Ronald Ower / Gary Mills
One method of culturing sclerotia for use as inoculum spawn is to fill a container with wheat or other vegetative material to between about 40 to about 80 percent of its volume. The wheat is then covered with a perforated liner, typically plastic film or metal foil, although other materials can be used, and the remaining 20 to 60 percent of the container volume is then nearly filled with moist soil. The volume of the container may range from about 50 ml to multiple liters, but is typically about 500 ml. The wheat berries or other vegetative material may be supplemented with additional nutrients consisting of both organic and inorganic nitrogen sources, other minerals, vitamins and carbohydrates which help to promote storage of the nutrients that are required during subsequent ascocarp development.
"The container is covered and autoclaved to kill possible contaminating organisms.
"
The soil layer of the sterilized container is inoculated with ascospores, with vegetative hyphae or with small pieces of sclerotia, and the jar is again sealed.
The container is maintained at a temperature of between about 50°F and about 86°F and preferably between about 64.4°F and about 71.6°F . Hyphae from the inoculum grow through the soil layer and colonize the grain. After about one week, a loosely compacted mass of hyphae appear in the soil layer. Microscopically viewed, the hyphal cells become highly branched, septate and swell to a barrel shape. This is then followed by the adhesion of adjacent cells to form a solid mass that is visible to the naked eye. It is the sclerotial hyphal cells which store the materials obtained from the colonized grain. The sclerotia at maturity are hard structures which can become quite large. Virtually all of the total soil layer can become enmeshed in the sclerotia.
"At this point, the sclerotia are harvested for use as spawn. Some of the developed sclerotia may be reserved as ""jar inoculum"" for producing additional sclerotia, or for other uses.
Storage at about 41°F is found to be satisfactory for long-term preservation. "
Preferred support substratum is nutrient poor, permitting the availability of nutrients to be controlled through application and subsequent removal of an external nutrient source to the substratum. Suitable substratum includes any standard bark, soil or sawdust compost or potter's soil with or without added minerals known to those skilled in the art. For example, Supersoil .RTM. (R.McL. Co., San Francisco) has been used successfully either directly from the commercially sold bag or leached two times with two equal (v/v) volumes of water. The substratum should allow adequate drainage, should provide buffering capacity, should have good water-retaining capabilities, and should provide adequate aeration to allow proper gaseous exchange. The substratum that is now being used is about 25% sand and about 75% organic material. A small portion of lime is also added. The organic portion of the soil is primarily ground fir bark (85%) and also contains 10% sphagnum and 5% redwood bark. The soil mixture has an available water content of 55% and an air capacity of 25%. It is expected, however, that a more optimal substratum may be developed.
The substratum is steam-pasteurized or hot water-pasteurized or autoclaved.
Pasteurized substratum is then typically mixed with water to produce a workable slurry. The slurry is poured into a tray that has holes in its bottom for drainage. After the slurry is added to the desired depth in the tray, it is allowed to drain until the soil is void of gravitational water; i.e., is below field capacity, allowing for maximum air spaces. This is advantageous in at least two ways. First, it allows for increased sclerotia production, and more specifically, sclerotia are formed throughout the substratum. Secondly, removal of standing water helps to minimize later microbial contamination problems. Also, as an alternate approach for tray preparation, trays may first be filled with the substratum as above, then pasteurized.
"As a convenient means for providing a removable source of nutrients, a nutrient-rich medium is placed onto the substratum, into which source hyphae can grow and from which source the hyphae can distribute nutrients throughout the mycelial colony. As one means of providing such a source, jars are prepared similar to those used to culture the sclerotia. Typically jars are nearly filled with organic material; a perforated heat resistant liner (usually metal foil) is placed over the organic material; and the liner is covered with soil to the top of the jar. The jar is again covered with another layer of perforated foil, further sealed with a sheet of metal foil and then sterilized.
"
In the first variation, sclerotia are divided into pieces between about 0.5 and about 4 cubic centimeter in size and inoculated into a thin layer of substratum which is typically between about 1 to about 4 cm deep. Good results occur when there are about 6 to about 30 cc. of divided sclerotia per square meter of substratum surface. Mycelial growth from the inoculum is enhanced by soaking sclerotial pieces in water just prior to inoculating them into the substrate.
"After the poured substratum is inoculated with sclerotial pieces,
the temperature around the tray is maintained between about
50°F and about 71.6°F, the relative humidity is maintained between
about 75% and about 95%, and the water content of the substratum
is maintained between about 50% and about 75%.
Soon after inoculation, hyphae grow from the sclerotia and completely colonize the tray in about one week.
As the mycelia develop, no further water is added, thereby allowing the substratum to dry, preferably
to a substratum moisture content of below about 75%.
Drying of the substratum before feeding is considered to be an important factor
in inhibiting growth of bacteria and other fungi which would harm or compete with the developing morels.
Better results are obtained if the sclerotia are maintained in the substratum and their mycelia allowed to colonize the substratum
for about 7 days under conditions similar to conditions during that period in the first variation when the sclerotia are maintained in the nutrient-poor substratum but before water is percolated through the substratum. Next, in a similar manner to the first variation,
water is percolated through the substratum, promoting initiation of primordia from the mycelia. "
In this first variation of the method, the top layer of foil is removed from the cooled sterilized jars, and the jars are inverted onto the surface of the substratum. Hyphae grow upward through the holes in the second layer of foil, gather nutrients and distribute the nutrients to the mycelial colonies.
It is desirable that as much sclerotia be produced within the substratum as is possible during this stage because there appears to be a direct relationship between the amount of sclerotia in the substratum and the total weight of ascocarps that develop per unit area of the substratum. Growth of sclerotia in substratum parallels growth of sclerotia in jars, and the same nutrient factors which enhance growth in the jars enhance growth in the substratum. Accordingly, the organic material may be supplemented with vitamins, minerals, additional protein and other substances.
"During feeding, the soil moisture is maintained at a level of between about 45% and about 70%,
the relative humidity is maintained at between about 85% and about 95% and the temperature
is maintained between about 50°F and about 71.6°F
Feeding continues for a period of between about 7 and about 40 days, typically about 16 days.
At the end of the feeding period, both conidia and sclerotia may be observed
in substantial numbers on the surface of the substratum.
"
"Having provided the mycelium and newly formed attendant sclerotia with substantially all of the nutrients needed for subsequent ascocarp formation, the nutrient source is removed. Removal of the nutrients is a necessary step for cultivation because the sexual cycle will not commence to any appreciable extent in the presence of excess nutrients that are external to the mycelium. The use of an inverted jar or the like containing nutrient material permits the immediate removal of most of the available nutrients, leaving the mycelia in a nutrient-poor substratum.
"
Following after removal of the nutrient source, a small amount of additional moisture is added to the substratum, e.g., about 1 liter per square meter of substratum surface, and vegetative growth is allowed to continue for a period of about ten days. During this period, the substratum moisture content is maintained at between about 45% and about 70%, the relative humidity is maintained at between about 85% and about 95% percent, and the temperature is maintained at between about 50°F and about 71.6°F After this period the sclerotia are mature.
PROPRIETARY SECRET
PROPRIETARY SECRET
"
"
"The mature sclerotia and associated mycelia, rich in stored nutrients but deprived of exogenous nutrients, are now ready for exposure to high amounts of water, which contribute to induction to the sexual cycle. Preferably the substratum and morel mycelium are hydrated by a slow percolation of water through the substratum for a period of between about 12 and about 36 hours. Water is added to the substratum at a rate of between about ¼ liter and about 1 liter per hour per square meter or 1.5 - 2.5 fl oz per square foot per hour of substratum surface area. The substratum and the percolating water are maintained at a temperature of between about 50°F and about 71.6°F
"
Following hydration in either the first or second variation, the substratum is allowed to drain, and the cultures may be aspirated to further remove water. The relative humidity is maintained at between about 85% and about 95%, and the temperature is maintained at between about 50°F and about 71.6°F The substratum moisture content is maintained at between about 55% and about 65% during this period.
At the end of this period, i.e., approximately 1-3 days after hydration, morel primordia start to form. Primordia are spherical hyphal aggregates which are about one millimeter in diameter. Within a few days, the primordia form protuberances which represent the first sign of ascocarp fundament formation.
A growth period extending from the initial appearance of primordia until the morel ascocarp reaches a height of about thirty millimeters represents an important period for ascocarp development. During this period, the temperature is maintained at between about 50°F and about 71.6°F and preferably about 64.4°F, the relative humidity at between about 85 and about 95 percent and the substratum moisture content at between about 50 and about 60 percent. Unless very favorable growth conditions are maintained, immature ascocarps are prone to abort.
It has been found that maximum yields of ascocarps are obtained when the air flow near the substratum is maintained at a substantially steady rate of between about 8 and about 16 inches per minute.
After the morel ascocarp reaches the height of 1.25 inches, conditions are maintained that are favorable to continued development and maturation. The temperature during this part of the maturation may range from about 50°F to about 80.6°F, the relative humidity may range from about 80% to about 95% percent, and the soil moisture may range from about 30% to about 55%. As the ascocarps continue to develop, they may turn a dark grey, and upon reaching maturity the ascocarp color changes from grey to a golden-brown, at which point the morels are mature.
In the second variation, the mature sclerotia which are produced in the jars are inoculated into a wetted, nutrient-poor substratum at a much higher rate, e.g., typically between about 1500 and 4000 cc per m.sup.2 of substratum surface. These sclerotia contain the stored nutrients that are necessary for hyphal proliferation and subsequent fruit body development. The sclerotia may be inoculated into the substratum whole or divided; they may also be inoculated directly from the jars or wetted with water first, e.g., typically an 18 to 24 hour immersion. Inoculation into the nutrient-poor substrate represents deprivation of exogenous nutrients to the sclerotia, one of the factors found to contribute to induction to the sexual cycle of growth.
The other factor found to contribute importantly to induction, i.e., exposure to high amounts of water, may commence at the same time with inoculation into the substrate or a relatively short period of time thereafter. The substratum may be thoroughly wetted at about the time of inoculation to provide the high amount of water which promotes induction.
"There are several advantages to the second variation of the method relative to the first variation. One of the more notable advantages to the second variation is the permissible depth of the substratum. For this method the substratum can be considerably deeper, typically between about 2.25 and about 6.25 inches. Cultures with a thicker substratum can contain more sclerotia and thus eventually support more ascocarps per unit area of substratum surface than can a thinner substratum layer.
However, the first variation may be preferred because it is more closely analogous to processes used to cultivate other types of fungi, and therefore, may be more adaptable to cultivation in existing facilities or with available apparatus."
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If you wanna ride, don't ride the white horse, if you wanna ride, ride the white pony
Edited by AcidHorse (07/24/06 09:21 PM)
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