Mushroom Cultivation And Application Course 226w3w

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Pressure cookers and steriuzers

A good pressure cooker (PC) is essential for successful cultivation. There are a variety of brands, de signs, and sizes available. Used ones can be found at thrift stores and garage sales though all parts should be inspected for cracks or excessive wear. A cracked pressure cooker can explode! All American is a popular brand with mycologists as they are well built and use a metal-to-metal system (instead a rubber O-ring that can wear out). The difference between the All American pressure cookers and their pressure sterilizers is, essentially, the pressure control and release system. Instead of using a rocker-top pressure release system used in pressure cookers, the sterilizers use a toggle system to better control the pressure. These toggles can be ordered online and used to replace a rocker top release. Pressure cooker dial gauges should be routinely checked for accuracy at your local county extension office. If scaling up, an electric, automated pressure cooker or pressure sterilizer such as those made by All American and Yamato Scientific will help avoid the hassle of monitoring pressure levels and constantly adjusting a heat source. PID-automated systems can also be home-made. Autoclaves are laboratory de vices that perform the same function as a pressure cooker; they may be small or enormous. Industrial mushroom farms use walk-in autoclaves that are able to sterilize hundreds of pounds of substrate. Propane tanks can be converted into pressure cookers. 55- or 85-gallon stainless steel drums or 60 gal lon steel wine barrels can be modified to serve as sterilizer units heated by steam that is either generated inside or piped in. In these home-made solutions to the bulk sterilizing question require great caution as a metal vessel that is cracked and/or under improperly controlled pressure can blow up! Lids and Filters The four main materials for filtering air for spawn jars or bags include: • Filter discs are a bit old school in my opinion as they are so prone to wicking moisture that they can easily allow contaminants to though them and onto the substrate. Once these filters are infected with mold they are best to be thrown out, adding to their already high cost. Alternately, they can be soaked in a 10% bleach solution then rinsed and dried between uses. • Micropore tape is adhesive tape with microscopic holes that allow for gas exchange. Commonly found in first aid kits, this tape can be purchased online and is not durable enough for repeated use. But it works.

• Tyvek can be purchased onUne from kite supply houses, bought as envelopes from office supply stores, or gotten from the Post Office in a pinch. This material is breathable and works well for a couple uses, but is cumbersome. A double layer is preferred. • Quilt Batting is a synthetic material used to stuff animals, quilts, and pillows. It does not absorb moisture and due to its malleable nature is versatile for many applications. "Airport" Style Lids

"Airport" lids were developed by Hippie3, founder of Mycotopia.org. They are a major innovation in the world liquid culture work. In essence, holes are drilled into canning jar lids and then plugged up with a glob of high-temp (a.k.a. RTV) silicone / gasket maker to become self-sealing injection sites that enable

needles to aseptically introduce or extract liquids. I prefer the simplicity of a 5/16" holes for the silicone and a 3/16" hole for an air filter. The fancy "Morelman" lid has a feeder pipe and its a more elaborate design. 25

Scheduling

and

Planning

If you wish to cultivate large quantities of spawn or mushrooms, it is helpful to gain a thorough famil iarity with the time requirements of each stage in the cultivation process. It is easy to get ambitious and spend a late night inoculating a bunch of grain jars only to find yourself overwhelmed two weeks later with mycelium and unprepared for the next stage in the process. Don't make this common mistake! Start small and slow and develop the habit of thinking ahead for all of your projects. • Agar growth: 5-21 days • Liquid culture growth: 10-30 days • Quart jar of grain growth: 7-14 days • 5 lb. nutrified sawdust kit inoculation to fruiting: 3-12 weeks • Oyster straw kit inoculation to fruiting: 1-4 weeks

Once you gain a familiarity with your cultivation process and develop a rhythm, it is possible to de velop a cultivation schedule that keeps your incubation and fruiting spaces full but not overcrowded. The best route to take is to first select your target species and gain familiarity with its growth habits and

then develop a cultivation calendar for that species that matches your storage capacity, climate, substrate availability, weather, and budget.

Other work scheduling factors to for include preparing, soaking, and cooking substrates; sourcing, ordering, acquiring, and unloading supplies; building infrastructure; cleaning and maintaining workspaces; and outreach/marketing. All of these processes and energy commitments quickly add up to the fact that moderate scale mushroom cultivation can be a relatively time-intensive practice. However,

working with others, using the best tools for the job, and acquiring proficiency and familiarity with tech niques all increase efficiency. Stick with it. It does get easier.

26

Treating

Substrates

Pressure Cooking Step-by-Step Good pressure cooker usage is essential for effective and safe cultivation. This is the first skill that must be learned before all others.

1. Put enough water in the bottom of the PC so that by the end of the pressure-cooking there is still some water left. For most runs, 0.5 inches (1.25 cm) of water is sufficient. Never run the pressure cooker dry! 2. Place your materials in the PC and securely close the lid. If using a "rocker top" pressure release system, leave the weight off the vent port. If using a toggle pressure release, open the toggle. Turn the heat source to a level that requires 15 minutes to before steam begins to flow from the vent. Heating the PC too rapidly can cause jars to break. 3. Allow a steady jet of steam to escape from the pressure release vent for 1-5 minutes and then place the weight on the vent port or close the toggle. This ensures that everything heats evenly. The PC will come up to pressure in 10-20 minutes. Once the desired pressure is reached (typically 15 psi), reduce the heat level on the burner until the pressure stabilizes. It may take a few minutes of ad justing the heat to get it just right. If using a rocker top, adjust the heat source so that the rocker maintains a slow, steady rocking motion and/or jiggles once a minute or so. 4. Once the pressure is stabilized, start your timer. Unless you are using an electric PC, you will need to sit with the PC during the entire run to make sure the pressure remains constant and to adjust the heat level as needed. Cook your materials for the specified run time. If you are above 2000 feet (6000 m) in elevation you will need to add 5% to the cooking time for every 1000 feet (300 m) (i.e. at 3,000 feet [900 m] add 5%, 4,000 feet [1,200 m] at 10%, etc). 5. When the run time is over turn off the heat source and walk away to let the PC cool and de-pres surize on its own. I do much of my PC work at night so that everything is cooled by the following morning. F r a c t i o n a l S t e r i l i z a t i o n / Ty d a l u z a t i o n If you do not own a pressure cooker, the process of fractional stmli^tion or tyndalli^tion is a slow, old school means of sterilizing substrates. Here, sealed vessels are heated in a steam bath for 30 minutes, three days in a row. Between steam treatments, the jars are kept warm (85-98°F [30- 37°C] is ideal, but room temperature will work). This heating and incubating process germinates heat-resistant endospore bacterium, making them vulnerable to latter heating stages. 1. Place vessels in a cold, shallow (2-4 inches [5-12 cm]) water bath inside of a pot with a heavy lid. Glass jars should be resting on a small towel or on canning jar rings to keep them off the bottom of the pot. 2. Bring the water to a boil to heat and steam the vessels at 212°F (100°C) for 30 minutes. Living bacteria and fungal spores are destroyed in this treatment but endospores survive. 3. Incubate the vessels overnight to germinate dormant endospores. 4. The next day, heat and steam at 100°C for 30 minutes to kill the germinated endospores. 5. Incubate the vessels for a second night to germinate the remaining endospores. 6. The next day, heat and steam at 100°C for 60 minutes to kill the remaining endospores.

27

Mttshroom

Nutrition

and

Substrate

Formulation

Fungi, like animals, do not produce their own energy by photosynthesis and must find it externally.

Unlike animals, fungi do not digest their food internally, but only absorb simple compounds from their environment. Most of the food cultivated mushrooms consume is composed of complex and insoluble

organic compounds, (e.g. cellulose, lignin, pectin, and starch). In order for this food to be utilized by the fungus, it must break these substances down into simpler soluble molecules that can be transported through the fungus' cell walls. As a cultivator we must ensure that the substrates we feed the fungi have a properly balanced nutri

tional profile. This process of balancing nutrients is called substrateformulation and is critical to successfully producing high yields. Below are short explanations of what nutrients fungi need to live. • Oxygen — Mushrooms are aerobic, meaning they require oxygen for their pimary metabolism. Oxygen often comes in the form of carbohydrates, alcohols, and amino acids. • Carbon — Carbon containing compounds are generally the "energy source" of organisms. Fun gi use the carbon sugars and amino acids for energy and to build their cellular structure. In plant waste, cellulose, hemicellulose, and lignin are the main sources of carbon for the fungi. • Nitrogen — Nitrogen forms the backbone of the numerous enzymes that fungi produce to de fend, digest, and metabolize their substrates. Enz5Tnes are a type of protein that accelerate and/ or catalyze chemical reactions; they are responsible for facilitating the vast majority of the chemi cal-based functions of fungal growth and digestion. Without nitrogen, fungi cannot perform these

functions or form chitin. The amount of available nitrogen in a substrate is thus a major limiting factor in the cultivation of mushrooms: when the substrate runs out of nitrogen, the fungus stops growing. To increase yields, cultivators intentionally add extra nitrogen to their substrates, often in the form of wheat bran or manure, depending on the species being worked with. Too much nitro gen can be counterproductive however as it can lead to abnormal growths, cause contaminants to proliferate, or enable the mycelium to grow so fast that it overheats in its container and kills itself. A concentration of 1-2% nitrogen is generally recommend for most substrate formulas. Organic forms of nitrogen, such as proteins and amino acids, are preferred. Urea should only be used to feed nitrogen to hot compost piles (don't pee on your mushrooms). • Sulfur — Fungi need trace amounts of sulfur to make the animo acids cysteine and methionine.

This can be provided in the form of biotin (vitamin B7) and thiamine (Bl). Many fungi cannot synthesize these compounds, so it needs to be supplemented in some form.

• Phosphorus — Used in ATP, nuclei acids, and cell membranes.

• Potassium — Critical to certain enzymatic process. • Magnesium — Critical to certain enzymatic process, including ATP.

• Vitamins — Fungi cannot produce as many vitamins as plants and need some supplementation, most notably with several B vitamins. The addition of yeast in media is helpful in this regard, and is only needed in low concentrations.

• Trace minerals — Zinc, Copper, Manganese, Iron, and Molybdenum are all needed in trace a m o u n t s .

This workbook contains several good starting points for substrate recipes. However, at the farm level, cultivators should design trials comparing various substrate recipes using available resources to determine the most cost effective means to achieving maximum yields from a given species and strain of mush28

room. Factors that can influence the nutrient content of a substrate include:

• The quality of soil the substrate was grown on — Soil devoid of trace minerals will produce plants (and plant "wastes") that are devoid of these important elements. • How THE PLANT WAS GROWN — The quality of your substrates translate to the quality of your mushrooms. If possible, organic ingredients are encouraged to discourage the accumulation of heavy metals or chemical residues in or on the mushrooms. • The method of processing the substrates — Many food processing methods and substrate preparation methods leach sugars or other nutrients out of substrates, requiring the cultivator to later add these missing nutrients back in the form of a co-substrate. • The actual plants or substances used — Some plants, on average, produce and/or retain high er levels of certain compounds than others. The measurement of acidity/alkalinity (pH) of a substrate is also important to mushroom growth. The pH scale goes from 0 (very acidic) to 7 (neutral) to 14 (very basic). Most mushrooms grow in the 4-8 range, with an average around 5.5-6.5. Some species are more tolerant of growing in a range of pH levels. Some are less tolerant and grow best in a narrow range. PH can be measured using a variety of test papers and meters. While the tried-and-true substrate recipes presented in this work book do not generally require pH measurement, the pH of novel substrate formulas should be tested using test papers or meters and ad justed as needed. Generally a substrate is too acidic and needs to have its pH raised with the addition of an alkalinizing supplement, such as hydrated lime. The digestive products of fungal digestion tend to acidify substrates. If the fungus makes their substrate too acidic, it will be no longer be able to grow. For this reason, cultivators may add a pH "buffer" that helps stabilize a substrate's pH regardless of the acidifying effects of the fungus. M e a s u r i n g a F o r m u l a ' s S u c c e s s W i t h B i o l o g i c a l E f fi c i e n c y The success of a given substrate formulation is measured by the quality and weight of the mushrooms it produces, relative to the weight of dry materials used to make up the substrate. This comparison of fresh mushroom to dry material weight is known as measuring the "biological efficiency" (BE) of the substrate. For example, if 5 pounds of dry materials produce 4 pounds of fresh mushrooms, the sub strate/strain combination is said to have an 80% BE rate. Six pounds of mushrooms produced from 5 pounds of dry substrate reflects a 120% BE. Some substrates and strains may produce a 200% BE,

though 100% BE is the minimum hoped for from most species/strains.

W

29

Stage 1: Agar Work Agar is a seaweed-derived, gelatin-like substance. For cultivation work it is mixed with water and other

nutrients, sterilized, and then cooled inside of small containers to form a semi-solid horizontal platform on which a mycelial network will grow two-dimensionally. Commonly referred to as plates^ petri dishes filled with nutrified agar are inoculated with spores or a piece of mushroom tissue. Once a contami

nant-free mycelial network is established (usually after a week or two), pieces of the myceliated agar are moved from the plate to another plate or to a different substrate.

Working with agar is one of the most cumbersome and contaminant-prone stages of the cultivation process and, personally, my least favorite. However, it does have several distinct applications that make learning the skill of working with agar indispensible to the experimental cultivator. These include the ability to: • Remove competitor microorganisms from cloned mushroom tissue.

• Begin multi-spore inoculations to isolate and develop individual strains for experimental purposes. • View, compare, and facilitate the various responses of mycelium to chemicals and other organisms. Agar Media Formulation

Agar is nutrient-poor and must be supplemented with carbohydrates, proteins, vitamins, and minerals to ensure healthy mycelial growth. Common sources of these additives include:

• Carbon: Commonly provided by dextrose (corn sugar), light malt extract (from beer-brewing supply companies), cereal flours, oatmeal water, or the broth made from boiling potatoes. Some

cultivators prefer to mix carbon sources for more robust recipes. • Proteins: Cereal flour, soy peptone, and potato water are most often used to provide different proteins and amino acids. • B Vitamins: Supplied by baker's yeast or nutritional yeast. • Minerals: I typically add a pinch of azomite dust. A few drops of a liquid trace mineral con centrate is an optional, experimental additive. Just be sure the concentrate does not include silver, which is antifungal.

For most agar formulas, it is recommended to add 3-5 grams of the grain and/or fruiting substrate that will later be used to grow the fungus. Introducing these substrates early on stimulates the mycelium into producing the digestive enzymes it will later need to effectively break down the substrates, leading to quicker myceliation and firuiting times. When cloning wild harvested mushroom tissue, an optional additive is an antibiotic. Gentamycin (at 20 milligrams per liter) and streptomycin are two commonly used antibiotics that can withstand autoclaving and thus can be added to the agar at mixing. Food grade 3% hydrogen peroxide is a cheaper antibiotic option, but it does not withstand the high temperature of the pressure cooker. Hydrogen peroxide (H2O2) must be added to the agar once it has cooled to below 140°F (60°C) at a rate of 6-10 milliliters per liter of agar. This temperature can be measured with a clean

thermometer or an infrared thermometer. Some cultivators add activated charcoal to their agar at a rate of 10 grams per liter as a bacterial suppressant and spore germination promoter. It is recommended to

change agar formulas at each transfer of the mycelium to prevent senescence and maintain mycelial vigor. Five hundred milliliters of agar medium will fill around 20 standard petri dishes.

30

T h e A g a r Ve s s e l

Agar should be cooked in a glass vessel that can withstand the high temperature and pressure environ ment of the pressure cooker. Consideration should be given to how the hot liquid and container will be handled after coming out of the PC and easily and quickly poured into the petri dishes without making a mess. Messy plates provide a route for outside contaminants to follow and enter your plates. Pyrex flasks and or long neck liquor bottles can be used. These should be stopped up with polyfil fiber and covered with aluminum foil if they do not have a screw on cap. Plate Options

Pyrex petri dishes are reusable and can sometimes be bought for a deal on eBay. These should be washed, dried, wrapped in aluminum foil and then PC'd with the agar between each use. Plastic one-time-

use dishes can also be used though are less sustainable. Baby food jars are used by some. As long as the vessel can withstand the pressure cookery and can be easily worked with to extract samples of mycelium, the choice is yours for where the mycelium grows. If you Hke, pre-poured agar plates can be purchased online. The cost is increased over making your own but this may offset the time and frustration spent on plates with high contamination rates. Prepoured plates are often made with only Malt Extract Agar. Cooking and Pouring Plates

1. Mix and dissolve the ingredients on the stove at a low heat. The liquid should then be PC'd at 15 psi for 20-30 minutes. 2. If using a rattle style pressure release system, do not allow this to rattle too much. This can lead to boil over or caramelization of the sugars, which is toxic to the mycelium. 3. Once the PC is turned off, allow it to sit for 2 hours so the can agar cool a bit. 4. At that point the agar should be poured into your plates inside of a clean glove box or in front of a flow hood as quickly and cleanly as possible. 5. Stack the plates as they are poured to reduce condensation on their lids. 6. Cover this stack in the plastic sleeve the plates came in and allow them to cool for an hour or so. Optionally, place a cup of hot water on top of the top plate to save the top plate from excess con densation as well.

7. Once cooled, plates can be wrapped with plastic food storage wrap and refrigerated for long-term storage or inoculated and then sealed with a single layer of Parafilm and placed in the incubator / spawn run space.

8. You may also opt to place the plates in an incubator for 24 hours to see if contaminants appear. Inoculating Plates Once your contaminant-free plates are cooled you are now able to begin growing mycelium! You now have three main options for how to inoculate agar: 1. Obtain a commercial culture of a tested strain in the form of a petri dish or slant. 2. Clone a wild-caught or store-bought mushroom (or a retail piece of grain or sawdust spawn). 3. Use a spore syringe or spore print to start a multi-spore culture of various strains.

31

Commercial Cultures

Commercial cultxires are an easy starting place for new cultivators to begin cultivating for a few rea s o n s :

• The strain has been tested and is known to be a heavy fruiter or otherwise valuable. • No time to wait for spores to germinate and then to isolate a strain from. • Less risk of contamination compared to cloning. The disadvantages are: • Based on the species, strain, and company, a given culture costs anywhere from $20-1,000 or more! • Depending on its age under sterile conditions, the strain may have senesced to a degree. • The strain may not be well-suited to your local environment or climate.

It issuggested to copy and back up any commerical culture before expanding it, so as to preserve the state of vigor it was obtained at. The steps to doing this are the same as a Plate-To-Plafe Tranter, described b e l o w. Cloning a Mushroom

Under aseptic conditions, a small piece of mushroom tissue can be excised from the interior of a wild or store bought mushroom and then placed on a petri dish. The mushroom tissue will then revert back to a vegetative mode and myceliate on the plate. Materials

• A fresh or slighlty dried mushroom • A scalpel and set of tweezers • A clean petri dish • Parafilm

• Bacti-cinerator and sterilizing materials • Optionally, a shallow dish of 3% hydrogen peroxide M e t h o d

1. Following aseptic practices, unwrap your petri dish. 2. Wipe the mushroom cap down with alcohol.

3. Flame-sterilize your knife and make a slight cut in to the center of the mushroom cap. 4. Set the knife down and open the mushroom like a book, exposing the sterile tissue inside. Keep your hands under the mushroom and do not your hand over the inner tissue of the mush

room. Alternately, tear open the mushroom if that is easier. Sterilize your scalpel or tweezers and wait several seconds for it to cool.

5. Excise a small piece of mushroom tissue from either just above the giUs (but not the gills them selves), the cap interior, or the stem interior. These are areas where cells are rapidly elongating and growth is active. With some species it may be difficult to pull tissue from anywhere on the mush room. Do your best.

6. Optionally, once the tissue is excised, dip it in a small dish of 3% food grade hydrogen peroxide for 5—10 seconds to clean its surface. Many wild harvested mushrooms contain bacteria that in

terfere with current cultivation protocols. In reality, these microbes are likely somewhat beneficial

to the fungus, hence their presence. This cleaning process is recommended for wild, woody, and/ 32

or thin-fleshed mushrooms and/or their mycelium cloned from wood chips, cardboard, or other naturalized substrates.

7. Quickly open the petri dish and place the mushroom tissue on the agar. Mycelium tends to stick to tools. A good technique to easily remove tissue from a tool is to cut through the mycelium and into the agar, then slide the tool back and through the agar, leaving the mycelium behind. 8. Optionally, repeat steps 5-8 two more times on the same plate to ensure that you obtain at least one clone that will regenerate without contamination. I prefer to use smaller petri dishes for clon ing, so as to conserve agar. 9. Wrap the plate once, label it with species, strain (or harvest location), and date, and place it in an incubator or in a warm space to encourage rapid myceliation. Regrowth should be visible within 1—2 weeks.

Spore Prints & Syringes

Working with spores enables the cultivator to create fresh lineages of mycelium. These novel com binations of genetic information provide for the possibility of creating more vigorous strains that may grow faster, yield higher and taste better than the parent mushroom they were liberated from. However, the selection process for finding a good strain can be time and labor intensive. Materials for spore print work:

• • • • •

1 clean petri dish 1 clean spore print Inoculation loop Plate wrapping material Tool sterilizing materials

Working with a spore print on agar:

1. Clean the transfer area and prepare to work under aseptic conditions. 2. If the petri dish was in cold storage, allow it warm to room temperature in the transfer space. 3. Unwrap the plate. 4. Open the spore print and sterilize the inoculation loop. 5. Open the petri dish like a clamshell and stick the hot inoculation loop tip into the agar to cool it off and to make it sticky with agar. CarefuUy pull the loop out of the plate without touching it to anything. Close the petri dish. 6. Wipe the loop across the spore mass to deposit spores on to the tool. If spores are visible, thou sands of spores are likely on the tool.

7. Open the petri dish as minimally as possible and wipe the spores across the surface of the agar in a zig-zag pattern. Spread the spores. 8. Remove the tool.

9. Close and wrap the plate with a single layer of wrapping material. 10. Label the plate with species and date and place it in an incubator or spawn run space to initiate spore germination. Return the inoculum to storage. Materials for spore syringe work:

• A clean petri dish • A spore syringe that has been in cold storage and not exposed to excessive heat • Parafilm

• Alcohol flame and sterilizing materials 33

Working with a spore syringe on agar:

1. Unwrap your plate. Expose the spore syringe's needle and sterilize the needle tip using a heat s o u r c e .

2. Squirt a small amount of liquid out to cool the needle. A hot needle can destroy spores. Flick the syringe with your finger to disperse the spores inside of the syringe. 3. Use the clamshell method to open the plate and insert the needle tip without touching any of the plate's surfaces. Deposit 1—3 drops of spore water on to the surface of the agar. Remove the needle without touching anything and close the lid. 4. Place the lid back on the needle.

5. Swirl the plate around to spread the spores.Label and place the plate in your incubator or in a warm area and the syringe in the fridge. In several days visible areas of mycelium should appear. Various sectors will develop delineating the different genetic combinations that have occurred. Selecting a strain from a multi-spore plate

When using a spore syringe or print on agar, numerous combinations of genetic material arise that will each have their own characteristics and performance patterns. Termed "strains," each of these pair ings should be compared based on a variety of factors to determine which one is to be worked with. Typical factors for selecting the strains include: • Recovery time from transfers or shaking. • Quality of mycelium. • Adaptability to substrates. • Dependence on microflora. • Time from inoculation to fruiting. • Duration between flushes.

• Temperature and cold shock requirements. • Number of primoria formed and percentage that mature. • Medicinal quality of mycelium and mushrooms. • Ability to degrade toxins. • Antimicrobial activity. • Appearance, flavor, texture, aroma, nutritional profile, and shelf life of mushrooms. Deaung with Competitors Whether you are working with spores or tissue, you will soon see pure mycelium growing for the site of inoculation 3—21 days after inoculation. However, it is also possible that you might see bacterial or fungal contaminants arising as well, especially if an antibiotic was not incorporated into the agar. Luckily there are several options for dealing with these inevitable moments: 1. Cut out a piece of clean myceuum and transfer it to a new plate: This process is known as suhculturing or subbing. This second plate might also develop the contaminant, as the transferred piece may be "dirty." A third (and hopefully) final sub might be needed to obtain a clean culture.

2. If possible, cut out the contaminant: Most molds produce a white mycelium during their initial growth. If the cultivator learns to recognize these colonies as competitors, they can be cut out and removed from the plate. If the mold has gone to spore (i.e. the mycelium has taken on a

distinct color), attempting to remove the mold might shake more mold spores around the plate. Subbing is preferred in this case.

3. Cover the mycelium and contaminant in hot agar: Just like it sounds. The mycelium will like ly climb up through this new layer faster than the mold. The mushroom can then be easily subbed. Alternately, when you clone a mushroom, flip over a piece of the plate's agar to cover the clone from the start. This can help leave behind contaminants that may appear. 4. Use antibiotic agar to kill off bacteria: Place a fresh piece of antibiotic agar from another plate on top of the contaminant in a manner similar to a Plate-to-Plate Tranter. Under this antibiotic agar, the microbe will die and the mushroom mycelium wiU continue to grow. 5. Do nothing: Often, the microbial war that develops on the plate between fungus and foe will result in the mushroom mycelium effectively outcompeting the competitor. Watching this process unfold is in itself a great learning opportunity and glimpse into the habits of the mushroom. If the mushroom overtakes the contaminant, the mycelium should not be used as an inoculum for aseptic cultivation. It should still be subcultured to ensure that a clean culture is obtained.

You can learn a lot about the quality of your transfer technique by observing where contaminants appear on the plate. If a contaminant shows up on the edge of the plate, it may have entered during the tissue transfer, during pouring and wrapping, or during storage. If the competitor shows up on or near where the mycelium was transferred, your mycelium or tool may have been dirty.

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Like a petri dish without the agar, sugar and nutrient rich liquid broth can be used as a 3-dimensionai media for mycelium to growth through. This process is commonly done industrially using large sterile fermentation tanks. For the smaller grower, canning jars with airport lids are an incredibly effective and inexpensive means to work with a liquid culture. The Pros

Compared to agar, LC has a number of advantages as an inoculum: • Liquid culture is cheap: No agar is needed, simple ingredients are used. The jars, lids, and s}^ringes used in the process are all reusable. Liquid culture broths can be sterilized in a hot water bath, avoiding the need for a pressure cooker. • Liquid culture is time efficient: There is no need to cook and cool agar for hours and there's non of the stress and mess associated with pouring plates. Once a healthy mother jar of LC is es

tablished, it can serve as inoculum for many months, essentially eliminating the need for constandy starting mycelial lineages with agar plates. • Liquid culture increases myceliation rates: Mycelium in a liquid suspension grows three-di-

mensionally, increasing growth rates when compared to the two-dimensional surface of an agar plate. When LC is aerated, the mycelial network breaks into a constellation of fragments, each with its own array of active hyphal tips. This is significant as, compared to an agar plate where the

mycelium is only active at the edge of a single colony, liquid culture inoculum is almost entirely comprised of leading edge mycelium. • Liquid culture inoculation rates are high: Liquid culture is sprayed onto substrates with a syringe. As the medium percolates through the substrate, the individual mycelial clusters distribute throughout the material to explode with growth at each point of . The result is a much

more even distribution of inoculum than that achieved by a piece or two of myceliated agar.

• Liquid culture work can be done anywhere: Using a syringe and airport lid, one can transfer mycelium to grain jars outside of an aseptic transfer space. Whole mushrooms can also be cloned in the open air using this method. This reduces the need for constantly preparing and maintain

ing a dedicated transfer space. This is perhaps the greatest benefit of this method over the many annoyances and costs associated with agar work. The ability to grow grain spawn in the absence

of a clean transfer space using liquid culture inoculum quickly translates to any home mycologist becoming a mushroom farmer. With this challenging step overcome, time and energy increases in supply, allowing for creativity to spur new applications for cultivation beyond the practices of food and medicine production. Liquid culture allows us to work, think, and grow outside the glove box. How will you work with its many benefits? The Cons

LC is my preferred inoculum for everyday use. However, it has some drawbacks compared to agar work: • It can be hard to tell if an LC is contaminated.

• If an LC is contaminated, it is very difficult - or near impossible - to clean it up. • Not easy to acclimate strains to novel compounds. • Not possible to isolate strains from multiple spore inoculations. Using spores in LC is also not

recommended due to a high chance of contamination. Preparing Liquid Media

1. Mix and dissolve ingredients in a pot over low-medium heat. 2. Once dissolved, fill a clean jar (or multiple jars) half-full with the solution. 3. Place a couple of marbles, a piece of broken glass, a crystal, a magnetic nail with its head cut off, or a magnetic stir bar in the jar. 4. Place an airport lid on each jar and cover them with aluminum foil. 5. Pressure cook the jar(s) at 15 psi for 15-20 minutes. 6. Once the pressure has reached 0 psi, the jars can be removed and allowed to cool. Alternately, allow the jars to cool inside the pressure cooker overnight. If you do not own a pressure cooker, boiling is an alternative means for sterilizing liquid media - just place the LC jars in a pot of cold water, submerging them half way. Cover the pot with a lid and bring the water to a boil. Boil the jars for 30 minutes then allow them to cool completely before inoculating. Be sure to run a blank jar with this method to confirm that you are effectively sterilizing the media. On Liquid Media Recipes

The most important points to consider are that the correct types of sugar are used and that the con centration of sugar is no more than 4% (4 grams of sugar per 96 mL water). This is barely sweet to the human tongue but is plenty sweet for the fungus. Any more can be toxic to the mycelium. Household sugar (sucrose) is typically not preferred by most species. Light malt extract and honey can be used alone, dextrose is not ideal as a sole carbon source. Additional nutrients can be added such as peptone and various flours, but they cloud the broth, making it difficult to determine the health of the mycelium and whether there are competitors present. Inoculating Liquid Media

Inoculating liquid media must be done with great care as a single bacterial cell or mold spore can ruin a whole jar of broth. Once a clear jar is established, working with the culture is much easier thereafter. Agar to LC

This process injects sterile water into a myceliated agar plate with a syringe, scrapes a small amount of mycelium off the agar with the needle and then sucks back up this mycelium water into the syringe to serve as inoculum.

Materials

• 1 jar with distilled water & an airport Ud • 1 jar with Hquid culture medium • 1 myceliated, clean petri dish • 1 syringe with 16 ga Luer-Lok needle • Aluminum foil • Flame sterilizer • Sterilization materials

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M e t h o d

1. Wrap the syringe and needle in aluminum foil. Cover both jar lids in foil. The foil prevents the filter from getting wet in the pressure cooker, which can lead to contamination problems later. Pressure cook both jars and the syringe at 15 psi for 15-20 minutes. Allow to cool. 2. During the cooking process oxygen was depleted from the liquid media and now needs to be reintroduced. This can be done using the methods described in the section The Growth and Aeration of a Liquid Culture, below. 3. Wipe both injection sites with an alcohol wipe or alcohol-sprayed cotton ball. Clean the sites well

but avoid excessive force as this may dislodge the silicone, enabling contaminants to enter the jar. Spray both silicone sites with alcohol. 4. Unwrap the syringe and draw up 5 milliliters of the sterilized water. Alternately, fill the syringes with water, wrap them with aluminum foil, and sterilize them with the LC.

5. In your transfer space, unwrap and open the myceliated agar plate. Quickly, and without touching any petri dish surfaces with the needle, point the opening of the needle down and inject 1 milliliter of sterile water onto a small portion of the leading edge of the mycelial mat. 6. Using the needle tip, scratch some of the mycelium off the agar, suspending it in the sterile water. Quickly and cleanly suck up as much mycelium and water as possible. 7. Spray the silicone port on the liquid media jar with alcohol and inject the mycelium water into the media jar. 8. Label and date the jar and place it in the incubation space. Mushroom to LC

In this approach we will be taking mushroom tissue directly from a fresh mushroom. If the mushroom is from the wild there is the risk of internal bacterial contamination.

Materials

• 1 jar with distilled water & an airport Ud • 1 jar with liquid culture medium • 1 fresh mushroom

• 1 syringe with 16ga Luer-Lok needle • Aluminum foil • Flame sterilizer

• Sterilization materials & alcohol wipe Method

1. Follow steps 1-4 for Inoculating Aâr to LC. 2. Wipe the mushroom stem or cap thoroughly with alcohol, spray the mushroom with alcohol and then insert the needle through the stem or cap. Or just tear it open and stab a clean piece of inner tissue.

3. Remove the needle and check to see if there is tissue in the needle shaft. If there is not visible

tissue, stab the mushroom again. Repeat until tissue remains in the needle shaft once it is removed from the mushroom. Try to be quick and avoid breathing on the needle.

4. Spray the LC jar port with alcohol again and insert the needle into the LC jar. Inject the piece of mycelium. 5. Label and date the jar and place it in the incubation space.

6. Optionally, consider sterilizing and inoculating multiple LC mother jars at once. This will help ensure greater success in the event that one of the jars becomes contaminated.Optional: Consider

sterilizing and inoculating multiple jars at once. This will help ensure greater success in the event that the one of the jars becomes contaminated. Growth and Aeration

Once the liquid medium is inoculated, I tend to let the jar sit undisturbed for 1-2 days, during which time the mycelium begins to recover from the shock of being transferred and reverts to vegetative growth. At this point the mycelium should begin to appear as a small cloud of tissue. As this network continues to grow it will begin to consume the dissolved oxygen in the liquid. If this oxygen is not re plenished, the mycelium may suffocate and rise to the surface of the liquid in search of air. To avoid this stress on the mycelium, LC jars should be oxygenated frequently. To dissolve oxygen in the liquid, swirl the liquid by hand or with the aid of the stir plate and stir bars. Take care to not get the lid's filter wet as this can enable contaminants to enter the jar. This agitation also breaks up the mycelium as it grows, thereby increasing growth rates and enabling easy extraction.

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Inoculum

Expansion

Plates and liquid cultures can be exponentially expanded to increase the mileage of your labors before shifting to grain or sawdust spawn. This saves the cultivator the time and risk of constantly pulling from reserve stocks.

Plate-to-Plate Transfers

An old method for inoculum expansion it to take a small piece of myceliated agar from a healthy plate and transfer it to a clean pre-poured plate. Ideally the new plate would have a different nutrient base than the first.

Materials

• 1 myceliated Plate • 1 (or more) clean, pre-pored plate(s) • Scalpel or metallic spatula • Flame and sterilization materials

M e t h o d

1. Under aseptic conditions in the transfer space, unwrap both plates. 2. Flame sterile your tool and allow to cool.

3. Open the myceliated plate and cut out a wedge of myceliated agar from the leading edge of the network.

4. Quickly open the new plate and transfer this wedge to its center. 5. Close the plate and wrap both with a single layer of Parafilm. 6. Repeat with more plates as desired. Depending on the size of wedge you cut, one plate can be expanded to 10 or more new plates. L i o u i d - t o - L i q u i d Tr a n s f e r s Like a plate, LC jars can be expanded exponentially, though this can be done outside of the transfer space. Again, changing the recipe of the medium is preferred. Materials

• 1 myceliated LC jar • 1 clean, sterilized, non-myceliated liquid media jar • Sterile syringe with large gauge needle (16 ga) • Flame and sterilization materials

M e t h o d

1. Select a healthly LC jar. 2. Wipe and spray the silicone ports on both jars with alcohol. 3. Flame sterile your needle and allow to cool. 4. Insert the needle into the myceliated jar, drawing out several CCs of mycelium rich LC. 5. Quickly insert the needle into the new jar of media and inject the LC. 6. Repeat with more jars as desired. 40

Culture

Storage

S h o r t Te r m S t o r a g e o f P e t r i D i s h e s a n d L C s

For short-term storage of plate cultures, place them in the refrigerator double wrapped in Parafilm. Plates tend to dry out within in a few months, so this method is not preferred for long term preservation of cultures.

Liquid culture jars can be stored in a fridge for 6-8 months (or longer). Some add a little H2O2 (approx. l-3cc) once to help prevent contamination. L o n g Te r m S t o r a g e S t r a t e g i e s A proper long-term storage strategy of fungal cultures is critical for ensuring projects and growing calendars are not interrupted by unforseen contaminant outbreaks of workplace disasters. Balancing be tween space efficiency for the cultivator and nutrient density for the fungus has lead to several approaches for long term storage. Slants

A common approach is to use test tubes filled with agar. These tubes have been cooled at an angle to provide a slanted surface for the myceHum to grow on (maximizing surface area in a concentrated vol ume). This works fine for 6 months or so, but many strains start losing viability when kept on a single nutrient for an extended period. Twice a year these cultures need to be moved to another slant with a different nutrient base. Some strains of Agaricus appear to start the dying process anyway, as though agar is not the media they prefer. Every 6 months or so the culture should be brought out of storage and allowed to sit out for 24-48 hours until signs of growth are apparent. A new plate is then inoculated with the slant and then a new slant from this plate, ideally each with a different nutrient base. Optionally overlaying the slants with sterile mineral oil keeps the sample from drying out and acts as an oxygen barrier. Inserting a small wood stick in the slant before PCing will provide a long term reserve for the mycelium in case it dries on the agar.

Sawdust / Spawn

Following the practice of making nutrified sawdust (described later), small baby-food jars can be filled 2/3 full with this blend, sterilized, and then inoculated under aseptic conditions. Once fully myceliated, the jars are refrigerated. A piece of this sawdust spawn can later be transferred to an agar plate to start a new mycelial lineage. Due to the substrate's densitty, this method can preserve cultures for more than a year. Distilled Water

Sterile and pour sterile distilled water agar plates (SDWA)(10g agar / SOOmL distilled water, 30 min. at 15 psi). At the same time sterilize small containers filled with distilled water (20 mL scintillation tubes work well).

1. Transfer leading edge mycelium from a healthy nutrified plate to the SDWA plate using a Plate-toPlate Transfer.

2. Once the mycelium has touched down and grown across the SDWA plate a little ways transfer a

piece of just the mycelium and SDWA (avoid the nutrient-rich piece that was transferred) to the container of sterile distilled water using a Plate-to-Plate Transfer. 41

3. Seal the container tightly and label it 4. Store the vials at room temperature away from direct sunlight. 5. In the distilled water environment, the mycelium enters a dormant state and remains in stasis. 6. To reanimate, simply dip a sterile tool into the sterile water and place a piece of the mycelium onto a nutrified agar plate. How I Back Up Cultures

I keep the following on hand at aU times: small nutrified agar plates sealed with plastic wrap, larger nutrified agar plates, sterile distilled water jars, distilled water agar plates, small nutrified sawdust jars, and slants that contain a coffee stir stick. I make up small liquid culture jars as needed as they require the greatest amount of storage space. To create multiple forms of backups, I first clone a mushroom or mycelium to a small petri dish. This helps to conserve agar in the event of contamination during cloning. I also clone to cardboard in case the mushroom doesn't regenerate on the agar. Once a pure culture is obtained (perhaps after several Plate-toPlate Transfers), I then make six backups from this young culture before moving on to spawn production. First, I inoculate a mother liquid culture jar, as this is the most sensitive backup, as well as a larger LC jar to be used as inoculum as soon as possible. Then I transfer pieces of leading edge mycelium into a nu trified sawdust jar, a distilled water agar plate, and three or four slants. If there is extra mycleliated agar I will also inoculate sterilized grains. Later, when the distilled water agar plate is barely myceliated, I transfer

pieces of this plate's leading edge mycelium into small jars of sterile distilled water. All of these backups

are kept in a dedicated refrigerator except for the distilled water jars. Replicates of slants are stored off site in the event that the main back-ups are damaged.

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Stage 2: Grain Spawn Work Once you have amassed some mycelium on a petri dish or in a liquid culture just, the time has come to transfer the mycelium to a jar of sterili2ed grains. These grains wiU provide a large amount of nutrients and minerals to feed the mycelium and, once myceliated, will provide a jar full of individual "seeds" to feed the next stage in the process. Selection of Grains

Rye berries, wheat berries, millet, and sorghum (milo) are common choices in commercial mushroom cultivation operations. Home cultivators also have success with spelt, popcorn, and whole birdseed. All of these grains are preferred due to their low levels of nitrogen and ease of preparation. Many other grains are too high in nitrogen, which can lead to overheating during mycelial growth or high contamina tion rates. Milo is preferred by some growers as it hosts over 30 types of vitamins and minerals as well as a small size, which provides for more points of inoculation for fruiting substrates. Preparation of Grains

Grains must be properly cooked and sterilized so that the mycelium can readily penetrate and myceliate them. There are many approaches to preparing grains for cultivation. Some people will simply mix water and grains in their jars and pressure-cook them immediately as such. Others prefer to soak their grains for 12-24 hours beforehand, as pre-soaking may help germinate dormant endospores of bacteria, making them more susceptible to the heat of the pressure cooker. Others rinse their grains several times to rid them of any dirt and debris that may make the grains stick together while also harboring contaminants. I myself have setded on a combination of these methods as it works consistendy for me. Depending on your altitude, environment, water and grain source, though, you may opt to adopt a modified version of this approach. However you choose to do it, these factors must be your guiding principles: • The grains should not be too hard. When you bite into a grain it should be a littie bit under cooked (al dente) for normal human consumption. There should be no hard center in the grain. In this state, the grain is fully saturated and supple enough for the myceUum to penetrate and digest it. • The grains should not be too soft. There should be a minimum of sprouted or burst kernels after sterilizing. Overcooked, burst, and/or overly wet grains make grains more prone to contami nation due to their protective outer layer being broken. • The grains should be easy to break up. Dirty or overcooked grains can stick together, making it difficult for the mycelium to grow and/or later be broken up for spawning. Pre-rinsing grains and adding gypsum helps to reduce stickiness. The following is a generic grain preparation process for 10 quart jars: 1. Measure out 10-11 cups of dry grains into a large pot. 2. Fill the pot with water and stir the grains to suspend any dirt and debris that is present on the grains. 3. Pour off this dirty water and continue rinsing the grains until the water runs clear. 4. Cover the grains with high quality water. 5. Cover the pot and let it sit for 12-24 hours. Some cultivators soak their grains in 50% strength coffee to add additional nutrients.

6. Place the pot on the stove and bring the grains to a boil for 5-10 minutes or until they are cooked 43

to the right consistency.

7. Drain the grains through a colander. This nutrient-rich water can be saved and used for incorpo rating into agar and liquid media recipes. 8. Toss the hot grains around until they have cooled and the excess moisture has steamed off of them. If you are cooking a large amount of grains, spread them on your substrate prep screen to speed cooling. 9. Add 1 tablespoon of g)rpsum evenly throughout the grains. Gypsum provides mineral supplemen tation while also helping to reduce the stickiness of the grains.

10. Fill each jar one-half to two-thirds full with the grains. A 16-ounce (475 mL) measuring cup and canning jar funnel significandy help to facilitate this process. 11. Seal each jar with an airport lid and cover the lid with an aluminum foil cap. 12. Pressure cook the jars for 60-75 minutes at 15 psi. Grains can also be tyndallized in the absence of a pressure cooker, but be sure to run a blank. 13. Turn off the stove and let the PC cool overnight. 14. Open the PC and remove the jars. Inspect each jar for cracks and/or an excessive number of burst kernels. Discard cracked jars. Notes on Grain Prep Soaking grains for 12-24 hours helps to germinate the dormant endospores of bacteria inside grains, making the endospores more susceptible to the heat of the pressure cooker. The amount of water used

to soak and cook the grains should be the minimum necessary for achieving properly cooked grains. If too much water is used while boiling, beneficial nutrients will leach out of the grains and be lost. Trial and error will help determine the proper amount of water needed for your grains. Fuel costs can be saved in this process if the grains are cooked to their proper state using a solar collector/pasteurizer. Smaller grains, such as millet, do not need to be cooked as soaking provides adequate hydration. These uncooked grains will be very wet and sticky on the outside and need to be dried off on a clean towel prior to ster ilizing. Brown rice, used for many commercial medicinal products, often turns out very sticky and needs extra attention. After soaking and cooking, spread the rice onto a clean towel and stir it occasionally with a spoon as it cools. Then load the rice into jars as gently and loosely as possible. After pressure cooking, lay the jars on their side to cool, occasionally turning and shaking the jars to minimize clumping in the rice. A small amount (5-10% by volume) of the fruiting substrate can be added to grain jars prior to ster ilization to help initiate the enzymatic expression that the mushroom will ultimately require to consume the substrate.

Te s t i n g m o i s t u r e c o n t e n t

If you are wishing to attempt a different method of grain preparation, consider measuring the mois ture content of your grain after preparation. To do this simply measure out 100 grams of prepared grains and them place them on a baking tray in a 350°F oven for 20 minutes or until bone dry. Weigh out the grains a second time. The difference in weight will correspond to the moisture content. Grain Inoculations

Once your grains are prepped, it is now time to introduce some mycelium that can grow on them.

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Plate-to-Grain

Much like a Plate-to-Plate transfer, this technique moves a wedge of myceliated agar to a cooked and cooled grain jar. Materials

• Myceliated petri dish (the mycelium should not be to the edge of the plate to avoid contamination) • Cooked, sterilized, and cooled jar of grains • Flame and sterilization materials

• Scalpel or spatula M e t h o d

1. Prepare your transfer space for aseptic work.

2. Shake the jar to loosen the grains. Shake the grains so that they are sloping in the jar. 3. Unwrap the myceliated plate and loosen (but don't open) the lid on the grain jar. 4. Sterilize your scalpel or spatula and allow it to cool. 5. Clamshell the petri dish open then cut out and extract a wedge of agar with your tool. Ensure that the piece has some amount of leading edge mycelium. 6. Close the petri dish lid. 7. Open the loosened grain jar lid as minimally as possible to drop the agar wedge in to the jar onto the lower end of the sloping grains. Do not place your hand over the opening of the jar. Try to not touch the tool to the jar. 8. Close the jar lid and tighten it down. 9. Gently shake the grains over the agar wedge so that they cover the mycelium. This move helps keep the mycelium from drying out as it recover from the shock of being transferred while also providing the fungus with easy access to the grains. 10. Wrap the plate. Label the jar with species and date and set to incubate.

After inoculation, grain jars should be left alone for several days. Soon, the introduced mycelium will begin visibly growing on and through the grains. Roughly 3-8 days later, when 25—35% of the grains are myceliated, the jars should then be shaken for 20—30 seconds to break up the developing mycelial network and distribute the myceliated kernels throughout the jar. This helps increase myceliation rates. While I prefer to only shake once, some growers shake their jars a second time at around 70% mycelia tion. Shaking at 90%+ myceliation often stunts the mycelium's growth, negatively impacting the success of later expansions. Liquid Culture-to-Grain

Using an airport lid, you can inoculate grains outside of the transfer space, lowering the cost of culturing while increasing the fun! Materials

Myceliated LC jar Sterile syringe with needle Sterilized grain jar(s) with airport lid(s) Flame and sterilizations materials

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M e t h o d

1. Clean the silicone ports on both jars with an alcohol wipe or alcohol dampened cotton ball then spray both ports with alcohol.

2. Insert the syringe needle into the LC jar and extract roughly 2-10 CCs of the liquid culture per quart jar of grains. Be sure you are drawing out mycelium and not just sugar water.

3. Withdraw the needle and insert it into the grain jar. Swirling the needle around gendy, spray the mycelium across the grains.

4. Repeat with each jar. If the transfers are taking a long time, spray the top of the uninoculated grain jars with alcohol again before inoculating. I prefer to use a large syringe for this process to reduce the number of times I enter the sensitive liquid culture jar. 5. Label each inoculated grain jar and set them to incubate. Grain-to-Grain

Grain spawn can be expanded to more grains before being moved on to final substrates. Whether spawning to more quart jars, Vz gallon jars, or gallon jars, an inoculation rate of 10-20% is recommended.

This process is very simple and may be done twice with relative ease for a given mycelial lineage, however pushing for a 3"^ or 4'^ grain generation is not recommended. Materials

• Jar of colonized grain spawn • Multiple jars of cooked, sterilized, and cooled grain spawn • Parafilm

Method

1. Under aseptic conditions in your transfer space, loosen all jar lids and arrange them for easy access during transfer. 2. Open the myceliated jar on its side to reduce ambient competitors from entering the jar.

3. Quickly and carefully open the lid of the first jar just enough to introduce 1/10-1/5 of the myce liated jar's grains. Rotating the jar as you pour helps in this process. 4. Close the lid on the freshly inoculated jar. 5. Repeat for each remaining new jar. 6. Tighten all lids. Label with date and species, shake to distribute the myceliated grains, and set the jars to incubate.

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Stage 3: Fruiting Substrates Depending on the species you are working with, myceliated grain spawn is transferred to a variable final substrate from which it can fruit. There are five main types of fruiting substrates: • Wood-Based Substrates:

• Plain, pasteurized sawdust • Supplemented sawdust (with or without wood chips) • Straw (with or without additives) • Miscellaneous organic wastes • Compost- and manure-based substrates Substrate Additives

Along with these primary ingredients, other additives can used to balance nutrient needs and increase yeilds. These additives are not always needed and can be overused, so it's important to understand what they are and why they help. • Gypsum (Calcium sulfate [CaSO]) — A flocculant, gypsum inceases fluffiness in substrates and reduces stickiness. It also provides sulfur and calcium, which help increase yield, speed, and myce lial health. It is typically dded at 2-10% by dry substrate weight. • Vermicuute or Coconut Coir — These are often added to compost and manure based substrates to increase texture, aeration, and moisture retention.

• Coffee — A fairly versatile additive, fresh coffee grounds can be added to most substates as a minor additive. Oysters do particularly well on this substrate alone. Weak coffee is used by some to hydrate substrates, such as grains. • Spent Malt — Distillery and brewery waste can be used as substrates. Brewery grains are often acidic and need to have their pH raised. The addition of dry vermiculite or newspaper may be an easy way to offset excess moisture levels. Lightly roasted grains are preferred for their sugar profile. • Agricultural Residues — Hundreds of byproducts from food and feed industries have been shown to be viable substrate options. • Worm Castings — Mostly used for compost and manuring loving species, castings add nitrogen, phosphorus, potash, and other nutrients. They are added at 10-15% by volume. • Chicken Manure — Mostly used for compost and manuring loving species, chicken manure is a rich nitrogen source and is only used at around 1-2% by volume. Substrate formulation is required when working with these alternative substrates. A quick search through journal databases can easily provide studies that utilized these and many other substrates for mushroom cultivation.

The following additives should be used in limited amounts: • Thiamin — One tablespoon per gallon of water. Adding too much B1 can throw off nitrogen levels.

• Humic/Fulvic Acid — Aids mushrooms digestion and stimulates growth. Use 1 tablespoon per gallon of water.

• Kelp — One-half to one teaspoon per gallon of pasteurization water. Adding too much will promote molds.

• Seaweed Extract / Seaweed — Adds minerals. Use up to 10% (by volume). • Pollen (not bee pollen) — Aids liquid inoculum growth. • Soybean / Sesame Meal — A long-term protein source and antioxidant with some anti-mold properties. Use at 2-4% of dry substrate weight.

• Soybean / Sesame Oil — Adds calcium, antioxidants, fats, and is possibly anti-mold. Only add small amounts (around 1 tablespoon per gallon of substrate).

• Vegetable/Canola Oil — Contain Upids and nutrients for mycelial growth. Use 1-2 teaspoons per gallon of substrate. Alkalinizing Agents

The various types of alkalizing agents all have different chemical properties. Care should be exercised with aU of the products as most are caustic and a skin and eye irritant. Carefully read and follow all man ufacturer directions exactly.

• Wood Ash —Just as it sounds. A cheap way to increase pH. Avoid questionable impurities from glossy papers, paint, adhesives, etc.

• Horticultural/Hydrated/Slaked Lime (Ca[OH] )2 — Produced by adding water to CaO. Causes rapid pH shifts, but is not long-lasting. When heated above 1077°F (580°C) it dehydrates, forming the oxide. Reacts with carbon dioxide to form calcium carbonate. • Pickling Lime — A food grade form of calcium hydroxide with no additives or preservatives. • Calcium Carbonate (CaCO )3 — Helps buffer pH for an extended time. It comes in the follow ing six forms: • Chalk — Soft in texture, chalk holds water well. Using a variety of piece sizes - from 1-inch

thick to dust - helps improve casing structure and provides the longest lasting buffering action. • Marl — Dredged from dry lake bottoms, marl is a soft lime similar to chalk but has the consis

tency of clay. It is a composite of clay and calcium carbonate with good water holding capacity. • Oyster Shell — Mainly calcium carbonate along with other minor ingredients. Ground oys ter shell is similar to limestone grit in its buffering action and its structural contributions to casings. Oyster shell should not be used as the sole buffering agent because of its low solubility i n w a t e r.

• Limestone — A sediment mineral composed mainly of calcium carbonate. It is similar to oyster shells.

• Ground Limestone — Generally, ground limestone is weaker than hydrated Ume, needing about 30% more to raise the pH by the same amount. Being cheap, it is the most widely used buffering agent for US Agaricus growers. • Limestone Grit — Produced in a fashion similar to ground limestone, limestone grit is rated

according to particle size after being screened through varying meshes. Limestone grit is an excellent structural additive but has low buffering abilities. A number 9 grit is recommended.

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Wood-Based Substrates Sawdust (Spawn]

From grain spawn the cultivator moves many of the commonly cultivated species on to either plain sawdust or to nutrified sawdust, depending on the course of action to be pursued. Five- to six-pound bags of this sawdust spawn are standard products from commercial farms. Two common options for making sawdust kits are: • Spawn grains to pasteurised plain sawdust that, once myceUated, is be used to lay outdoor beds, plug logs, or make bulk naturali2ed spawn. Some mushrooms can fruit directly from plain sawdust though yields are often not high. • Spawn grains to supplemented and sterilized sawdust (with or without wood chips added) which, once myceliated, is fruited from directiy. This route results in greater indoor yields compared to fruiting from non-nutrified sawdust, but is more sensitive to contamination. Pasteurized Plain Sawdust Kits If you do not plan to add supplements to your sawdust, you may simply pasteurize the sawdust, saving fuel costs and enabling inoculation to occur outside the transfer space. Pasteurized sawdust should be given a higher inoculation rate (closer to 20%) to compensate for the lack of readily available nutrients (especially nitrogen) in the wood. Once mycelaited, pasteurized sawdust spawn is not used in sterile work, but can be applied outdoors. Pasteurizing means that your substrate is only heated to 140-170°F for an hour, as opposed to the high (250°F) sterilizing temperatures obtained by pressure cooking. Pasteurization kills the mesophilic organisms that normally Uve in the 68-113°F temperature range. Pasteurization kills many immediate competitors that may either overwhelm your mycelium or slow down their growth while leaving aUve beneficial mi croorganisms (mainly bacteria) that help guard the substrate against other contaminants, such as molds. Some bacteria and molds still survive however, hence the high inoculation rate to ensure rapid myceliation. Further, pasteurized substrates should be inoculated as quickly as possible to avoid "spoilage." Pasteurizing Sawdust Materials

• Appropriate sawdust type • Non-chlorinated water source • Wire screen unit

• Heat tolerant bags • Pot of water

• Weight • Thermometer

M e t h o d

1. Set up your wire mesh unit and lay a 2-6" layer of sawdust on top of it. 2. Alternating between sprinkling water on the sawdust and mixing, obtain a rough field capacity W moisture content for the sawdust. 3. Allow the sawdust to sit for 20-60 minutes.

4. If the sawdust feels too dry at this point, add most water. If too wet, add more dry sawdust. 5. Once the proper moisture content is achieved, load the sawdust into heat tolerant bags and wipe 49

down the top of the bag's interior with a clean cloth. Roll down and tie off the tops of the bags. 6. Place the bags in a pot of water with a weight on top and achieve an internal temperature of 140160°F for 1 hour. This is best done by turning off the water once the temp reaches 120°F. It will continue to climb and should stabilize in the correct range. 7. After an hour, remove the bags and allow then to cool in a clean environment. 8. Spawn away! Steam Pasteurizers

For larger quantities of bags, a steaming unit can be used to treat larger volumes. One may also set up a small electric steam filled chamber to achieve pasteurization. The benefits of steam pasteurizing over hot water is that steam does not affect the moisture content of the substrate as much as water submersion.

Also, this setup can easily be automated with a timer. Simple designs rig up power steamers (such as the 705 Model Wagner Power Steamer) through heat tolerant PVC (VC) or copper pipes into a cooler, repurposed deep freezer, or insulated tote. A remote thermometer allows for monitoring of substrate temperatures without opening the unit. Inoculating Pasteurized Sawdust Pasteurized sawdust can be inoculated with myceliated grains outside of the transfer space. This is be

cause the sawdust is not sterile and thus not a blank slate. Plain sawdust also does not provide the readily available nitrogen that competitors need as the nitrogen is locked up in the lignin of the wood and the grain spawn is protected by a mycelial coat. Still, transfers should be done quickly in an environment that is as clean as possible to avoid contamination, which can occur with this technique. Materials

• 1.5-inch (4 cm) piece of 2-inch (5 cm) diameter PVC collar, or a paper towel tube • 1 myceliated grain jar

•Jars or bags of pasteurized sawdust • Rubber bands

• Synthetic fiberfil M e t h o d

1. Shake the grain spawn to break up the grains for transfer. 2. Open the sawdust bag and fold over its top as you set up. 3. Open the grain jar and quickly inoculate the sawdust. I use around 0.5-1 cup (100-250 mL) of myceliated grains for every gallon (4 L) of pasteurized sawdust. Close the grain jar.

4. Close the sawdust bag and shake the contents to evenly distribute the grains throughout the bag. 5. Insert the top of the bag through the collar. Fold the bag opening over and fill the hole with syn thetic fiberfil. Use a rubber band to secure this filter.

6. If you are inoculating multiple bags, open them all up first and quickly inoculate them, one after the other.

7. Label the bag and set to incubate. Inoculated sawdust kits can be stacked around 5—8 bags high in the incubation space to myceliate. Some species will form thick mycelial mats around the sawdust block while others do not. These kits take from 3 weeks to 3 months to fully myceliate, depending on the species. I do not shake the sawdust while it is myceliating as most species do not recover from this disturbance. 50

S t e r i u z e d N u t r i fi e d S a w d u s t ( a n d Wo o d C h i p ) The most common fruiting substrate for wood loving edible and medicinal mushroom species is a mixture of sawdust, gypsum, and a nitrogen supplement, such as bran cotton seed hulls, or other agricul tural wastes. This method produces higher yields compared to pasteurized, non-supplemented sawdust However, as the nutrient-rich bran is prone to contamination it must be sterilized and handled under aseptic conditions, adding time and fuel cost to the preparation and inoculation process. Preparing and Sterilizing Supplemented Sawdust

1. If adding woodchips (added 50:50 with sawdust), presoak them for 24 hours. Drain and save this liquid if you like for making grain spawn or agar later on. 2. Separately mix the dry ingredients of sawdust, gypsum, and bran in a bowl or bucket and slowly add water, mixing and testing often, until field capacity is reached. 3. Mix the chips with this moist mix. 4. Load into clean, autoclavable bags or jar/bottles. 5. Clean the top of the container's interior and close.

6. Place the containers into a your pressure cooker, using lid rings to space the bags from each other and the side of the cooker to allow for even steam penetration. 7. Pressure cook at 15psi for 2 hours. 8. Turn off the stove and allow to entirely cool before inoculating. A modified 55-gallon drum retrofitted with racks can hold 25-35 sawdust bags. With the drum lid in place, but not sealed, the bags are then steamed ("ultra-pasteurized") in the drum for 8-10 hours to ster ilize them. This steam can be produced from 7 inches (18 cm) of water in the bottom of the drum, or it

can be piped in from a pressure cooker or another metal drum. These kits can also be tyndallized for two hours at a time on three sequential days. 55-gallon drums can also be modified with pressure regulating systems and used Uke a large autoclave. These drums can only maintain a pressure of around 3 psi, which cuts cooking time down to six hours. A drum lid can also be rigged with a pressure gauge and petcock. This low-cost, low-pressure autoclave must be monitored as the drum could potentially blow up if left unattended! I n o c u l a t i n g a n d I n c u b a t i n g N u t r i fi e d S a w d u s t K i t s

Nutrified sawdust must be inoculated under aseptic conditions. As inoculating many tall bags in a small glove box can prove challenging, this is one process that greatly benefits from the use of a flow hood. Materials

• Jar of myceliated grain spawn • 5-pound (2.25 kg) bag of sterilized and cooled nutrified sawdust • Wire, impulse sealer, or electric food preservation sealing preserving unit M e t h o d

1. Shake the grain jar to break up the grains. 2. Under aseptic conditions, open the sawdust bag and grain jar. 3. Quickly pour approximately 1 tablespoon of myceliated grains into the sawdust bag without plac-

ing your hands over the opening of the bag. RolHng the jar while you pour helps the grains easily fall out of the iar. 51

4. If using a flow hood, fiU the bag with a plenum of air.

5. Seal the bag. This can be done by simply rolling down the bag and tying it with a piece of stiff wire. For larger operations, bags can be quickly sealed using a heating element such as those in an

impulse sealer or food preservation unit. Check for a proper seal by gentiy squeezing the bag and listening for a hiss. Reseal the bag or patch any holes with tape if needed. 6. Shake the bag to distribute the grains. 7. Label with date and species/strain and set to incubate. With the additional nitrogen, around 1 tablespoon of grain spawn is all that is needed to inoculate

these kits. In front of a large flow hood, an assembly line can be set up with multiple people to increase the efficiency of this repetitious process. Supplements kits are incubated standing up and with a slight air gap between them to minimize overheating.

Straw-Based Substrates A handful of mushroom species are commonly fruited from pasteurized, non-supplemented straw. These include the Oyster complex, Ekn Oyster, Pioppino, Paddy Straw, and some Shiitake strains. Straw

is an ideal substrate due to the physical structure of the straw shaft, which can both retain a high amount of water and yet be tightiy packed into a container without impeding airflow or mycelial growth. The following preparation concepts apply to other agricultural waste streams as well. If using alternative sub strates, be sure to review the earlier section on substrate formulation.

Shredding

Straw

Straw should be shredded to 1-3 inch pieces to provide the mycelium with maximal surface area. Creat

ing pieces any shorter risks creating dead-air zones inside the substrate where anaerobic rotting can occur. The most common ways to shred straw are: • With an electric, gas, or bike powered yard debris shredder. • With a lawn mower on a driveway/concrete slab. • In a large plastic tote or trashcan with a weed whacker. • With an inverted lawn mower fitted with a trash can basin.

Pasteurizing Straw

Straw must be treated before inoculation. For a home scale grower there are several options to pre-treating straw: • Pack shredded straw into a pillowcase and pasteurized in a pot of hot water on a stove. Maintain an

internal temperature on the straw of 140-160°F for 60 minutes. Once done, hang and allow to drip and cool for 1-2 hours. Do not squeeze. The drained water may be retained and used for soaking grains.

• Pack shredded straw into a burlap sack or large metal cage and place it in a 55-gallon drum filled with water heated from a propane burner below. Water should be heated to 180°F before the straw

is introduced, which will drop the temperature considerably. Lift the burlap sack or cage out of the drum and place 2 2x4s below it to suspend it above the drum. Allow the straw to drain and cool for

1-2 hours. This can be done with any large container fiUed with hot water and straw - get creative! 52

^

• Hydrogen peroxide soaking methods have been developed using high (27%) strength H2O2. I've heard mixed results and do not choose to pursue this approach. The method was developed by Rush Wayne and his booklets can be found online. • Hydrated lime baths are popular as they require no fuel and the lime water can be reused multiple times. Two or three burlap sacks of straw are placed in a 55-gallon barrel. One gallon of hydrated lime (with magnesium content below 10%) is mixed with water in a 5-gallon bucket, then slowly added to the 55-gallon drum as it is filled with water. Once the straw is submerged, it is left in the 12—13 pH water for 16 hours. • Cold fermation requires no heat or chemical additives. It is explored in the non-sterile cultivation section.

Straw Additives

Many industrial Button Mushroom farms hot compost straw with chicken manure and gypsum to create a cheap and consistent substrate. Moistened worm castings and leeched horse manure are other common straw additives used by compost/manure-loving species growers. As with the straw, these addi tives are pasteurized and not sterilized, so as to maintain their beneficial microflora. • Chicken manure is very rich in nitrogen and should only be used up to 1-2% by volume. • Horse or cow manure should be dried/leeched (to remove the ammonia from urine) prior to use. 30-40% "Hpoo" to 60-70% straw is a simple blend. Hpoo nuggets should be thoroughly broken up before being saturated to field capacity and pasteurized. • Worm castings can be added up to around 10% by volume. Spent mushroom kits can also be fed to worms, closing the loop of production. Preparation of these manures follows the same process as for pasteurizing sawdust or straw. It is best to reach field capacity beforehand and place the additives in to high temp-tolerant bags or jars covered with aluminum foil. Place these jars in cold water and bring up their internal temperature to 140-160°F for 1 hour. Once cooled, mix with the pasteurized straw, and inoculate and pack as normal. Inoculating Straw Once your straw is chopped, pasteurized, drained, and optionally mixed with an additive, it is ready for packing and spawning! Of the many innovative and creative forms that straw can be spawned and packed in to, below are two common routes. P l a s t i c B a g s a n d T u b e s Te k Materials

• Pasteurized and drained straw

• Clean plastic bag • Oyster grain spawn (4-12 quarts per bale of straw) • Knife or arrowhead

M e t h o d

1. If using a plastic tube roll, cut off about 3 feet of material and tie a knot on one end and cut a few small slits on this end. Zip tie or impulse seal the end closed. 2. Add a layer of straw to a depth of a few inches. As you fill your log with straw, hold the straw in

your hand, and gently sprinkle it down into the tubing, making sure it spreads evenly. You don't 53

want dense clumps of straw. These will produce anaerobic pockets in the tube and/or air cavities between them, which encourage fruiting inside of the tube.

3. Add a sprinkling of grain spawn (around 15-30 myceliated kernels) and then a couple more handfuls of straw.

4. Repeat this process. After each handful or two of straw, stop and pack the contents down really tight with your hand. It's important that the straw is packed tightly into the tube to enable the mycelium to move from one piece of substrate to the next easily. There's no need to stand on the contents or use equipment. Just push as hard as you can with your hands.

5. Continue to add spawn and straw until the bag/tube is nearly filled then tie it off, maintaining com paction. Tie it as tightly as possible. 6. Using a clean arrow head or sharp knife, cut 1" long, X-shaped slits around the entire surface of

the tube. Space these slits about 2-3" apart in every direction to allow for adequate airflow. Pucture boards can also be made to serve the role.

7. Label the bag/tube with the species and date then place it in the final fruiting space. Do not disturb the tube during incubation. Once pins begin to develop, turn up the humidity and air flow follow ing the principles outlined for Stage 4 and provide the cultivation parameters for the given species. 5 - g a l l o n B u c k e t s a n d To t e s Te k

Materials

• Pasteurized straw

• Oyster grain spawn • Clean 5-gallon bucket • Moisture containing system M e t h o d

1. Drill 15-20, equally spaced, 0.5-1" holes around the bucket along with 4, V4" holes in the bottom. 2. As with plastic bags and tubes, layer de-clumped straw and spawn inside the bucket.

3. Once full, apply weights to the top of the basket to keep the straw compressed then place the lid on top.

4. Place in a humid chamber or under a plastic bag loosely draped to provide some air exchange. 5. Mist as needed depending on the chamber and provide fruiting requiremens once pins form in several weeks.

Oysters by the Season

The various species/strains in the Oyster complex vary widely in their fruiting temperature require ments. Some require very low temperatures to fruit, while others are sub-tropical and will die under such chilly conditions. If you wish to grow Oysters year-round, the best way to avoid the excessive electrical

costs of temperature control is to grow with the seasons. The following is a generic calendar for fruiting some common species/strains in the temperate climate of the Pacific Northwest U.S. Adjust these time frames to match your local climate. • October — Jan: Brown, Cold Temp. Blue • February - March: Brown, Warm Temp. Blue • March - April: Warm Temp. Blue, Elm, Golden • May - Sept: Warm Temp. Blue, Elm, Golden, Pink, Phoenix 54

Manure-Based

Substrates

The same species that do well on straw supplemented with manure (e.g. Agaricus and Psikgihe spp) tend to do well on manure-dominant substrates. Additives are often provided here to increase aeration, moisture retention, or pH stability. For aeration, the most commonly used additives are coconut coir and vermicuUte. Coir is the shredded fiber of coconut husks. It holds many times its weight in water but does not decompose for years. It is pH neutral and inexpensive. Hydroponics stores and hardware stores sell it in compressed bales and pet stores sell it in dried bricks. Vermiculite is an artificially made water-absorbing material. For nutrients, gypsum, coffee, worm castings, oils, and other ingredients are often added, depending on the grower's resources and the species response to formulas Refer to the additives list earlier for more details on common additives. Of the many recipes availabe, the following are good starting places for further elaboration:

Inoculating Manure-Based Substrates Manure-based substrates are often spawned in shallow, horizontal trays as opposed to the bags and jars discussed thus far. Home cultivators often use plastic storage trays or baking dishes that have been washed with soap and water and then cleaned with alcohol. Larger operations use large trays that are of ten lined with clean plastic. These trays may be plastic, metal, or wood and 2-12 inches (5-30 cm) deep. A deeper tray can more substrate and thus a greater yield for the same amount of surface area. Trays are generally inoculated lasagna-style with thin layers of grain spawn between 0.5-inch (1.25 cm) layers of substrate. Alternately, grain spawn can be shaken into the substrate inside of a bag or large con tainer (similar to how pasteurized sawdust is inoculated) before being poured into the tray. Do not fill the tray to the top; leave 1-2 inches (2.5-5 cm) of headspace. Cover the tray with aluminum foil and poke a tiny hole in the foil every 4-6 inches (10-15 cm) to allow for modest gas exchange. Label the tray and set it to incubate. Once the substrate is fully myceliated in 4—10 days, a casing layer is generally applied to facilitate the greatest fruiting. Casing

To initiate pinning and mushroom formation, some mushroom species require the addition of a "cas ing" layer on top of their substrate. The best casing formulas are designed to: • Reduce water loss via evaporation off the top of the substrate. • Provide a humid microclimate that assists in the initiation of primordia. • Signal to the mushroom that it is running out of food and should fruit. • Provide a water reservoir for the maturing mushrooms. • And, in some species, to provide a bacterial microflora necessary to initiate primordia formation. Casing pH

Casing layers are made from a variety of highly absorptive, non-nutritive substances such as peat moss, vermiculite, and coconut coir. Maintaining the proper pH in a casing is important for fruit body devel opment as a pH out of range can impede a fungus' ability to grow properly, significantly reducing yields. Even if all prior steps in the cultivation process were successful, applying a casing layer that is too acidic or dry can ruin the project's yield. For most species, the casing pH should initially be in the 6.5-8 range. Most casing materials are too acidic and need to have their pH raised with a quick-acting alkalinizing ingredient such as hydrated lime. 55

Casing pH gradually falls to a less than optimal level after the first few harvests due to acids secreted by the mushroom mycelium. Adding a long-lasting, slow-release pH buffer mitigates this acidifying process and extends the harvest. On Casing Recipes

Peat moss is a main ingredient of most commercial casing mixes. However, peat is overharvested throughout the world, threatening the boggy ecosystems that it grows in. The next best alternative many home cultivators use is coconut coir, though many growers have issues with this material causing exces sive overlay. Biochar provides the same functions as these materials and can be experimented with as a casing ingredient. Mixing, Pasteurizing, Applying, and Incubating Casing Layers

Casing ingredients (not including pH adjusting additives) are hydrated to field capacity separately, combined, and finally pH adjusted using the slow addition of alkalinizing agents. Once the casing is pH adjusted, it is pasteurized in aluminum covered jars or, for large volumes, in heat tolerant bags in a steam er unit. Once cooled, the casing is applied to the trays of myceliated substrates to a depth of 0.5-1.5 inches (1.25-4 cm) for each tray. Deeper casing layers encourage a greater number of flushes but require a longer incubation time. Once applied, casing layers are covered with perforated plastic or aluminum foil and placed back in the incubation space. Over the following week or two, the mycelium will enter and myceliate the casing from below.

The cultivator must pay close attention to the casing and rate of myceliation. The fungus should ide ally myceliate the casing evenly. Fruiting should be initiated once the mycelium is just visible across the

surface of the tray. If any mycelium surfaces in one area of the tray sooner than in the rest of the tray, this visible mycelium should be covered with a small amount ( a "patch') of freshly pasteurized casing material to ensure even myceliation. Even myceliation of the casing layer ensures even primordia forma tion ("pin set") once fruiting is initiated, leading to higher yields. Overlay

If the cultivator waits too long and does not move the tray to the fruiting space as soon as the casing is evenly myceliated, the mycelium will grow up and over the top of the casing in search of food. Soon,

the mycelium will form a hard, dense, water-repellant mat known as overlay, which can significantly re duce yields. Overlay can also occur in the fruiting environment due to prolonged high C02 levels and/or

excessive humidity. If over watered, the overlay will become matted, or will form a dense layer of dead cells on the casing surface. Casing experiencing overlay will shrink and pull away from the sides of the container. It will also be come unreceptive to water and puddles may form on the surface after misting. If any primordia form,

they wiU likely do so at the edges of the casing. Most of the primordia will abort, and only a few mush rooms will fiiUy mature. Once this has happened, the casing layer really isn't a casing layer anymore. It is no longer serving its main functions, and has in essence become a second layer of non-nutritive substrate.

If your casing becomes overlaid, a rescue effort can be made by scratching the surface of the casing to a depth of a few millimeters with a fork or similar device. The entire tray can be also covered in a

thin fresh layer of casing in the hopes that the fungus will continue to grow and myceliate this new layer.

56

Pinning

and

Fruiting

Once your substrate is myceliated, environmental conditions are typically changed to signal to the mycelium that it is time to sporulate. The metabolic shift that causes a mycelial network to condense and form the highly structured mass of a fruiting body is a bit of a mystery. As such, current practices for bringing about this transformation rely on reproducing the environmental changes that trigger fruiting in the wild. These include:

• An increase in the relative humidity. • An increase in the fresh air (O2) / decrease in the CO2 concentration. • A decrease in the ambient temperature. • An increase in ambient light levels.

The first two factors are the most important in assuring high yields and the full maturation of fruit bod ies. In the design of all fruiting spaces, it is best to provide enough fresh air for the mushrooms to metaboli2e adequately, but not so much air that the humidity drops and the mushrooms dry out. If humidity levels are not maintained at the recommended levels, primordia may not develop or will abort prior to maturation. Humidity

As a fruit body swells from a cluster of cells into a mature mushroom, water is lost through evapora tion from its surface. If the surrounding air has a low level of humidity, water will evaporate too quickly from the mushroom, inhibiting maturation. Thus, control of humidity in the fruiting space is critical for obtaining a high yield. For many species, 90-95% relative humidity is recommended to initiate primordia

K ^ > formation, while 80-90% rh is needed for fruit body maturation. Some woody conks (e.g. Reishi) can mature at much lower humidity levels. There are three ways to measure and control humidity: • Hygrometer: Using a high quality human or synthetic hair hygrometer that is certified and accu

rate within +/- 5% is a fairly good and inexpensive means to monitoring humidity levels. Using this meter as a guide, humidity^ levels are adjusted in the fruiting space with timers connected to hu midifiers. Most hygrometers found in horticultural shops are inaccurate at higher humidity levels. • Eye it/lung it: Experienced cultivators can learn to tell when a fruiting chamber is in the 80-85% range. This appears as light condensations on the walls of smaller chambers along with an occa sional streaking of water. When inhaled, the air inside larger environments feels heavy and moist. • Humidistat: Devices that measure humidity and control electric humidification systems are used in many operations. Low cost humidistats tend to malfunction at the high humidity levels required for primordia formation, while more costly humidistats also get damaged by heavy spore loads. My advice: get a good humidistat if you can afford it, and keep the controller outside the fruiting space (the sensor would feed into the fruting room). For the technically included, automated Arduino systems can be used to control temperature, humidity, and C02 levels. Air Exchanges

Maturing mushrooms require a significant drop in ambient CO2 levels and an influx of O2 to properly develop. This increase in fresh air provides a clear signal that the mushroom should now develop a fruit body to sporulate. Many consider this environmental change to be the main trigger for initiating fruiting. Some species (such as Oysters) require a significantly greater amount of fresh air than other species. Other species, such as Reishi and Enoki, are intentionally fruited in high CO2 conditions to encourage the development of elongated stalks. 57

Lighting

Though mushrooms do not photo synthesize, many species need some amount of light to initiate fruit

ing and to provide a direction to orient toward. Some mushroom species are stimulated by specific wave lengths of light. Experience has shown that bright light with a color temperature of 5000-7000 K, such

as that produced by a 15 watt, full spectrum ("daylight"), compact fluorescent bulb is adequate. Lights are generally set on a 12-hour on/off cycle. Indirect sunlight also works as long as the mushroom does not heat up or dry out. Sunlight decreases energy use while also increasing the pigmentation and vitamin D content of some mushroom species. LED strings that produce 6500 K light can also be used. LEDs

lighting systems can run off of 12-volt power sources, providing the option for discrete, solar-powered, off-the-grid, sealed lead acid battery pack power based systems. Te m p e r a t u r e

Many species will grow and fruit at the same temperature, but some mushroom species/ strains require a drop in temperature of IG^F (5.5-C) or more to initiate primordia formation.

Or, they may need to be placed within a specific temperature range to initiate fruiting. Many species develop a darker color and more umami-rich flavor when grown in cold temperatures. For the small-scale grower, controlling the temperature of an incubation and/or fruiting space can incur undesirable costs. Simple strategies for helping drop the temperature at fruiting include: • Incubating spawn above a refrigerator or on the top shelves of a closet where the air is warm. At firuiting, move these kits to a cold room, basement, or shed. • "Cold shock" the mycelium by placing it in a refrigerator overnight. This is commonly done for some species, such as Shiitake. • Grow mushrooms seasonally, as noted earlier for Oysters on straw. The Fruiting Process

Once you have determined the scale and design of your fruiting space, the next step is to de termine how and when to move spawn kits from the incubation space to the fruiting environ ment. There are two basic options for making this crucial decision:

• Let the mushrooms fruit — Some species/strains will begin to fruit in the bag/ jar ("in vitro") once they have run out of food. Once fruiting is self-initiated, the bag is opened and moved to a fruiting environment set to the proper parameters. For mushrooms that form stalks and fruit vertically, all but 1-2 inches (2.5-5 cm) of the top of the bag is cut off, proving a slightiy C02-rich

environment that encourages longer stalk formation. For mushrooms that form horizontally, the bag is punctured and rolled down, while slits or X-shaped holes are cut in the side of the bag to allow the mushroom to develop unimpeded. These holes should be cut as soon as the substrate is

fully myceliated to minimize growth inhibition. Shiitake, Agrocyhe spp., Nameko, and Brick Cap can be entirely removed from their grow bags to the formation of more fruit bodies.

• Trigger the mushrooms to fruit — Some species/strains require a cold shock, overnight sub mersion in water, or both to initiate primordia formation. Otherwise, changing environmental conditions triggers fruiting. Harvesting

For many mushrooms, the ideal harvest time is just before or soon after the cap begins to uncurl or

the partial veil starts to tear away from the cap margin. Generally, the smaller the fruit body, the richer its 58

flavor. To harvest, twist the mushroom or cluster off of the fruiting substrate, then cut the stem butt off to dress the mushroom for consumption or market. Cutting the mushrooms in place will leave the stem bottom behind, which can rot and quickly cause contamination problems. After a fruiting kit produces its first flush, it is generally left in the fruiting space where, within a week or three, it will likely produce a second flush. A third or fourth flush may be possible, but most farms do not use their limited shelf space for these older kits due to the low yield gained from these later flushes. Pushing the Yield

A few tricks have been developed to get the most mycobang for a substrate's buck: • Dunking — Some species/strains will produce a greater yield if submerged in a bath of cold water for 6-12 hours prior to the first flush and/or between flushes. This water bath increases the substrate's water content, helping increase yields. This technique is frequently used for Shiitake as this species produces a hard crust of mycelium that can withstand such a degree of manipulation. Other species that form strong myceUum, such as the Oyster complex, Turkey Tails, Reishi, and even Psilog/he cubensis, are also commonly dunked. Most other species cannot easily withstand dunk ing as their mycelium is not very tenacious. • The rez effect — After flushes have ceased, myceliated substrates can be broken up, mixed with

additional nutrients (such as a pasteurized nitrogen source) and/or water retaining additives, and repacked into a vessel. If all goes well, this last ditch effort may result in a remyceliation of the substrate and further yields.

• Substrate sequencing — Once a primary decomposer has finished fruiting from a nutrified sawdust kit, a large amount of nutrients remain in the substrate. These spent kits can be broken up, supplemented with a nitrogen source (e.g. 10-15% bran), rehydrated, sterilized, and inoculated with another species to mimic the ecological succession of decomposition. After wood-loving species have been run through the substrate, the remaining material can then be used to make com

post for compost-loving species. Examples of sequencing include Lion's Mane/Shiitake/ Nameko to Oyster complex/Maitake/Enoki/Turkey Tail to King Stropharia/Shaggy Mane to Agaricus spp.

59

Making Spore Prints and Syringes Once your harvest has matured, select a few choice specimens to take spore prints from. Unless your mushrooms fruited in a closed sterile bag, the prints you take will often be contaminated to some degree. However, following the strategies below you will have the best chance for success. Spore Prints

spore prints are best made direcdy on clean aluminum foil. Materials

• • • •

Fresh mature mushroom cap Toothpicks, aluminum foil, alcohol Clean bowl, small zip lock bag Clean rag / lint-free cloth

M e t h o d

1. Using aseptic conditions, clean your workspace and wipe down with alcohol.

2. Place a fresh piece of aluminum foil down and spray with alcohol and allow to evaporate. 3. Place two fresh toothpicks on the foil 1-2" apart. 4. Wipe down your bowl's interior with alcohol.

5. Being as clean and quick as possible, cut your mature mushroom cap off the stalk and place on top of the toothpicks. This keeps the mushroom off the foil, reducing excess moisture that can cause spore germination.

6. Cover with the bowl and let it sit for 4-24 hours, or until a noticeable spore load has developed. 7. Fold the foil to seal it or place it in a small plastic bag.

8. Label with date and species/strain and store in a cool, dark, dry place until ready for use. Spore Syringes While sterile water could be injected into the spore print baggie you just made and then sucked back up to make a spore syringe, this method is a bit cleaner. Materials

• Fresh, clean, mature mushroom cap • Jar of sterile water with suspension wire cage or wire mesh cover • Airport lid and aluminum foil • Sterile syringe and needle M e t h o d

1. Pressure cook your jar of water with cage inserted and covered in foil for 15 minutes at 15 psi. Arguably this does not need to be pressure cooked but can also be brought to boil on the stove as there are no nutrients in the water. AUow to cool in a clean environment.

2. Once cool, open the foil and follow aseptic conditions, place your mushroom cap on the cage/ mesh.

3. Recover with foil and allow to sit 4-24 hours as the spores drop. 4. Cleanly remove the foil, cage, and cap and cover with a clean akport lid. 5. Flame-sterilize your needle and suck up the spore water through the airport.

L o w - Te c h

Cultivation

This section presents techniques that, while built off the concepts and theory behind aseptic cultiva tion, bend or break these "rules" to allow the home cultivator to explore other options for achieving the same end: mushrooms!

Spore Sprays Here, the spores of desirable mushrooms are collected, placed in water, and then spread across a sub strate in the hopes of establishing a mycelial network. This technique is simple, cheap, and easy to apply but, as with all spore work, has an indeterminate outcome. Some attempts will produce vigorous mycelial networks and substantial yields of fruit bodies. Other times, very little is wimessed after months of wait ing. Thus, I prefer to apply this technique when working with old or experimental substrates. For higher quality substrates, aseptic or bulk spawn is a better choice of inoculum. To bulk up a spore collection, develop a habit of collecting spore prints from all of the edible and medicinal mushrooms you harvest or cultivate. As these spores will not be used for aseptic work, their collection can be relatively dirty. Just be sure to keep the spores dry until you apply them, so as to avoid pre-germination. In the spring, scrape these spores into a watering can or sprayer filled with non-chlori nated water, and two percent dextrose by volume (e.g. 2 grams per 98 milliliter water). Allow the mixture to sit at room temperature for two days. During this time the spores wiU begin to germinate in response to the dextrose. After this incubation period, apply the serum to the desired substrate. I prefer to spray the mixture instead of pouring it, so as to spread the spores farther. Spraying also reduces the chance of the spores being washed away in a heavy flow of water. If you are working with a mixture of substrates or contaminated substances, try combining the spores of different species and/or strains to increase the genetic diversity of the mix and the ultimate chance of obtaining a tangible outcome. Spore sprays can be applied in uncommon outdoor settings in an attempt to develop strains tolerable of that environment. If luck is on your side, a fruiting strain will arise from a spore spray applied under such conditions. The spores from these first generation mushrooms can then be harvested and applied under the same conditions to create an offspring strain that is perhaps even more tolerable of the new environment than the parent mushroom. This self-selecting breeding process can be repeated over mul tiple generations in an effort to "speed up evolution" and develop strains that are highly specified to a given substrate or climate. Cardboard spawn This cheap and simple technique can be attempted for any species of saprophytic fungus. The glue in corrugated cardboard is corn starch-based and, thus, a viable food source for decomposer mushrooms and surprisingly not much else. What this means is if you follow the simple steps below you will get a bit of mycelium in not a lot of time, for not a lot of dough, and with limited risk of contamination. Great! This technique is best used with fresh mushroom pieces (preferably the stem bottoms, though any part

of the mushroom will likely work) or mycelial fragments. Done right, this technique is quite successful and easy to practice. Materials

• Mushroom stem bottoms and mycelial strands • Tape and ink free corrugated cardboard from the US or Canada • Plastic bag or food storage container • Optional coffee grounds or moistened sawdust 61

Method

1. Soak the cardboard in hot water until it is thoroughly saturated (approximately 30 minutes). 2. Remove the cardboard from the water and let any excess water drip off. 3. Remove one side of the cardboard, exposing the corrugations.

4. Evenly disperse your fungus throughout the cardboard (say, one piece every 3").

5. On top of this fungus layer, now place another layer of saturated corrugation and backing such that the fungus is sandwiched between two layers of corrugation. Roll the sandwich up. 6. Store the rolled cardboard in a dark, warmish place where it will get air exchange, retain its humid ity, and not dry out. One example would be in Tupperware container kept above the refrigerator (this space is often warm due to rising heat of the refrigerator's motor). Be sure to circulate the air

in the container at least once daily (if not more often) and to spray with water as needed to keep moist.

7. In several days or weeks the mycelium should start to grow out onto the cardboard, appearing as distinct or fluffy white lines.

8. Once the myceUum is actively growing is should soon be moved on to another food source. In my experience, this technique works for a short period of time before the mycelium begins to stagnate. Adding a sprinkling of coffee grounds to the cardboard can help provide an additional nutrient

boost and mycelial growth. However, I would recommend using cardboard spawn as a short intermediary before quickly moving on to bulk spawn (described below). N o - Te c h S p a w n

Following the basic principles and process of working with Plain Pasteurized Sawdust, I have had suc cess introducing myceHated grain spawn to hydrated, but non-heat-treated sawdust. This method works

well for King Stropharia, Turkey Tails, and the Oyster complex of species. As no heat is being introduced, one advantage to this method is almost any clean containers can be used in place of specialty bags or other containers.

Cold Fermentation

Simple and scalable, this method is a breakthrough in the field of low-tech cultivation techniques. This method kills off competitors to your spawn by the simple act of submersion in water over a period of days. During the submersion, the anaerobic bacteria present on the substrate thrives by eating all the aerobic (oxygen loving) bacteria and mold spores. When the water is removed after a week, the anaerobic

bacteria die, leaving "clean" substrates ready for inoculation. This practice is best for straw. Excessively soaked sawdust is too wet to use as a substrate.

Materials

• Chopped straw • A garbage can, 5-gallon bucket, trashcan, or similar vessel • 3 mil trash bag • Non-chlorinated water

• Clean stone or heavy weight Method

1. Line a garbage can (or any hard, upright container) with a heavy duty (3 mil) trash bag. 2. Wet the inside of the bag down with a bit of water. Ideally, this would all be non-chlorinated water 62

or, better yet, well water. 3. Fill the bag with dry straw that has been chopped into 2-3" pieces. 4. Place the chopped straw in the can with the trash bag and fill with water, covering the straw. 5. Add a clean weight on top to of the straw to keep it submerged. 6. Put a Hd on the can and keep it in a warm place. 7. Wait 7-14 days. 8. At this point the water should be discolored and stinky. This is good. You will now want to turn the can upside down and drain the water off. Once empty, twist up the top of the trash bag and turn the can upside down to allow the remaining water to drip off for two additional days. 9. At this point the straw is ready to be used direcdy as a substrate or in a mix of bulk spawn. Bulk Spawn

Here, bulk substrates and inoculums are placed into a plastic tub and stacked in a shaded area to myceliate over a period of months. Materials

• Drill and drill bits

• Fresh coffee grounds • Gypsum • Hydrated wood chips • Ink- and tape-free corrugated cardboard • Large tarp • Pasteurized or fermented straw

• Plastic container(s) • Sawdust spawn

• Tape and pen for labeling M e t h o d

1. Place the wood chips in a wire cage or several burlap sacks and soak them in non-chlorinated water for 24 hours. Optionally, add diluted liquid manure or the water left over from hot-water-pasteur ized straw to this water. If you add one of these nutrients to the soak water, make sure the concen tration is very weak so as to avoid contamination issues. 2. After 24 hours, remove the chips and allow them to drain off excess water. 3. Shred the cardboard into pieces approximately 8x8 inches (20x20 cm). Soak these in water for 30 minutes until thoroughly saturated. 4. Lay out the tarp and clean it thoroughly. I tend to just spray my tarps down with water. A very dirty tarp should be cleaned with soap and water. 5. Spread the pasteurized straw and hydrated wood chips on the tarp. Add gypsum at a rate of ap

proximately 1-2% by volume. Break up and sprinkle the majority of the sawdust spawn across the

surface of the substrates, reserving a small portion of the spawn for step 7. 6. Lift the ends of the tarp and roll the substrates and spawn back and forth to thoroughly mix them together. Alternately, these materials can be mixed in a substrate tumbler.

7. Strip open the cardboard pieces from step 3 to expose their interior corrugations. Introduce a small piece of sawdust spawn to these corrugations along with a pinch of coffee grounds and gypsum and roU them up in the cardboard like a burrito. Make a dozen or more of these mycoemhers.

8. Place the mixture ftom the tarp into the plastic container. As you do so, introduce 2-3 of the my63

coembers. Once the container is full, close the container's lid. Drill drain and aeration holes around the container as needed.

9. Stack these containers on the ground or on a pallet outside and in the shade.

From here the myceUum will hopefully do rather well. Like building a fire, the mycelium will quickly jump from the cardboard to the straw to the wood chips until, in time, the entire bag becomes consumed in mycelium as the fungus digests the organic material. Mushroom Beds

If applied in a shady, moist location in the spring or fall, a mushroom bed can be a great source of yearly harvests. The process is simple and requires little time for set up and maintenance. Materials

• Biochar

• Cardboard and sharp knife OR burlap • Gypsum • Hydrated lime • Sawdust, coffee, or bulk spawn • Shovel

• Straw or other mulching materials • Wood chips (optionally soaked in [nutrified] water) M e t h o d

1. Place the wood chips in a wire cage or several burlap sacks and soak them in non-chlorinated water

for 24 hours. Optionally, add diluted liquid manure or the water left over from pasteurizing (not fermenting) straw. If you add one of these nutrients to the soak water, make sure the concentration is very weak so as to avoid problems with contamination. 2. Clear the ground of debris and dig out 4-8 inches (10-20 cm) of soil from the entire site. 3. Protect the bed from soil-dwelling fungi by laying multiple layers of burlap or punctured cardboard

along the bottom and up the sides of the depression. These materials allow for drainage while also serving as eventual substrates for the mycelium. 4. Add a thin layer of spawn on top of the cardboard. Bulk spawn is arguably ideal as it is naturalized to ambient competitors. 5. Apply 2-4 inches (5-10 cm) of hydrated wood chips.

6. Distribute a thin, nearly contiguous layer of spawn and a light dusting of gypsum and hydrated lime across the wood chips. If you have biochar (discussed later in this chapter) available, soak this

material in water or diluted fertilizers and apply at a rate of 5-15% by volume. Thoroughly mix the spawn and supplements into the wood chips using your hands or shovel. 7. Apply another thin layer of spawn on top of this mix.

8. Repeat steps 5-7 to create a bed that is approximately 8-10 inches (20-25 cm) deep. Optionally, steps 5-7 can be repeated a third time. Beds should not be deeper than 18 inches (46 cm) as anaer obic conditions can occur in the center of the pile.

9. Cover the bed with a layer of mulch to reduce desiccation of the substrate and mycelium. Cardboard and/or burlap can be used as a mulch while the mycelium is establishing itself. Once the fruiting season approaches, these materials should be replaced with 6-8 inches (15-20 cm) of fresh straw. Straw is the preferred mulching material as its structure naturally produces a humid micro64

climate that helps initiate primordia formation. Straw can be applied at spawning but tends to lose this beneficial structure before the fruiting season approaches. 10. Optionally, once the mycelium is thoroughly established, incorporate a light sprinkling of fresh coffee grounds into the bed several weeks before the fruiting season to supply a nitrogen kick that will greater fruiting. These beds can be applied as pathways in gardens, as a beneficial living mulch, and in a blend of spe cies to maximize yields in a minimum of space. Room for experimentation is encouraged but selecting species that fruit at different times of the year is a good starting place. Some mushroom beds serve as companions to certain plants. Hjpsi^gus ulmarius is known to greatly benefit Brassica plants as is King Stropharia with corn or Cannabis. Beds should be fed fresh wood chips every other year or as needed. Chunks of established beds can be used as inoculum to start other beds. If the weather is particularly dry and hot, the beds should be watered.

Mushroom logs and stumps

Hardwood logs and stumps can be inoculated as a source of long-term fruitings. The method ap proached for both is the same. Logs would ideally be sourced late winter, when the sap is high and the leaves are still off the tree. Logs should be used 2 weeks-2 months after felling. Stumps should ideally be inoculated in the same time frame. Logs should be 4-8" in diameter and 2-4' long. Materials

• Ballpoint pen • Electric drill or angle grinder • Empty soda or beer cans • Log of the appropriate size and species • Paint brush

• Plug spawn and a rubber mallet OR sawdust spawn and a palm inoculator • Slow cooker or countertop deep fryer • Soy or bees wax or wheat paste • Standard drill bit set or specialty log plugging bits • Stiff wire or nails

• Tin snip M e t h o d

1. On one end of the log draw several equally spaced marks with a pen to signify the lines along which holes will be drilled down the log's length. For 4-inch (10 cm) diameter logs, mark 4 lines; for 5-6-inch (13-15 cm) diameter logs, mark 6 lines; and for 7-8-inch (18-20 cm) diameter logs, mark 8 lines.

2. Starting with one of these marks begin drilling holes down the length of the log. Start near the end of the log and space each hole 3-4-inch (7.5-10 cm) apart (approximately fist width). If you will be inoculating the log with plug spawn, use a 5/16-inch (0.8 cm) drill bit and drill down 1.25-inch (3.2 cm). If you are going to be inoculating with sawdust spawn, use a 7/16-inch (1.1 cm) bit and

drill down 1.25-inch. An electric drill can be used, though an angle grinder with a modified drill bit works much faster. Specialty drill bits designed for log plugging can be purchased as well.

3. Once the entire length of the log has been drilled, start down the next line. Stagger this second row of holes in relation to the first row to form a diamond pattern. Repeat this pattern across each row 65

until the log is evenly drilled out. Drill several more holes around both ends of the log. 4. Fill the holes with spawn. If using plug spawn, gendy tap the plugs in with a rubber mallet until they are flush with the surface of the log. If working with sawdust spawn, you will need a clean

palm inoculator. This tool is used to poke the sawdust spawn, filling its shaft with spawn that is then injected into the drilled hole. If using a palm inoculator, pack the holes relatively densely with spawn but do not cram it in, leaving a slight amount of aeration in the spawn holes will help the mycelium more readily establish in the cavities. 5. Cover all holes and any areas where bark is missing with a sealant. Melted soy or beeswax are com

monly used, but wheat paste works as well. The wax should be very hot (slightly smoking) when applied to ensure a good seal. This temperature can be quickly accomplished with a countertop deep fryer or more slowly with a crockpot. Covering the ends of the log with wax is optional, but recommended.

6. Label the log with species/strain, wood type, and date. Weather-resistant labels can be made by inscribing information on the inside of an aluminum can with a ballpoint pen. These metal tags are then tied or nailed to the log. Once inoculated, logs are set to incubate in a shaded, moist location where the drying effects of wind

and sun exposure are minimi2ed. Logs can be stacked in open towers but should not be packed too tight ly as stagnant air in the towers can encourage contaminant growth. While incubating, the logs must be monitored to ensure that they are not drying out or cracking. Occasional watering with a hose or sprinkler or by soaking in a dunk tank can help maintain high moisture levels in the logs, especially during warmer months. Ravines, streams, and other water sources provide a naturally cool, moist environment that is ideal for cubation. Shade cloth can be strung above the logs or laid over them as well to limit sun

exposure. For all these shading practices, be sure to create adequate ventilation among the logs to discourage contaminant growth.

Logs need to incubate before they are ready to fruit. The length of this incubation period is dependent of the species/strain of mushroom and wood worked with as well as the inoculation rate, log size and quality, and ambient temperatures. Phoenix Oyster mushrooms grown on alder logs may be ready to fruit within just a few months while Shiitake grown on oak make require an incubation period of 18 months or longer before fruitings can be initiated. These rates can vary widely. I have met Shiitake farmed who have obtained fruitings off of oak logs 6 months after inoculation, a very lucky feat. Fruiting Logs

When the log has incubated for the recommended wait time, or if you are just curious to see if it will fruit, the most successful way to obtain a flush from a log is to soak it in cold water for 24 hours. This soaking is required to obtain significant yields as it provides a large influx of the water required to form fully developed fruit bodies. Cold water is preferred, especially for Shiitake, as it helps stimulate primordia formation by simulating autumn temperatures. Soaking also helps remove CO2 captured in the wood, drawing in air that also triggers pinning. After soaking, logs are stood upright on a cross-beam or fence. If the log is thoroughly myceliated, a

flush should appear within a week or so. If the log has not incubated long enough, a flush will not appear and the log should be laid to incubate for several more months. Once the first flush is obtained, logs can

be laid to rest for a period of 6-8 weeks and then soaked again to initiate another crop. This resting/ fruiting cycle can be repeated 3-5 flushes per log during the growing season. The fruiting season varies for different species and strains. Many farms rotate through warm and cold weather strains to ensure consistent production throughout the year. 66

Mycorrhizal

Fungi

Mycorrhizal fungi form symbiotic relationships with plant roots. These fungi tend to fall into two broad categories based on how their mycelium interacts with the roots. Many gourmet wild harvested mushrooms (e.g. Chanterelles, Boletes, and Matsutake) are ectomjcorrhiâl (ECM). These fiingi are usually associated with specific plants (mosdy trees) and most cannot be easily cultivated under controlled con ditions.

B.ndomycorrhit(al (a.k.a. glomeromycotan fungi, arbuscular mycorrhizal fungi, AMF, or just AM) species generally do not produce macroscopic fruit bodies but can be cultivated to benefit plants and soils. AM fungi are generalists that associate with many plants simultaneously. All AMF are placed in the phylum of the Glomeromycota (approximately 169 morphospecies) and associate with at least 90-95% of all plants in the world. These include shrubs, wildflowers, and broad leaf trees, grasses, palms, almost aU bulb plants, anything related to roses, apples, peaches, pears, strawberries, etc. (the rose family), most tropical plants (apart from orchids), and the great majority of horticultural species. Almost all crop plants (apart from the mustard and spinach families), associate with AM fungi. Redwoods, cedars, and junipers are the main confiers that form AM assocaitions. It is easier to list the non-host species (no mycorrhizae) than the ones that have AM.

Most forest timber trees form ectomycorrhizal associations. These include pines, firs, Douglas fir, spruces, larch, oaks, birch, beech, and some willows and cottonwoods. A few nut crops host ECM, in cluding chestnut, hazel, and sometimes walnut and pecan. ECM are much more specific in their choice of host plants, and most have not yet been successfully fruited on a mass scale. ECM will not grow on the roots of garden plants such as maize, beans onions, etc. However, soil collected from the root zone

of tree species that associates with an ECM should contain propagules of the fungus. While there are no current sure-fire ways to cultivate gourmet ECM, two simple experimental methods are as follows:

Plant AND Transplant

1. Identify a wild tree that is known to host the desired mushroom. This can sometimes be deter mined by the fact that the mushroom is fruiting from the base of the tree. However, as root sys tems as well as mycelial mats extend in all directions it may be difficult to determine exactly which tree a given mycorrhizal mushroom is associating with. Use your best judgment. 2. Plant a sapling of the same species in close proximity to the host tree. Be sure to observe best practices for forest management throughout this process. 3. After 2 or more years, transplant the sapling to your desired location. At the time of planting, apply aerated compost tea that has been inoculated with soil gathered from the base of the original host tree. This will provide soil microbes from the host tree to your new planting that may be required to initiate fruiting. Adding fish bones or rock phosphate nearby will provide a source of phosphorus, which the fungus can channel to the plant, encouraging the sustainment of the symbiosis. Spore Sprays

1. Obtain a tree sapling of a species that is known to associate with the mushroom you wish to grow. 2. Create a spore spray as described earlier. 3. Plant the sapling in a properly lit location. Spread the spores around the plant roots.

67

Arbuscular (Endo)Mycorrhizal Plants The majority of the world's plants (90% or more) associate with AM fungi. These include: • Almost all groups of Pteridophyta. • Most groups of Gymnospermae. • The majority of families in the Angiospermae. • All palms. • All plants in the Bryophyta. • Almost all bulb plants. • Anything related to roses, apples, peaches, pears, strawberries, etc. • Most tropical plants (apart from orchids). • The majority of horticultural species. • Almost all crop plants. • All cultivated grasses (but not all weedy grasses). • Shrubs and foliage plants except for Rhododendron, Azalea, and Heath. • Berries except for blue-berries, cranberries, and lingonberries. • Nut trees except pecan, hazelnuts, and filberts. • Fruit trees including tropical fruits. • Many wetiand/aquatic species except rushes and horsetails. Ectomycorrhizal (ECM) Plants The primary ectomycorrhizal plant families are the Pinaceae, Fagaceae, Betulaceae, Salicaceae, and

Dipterocarpaceae. Some species in the Cupressaceae and most species in the Myrtaceae and Caesalpinoideae also form ECM. Some plant groups that assocaite with ectomycorrhizal fungi include:

68

Alder

Basswood

Linden

Birch

Eucalyptus

Madrone

Chestnut

Beech

Manzanita

Arborvitae

Filbert

Oak

Chinquapin Arctostaphylos

Fir

Pecan

Hazelnut

Pine

Cottonwood

Hickory

Aspen Douglas-Fir

Hemlock

Poplar Spruce

Larch

Willow

Non-AM/EctoMycorrhizal Plants

The following plants or plant groups do not associated with endo or ectomycorrhi2al fiingi: Brassicaceae • Broccoli

Others • Beet

• Brussels

• Carnation

• Cabbage

• Mustard

• Cauliflower

•

Protea

• CoUards

•

Rush

•

• Sedge • Spinach

Kale

• Rutabaga

69

Cultivating

AM

Fungi

S o m e B e n e fi t s o f A M

AM-plant relationships are by far the most studied plant-microbial interactions. As such, there is a

rather incredible wealth of information ing the value and benefit of introducing AM into any plant ecosystem. NLrmmoN

Disease

Drought

and

Saunity

• Increases nutrient efficiency • Suppression of pathogens • Increases efficiency of water (especially phosphorus) • Increases plant health use

• Decreases run off and leaching • Decreases pesticide use • Decreases crop loss • Improved Water Quality • Increases acreage of farm land E m C I E N C Y & P r o fi t a b i u t y

Soil Quality • Improves root growth and sur- Product Quality • Improves soil structure & sta- vival • Alters phytochemical attributes

bility via the sticky protein • Decreases production time • Increases flowering Glomalin • Enhances plant marketability • Enhances nutritional value • Decreases erosion and topsoil loss • Enhances nutrient retention

The mycelium of AM fungi cannot be cultivated in isolation; they are obligate symbionts that must be

grown in symbiosis with plant tissue, typically by introducing AM spores to the root system of a plant. The plant should be in a nutrient-deprived state as this will encourage the plant to accept the fungus as a beneficial partner.

If successful, the mycorrhi2al symbiosis should establish in a short time, at which point the partner ship is left to grow in a container for several months. At the end of the growing season, the plant is cut back, sending a signal to the fungus that it should produce spores. The following spring, the growing medium is harvested from the container as it is now filled with mycelium, myceliated root fragments, and spore packets. All of these components are viable AM inoculum for future plant crops.

Due to the expense of producing, transporting, and applying high quality AM inoculum - along with the uncertainties of introducing foreign species/strains - broad-scale cultivation is not currently practical on an industrial scale. Smaller scale, on-site production of locally-adapted AM inoculum is a much more appropriate and resilient practice for the average gardener or farmer. Materials

• 16 7-gallon (26.5 L) plant containers

• 16 cubic feet (0.45 m^) vermiculite • 240 cubic inches (4,000 cm^) coarse (swimming pool filter) sand • 4 cubic feet (0.11 m^) compost • Seedling trays or conical plastic pots • Seeds of a suitable plant • Wild harvested soil

70

M e t h o d

1. Foiir months before the last frost date, germinate the plant seeds in vermiculite or a low-nutrient seed starting mix. 2. One month later, mix the soil and sand at a ratio of 1:3 by volume. The soil should be collected from an intact soil system that hasn't been disturbed in at least two years (such as a forest, wood lot, or fence row). Collect samples from rom the top 4 inches (10 cm) of the soil and from 5 different sites to ensure that a diversity of AM species is obtained. Screen the soil to remove large roots and rocks.

3. Transplant the seedlings into small pots or cone cells filled with the soil/sand mixture. 4. After the last frost, fill the grow bags or pots three-quarters full with compost and vermiculite (mixed at a ratio of 1:4 by volume). If biochar is abundantly available, you can experiment with substituting some or all of the vermicuUte with this medium. 5. Add 0.5 cups (120 mL) of field soil to each container and mix thoroughly. 6. Transplant 5 host plants into each container. Place the containers in an area where weeds are well controlled.

7. During the growing season, water the plants and weed as needed. 8. At the end of the growing season allow the plants to die from frost. If the plant is cold tolerant, cut it back at the root level. Leave the containers outside over the winter.

9. The following spring, cut back and discard any plant matter from the tops of the bags. 10. Harvest the roots from the soil mix and chop the roots to 1-1.5-inch (2.5-4 cm) segments. Save the soil mix and root fragments. The most commonly cultivated AM species are in the genera Glomus and Gigaspora. This is due to the J global distribution and climatic tolerance range of these species as well as their minimal requirements for successful cultivation. Gigaspora margarita probably has a worldwide distribution. Studies suggest that working with a blend of AM species incurs greater benefit to the soil ecology and overall plant health

when compared to inoculum that use a single species. This is likely due to differences in each morphospe-

cies' growth pattern inside the plant tissue, mode of spatial exploration for phosphorus in soils, level of glomalb production, and in their ability to induce growth responses in different plant species. Compared

to imported species/strains, locally-adapted AM fiingi seem to be more effective at promoting plant growth, especially when applied to the same plant species that they associate with in the wild. Which Plant Partner?

The plant host for AM should be able to form the AM symbiosis and not be of the same family that the inoculum will be eventually applied to in the field or garden, so as to avoid the spread of pathogens. Deep rooting, quick growing grasses are an easy and low-cost option. The tropical grass Paspa/um notatum is fast growing and has a low tolerance for frost, increasing greater spore yields and decreasing the likeli

hood of it becoming invasive in colder climates. Local grasses can also be used but should be thoroughly cut back and have their watering stopped at the end of the growing season, thereby ensuring that the plant dies and that the highest spore production is encouraged in the fungus. A combination of plants may the development of a wider range of mycorrhizal species. Such a combination of plants could include a grassy species (including cereal crops), an allium (e.g. onion, leek), and a legume (e.g. beans, peas, lentils, alfalfa, or clover).

71

The phosphorus and nitrogen concentration in the compost used for step 4 can significandy impact the rate of mycorrhizae formation. Composts made primarily from yard clippings (such as those from municipal composting facilities) or dairy manure and leaf compost tend to be high in nitrogen, low in phosphorus, and with moderate potassium levels, leading to a higher rate of mycorrhizae formation. Composts that are high in phosphorus, low in nitrogen, and have moderately high potassium levels must be diluted to rates of 1:19-1:49 to ensure that the plant is not over fertilized. AM Inoculum Storage It is unknown how long vegetative hyphae survive in root fragments. As such, it is recommended to

apply AM inoculum directly to crops in the spring to maintain the highest concentration of living propagules. Inoculum can be dried and stored in a cool, dry location until use. It is best to use the inoculum within 6 months but it can potentially retain viability for 2-3 years. AM spores are more resistant to environmental stress and desiccation than their mycelium. However, spores do not produce mycorrhizal associations as quickly as the mycelium of living mycorrhizal root fragments. AM Application

Mycorrhizal symbiosis will only form if the AM inoculum is in close proximity to the roots of the host plant. The easiest way to apply the inoculum is to mix it into planting soils and composts at a rate of 5-10% by volume. Depending on the dilution, the above protocol will produce between 200 and 400

ft.^ (5.6-11.3 m^) of planting medium. Creating a phosphorus-deprived environment for plants will encourage the formation of mycorrhizae. If you are using an organic planting mix that typically requires phosphorus fertilization, use materials that

are low in phosphorus, such as fish hydrolysate. Chemical-based phosphorus fertilizers should be applied at a rate of 3 ppm or less for no more than 3 times a week. A p p l y i n g C o m p o s t Te a Most AM species from genera other than Glomus and Gigaspora have not been successfully cultivated. This is likely due to a limited understanding of their environmental requirements. Other studies have shown that AM spore germination is decreased in sterile soil and increased in the presence of microbes. As such, I suggest the application of actively aerated compost tea at the time of inoculating plants with mycorrhizal fungi. Part of the ingredients used to make the tea should include soil sourced from the

natural habitat of the AM species being worked with. This will help bring in the nitrogen-fixing and phosphorus-solubilizing bacteria that are linked to AM symbioses. T e s t i n g E f fi c a c y

Before any large-scale application of AM is applied to a landscape, it is recommended to determine the various impacts that an inoculum will have on a living system as well as the degree of maintenance that its introduction will require. Small test plots can be established to monitor the rate of mycorrhizae formation, the assemblage of indigenous AM and pathogens before and after inoculation, and the effects of tillage and fertilizer application over short- and long-term intervals.

Several weeks after inoculation, harvest the roots of several plants to check for the formation of my corrhizae. Mycelium may be visible to the naked eye. Arbuscules can be detected under a microscope using simple staining practices. If plant fertilization must be increased, monitor the percentage of my72

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Recommended

Reading

C u LT f VAT i o N Te c h n i q u e s a n d S p e c i e s P a r a m e t e r s

• Radical Mycology - Peter McCoy • http: //www.shroomery.org/11327/ Gourmet-Mushrooms • Let's Grow Mushrooms - Video series by Marc Keith • Organic Mushroom Growing and Mycoremediation - Tradd Cotter • Growing Gourmet and Medicinal Mushrooms - Paul Stamets • Mycelium Running - Paul Stamets

Websites and Online Forums

• Radicalmycology.com • Shroomery.org (Etiquette tip: use the search function before posting) • Mycotopia.net

• Permies.com/forums/f-39/fiingi

74

Prima

• Build a Stir plate. • Grow Stropharia spawn on non-heat-treated sawdust. • Grow Oyster spawn on pasteurized sawdust. • Create three medicinal products from grain spawn and/or locally harvested mushrooms. • Fruit Oysters on coffee and cardboard in a 5-gaIlon bucket. • Fruit Oysters on fermented straw in a 5-gallon bucket. • Fruit Shiitake on outdoor logs. • Fruit Turkey Tails from pasteurized sawdust blocks. • Inoculate outdoor substrates with a spore spray of local species. • Install a King Stropharia wood chip bed. • Set up a paper digester with a local Oyster strain. Segunda

• Build a steam pasteurizer / sterilizer. • Build an automated fruiting environment. • Build a grey water or microbial water filter. • Bulk up naturalized King Stropharia spawn in totes. • Collect liquids dripping from a leaking engine on cardboard and inoculate with Pkurotus ostreatus or Pleurotuspulmonarius in a 5-gallon bucket. • Cultivate Agaricus bhfêi on compost or manure-based substrates. • Cultivate indigenous arbuscular mycorrhizal fungi. • Fruit Blewits on spent oyster straw. • Fruit King Oysters from All-in-One Jars. • Fruit Reishi in All-In-One bags. • Fruit Reishi in a monotub with pasteurized sawdust as the substrate. • Fruit wood loving species from nutrified sawdust blocks. • Grow mycelium into a form. • Organize and maintain a culture library. Te r c i a

• Determine the best substrate formula (highest BE) for a local mushroom strain. Develop a coffeeor chemical-tolerant strain. • Fruit Maitake indoors.

• Fruit Morels (indoors or outdoors). • Fruit Shaggy Manes indoors on high quality compost. • Grow mycelium into functional products (e.g. insulation, flip flops, bike helmet). • Propagate Pleurotus ostreatus on used cigarette filters.

W

75

Media

Cookbook

Helpful Conversions 1 cup

= 8 oz

= 16 Tbsp.

3/4 cup 2/3 cup 1/2 cup 1/3 cup 1/4 cup 1/8 cup 1/16 cup

= 6 oz

= 12 Tbsp.

= 5 oz

= 11 Tbsp. = 8 Tbsp. = 5 Tbsp. = 4 Tbsp. = 2 Tbsp. = 1 Tbsp.

= 4 oz = 3 oz = 2 oz = 1 oz

= 0.5oz

= 48 tsp. = 36 tsp.

= 237 ml

= 32 tsp.

= 158 ml

= 24 tsp.

= 11 8 m l

= 16 tsp.

= 79 ml

= 12 tsp.

= 59 ml

= 6 tsp. = 3 tsp.

= 30 ml

= 177 ml

= 15ml

1 ml (Icc) of water = 1 gram 1 tablespoon dextrose = ~10 grams 1 tablespoon light malt extract = ~10 grams 10 ml honey = 14 grams 1 tsp (5 ml) honey = 7 g 1 tbsp (15 ml) honey = 21 g Agar Media Recipes

Unless noted, all agar recipes start with 500 milliliters of water and 10 grams of agar. Never use the following antifungal ingredients in media recipes: coconut, garlic, pomegranate, cinnamon, cloves, curry, and cilantro. Malt Extract Agar (ME) • Dry, light malt (barley) extract - 10 g Malt Yeast Extract (MYA) • Malt - 10 mL • Yeast - 1 mL

Malt Extract with Activated Carbon (MEAC) • Malt - 10 g • Activated carbon - 1 g Malt Extract with Peptone (MEP) • Malt - 10 g • Peptone - 1 g

Malt Extract with Yeast and Peptone (MYAP) • Malt - 10 g • Yeast - 1 g • Peptone - 1 g 76

Malt Extract with Yeast, Peptone, and Acti vated Carbon (MEPYAC) • Malt — 10 g • Peptone - 1 g • Yeast - 1 g

• Activated carbon - 1 g Malt Extract with Milo (MEM) • Malt - 10 g • Ground milo - 10 g Malt Extract with Corn (MEC) • Malt - 10 g • Ground corn - 10 g Malt Extract with Honey (MEN) • Malt - 10 g • Honey - 16 g AT T C M e d i u m 5 9 7

• Malt - 3.75 g • Yeast - 0.3 g • Peptone - 0.5 g MYAP WITH Multi-vitamin (MYMP)

• Fresh ground crickets - 8 g • Multi vitamin - 0.01 g

• Peptone - 1 g • Yeast - 1 g • Malt - 10 g Add multi-vitamin after sterilization.

EntheoGenesis Agar (EA) • Amaranth flour — 20 g • Soy flour — 20 g

• Brown rice flour - 10 g • Potato flour - 10 g

MYAP WITH ELEMENTS (MYPMCZ) • Yeast — 1 g • Peptone - 1 g

• Calcium - 0.01 g • Magnesium - 0.01 g • Zinc - 0.01 g

Potato Dextrose Agar (PDA) • Diced and boiled potatoes — 150 g OR Potato starch - 10 g • Dextrose - 7 g In 500 mL of water, boil 150 g of potatoes. Strain out the solids, bring the solution back to 500 mL with water, then add remaining ingredients. Five grams of potato flakes can be used as a sub stitute.

Potato Skin Dextrose Agar (PSDA)

^ ^ ^ • Dried potato skins - 10 g • Dextrose - 7 g

• Malted barley - 2 g M o o n fl o w e r ' s R i c e M a l t - A l f a l f a - B r e w e r ' s

Yeast Agar (MRMABYA) Soak 1 cup of alfalfa and 2 cups of rice in 1.5 quarts of clean water for 2 hours at room temp. Stir occasionally. Filter out solids. Pour a pack- et

of baker's yeast in water. After 30 minutes, filter out the solids. Combine liquids and add 1 tablet of crushed dolomite. s luxuriant mycelial growth and germinates spores. Spore Germination Agar #1

• Cornmeal - 10 g • Dextrose — 1 g

• Yeast - 0.5 g Spore Germination Agar #2

• Cornmeal — 10 g • Dextrose - 3.5 g • Sucrose - 5 g

Barley Flour Malt Extract Agar (BFMA) • Barley flour — 40 g • Malt extract - 2 g

• Yeast extract (optional) - 1 g Oatmeal Agar (OA) • Filtered, cooked oatmeal - 30 g

• Yeast - 0.5 g •KH2PO4-0.5g Spore Germination Agar #3

• Cornmeal - 10 g • Malt - 0.8 g Spore Germination Agar #4

CoRNMEAL Malt Agar (CMMA) • Cornmeal - 10 g • Dextrose - 7 g

Dog Food Agar (DFA) • Dried, ground dog food - 10 g • Amaranth flour — 10 g • Dextrose or malt extract - 2 g Amaranth Soy Agar (ASA) • Amaranth flour — 20 g • Soy flour — 20 g

• Cornmeal — 10 g • Glucose - 1 g • Sucrose - 1.5 g • Yeast — 0.5 g Manure Agar

• Ground horse manure - 50-60 g Boil manure in water for 10 minutes, let sit for

16-20 hours, filter out solids, bring back to 500 mL and add agar.

77

Compost Agar

Air-dry and then grind hot compost obtained during its peak state. Mix 50 g of this dried com post powder to 750 mL of water. PC for 1 hour at 15 psi. Filter twice through cheese cloth. Bring volume up to 500 mL. Add 10 g agar and PC as normal.

Complete Media (CM) • Sucrose — 15 g

• Ammonium tartrate (NH4-) - 2.5 g • Ammoniumnitrate (NH4NO3)-0.5g • Magnesium sulfate heptahydrate (MgSO4-7H2O)-0.25g • Sodium chloride (NaCl) - 0.5 g • Calcium chloride (CaC12) - 0.065 g • Yeast - 0.5 g • Monopotassium phosphate (KH2P04) - 0.5 g Distilled Water Agar (DWA) This formula should be made solely with dis tilled water and agar. Liquid Media Recipes

AU recipes are for 500 mL of water (approxi mately 2 cups). I often add a pinch of gypsum to every jar as well. Complete LC (CLC) • Malt - 10 g • Peptone — 1 g

• Yeast - 0.3 g • Vegetable oil - 5 drops • Ground grain - 1 g Malt Extract Dextrose LC (MDLC) • Dextrose - 1 Tbsp. • Light malt extract - 1 Tbsp. Honey LC(HLC) • Honey - 2 tsp. or 10 g Corn Syrup LC (CSLC) • Corn Syrup - 2 tsp or 10 g

78

Lysogeny LC (LLC) • Dextrose — 10 g • Peptone - 5 g • Yeast - 2.5 g Manure-Based Substrates Recipe 1 • 60% Dried horse manure • 25% Coconut coir • 1 0 % Ve r m i c u l i t e

• 5% Coffee

Recipe 2

• 12 parts horse manure • 6 parts vermiculite • 4 parts dry whole bird seed • 4 Tablespoons dry kelp meal • 6 Tablespoons Vegetable oil Recipe 3

• 1 part Dried horse manure • 1 part Coconut coir • 1 part Coffee • 1 part Straw

• 1 part Coffee grounds • 1 part Leached cow manure • y4 part Worm castings • Va part Gypsum

• Vegetable oil at 1 tsp./gal of substrate Recipe 4

• 2 parts Leached cow manure • 2 parts Coconut coir • 2 parts Worm castings • 2 parts Coffee grounds • Vz part Chicken manure • Va part Hydrated lime Casing Recipes Recipe 1 • 6 0 % Ve r m i c u l i t e • 40% Coconut coir

Use approximately 5% Calcium carbonate to raise pH as needed

Recipe 2

Recipe 4

• 12.5 parts Peat moss

• 4 quarts Peat moss

• 12.5 parts Vermiculite • 1 part Hydrated lime • 3 parts Calcium carbonate

• 4 quarts Vermiculite • 1 quart Hydrate coconut coir • 1 cup Oyster shell flour • 5 tablespoons Hydrated lime

Recipe 3 • 25% Peat moss • 2 5 % Ve r m i c u l i t e

• 30% Hydrate coconut coir • 10% Oyster shell

Mix dry components, add water slowly until field capacity is reached. Pasteuri2e at 140-170°F for 1 hour in aluminum foil covered jars.

• 10% Calcium carbonate

79

Commonly Cultivated Species Common Edible Wood Lovers

AA - PioppiNO / Black Poplar {Agrocybe aegerita) A clustering, chestnut brown mushroom. Good, umami-rich flavor. ASp - Wood Ear (Auricula Spp.) Chewy, folded fruitbody that swells in water. Highly medicinal and with a neutral flavor. The "black mushroom" in black mushroom soup. FV - Enokitake (Flamulina velutipes) Cultivated form with long stalks, small caps, and lack of pigmentation due to being grown in colored

bottles under low light. Crunchy texture and mild flavor. Commerical strains often fast growing. Wild form with black fuzzy stalk and orange cap. GF - Maitake (Grifola frondosa) Layered mass with strong, rich flavor and high medicinal value (good for blood sugar, diabetes, and more). Historically hard to grow but newer commerical strains produce better yeilds. Needs lots of hu midity and proximity to the ground to mature. Wild form much larger and more flavorful. HE - Lion's Mane (Hericium erinaceus)

White, densely branching mass of long teeth. Crab/seafood-like flavor and powerful neurological benefits (helps rebuild neuron insulation [myelin] and stimulate production of Nerve Growth Factor). HT - Bunashimeji (Hypsizygus tessulatus) Small clustering cap-and-stalk mushrooms with strong almondy flavor. White and brown strains com mercially available. HU - Elm Oyster [Hypsizygus ulmarius)

Meaty, stalked mushroom with good flavor. Aggresive and quick growing — will grow on a range of organic substrates. LE - Shiitake [Lentinula edodes) Delicious and highly medicinal. Many commerical strains available, each with different appearances, maturation timeframes, and fruiting temperature ranges. After fully myceliating, sawdust blocks tend to

get bumpy, then form a brown crust. Blocks are then smacked/tapped, removed from bags, and soaked

in cold water for 8-12 hours (avoid waterlogging, which reduces mycelial integrity) before being moved to the fruiting room. After harvest, remove the block and allow to dry and rest for 2—3 weeks before

repeating the smack/chill/soak/fruit process. PC - Yellow Oyster [Pleurotus citrinopileatus) Fast growing, warm temperature preferring Oyster that is starting to naturalize in the U.S. W

PlO - Abalone (Pleurotus cystidiosus]

Small Oyster species that is medicinal and choice, but less commonly cultivated. Grows well on agri cultural residues. 80

PD - Pink Oyster {Pleurotus djamor) Fast growing, warm temperature preferring Oyster. Colors fades with cooking and flavor is not as preferrable by some (when compared to other Oyster species). PE - King Oyster {Pleurotus eryngii] Large, bulbous fruitbody with dense tissue and meaty flavor. Commonly grown in bottie culture. PA - Chestnut {Pholiota adiposa) A newer commerical species. Interesting appearance, good flavor, and showing some medicinal ben e fi t .

PN - Nameko [Pholiota nameko)

Forms slimy-capped, stalked fruit bodies that prefer coolder temps. Good flavor, common in many Asian dishes.

PO - Pearl Oyster [Pleurotus ostreatus) The classic Oyster. Many commerical strains available with different appearances and fruiting tempera ture ranges. Easy to grow on a wide range of agricultural residues and urban wastes. PP - Phoenix Oyster [Pleurotus pulmonarius) Aggressive and very fast growing, this species prefers warmer temps than many P. ostreatus strains. SRA - King Stropharia [Stropharia rugosoannulata) \^/ The quitenessential outdoor mushroom. SRA grows slow on sterile media, thrives in with mi crobes. Can be fruited indoors if cased with pasteurized soil.

Common Woody Med/cinal Wood-Lovers

AC - Stout Camphor (Antrodia camphorata) Commonly used as an anti-cancer, anti-itching, anti-allergy, anti-fatigue, and liver protective herb in Taiwanese traditional medicine.

GL - Reishi [Ganoderma lucidum) The "mushroom of immortality." Indoors, commerical strains grow well on supplemented sawdust. Can form antlers "in the bag" (in vitro), making for easy harvests. To form conks, cut slits or open the bag to let stalks "breathe." Fruit bodies can take weeks-months to fully mature. Tolerant of lower humid ity levels. Outdoors, does best on buried logs in a shade/hoop house. 10 - Chaga [Inonotus obliquus) Traditional medicinal sclerotia grow primarily on birch in the wild. A viable commerical sclerotia pro duction method has yet to be established. PL - Black Hoof (Phellinus linteus) Well-studied mushroom from various traditional Asian medicinal practices.

81

PU - Umbrella {Polyporus umbellatus) A white-to-gray mushroom that grows in dense rosettes from a single stem. Regarded for its medicinal functions (hepatoprotective, and immunomodulatory). TV - Turkey Tail [Trametes versicolor) Fast growing, highly medicinal, rubby fruit bodies that come in a range of color patternations that are influenced by sustrate. Grows well on many kinds of wood. WE - Tuckahoe (Wolfiporia Extensa) The most commonly prescribed mushroom in Traditional Chinese Medicine. Sclerotia are harvested from pine logs that were inoculated and buried two years prior. XN - Stag's Horn (Xylaria nigripes) Known for its anti-depressant effects and help with insomnia. Common Woody Medicinal Wood-Lovers

CSp - Bird's Next (Cyathus spp.) FF - Amadou (Fomes fomentarius) GA - Artist's Conk {Gannoderma applanatum) PB - Birch Conk [Piptoporus betulinus) Uncommon/Experimental Edible Wood-Lovers HA - Comb Tooth (Hericium abietes) HC - CoNFiER Tuft {Hypholoma capnoides) HS - Brick Cap {Hypholoma sublateritium) LSp - Chicken-Of-The-Woods {Laetiporus spp.) PS - Glowing lus {lus stipticus) PT - Tuber Oyster (Pleurotus tuber-regium) SC - Cauliflower (Sparassis crispa & relatives) XR - Black Foot (Xerula radicata) Psychoactive Wood-Lovers

GJ - Big Laughing Gym (Gymnopilus junonius) PA - Azures (Psilocybe azurescens) PsC - Wavy Caps (Psilocybe cyanescens) PsS - Blue Ringers (Psilocybe stuntzii) Common Edible Non-Wood-Lovers AB - White Button/Porcini/Portobello {Agaricus bisporus) The most commonly cultivated mushroom species in the world. Often grown industrially on compost ed straw and chicken manure.

82

CI - Milk Mushroom (Calocybe indica) Stout, productive, warm-temperature loving, and medicinal mushroom with a long shelf life. Grows well on pastueri2ed straw and supplmented sawdust blocks, but needs peat-based casing to fruit well. GSP - CORDYCEPS (CORDYCEPS SPP.) Highly medicinal entomopathogenic species. C. sinensis is revered, but cannot yet be fruited under constrolled conditions. C. militaris can be fruited on simple ingredients and contains appreciable amounts of cordycepin. The following is one fruiting formula that can be inoculated with an LC of C. militarisr. • 3 cups of Grains (Rice, Millet, Local Wild Grass Seed, Popcorn) • 6 cups Water • 2 tbsp Sucrose

• 1 tbsp Corn or Potato Starch • 1 tbsp Yeast Extract or Nutritional Yeast (Nitrogen Source) • 1 tsp Azomite W - Paddy Straw {Volvariella volvacea) A fast-growing, warm-temp, high protein mushroom that is commonly grown in SB Asia on hydrated straw (with or without pasterization). Medicinal Non-Wood-Lovers

AB - Almond Portobello {/^garicus subrufrescens) Strong, almond-like flavor and high medicinal value mushroom that is grown with techniques similar to A. bisporus, yet it is not commonly cultivated. TA - Golden Ear (Tremella aurantialba) An immunostimulating, yellow jelly fungus. TF - White Jelly Fungus (Tremella fuciformis) Flavorless, but commonly cultivated for use in sweet dishes that pair well with the jelatinous texture. Needs to be co-inoculated with a host fungus, often Annulohypo>ylon archeri. Uncommon/Experimental Edible Non-Wood-Lovers CO - Shaggy Mane (Coprinus comatus) OR - Shaggy Parasol [Chlorophyllum rachodes) ON - Blewitt {Clitocybe nuda) MP - Parasol (Macrolepiota procera) MSp - Morel (Morchella spp.) Psychoactive Non-Wood-Lovers

PaC - Pan Cyan (Panaeolus cyanescens) PsC - San Isidro (Psilocybe cubensis) PsT - Philosopher's Stones (Psilocybe tampanensis) PsM - Teotlnanacatl (Psilocybe mexicana) 83

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Total Number of

Average BE Per

Containers

Container

Contamination Rate AND Notes Harvesting and Processing Notes

Plans For Future Lineages

Outdoor

Application

Where and How

Biodynamic

Applied

Details

Location Details

1 Month Progress

6 Month Progress

Feeding Regimen

3

12 Month Progress R e s p o n s e To Feeding

Future Plans

Spent Spawn Notes on Spent

Spawn Application Medicine

Preparation Notes Effects of Medicine

Month

Progress

Mushroom Cultivation And Application Course 226w3w

Overview 4q3b3c

More details 26j3b

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