Mushroom Growing

Understanding and manipulating mushroom cultures to produce has been a long and sometimes frustrating task that has taken lifetimes of research. Once it was found that a profit could be made in the cultivation of mushrooms, the successful cultivators kept their techniques and methods to themselves as to keep out possible competitors and therefore increase profit potential. As a result, until recently families passed on the secrets from generation to generation. Still today with only limited works on the subject those with the resources are becoming the major producers, those who inherited the knowledge from their parents are not using the information as often as previously. Those who would enjoy the cultivation find the research too much of a hassle to continue in their efforts. As a result, the number of home cultivators is very small in comparison to most any other crop, and the commercial growers probably enjoy this situation.

The techniques for growing mushrooms are essentially the same for all species even though growth parameters, pH preferences, or substrate preferences differ. The following is a summarization of the basic steps to follow:

1)     Prepare and pour agar into Petri dishes.

2)     Germinate spores and isolate pure mushroom mycelium.

3)     Expand the growth of the mycelium on the agar media.

4)     Prepare the grain medium.

5)     Inoculate the grain.

6)     Incubate the inoculated grain spawn.

7)     A. Lay out grain spawn into trays; or

        B. Inoculate the grain spawn into bulk substrates

8)  Case with a soil-like mixture.

9)  Initiate the formation of pinheads.

10)  Influence cropping. This is the start of the flushing pattern.

11)  Continual harvesting and recasing.

Modest harvests can be made from mushrooms grown and fruited on cased grain for most species; maximum harvests can be realized if the mushrooms are grown on compost, straw, or wood. Whichever method one chooses to utilize, there must be adequate moisture and humidity that can be controlled for the grower’s benefit. Without enough moisture, mushrooms do not grow.

Mushrooms are part of the fungal kingdom Eumycota. Mushrooms are known as the “fleshy fungi” and they constitute only a small fraction of the tens of thousands of species known. Fungi are non-photosynthetic and are believed to have evolved from algae. The primary role of fungi is to decompose dead organic matter.

All gilled fungi are members of the class Basidiomycetes which are characterized by the production of spores on club-shaped appendages called basidiospores. Most of the conspicuous fungi that one encounters (i.e. mushrooms, puffballs, and bracket fungi) are members of the subclass Homobasidiomycetes. Of the members of this subclass, the gilled mushrooms are placed in the order Agaricales. The basidiospores germinate to form monokaryotic hypha. A hypha is a tubular filament; aggregations of these hyphae collectively comprise a mass of thread-like filaments referred to as the mycelium. The mycelium comprises the main body, the thallus, of the fungus. The stalked, capped structure, which we call the mushroom, is actually only the fruiting body or the spore-producing reproductive structure, and constitutes only a small portion of the total mass of the fungus. The great bulk of the organism exists underground in the form of a network of mycelium that occasionally fruits under the appropriate conditions.

The basidiospores germinate to produce a monokaryotic mycelium which is a mycelium having only one nucleus per cell. This mycelium grows outward until it encounters another monokaryotic mycelium, which germinated from another spore and is of a compatible mating type. If the monokaryotic mycelium does not contact a compatible monokaryotic mycelium, it will eventually die. If the mycelium does contact another compatible monokaryotic mycelium a process called somatogamy (the fusing of the somatic cells of the two mycelia) takes place, but fusion of the nuclei does not take place. The result of this junction is the formation of a dikaryotic mycelium which is a mycelium containing two nuclei.

The dikaryotic stage is the most prolonged portion of the life cycle and is the main assimilative stage of the fungus. The dikaryotic mycelium can propagate vegetatively indefinitely without going through a sexual stage or if given the appropriate conditions the mycelium can be induced to fruit. The undifferentiated mycelial thallus of the fungus begins to weave itself into an articulated spore-bearing, fruiting body, in this case a mushroom. The mushroom continues to enlarge and thrust above ground incorporating more and more mycelium while at the same time expanding by the absorption of water. At a certain stage in the growth of the mushroom, basidiocarp, club-shaped basidia form on the underside of the gills. At this point karyogamy (the fusion of the two nuclei of the dikaryotic mycelium) takes place within the basidia. This is the only diploid (2n) stage in the life cycle of the fungus and is the briefest stage. Meiosis (the reduction division of a diploid, 2n, nucleus to four haploid, n, nuclei) occurs immediately following karyogamy. The result of meiosis is the production of four haploid nuclei within the basidium. These are then pushed out of the basidium and become surrounded by hard sheaths to form the basidiospores. The result is the basidium bearing four basidiospores on its outer surface. These basidiospores eventually detach from the basidium to begin the life cycle again.

With the life cycle in mind, there is competition between the organism we are intending to grow with other fungi, viruses, bacteria, as well as many other microscopic particles and this competition usually dictates the success or lack thereof of a crop. To keep competition to a minimum, an understanding of the nature of contamination is needed as well as a sterile or “semi-sterile” controlled workspace, laboratory, with good hygienic maintenance measures taken. Whereas the actual shaper and construction of the workspace will vary from cultivator to cultivator, the same idea is built into each of them, keep contamination out and yet still provide a suitable environment for the mushroom crop.

There are five primary sources of contamination in mushroom cultivation: the immediate surroundings, the culture medium, the culturing equipment, the cultivator and his or her clothing, and the mushroom spores or the mycelium. To control contamination the first measure in preparing a workspace would be to remove all carpeting and other cloth-like materials that can harbor enemy spores. Next, an anti-chamber should be built and an air box constructed with controls to control temperature, humidity, CO2, airflow, and contamination inlet. The room and its contents should be thoroughly cleaned and sterilized with a ten percent bleach solution, Lysol, or isopropyl. Any materials to leave and reenter the workspace should be resterilized. A strict regimen of hygiene should be kept at all times, just remember that it is much easier to prevent contamination than to eliminate it after it has occurred. Some cultivators get by in what seems to be the most primitive conditions, working out of Styrofoam boxes, while others battle contamination working in commercially set-up laboratories. Each event of contamination dictates an appropriate counter measure. Whether you are the one working among primitive conditions or within the most that technology has to offer, the problems are similar, differing not in kind but rather in degree. In either case, sterile work requires patience, concentration, and a steady hand. Work for reasonable amounts of time and not to the point of exhaustion. Never leave an alcohol lamp or Bunsen burner unattended. Lysol and other such products are extremely flammable. Moreover, always be conscious of the fact that an airtight space is quickly depleted of oxygen.

Once the laboratory is complete, the next step is preparation of the agar media. There are extensive amounts of agar recipes suitable for growing mushroom mycelium and this can result in a personal study producing deeper understanding of the inner processes of the mushroom nutritional needs. However, this is not necessary since one can buy a standard formula of either potato dextrose agar or malt extract agar, which is suitable for most mushroom species. To the though, one may supplement the media with peptone or neopeptone, protein sources. If there is an enormously high rate of contamination from bacteria, antibiotics can be introduced to the medium. Most antibiotics like streptomycin are not autoclaveable and must therefore be added after the medium has been sterilized and while the medium is still molten. Gentamycin is an antibiotic that will survive autoclaving. Antibiotics adversely affect some mushroom species so to a certain degree experimentation is needed. The agar medium is sterilized by use of an autoclave or by using a pressure cooker (cook for sixty minutes at fifteen psi, do not allow the temperature to exceed 250 degrees Fahrenheit as this will cause the media to caramelize, inhibiting mycelial growth and promoting genetic mutations). In the case of the pressure cooker, a layer of petroleum jelly can be used around the opening where the lid and pot come into contact to prevent entering contaminants and it helps to stabilize the pressure by not letting air seep out.

Once sterilized, the pressure cooker is brought into the laboratory and the media is poured into Petri dishes. Glass Petri dishes can be autoclaved, removing the addition of the preceding step. In pouring the media, the following steps are used: allow media to cool to a temperature where it is comfortable to handle, vigorously shake the media to evenly distribute the contents, flame the lip of the container, pour the media into the Petri dishes, stack the Petri dishes to minimize condensation which can hold and promote contamination, allow the media to solidify before inoculating with either spores or living tissue.

Once one has obtained a spore print, the monokaryotic mycelium can easily be obtained by germinating the spores on the appropriate medium. Spores can be transferred most readily by using a clean #11 scalpel, inoculating loop or similar instrument. Flame the instrument to be used and wear latex gloves during the transfer. The inoculating loop can be used to scrape the spores onto the medium or with the scalpel  cut a 2-3mm square in the medium, spear the square, use it to lightly touch the paper or slide with the spores, then quickly replace the square and Petri dish cover. Another method uses spores diluted in water transferred by syringe. Incubate the spores at 86 degrees F for 24 to 36 hours. This will break dormancy and force the spores to germinate faster. Spores will germinate without incubation but it may take up to 24 days and this increases the chances the culture will be contaminated. During this time, the spores will germinate and monokaryotic mycelium will begin to grow radially outward from each point of inoculation. The plate should be left undisturbed until the mycelia from two different spores have grown together and made contact. A few days after contact has been made it is reasonable to assume that somatogamy has taken place and that a dikaryotic mycelium has been established. In practice, one transfers many pores at a time and therefore it is necessary to isolate a single strain. This can be done in the same manner as the scalpel/square technique used in inoculation. The dikaryotic mycelium thus isolated will grow outward in all directions from the point of inoculation on the new plate, covering it within eight to twelve days. Then one can make further transfers being reasonably sure that one is working with a single strain and that the mass of the culture will grow at the same rate, requiring the same environmental changes, at the same time. Deviations indicate contamination.

It is advisable to isolate several different strains. The different strains should be labeled and their characteristics compared. The strain with the most favorable characteristics should then be used exclusively thereafter. The mycelium being ropy in appearance is the most favorable and those strains with contamination or cottony sectors should be discarded. The ropy morphology is caused by the formation of rhizoids, thick strands of hyphae, which are similar to the rhizoids formed prior to and during the fruiting stage. The rhizoid’s function is to transport water and nutrients to the developing mushrooms.

Once one has successfully grown several mycelia cultures on a solid agar medium, it is time to move on to the next step, inoculating and growing on sterilized grain. Many grains are suitable for expanding the mycelia mass including rye, wheat, barley, triticale, oats, rice, sorghum, millet, and buckwheat. However, rye is recommended since it works as well as any and yet it is less expensive. One must be sure, however, that the rye is suitable for human consumption. Feed rye is usually treated with a fungicide. 160 ml of rye to 130 ml of water is mixed in a quart mason jar as sterilized, as was the agar medium. In a single gram of rye there is an estimated cell count of 50,000-100,000 bacteria, more than 200,000 actinomyces, 12,000 fungi, and large numbers of yeasts, which is why it is necessary to sterilize. After the jars have been sterilized, checked for imperfections, and cooled, one can inoculate the grain. A scalpel is used to cut a grid of nine to twenty squares in the Petri dish of agar and mycelia and each square is transferred to an individual jar, always flaming the scalpel between transfers. During this time, a square from two different strains can be introduced to one jar to create a hybrid strain. By the eighth to the fourteenth day, the mycelium will have grown to about the size of a fifty-cent piece. The next step is to shake the jars. This will loosen the rye for easier penetration and allow for air exchange while at the same time this will break up the mycelium into smaller sections throughout the jar, which creates more points of inoculation, and faster, even growth throughout the jar. Allow the jar to recuperate for three to four days then shake again on the fourth, sixth, eighth, and if necessary the tenth day. During this time, discard any contaminated jars. Anywhere from the eighth to fourteenth days the entire jar will become completely permeated. In many circumstances, complete permeation may take much longer but the factor this is contributed to is temperature. The most likely contaminants are penicillium and aspergillus, which are almost impossible to eliminate. Rhizopus also seems to be another contaminant so commonly present that it escapes most sterile techniques. The amateur mycologist may become quite frustrated despite his or her most dedicate attempts. Nevertheless, if it is any recourse, this step is the most difficult step. In addition, if one jar has no contamination, then the mycelia clump can be broken up and divided into ten other jars with grain and the shaking process repeated until these ten new jars are permeated, then these ten can inoculate one hundred jars and so on indefinitely until contamination stops the process.

When one or more jars are completely permeated one can move on to the next step, casing. It should be noted that this is where personal choice enters the picture for people differ from here on out with individual techniques. Some employ the use of compost, an exact art requiring much time, resources, knowledge, as well as experimentation but usually maximum yields are realized. Since a discussion on compost would warrant a report in itself and since I have not used compost in the production of mushrooms, I shall omit further discussion. Other cultivators vary in the use of containers but the following discussion will follow, arguably the simplest of techniques, casing in the grain master jars. The basic functions of the casing layer are to protect the colonized substrate from drying out, to provide a humid microclimate for primordial formation and development, to provide a water reservoir for maturing mushrooms, and to support the growth of fructification enhancing microorganisms. A correctly balanced casing layer promotes beneficial microflora. The selection of the casing layer is dependent upon the following properties: water retention, structure, microflora, nutritive value, pH, and hygienic quality. The casing layer is now a soilless medium since peat moss has been introduced. It supplies nitrients and has an acid vase in which most contaminants cannot survive. Standard formulas are provided below:

#1

Course peat: four parts

Limestone flour: 1 part

Limestone grit: ½ parts

Water: approximately 2 – 2¼ parts

#2

Coarse peat: two parts

Chalk or Marl: 1 part

Water: approximately 1 – 1¼ parts

The process of casing is to place a one half inch layer, a half cup for quart jars, of remoistened, sterilized casing medium over the substrate. During the casing period, the daily transpiration and evaporation cycle is very important in order to have vigorous fruiting, healthy cultures. Maintaining the proper moisture and the evaporation rate is a complex interplay between temperature, aeration, and evaporation. If temperature or aeration is excessively high, the soil will dry out. However, this does not mean that the soil should be allowed to become waterlogged either. With temperatures at their optimum, and air exchanges as required by the cultivar, each jar should need only two to three good squirts from a fine mist sprayer daily. Watering is a critical matter at this stage to provide the first flush just enough to produce a normal flush. The first and second flushes will be the largest yielding and the following flushes grow from those before them. If the first flush is bad, then it will not get better for the following flushes. Mushroom mycelium thrives in a moist, humid casing sending out minute branching networks that expand and grow, absorbing water, carbon dioxide, and oxygen from a nearly saturated casing. The overall aspect is lush and dense. When the layer is examined, it should be held together by mycelium but should separate easily. In a dry casing, fine capillary-like mycelia will eventually permeate the entire casing layer and cause overlay, a condition in which mycelium covers the surface of the casing layer, dries out, and effectively seals the casing from water. Therefore, even though one may provide plenty of water later and even to the point of forming puddles being formed, the mushroom mycelia will not be able to utilize the resource. In a saturated casing, the mycelium grows coarse and stringy. Mycelial growth is sparse and slow, if there is any growth. A saturated casing often leaches into the substrate, which inhibits further growth and promotes contamination.

During the next two to three weeks, the mycelium will grow into the casing layer gaining more and more intersecting nodes that are visual at the interface between the glass and the casing layer. By the fourteenth to twentieth day, these nodes differentiate into tiny white dots along the surface and perimeter of the surface of the casing layer. These tiny white dots are small, young mushroom primordia. Gradually they enlarge and incorporate more mycelium and take on the appearance of small, squat mushrooms with tiny heads. This is the beginning of the pin stage. The pins gradually enlarge and some will begin to thrust above the surface. It will take another five to ten days to reach maturity.

Once the mushrooms have reached the fruiting or flushing stage, the awareness is shifted to a new class of pests and contamination, flies and mites. The first protective measure is to have screens in the air box but this will not prevent mites and flies. Second, one must be able to identify the mites. Mites are most visually active during the mid through late afternoon. They appear as tiny specks discernable from the casing layer only by their movement. Once spotted, the first recourse should be to discard contaminated jars. Next, if this is not working to one’s satisfaction would be to use Malathion ½ solution. Malathion is one of the least toxic and most rapidly degraded of commercial insecticides. However, I would recommend not harvesting for at least a week after application to allow the chemicals to dissipate. Never use the miticide Keldane because it is fatal to mushrooms.

When the veil separates from the cap margin, the mushroom is ready to harvest. The way one harvests can dramatically influence the yield of the next flush. When picking, one must be careful not to harm resting pinheads or the casing layer. Any pinhead disturbed was a potential mushroom. The best way to pick the mushrooms is with a good frame of mind. Be meticulous, unhurried, and treat the mushrooms with care. Equipped with a short bladed knife and a basket or paper bag, grasp the base of the stem and with a twisting motion, pull the mushroom from the casing layer being careful not to disturb the surroundings. Use the knife to remove, by scraping, attached casing or substrate. Then remove scrapings from the growing area and use or store your mushroom harvest.

Reference:
1. Fungiculture
2. Growing Mushrooms at Home – North American Mycological …

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