Arbuscular mycorrhizal fungal spores.
MYCOLOGISTS ARE BUSY developing ways to improve methods of producing inoculum, and they are creating systems for easier reproduction of arbuscular mycorrhizal fungi. Most of these efforts stem from a desire to conserve the use of fertilizers and water for environmental and economic reasons. The result is increased availability of commercial mycorrhizal fungi products for agriculture, silviculture, horticulture, and home gardening, as well as a number of do-it-yourself systems for producing mycorrhizal fungi for inoculation.
To grow your own arbuscular mycorrhizal inoculum, you can start with spores, hyphal fragments, or colonized root fragments. Large, arbuscular mycorrhizal fungi spores, at 30 to 500 micrometers in size, can be seen with a microscope or a hand lens and can be collected easily. Moreover, these fungi can reproduce from spores or from vesicles created inside a colonized plant root. When the root dies, the vesicles germinate and develop hyphae, just as spores do.
You can use commercial propagule mixes with sufficient mycorrhizal fungal material and appropriate strains to match the specific crops for which the inoculum will be used. You can also gather your own propagules from soil collected from a field or from areas adjacent to a field, such as fence rows or woodlots. After you collect soils from several areas, mix them together and use a sieve to remove sticks, rocks, and other debris. This soil should contain a large and diverse population of indigenous mycorrhizal fungi to use as an inoculant; some studies suggest that indigenous mycorrhizal fungi perform better for their host plants than introduced species. If you are unsure about the soil, you can send samples to a lab to test and determine the presence and amount of propagules.
To extract spores from soil in the lab, scientists place the soil into solution, which is then centrifuged. Spores can also be mixed with adhesive chemicals that cause them to float in a single level. This process requires laboratory chemicals to which most lay people do not have access. But even without access to or training in using lab equipment, you can use a small-to-smaller series of mesh screens to isolate the spores from the soil in solution. Relatively inexpensive kits of soil sieves are available from scientific supply companies. To do the job, you’ll need a 750-micrometer mesh sieve to catch the big stuff, a 250-micrometer sieve for the next pass, then a 100-micrometer sieve, and finally a 50-micrometer sieve.
The final collection is flotation-separated: in a liquid solvent (such as water and glycerol or sucrose, also available from a scientific supply company) the bits of soil in the solvent are suspended at different levels. You can use a density gradient to measure the various densities of the materials. The fungal spores are heavier than most of the other organic matter in the soil, though not as heavy as minerals.
Based in Pennsylvania, the Rodale Institute has been studying organic farming and gardening methods since it was established in 1947. Its founder, J. I. Rodale, was heavily influenced by the food-growing techniques of British organic gardening pioneers Eve Balfour and Albert Howard. He wanted to promote a holistic system of agriculture that offered benefits for human health and respect for the environment. In addition to publishing several magazines and books, the Rodale Institute engages in agricultural research, partnering with private groups, colleges and universities, and the USDA.
Although the mycelium, root coverings, and fruiting bodies of some ectomycorrhizae are visible to the naked eye, it takes a dissecting microscope to view arbuscular mycorrhizae. Because the natural pigments of the plant roots are dark, obscuring the mostly transparent fungi from view, these pigments must be removed and then a stain that sticks to the fungi applied to reveal them under the lens.
To view mycorrhizal fungi in the lab, scientists wash freshly collected roots in water before bathing them in a hot solution of 10 percent potassium hydroxide (KOH) to clear out the plant parts. This is acidified with hydrochloric acid (HCl) and then finally stained.
Traditional stains are highly alkaline, which makes them too dangerous for the untrained lay person to use. Scientists take extensive safety precautions before handling them, because each poses great dangers. For this reason, the reader is not advised to use these chemicals without adequate training and supervision.
Other, slightly safer, methods can also be used for staining and viewing these small organisms. Diluted potassium hydroxide, at 2.5 percent, is a bit safer to use, though it is a hazardous material and, as such, you must be extremely careful when handling it and follow the safety guidelines on the label. Samples first need to be for soaked in water for at least 24 hours in a test tube or similar vessel. Then the samples are soaked in a 1-to-1 mixture of hydrogen peroxide-to-water. An India ink and vinegar mix works well as a stain. Further soaking in glycerol for two or three days will make the fungi even clearer.
As interest in growing mycorrhizal fungi increases, so will the need for simple testing procedures. Current staining techniques take too much effort and require too many skills to be conducted safely by the untrained person, however. In the meantime, biological labs will test and identify the existence and numbers of mycorrhizae for you. Identification by DNA or RNA will eventually be economical as well.
In 2002, the institute partnered with the USDA Agricultural Research Service to develop a system that would enable farmers to produce arbuscular mycorrhizal fungi. The system needed to be simple, to use readily available and inexpensive materials, and to produce a spore-rich inoculum that was less expensive than commercial brands.
Rodale and USDA scientists spent eight years studying and developing several ways to grow arbuscular mycorrhizal fungi. In 2010, they published a description of a system they used to grow fungi that would make it more available and affordable than expensive commercial formulations. In addition, the group developed a series of recommendations with regard to standard practices that ensure the viability of the produced fungi once put to use.
The On-Farm Arbuscular Fungus Inoculum Production System was created for farmers and designed to avoid the need for complicated equipment or expensive, hard-to-find materials. (For more specific information, consult the Rodale website at rodaleinstitute.org/quick-and-easy-guide-on-farm-am-fungus-inoculum-production.)
One of the findings of the Rodale/USDA study: soil transfers run the risk of the introduction of organisms such as root pathogens. Using a host plant that is different from the crop plant prevents the inadvertent growth of pathogens associated with the final crop.
In the study, (100 cubic centimeters) of soil was placed into individual grow bags. Rodale has experimented with the mixture used in the grow bags. Pure compost, pure sand, and perlite or vermiculite all supported mycorrhizae, but these were not successful media when used alone. They were either too low in nutrients, or, in the case of the compost, too high, particularly in phosphorus and/or nitrogen.
Compost with high nitrogen, low phosphorus, and moderate potassium levels work best. The study referenced different composts made from yard clippings, dairy manure, and leaves. The best dilution rate with the compost varied, depending on the mycorrhizal fungi used as well as the diluting media, but a 1-to-4 mixture of compost-to-diluting media worked best.
The study showed the 1-to-4 mixture of yard-clipping compost–to–vermiculite produced an average of 30 spores per cubic centimeter. The additional propagules come from the colonized roots, and the vesicles in them can develop mycorrhizae, too.
This method of developing inoculant can be adapted using smaller bags and different host plants. Rye (Secale spp.), fescue (Festuca spp.), corn (Zea mays), and other grasses will support mycorrhizae for collection of spores, propagules, and roots. With the ability to test results and identify specific fungi if need be, you can tweak the process to specific needs.
Inoculant can also be produced in greenhouse conditions using various host plants. Over the years, nursery growers and farmers have had success using corn (Zea mays), onion (Allium cepa), strawberry (Fragaria spp.), peanut (Arachis hypogaea), sorghum (Sorghum spp.), and big bluestem (Andropogon gerardii) as hosts. Warm-weather plants can also be used as host plants, including pencilflower (Stylosanthes spp.), bahiagrass, and kudzu (Pueraria phaseoloides).
To grow your own mycorrhizal inoculum, you can start with any commercial soil mix that does not include added fertilizers or manures. Sterilize the commercial mix in an autoclave or oven for about an hour and a half at 260°F (121°C). (Sterilizing soil in an oven can create unpleasant odors.) After a week or two, mix 1 part field-collected soil with 3 parts sterilized soil, or use commercially available mixes of fungi instead of field soil to prevent pathogens.
The best soil and propagule collection site is a field that has been in production previously or is presently in production and that offers soil rich in organic material. The better the crop grown in the soil added to the mix, the more likely crops inoculated with the propagule mix will thrive. In addition, the higher the diversity of the field soil’s mycorrhizae, the better. You can have the field soil tested for propagules before using it.
You can also gather propagules from strawberry plants using field culture. First, transplant a strawberry plant from the field to a pot filled with sterile soil. Or establish a runner in potted field soil and transplant it into a pot with sterile soil. Nurture plants until they are mature and well established. Then remove the plants and roots, save the soil clinging to the roots, and chop up the roots. The resulting products can be used to inoculate plants started in sterile soil.
These methods would likely be viable for use with other crops as well. Experiment to find out.
Ectomycorrhizal fungi are easy to produce provided you have an appropriate host plant. Amateurs and professional foresters and landscapers can help plants develop ectomycorrhizae using several methods.
Studies have shown that soil containing ectomycorrhizal fungi taken from a forest at one location can be successfully used to associate with host trees at another. One such study used soils taken from New Zealand, India, Italy, Wisconsin, and the Alaskan tundra to establish successful mycorrhizal associations with the same tree hosts in one plot. Conifers and deciduous trees respond equally to these treatments.
Collect the fruiting bodies (such as mushrooms) of ectomycorrhizal fungi to use as propagules. This takes some preparation and a bit of knowledge—based on the season and conditions, the appearance and thus availability of fruiting bodies can vary. The sporocarps are loaded with spores. Experienced mushroom hunters know how to make a spore print to identify a mushroom by removing the stem and placing the cap, spore side down, on a piece of paper or glass for 24 hours. Spores will be deposited onto the paper or glass and can be collected.
To establish ectomycorrhizal colonies, add propagules directly adjacent to plant roots or spray seeds with spore suspensions. For the home gardener and small grower, a much easier method is to cut the fruiting bodies into small pieces and mix them into potting soil. You don’t need more than a few spores to inoculate the soil, and it is impossible to use too much. This is a great way to grow a single strain or to make your own mix.
