrial ag- frequently tilled, heavily
fertilized, fungicide use)
degraded soils (remediation of mine sites and brownfields, ecological restoration and reforestation efforts)
nurseries (and hydroponic or container gardens)
You can restore mycorrhizal fungi through agricultural and land management practices or through introduction of mycorrhizal inoculum. Management practices that encourage mycorrhizal fungi:
Crop rotation including deep rooting of mycorrhizal plants
Cover cropping - do not leave soil bare
Reduced/no till
Deep plowing
Reduced fertilization (esp Phosphorus)
Where to source your MYCORRIZAL SPORES?
You can purchase commercial mycorrhizal spores to inoculate plants and soil but recent meta analysis has shown they are less effective than using methods that introduces native, cultivated mycorrhizae. Mycorrhizal spores, pieces of mycorrhizal roots and viable mycorrhizal hyphae are active propagules that can be used as inoculum.
Methods OF CULTIVATING AND AMPLIFYING Native mycorrhiza
Whole Soil Method
Trap Culture Method
Nurse Plant Method
Spore Slurry Method (EMF)
Species-specific AMF Method
Whole Soil METHOD
Takes soil from a healthy reference ecosystem as inoculum
It inoculates with intact rhizosphere soil containing the complete array of the soil community including AM fungi, beneficial bacteria such as nitrogen fixing Rhizobia, pathogens, soil-dwelling insects, nematodes, plant roots, and even seeds.
Destructive of healthy ecosystems and requires huge amounts of soil (150-10,000 gallons per acre).
Workshop series with Danielle at Circle Acres on how to cultivating bioregional mycorrhizal fungi
Trap Culture Methods
The following method is what we are using for our Healthy Soils, Healthy Trees research.
Works to amplify the volume of whole soil microbes so that a few liters of whole soil inoculum can be used to inoculate hundreds of plants.
Trap cultures are a way of ‘bulking up’ the AM fungal community present in whole soil before it is used as inoculum in a restoration. The AM fungi + plant dependent soil microbes are amplified by growing with plant hosts in a pot and this allows their propagation for future use.
StepS for producing AMF inoculum via trap cultures
Materials needed:
Containers: One gallon or larger pots can be used to produce your "pot culture." Allow for drainage so that water does not accumulate in the bottom of the pot.
Growth Medium: A good growth medium for inoculum production consists of sand (construction blue sand) or a pasteurized sandy soil with good drainage.
AM Fungi: Collect fine roots or soil from the root zone of native vegetation from the habitat you are restoring or from specific plants that are likely to be mycorrhizal [list of plant species that form AM associations online].
Seeds of a "Host" Plant: A fast-growing plant species different from the plants that will be grown in agriculture or restoration should be used as a host plant. Corn, bahiagrass are often used.
Low-Phosphorus Fertilizer: For best results, use fertilizer low in phosphorus or slow release fertilizer pellets (e.g. 17-6-10 with micronutrients).
Collecting soil from mother trees in the winter.
Step 1: COLLECT the AMF fungi you want to cultivate
Dig up rhizosphere soil (attached to roots of plants), remove shoots and chop roots into small bits from a healthy and mature plant. Get enough soil so you can mix it 1:1 with your sterile soil.
Step 2: Sterilize POTTING soil OR MIX STERILE POTTING SOIL
Additional potting soil is mixed with your collected AMF soil. It should be sterilized of soil organisms including other AM fungi prior to inoculation. Soil can be sterilized in a variety of ways. Soil temperatures of 140 F for one hour or longer are required to kill most soil pathogens.
Autoclaving (2 hrs 2x)
Steam aeration (4 hrs 2x)
Conventional oven (170F for 2 hrs)
Solarization (170F for 2 hrs)
Or you can buy and make sterile potting soil – make sure it is low or no nutrients!
For the Healthy Soils, Healthy Trees research we chose to make our own sterile potting soil. This soil mix consists of one-third coco coir for moisture retention (make sure brand has no AMF or EMF fungi added), one-third vermiculite for aeration and moisture retention, and one-third course sand to enhance drainage and structure.
Step 3: Mix inoculum with STERILE SOIL AND TRANSFER TO pots
Mix the sterile soil and inoculum 1:1 then is transferred to a clean 1-gallon plastic pot.
Step 4: Seed host plant into pots
Seed pots with a highly dependent host plant- people often use grasses like Sudangrass/Sorghum, bigbluestem (tolerates hot greenhouse temperatures) and corn is also used. Overseed (80-100 seeds/pot).
For the Healthy Soils, Healthy Trees research we grew winter rye grass and Sudangrass/Sorghum in the late spring/summer. Make sure you read the growing guides and understand seasonality of plants you choose.
Step 5: Let plants grow for 3-4 months
Cultures are grown in a greenhouse or grow room for at least four months.
Fertilize as little as possible, only as needed.
Water as normal.
Step 6: Stop watering, let plants dry out
Pots are left to dry undisturbed for 2-3 weeks.
This causes plant partner to invest carbon in AMF partner, and as plant dies AMF sporulates.
Then, cut off the shoots above the soil level.
Step 7: Harvest, store and use inoculum
Chop the roots and mix them in with clean sand. This root and sand mixture is your concentrated AM fungal inoculum. The inoculum will remain effective for at least one year if stored in plastic containers and kept in a cool, dry area. You can use this inoculum to produce more "pot cultures," to add to potting mix to raise mycorrhizal plants in the greenhouse, as well as to inoculate ‘nurse’ plants that are being transplanted to the field.
For the Healthy Soils, Healthy Trees research we will store all of the soil in 5 gallon pots indoors until it is time to plant trees in the late winter. Watch recording of workshop where we harvest the roots and validated that we cultivated AMF.
Variations:
INVAM method: International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Collection at West Virginia University.
Rodale-USDA method: On Farm Arbuscular Mycorrhizal Inoculum Production. Rodale Institute.
Low-Tech, In-Ground Trap Cultures: In-Ground Trap Cultures: Missouri Botanical Garden and the Restoration EcologyLab at Virginia Tech
Fill a pit (150 cm long × 50 cm wide × 30 cm deep) lined with sacks with topsoil collected from around the roots of three native tree species
Then grow maize and beans in this soil for three months before cutting these plants down
Letting the substrate dry out for two weeks. The substrate remaining in the pit is the inoculum
Use by adding one tablespoon inoculum to each seedling container.
The nurse plant method
Inoculating potted seedlings with AM fungi before planting into a site allows them to act as nurse plants
Enhances the likelihood that the inoculum will remain viable in the field
AM fungi can spread to nearby un-inoculated plants, over distances of up to two meters within a single growing season
SPORE SLURRY METHOD FOR Cultivating native Ecomycorrhizal fungi (EMF)
REQUIREMENT: Mushrooms (that you know are ecto) must be gathered from near known ecto-mycorrhizal fungal trees.
Spores or macerated fruiting bodies of some ectomycorrhizal mushrooms, puffballs, or truffles (and false truffles) provide good inoculum. Truffles (Ascomycotina) and false truffles (Basidiomycotina), from now on together referred to as truffles, are uniquely suited for this because their fruiting body tissue consists mostly of spore-bearing tissue and the fruiting bodies can be quite large. We recommend using fresh spores whenever possible, but have stored spore suspensions of various Rhizopogon species up to 3 years without a significant reduction in inoculum effectiveness (Castellano 1987).
Rinse freshly collected fruiting bodies with tap water to remove adhering soil or organic matter
Cut into pieces (1 - 3 cm) and blend with tap water at high speed for 2 to 3 minutes, until all pieces are thoroughly blended. Note: The final consistency is similar to thick chocolate milk.
It is unnecessary to purify spore suspensions. Li and Castellano (1987) and Li (1987) have found beneficial microorganisms within and on the surface of mature fruiting bodies of various ectomycorrhizal fungi; these organisms should be encouraged, not excluded.
Spore concentrations within the resulting suspension are determined with a hemacytometer (blood cell counter).
Store slurry in the dark in the refrigerator until used (up to 3 years) at up to 5 °C or 41 °F).
To then inoculate plants:
In a nursery or for nurse plants, apply spore slurry 6 to 12 weeks after sowing, either with a standard watering can or through the existing irrigation system. Most truffle spores are less than 50 μm in diameter and will pass freely through most filters and nozzle tips.
The desired amount of spores is mixed into a watering can containing sufficient water to cover a certain number or area of seedlings (Styroblocks® or racks of plastic tubes).
Applying spores twice, 2 to 3 weeks apart, works best to assure even distribution (fig. 5.2.53), especially when using the irrigation system instead of watering cans. Alternatively, spores can be applied to the seed before sowing. Although we have not tried this method, it may prove more effective than the watering can method in inoculating each seedling. Seed treatment would also allow finer control in matching ecotypes of fungi to specific seed sources.
Species-specific AMF method
Arbuscular mycorrhizal (AM) cultures contain only living propagules of single or multiple mycorrhizal fungal species). Cultures of a single species usually must be started from carefully selected spores. These spores can originate from a field soil if they are healthy. Step by step process for Establishment of Monospecific Cultures from INVAM
RECORDING: Cultivating mycorrhizal Fungi Workshop with Danielle Stevenson (THREE Parts)
PHOTOS: Cultivating mycorrhizal Fungi Workshop with Danielle Stevenson
Resources
The Container Tree Nursery Manual, Volume 5. Agric. Handbk. 674. Castellano, M.A.; Molina, R. 1989. Mycorrhizae. In: Landis, T.D.; Tinus, R.W.; McDonald, S.E.; Barnett, J.P. Washington, DC: U.S. Department of Agriculture, Forest Service: 101-167.
Use of Mycorrhizae in Restoration of Hawaiian Habitats. Gemma, J.L., R.E. Koske. Hawaii Conservation Alliance.
The Use of Mycorrhizae in Native Plant Production. Lorraine Brooks, Deborah Brown, Sierra Smith, and Samantha Sprenger. June, 2006: University of Washington.
A Practical Guide to Inoculation with Arbuscular Mycorrhizal Fungi in Ecological Restoration. Liz Koziol, Peggy A. Schultz, James D. Bever. University of Kansas, Lawrence Geoffrey House, Jonathan Bauer Indiana University, Bloomington. Elizabeth Middleton, Missouri Department of Conservation. June 2017.
Trap Culture Methods. International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Collection at West Virginia University.
On Farm Arbuscular Mycorrhizal Inoculum Production. Rodale Institute.