| By Marc-Andrew Donsky
Perhaps this will be the year when you devote a corner of your garden to growing your own portabelas, criminis, shitakes, shaggy manes, Lepiotas, oysters, enokitakes. In this column we will give a basic outline of mushroom cultivation techniques and try to answer your questions about cultivation.
Each column will briefly cover a different aspect of mushroom cultivation:
Growth on Agar
Growth on grains
III. Growth on Compost
Pests and Diseases plus culture maintenance
Growth on Logs
I hope cultivation corner will provide stimulus and help you establish your own cultivation corner. Happy hunting and growing!
Growth on Agar:
The first phase of mushroom cultivation is the
isolation of a pure culture of mycelium. Nutrient agar provides the medium on which this takes place. The two most commonly used nutrients agars for mushroom cultivation are Potato Dextrose (Yeast) Agar (PD(Y)A) and Malt-Extract Agar (MEA). Potato Dextrose (Yeast) Agar is most often used for tissue culture and Malt-Extract Agar is usually the medium of choice for spores.
PD(Y)A should be sterilized for 45 minutes at 15 pounds pressure. MEA should be sterilized for 30 minutes at 15 pounds pressure. Sterilization of MEA for more than 30 minutes can lead to
caramelization of the malt sugars. Most mushrooms prefer a neutral to slightly acid pH. That is a pH of 5.5 to 7.0. These two recipes should result in medium within this pH range without further adjustment. Petri dishes, baby bottles and baby food jars all make good containers for agar.
Potato Dextrose (Yeast) Agar (PD(Y)A):
Potato Water 1 liter
Dextrose 10 grams
Nutritional Yeast or Yeast Extract 1-3 grams (optional) Agar- agar 20 grams Potato water is prepared by boiling a large, washed but not peeled, thinly sliced potato in water for one hour. The potato water is strained, brought to one liter of volume and the other ingredients then added prior to sterilization. Malt-Extract Agar (MEA) Malt- extract 15 grams. Agar-agar 20 grams
Water 1 liter Optional: Potassium biphosphate 0.1 gram and Calcium carbonate 0.1 gram Inoculation of Agar for Culture Isolation: Mycelia can be gotten from spores or tissue culture. Tissue cultures may be taken from any clean portion of the mushroom using a sterilized exacto knife or surgical blade. Spores may be started form pieces of gill tissue, spore prints, or direct spore drops onto to agar made by placing a portion of the mushroom with the gills toward the agar surface on the roof (cap, top,) of the petri dish using a touch of Vaseline.
Commonly used spore isolation techniques include:
A. Streak Method:
Spores are transferred to one portion of the
agar plate which is then streaked across with an
inoculation loop to another portion of the plate in order to separate individual spores and make the single colonies available for isolation.
Spores are soaked in cooled sterilized distilled water for several hours. Stir an inoculation loop in the spore solution and streak the petri dish, or using a sterile eyedropper put a drop of the spore solution onto the petri dish and spread. Agar to agar inoculations can be easily done using sterile technique with an exacto knife, surgical blade or an inoculating loop.
Once a pure culture of mycelium has been isolated, it is allowed to take over the agar dish. With mycelium grown from single spores it will be necessary to co-incubate two monokarotic cultures in order to generate the dikaryotic culture needed to yield mushrooms. This can be done by inoculating two pure spore cultures onto the same agar plate, or by placing the two cultures into the same jar of grain. If mycelium has been isolated by tissue culture of a carpophore, you are now ready for transfer to grain media.
Growth on Grain
For growing mycelium for spawn production and/or fruiting many types of grain can be used; rice, rye, milo, wheat, rye grass seed. One spawn recipe uses bran and perlite as a base. Again, many types of bran are acceptable. Rye grain is one the most preferred grains for spawn production because it does not tend to cake together as easily as does rice or wheat berries. It is also one of the least expensive grains and is quite suitable to mycelial growth. Rice tends to form solid cakes when sterilized. These "patties" are excellent as fruiting tests for many varieties of mushrooms. Caking can be avoided to some degree by the addition of gypsum (calcium sulfate) (1-4 grams per 1/2-3/4 cup of grain) to the rice prior to sterilization. Another method used to avoid caking is to precook the grain, mix in the gypsum and then sterilize the blend.
The optimum moisture content for spawn grain is between 49-54% (not counting the water in the uncooked grain). For example, with rye grain use1 cup of grain plus 3/4 cup of water (236 ml/cup) are placed into a one-quart jar. The lids are loosely placed on the jars and the filled jars are sterilized at 15 psi in a pressure cooker for 45-120 minutes. It is important when using standard Mason jar lids to invert the inner dome so that the lid does not seal when the jars cool. Sealed lids cause the inhalation of ambient contaminants upon opening.
Calcium carbonate (chalk) can be added as buffer in the amount of one to three grams per jar, but its use is optional. Stamets recommends the addition of 1% (w/w) of a 1:4 chalk/gypsum combination. That is one gram of the chalk/gypsum to one hundred grams dry grain. When using these calcium buffers the volume of water should be increased by 10%. Stamets also advises soaking the grain for 12-24 hours prior to heat sterilization. This initial wetting will germinate heat resistant endospores. 4-10 hours soaking should be adequate. With healthy grain, the seeds will begin to sprout in 12-24 hours.
After the jars of grain have been sterilized, they are allowed to cool. They can then be inoculated with pieces of mycelium overgrown agar or with portions of sterile grain or sterile sawdust grown spawn. Immediately after inoculation, the jars are shaken to spread the mycelium throughout the grain. The jars are then stored in the appropriate environment (humidity, temperature) with the lids loose, to permit the exchange of gases. After 5-6 days, if growth seems slow or restricted to certain areas of the jars, the jars are again shaken to disperse the cells. Sometimes a third shake after 5 more days is required to ensure a saturated growth. Once the grain is saturated with pure mycelium it is ready to be used as inoculum for more grain, compost or sawdust medium, or it can be cased to induce fruiting.
Casing: The term casing refers to a non-nutritive soil-like layer which is put on top of a mycelium saturated grain or compost media. The casing layer helps to induce fruit formation, support the developing mushrooms and increase the fruiting yields. The casing also provides the moisture essential to the developing mushroom, and helps to maintain the appropriate humidity. A typical casing recipe is: 1 part peat 1 part vermiculite 1 part lime (calcium carbonate) This mixture is moistened to 70% water content and is then applied to the beds or jars to a depth of 1-2 inches. The casing is kept moist by light spraying, as needed; taking care that water does not soak into the mycelium below. We will discuss fruiting further in a later column.
III.Growth on Compost
By preparing compost, you are creating an ideal medium for mycelial growth. Basic mushroom compost is made up of wheat straw, horse manure and gypsum (calcium sulfate). There are a variety of optional ingredients that may be added. A brief outline of some materials used in making composts follows:
serves as a carbon source (carbohydrate) source wheat - considered the best - contains xylan oat, barley - break down more rapidly than wheat rye - breaks down slower than wheat also corn cobs, oak and beech leaves, etc.
Other Carbohydrate Sources:
Rice straw, molasses, brewer's grains, cottonseed meal (provides the fatty acid - linoleic acid -which is reported to stimulate yields.)
nitrogen source, provides organisms essential to composting horse - most commonly used, fresher the better poultry - higher in nitrogen and phosphorous than horse, not so rich in potash (provided in wheat straw), faster and hotter than horse, use dry pig and sheep - must be used before they become sticky - used partly dry
Other Nitrogen Sources:
Blood meal (dried blood), bone meal urea, ammonium sulfate ((NH4)2SO4) Gypsum: calcium sulfate (CaSO4) - essential to mushroom compost preparation - prevents the compost from becoming too "greasy" - by forming an equilibrium matrix with the water, also helps the colloids to flocculate producing a compost with a more granular structure with increased water holding capacity: provides Ca++ ions; a mineral essential to mushroom growth: helps to prevent the loss of nitrogen (from the breakdown of proteins during the act of composting) by chelating the ammonia
Optional Mineral Sources:
Superphosphate; is said to promote vigorous mycelial growth, but an excess may make the beds too acid too soon which depreciate the crop. 14 lbs./ton of compost should be added at the last turn. It should not be used if there are a lot of droppings 9 fresh) in the compost.
Sulfate of potash; used in synthetic composts. the ubiquitous calcium carbonate.
Activators; compost "activators" can be obtained from nursery and garden stores and assures the presence of the organism essential to composting.
The following recipes create about one half ton of compost. One half ton of compost will provide enough compost for about 60 square feet of beds (surface area). At least one quart of grain spawn per 15 square feet of bed surface should be used.
Sample Compost Recipes:
5 bales wheat straw, half a pickup (half ton) horse manure, third of a pickup of horse manure, 30 lbs. gypsum, 2 lbs. activator, 70 lbs. chicken manure, 4 lbs. Blood meal and 30 lbs. gypsum.
To prepare compost, the straw must be soaked for several days until it just about, but not quite, squeezes water out in your hands. The compost pile is then built by stacking alternating layers of straw, activator, manure and gypsum until all the materials are used up. The stack should be 4-6 feet high.
In about 48 hours the heap will begin to generate heat and will sink somewhat in height. By the fourth to sixth day the temperature in the interior of the pile should reach 160°F (71°C). Temperatures of up to 160°F are due to thermophilic organisms. Temperatures over 170°F are due to chemical bonds being broken as well as other chemical reactions. Temperatures over 160°F are undesirable. After the pile reaches a peak temperature the temp will then begin to fall and the pile should be turned. The pile is turned by moving the middle half third to the bottom, the top and sides to the middle, and the bottom to the top. If any parts appear excessively dry, water should be sprinkled on those parts at this time. There should be no need to add any water after the first turn.
The heap will again heat up and be ready for a second turn after six more days. It should now be turning a rich brown color. With the second turn, no water should be given unless there are very dry patches - wet sparingly. One more turn should complete the mixing but if the temp (peak) is above 130°F a fourth turn may be necessary, (some authors recommend even another turn). If on the final turn the compost is too wet or has a greasy appearance, more gypsum may be added.
When done, the pile should be brown to gold in color, open in texture, and have a rich humus smell. The straw should break readily when twisted, and the compost should be just moist enough to bind together when squeezed in the hand. Initially the compost will have an alkaline pH. When mature and ready for inoculation the pH should be between 7.0 and 8.0. The heating of the compost has pasteurized the compost by the action of the thermophilic organisms. These organisms will not grow at the lower temperature at which mycelium grows. With proper composting the resulting compost will be free from competing organisms. Insects in all their forms will be absent from the medium and the rapid growth of the thermophilic composters will have also eliminated bacterial and fungal competitors.
Inoculating Beds: The compost is then filled in boxes about 10-12 inches deep. The temperature should be 80°F or less and there should be no ammonia fumes present when the boxes or beds are inoculated (spawned). The compost is inoculated with grain spawn either by mixing throughout the compost in the bed or box or by sprinkling a tamped down box with spawn and then covering thin layer of compost. In either cased the compost and spawn are then tamped down and covered with moist newspapers or a sheet of plastic to retain the humidity. The inoculated compost should be allowed to sit for 2-5 weeks (until the mycelium has taken over the compost). It may be necessary to moisten the newspapers occasionally during this time.
When the compost is permeated with mycelium it is then cased for fruit initiation. A drop in temperature and increase in ventilation induce fruiting. As the mushroom and mycelium grows there will be a drop of pH from the excreted metabolites until the pH reaches 5.0-5.5 at which time mushroom production will cease. At this time, the boxes/beds should be removed and the area thoroughly cleaned and sanitized.
IV. Pests and Diseases plus culture maintenance
Pests and Diseases: Once you have gotten to the stage of mushroom production, you will now be faced with a continuing battle with pests and diseases. Among the pests, you will find insects, mites and nematodes.
Among the diseases and competitors, you will find many fungi and bacteria. [Note: The author believes that an organic approach to mushroom cultivation and pest control is best. He feels that proper growing conditions and good strain selection can produce healthy, robust mushrooms that resist disease and invasion by pests. He does not recommend the use of copper salts, fungicides or other pesticides. The use of these poisons can be avoided by proper composting and maintaining proper growing conditions. Information on the use of pesticides is provided because of their proven efficacy, reference within the mushroom culture literature, and common use by the commercial mushroom agricultural industry.] Most pests can be avoided by well done, clean composting. It is impossible to over emphasize the importance and benefits of strict adherence to tidiness and sanitation of the work area and continued attention to the cleanliness of the growing areas.
The compost should be constructed on a cement slab, or by some means kept from direct contact with the earth; sheets of plywood, plastic pools, flattened van tops can all serve as a barricade to soil bacterial and fungal contaminants and competitors, and help to minimize invasion of the compost by insects and other pests.
Peak heating of the compost will eliminate most fungal problems and insure that you start your cultivation with a medium free of insects. The addition of 1 lb. of copper sulfate per ton of compost will help prevent those diseases not killed by peak heating. Zineb is a selective fungicide that is often used.
Bacterial problems are generally a result of the combination of too high a humidity with a
lack of adequate ventilation. The mushroom caps must be allowed to dry after (between) misting/sprayings or bacterial contamination can result. Sometimes a weak (2%) chlorox solution will help in extreme cases.
Insect pests can be a constant source of irritation. Everything within ones' power should be done to prevent insects' access to growing rooms. Diazon can be used as a dust on casing for mites and flies. Malathion can also be used but tends to cause pinhead abortion when sprayed. Fly paper and bug lights may be helpful for those of us wishing to avoid the use of chemical insecticides but where there are flies in the air, there are larva in the mushrooms and/or medium. The use of diatomaceous earth in the casing layer can be attempted as a preventative approach to larva infestation. The immediate removal of contaminated boxes is highly recommended.
Mushroom Growing Today, by F.C. Atkins and Mushroom Growing for Everyone, by Roy Genders both have fairly good chapters describing the pests, competitors, and diseases, although both books rely on heavy applications of chemical pesticides to control these problems.
The Mushroom Cultivator, by Paul Stamets and J.S. Chilton devotes a large section to the description, causes and prevention of the contaminants and pests of mushroom culture. Stamets reports the use of tree frogs for insect control in his growing rooms in Washington State. Again, it should be stated: Most pests can be avoided by well done, clean composting. It is impossible to over emphasize the importance and benefits of strict adherence to tidiness and sanitation of the work area and continued attention to the cleanliness and environmental conditions (humidity and ventilation) of the growing areas.
Maintenance of Cultures:
Cultures can be stored on agar plates or slants, sterilized grain, or sterilized compost. Sometimes the cultures are submerged under sterilized mineral oil. The stored cultures are generally refrigerated and can be kept viable for years.
V. Growth on Logs
One of the first methods developed for cultivating mushrooms was the cultivation of Shitake mushrooms on logs-the first written record being that of Wu Sang Kwang of the Sung Dynasty of China (960-1127 AD). The advantage of log cultivation is the relative ease of the method and care of the logs, and the life span of the fruiting logs (2-3+ yrs.). The disadvantage is the unpredictability of the method (yield) and the length of time between inoculation and fruiting (6-12 months).
Some mushroom species for which log cultivation techniques are applicable include: Reishi or Ling Chi -Ganoderma lucidum; Maitake or Hen of the Woods-Grifola frondosa; Lion's Mane-Hericium erinaceus; Shitake-Lentinula edodes; Chicken of the Woods-Polyporus (Laetiporus) sulphureus; Oyster Mushrooms-Pleurotus sp.
Log cultivation involves the placement of "plug" spawn into suitable logs. Plug spawn is most conveniently to be found on fluted hardwood dowels. You can produce your own plug spawn by weighing a quantity of dowels into a mason jar; adding an equivalent weight of water; sterilizing as with grain spawn; and inoculating the dowels with mycelium covered agar, grain, or sawdust. If the logs to be inoculated are a different wood than the dowels then 1or 2 grams of the log's wood should be placed with the dowels. Sawdust spawn can also be use to inoculate logs; a special tool called a "sawdust palm inoculator" can be used to facilitate the process.
Hardwoods such as oak, cottonwood and elm are often recommended as standard for log cultivation. Thick-barked hardwoods are preferred over paper-bark woods such as birch. Maple and alder are also frequently seen in the literature. Plum wood is given mention in the cultivation of Reishi Mushrooms. Logs should be cut to lengths of 3-4 feet, three weeks to three months prior to inoculation (plugging). Stumps can also be used. The period of time between cutting the logs and inoculation allows the naturally released anti-fungal (wound compounds) terpenes and (poly) phenols to degrade so that they do not inhibit the colonization of the log by the mushroom mycelium. Logs and stumps can be inoculated any time between last and first frost. Holes, 2 inches deep, of the same diameter as the plugs being used are drilled into the logs, 2-4 inches apart. Stumps are inoculated around their circumference into the sapwood (between the bark and the heartwood). One mycelium-covered dowel per hole is then pounded into each hole in the log or stump.
Holes can be painted with cheese wax or beeswax to protect the mycelium during incubation. The ends of the logs can also be painted with wax if moisture retention is a concern. (Note: in higher temperature climates, such as our own, the wax can melt into the dowel holes and potentially cut of the availability of air to the mycelium. Thus, there is a toss up between the advantages of moisture retention/mycelium protection vs. adequate aeration of the developing mycelium.) After the logs are plugged, they are stacked in a suitable location in crisscross piles called "ricks" for incubation over the next 6-12 months. The rick stacking helps to maintain an even moisture content/environment. The logs can also be covered with tarp (not with plastic) to help conserve moisture. Beds of (and insulation with) sawdust and/or wood chips is a very effective and simple method of environmental control. During incubation, logs should be watered once or perhaps twice a week as required to maintain an optimum humidity.
After 6-12 months of incubation, the logs are checked for mycelia growth by chipping away an area of bark around one of the plugs. If mycelia growth is evident fruiting is initiated by soaking the logs by submerging them in a tub or tank (or watering with a sprinkler) for 24 hours. Chlorinated water should be avoided for fruit initiation although it is acceptable for general watering of your logs. The logs are restacked in an arrangement that allows fruiting from and access to all sides of the logs. Watering is continued 2-3 times a day (2-3 times a week for partially buried logs and stumps) or as required- depending upon the prevailing weather conditions. Mushrooms should begin to form two weeks after initiation. After each flush, allow the logs 2-3 week dormancy period before reinitiating fruiting. This cycle of fruiting and rest can be continued throughout the growing season. Some hardwood logs have been reported to bear fruit for upwards of 3 or more years although the size of the crop decreases each season.