United States Patent (19) (11) Patent Number: 4,757,640

Ower, deceased et al. 45 Date of Patent: Jul. 19, 1988

(54. CULTIVATION OF MORCHELLA Mushroom Volvariella Volvacer' Hortscience vol. 7(5)
(75) Inventors: Ronald D. Ower, deceased, late of San pp. 461-464, Oct. 1972.
Francisco, Calif., by George A. Bels, R. J. et al, "The Challenge to Agaricus Bisporus
Yeoman, executor; Gary L. Mills, from Other Fungi' (paper to) NGA Brighton Confer
East Lansing; James A. ence, Oct. 1973.
Malachowski, Haslett, both of Mich. Lambert, E. B. "Principles and Problems of Mushroom
73) Assignee: Neogen Corporation, Lansing, Mich. Culture' Botanical Review, vol. 4, Jul. 1938, pp. 419
and 420 only.
(21) Appl. No.: 872,823 R. Ower, "Notes on the Development of the Morel
(22 Filed: Jun. 11, 1986 Ascocarp: Morchella Esculenta,' Mycologia,74(1): 142
(1982).
Related U.S. Application Data Primary Examiner-James R. Feyrer
63 Continuation-in-part of Ser. No. 728,176, Apr. 29, Attorney, Agent, or Firm-Fitch, Even, Tabin &
1985, Pat. No. 4,594,809. Flannery
51) Int. C. ............................................... A01G 1/04 57) ABSTRACT
52 U.S. C. ....................................................... 47/1.1 The invention pertains to culturing ascoscarps or fruit
58) Field of Search ................... 47/1.1, 1.3, 58; 71/1, bodies of species of the genus Morchella. Mycelia are
71/3 provided with nutrients and subsequently produce nu
56) References Cited trient-primed mycelia, such as nutrient-rich sclerotia or
U.S. PATENT DOCUMENTS nutrient-rich hyphae, in which are stored sufficient
nutrients to supply the ascocarps that develop later. The
3,942,969 3/1976 Carroll et al. ... ... 47/1.1 fungus is induced to give rise to ascocarp development
4,164,405 8/1979 Pinkard ..................................... 71/3 by initially maintaining the fungus in an environment
4,229,442 10/1980 Pinkard ..................................... 71/3 that is poor in exogenous nutrients, and by exposing the
4,370,159 1/1983 Holtz........ ... 47/.1 fungus to a high level of water. After induction, primor
4,594,809 6/1986 Ower et al. ............................ 47/1.1
dia appear. The period from primordia appearance until
FOREIGN PATENT DOCUMENTS midway to maturation of the fruitbodies represents a
01.07911 5/1984 European Pat. Off. ............... 47/1.1 critical period during which the fruitbodies are prone to
0112156 9/1978 Japan ................... ... 47/1.1 abort. During this critical period, particular attention is
0124678 10/1978 Japan ................... ... 47/1.1 directed to maintaining favorable conditions. The fruit
8500002 1/1985 World Int. Prop. O. ...........4i/ii bodies, which may be grown to maturation, are ulti
OTHER PUBLICATIONS mately harvested.
San Antonio, J. A. et al, "Cultivation of Paddy Straw 14 Claims, No Drawings
 1.
4,757,640
2
Condiophore-a simple or branched hypha arising
CULTIVATION OF MORCHELLA from a somatic hypha and capable of bearing at its
This application is a continuation-in-part of pending tip or side one or more conidiogenous cells.
application U.S. patent application Ser. No. 728, 176 Conidium-(pl. conidia) sometimes called conidios
filed Apr. 29, 1985, now U.S. Pat. No. 4,594,809. pores, a nonmotile asexual spore usually formed at
The present invention is directed to cultivation of the the tip or side of a cell; in some instances a pre
morel fungi i.e., species of Morchella, including their existing hyphal cell may transform into a conidium.
mature, edible ascocarps. Hypha-(pl. hyphae) the unit of vegetative structure
10 of most fungi; a tubular, filamentous cell containing
BACKGROUND OF THE INVENTION asexual nuclei.
The genus Morchella contains the species of mush Mycelium (pl. mycelia) mass of hyphae constituting
rooms known as morels or sponge mushrooms. They the body (thallus) of a fungus.
belong to the ascomycetous fungi. True morels are Primordium (pl. primordia) the beginning stage of
edible and delicious. Indeed, some consider them the 15 any structure.
most delectable of all the fungi. While the taste of these Sclerotium (pl. sclerotia) a hard surfaced resting body
mushrooms is known and loved by those who search of fungal cells resistant to unfavorable conditions,
the forests in the early spring, morels are unavailable to which may remain dormant for long periods of time and
the general population because heretofore they have resume growth on the return of favorable conditions.
defied cultivation such as would be practical for com 20 Substratum (pl. substrata) for the purpose of this
mercial production year round. document substratum will be defined as the soil-like
To the connoisseur of mushrooms, morels are known material which serves as the habitat in which the fungus
by their ascocarp or fruitbody (the visible mushroom). grows and from which the fungus produces fruitbodies.
One would suppose that if these fungi grow freely with Nutrient-rich hyphae is defined herein as asexual
out cultivation in the wild or natural state, cultivation 25 hyphae developed in the presence of a nutrient source
methods would have been developed to maximize their either in an aqueous medium or in a substratum.
production. This, however, has not been the case. There Nutrient-primed mycelia is defined herein as asexual
are reports of growing morels outdoors; however, no mycelial growth material containing sufficient stored
one has succeeded in cultivating morels, like the com nutrients so as to be inducible into the sexual cycle of
mon Agaricus species or other edible forms, in environ 30 Morchella growth, this term includes both nutrient-rich
mentally controlled rooms and harvesting them sclerotia as defined above and nutrient-rich hyphae as
throughout the year. defined above.
Ascocarp or fruitbody production is the mature em
bodiment of the sexual reproduction cycle of the morel. SUMMARY OF THE INVENTION
The mature ascocarp containing ascospores or germ 35 The invention provides for the culturing of species of
spores represents the culmination of a life cycle high the genus Morchella to produce mature ascocarps or
lighted by an internal mating of two haploid nuclei to fruitbodies. Vegetative mycelia are fed nutrients for
form a diploid nucleus which undergoes meiosis to form development into nutrient-primed mycelia, such as nu
new haploid ascospores. Both autogamous and heterog trient-rich hyphae or sclerotia, in which are stored suffi
amous pairing prior to meiosis have been reported for cient nutrients to supply substantially the entire nutrient
Morchella. An alternative life cycle is an asexual pro requirements for subsequent development of fruitbo
cess in which conidia (asexual spores) are produced and
from which new mycelium, containing haploid nuclei, dies. Subsequent to feeding, the environment of the
nutrient-primed mycelia is substantially altered in order
can be grown.
to
Also, as a means of protecting the species under cer 45 carpspromote the sexual cycle of growth in which asco
tain conditions, the vegetative mycelia coalesce into to this(visible mushrooms) are produced. Contributing
process is removal of available exogenous nutri
hardened bodies known as sclerotia which may lie dor ents from the nutrient-primed mycelia. Also contribut
mant during periods of unfavorable conditions. Accord
ingly, fruiting of the morel occurs during select condi ing to this process is exposure of the nutrient-primed
tions; a situation recognized by mushroom hunters who 50 mycelia, and additional mycelia that grow therefrom, to
have experienced "bad years' for morel gathering. high levels of substratum water. The sexual cycle of
It is a general object of the invention to provide a growth is first evidenced by the appearance of primor
method for culturing morels in a manner suitable for dia and culminates in mature fruitbodies. The growth
commercial production of ascocarps throughout the period from primordia appearance to about the time of
year under controlled conditions. 55 fruitbody maturation is an especially critical time of
DEFINITIONS
development, and conditions are carefully controlled to
minimize abortion of the developing fruitbody. One
For purposes of clarity, terms used in this application important factor in minimizing abortion of the develop
are defined as following in C. J. Alexopoulos and C. W. ing fruitbody is ensuring previous storage of sufficient
Mims, Introductory Mycology, 3rd Ed., John Wiley & nutrients, particularly neutral lipids, in the nutrient
Sons, New York (1979).: primed mycelia to support subsequent fruitbody devel
Ascocarp-a fruitbody containing asci. opment and maturation. Other important factors are the
Ascospore-a meiospore borne in an ascus. maintainance of correct air humidity and substratum
Ascus-(pl. asci) a sac-like cell generally containing a moisture during fruitbody development and proper
definite number of ascospores (typically eight) 65 ventilation during fruitbody development. Other fac
formed by free cell formation usually after karyog tors include maintaining optimal air velocity relative to
amy and meiosis; characteristic of the class Asco the habitat and maintaining a daily water loss from the
mycetes. habitat.
 4,757,640 4.
3
DETAILED DESCRIPTION OF THE layer. Microscopically viewed, the hyphal cells become
PREFERRED EMBODIMENTS
highly branched, septate and swell to a barrel shape.
This is then followed by the adhesion of adjacent cells
The invention provides for the culturing of morels to to form a solid mass that is visible to the naked eye. It is
produce ascocarps or fruitbodies. The spawn that are 5 the sclerotial hyphal cells which store the materials
used for morel cultivation are nutrient-primed mycelia, obtained from the colonized grain. The sclerotia at
including sclerotia, which are resting bodies and nutri maturity are hard structures which can become quite
ent reservoirs that are somewhat resistant to unfavor large. Virtually all of the total soil layer can become
able conditions, and also including nutrient-rich hyphae enmeshed in the the sclerotia.
such as that developed in an aqueous nutrient medium. 10 At this point, the sclerotia are harvested for use as
Such nutrient-primed mycelia, depending upon envi spawn. Some of the developed sclerotia may be re
ronmental conditions, may either sustain additional served as "jar inoculum' for producing additional scle
asexual vegetative mycelial growth or give rise to the rotia, or for other uses.
sexual cycle and mature ascocarps. Nutrients, particu An alternative method is to culture aqueous
larly neutral lipids, in the form of triglycerides, are 15 developed mycelia for such spawn. For example, myce
stored in the nutrient-primed mycelia, and during the lial inoculant is grown statically at room temperature,
sexual cycle, substantially all of the nutrients necessary i.e., about 22 C. for 3-4 weeks in a liquid nutrient me
for fruitbody development are drawn from these and dium. Although many different media can be used, one
other stored nutrients. such medium is potato dextrose broth, a liquid medium
Accordingly, the invention provides for production 20 commonly used to culture many different fungi. Hy
or cultivation of nutrient-primed mycelia which contain phae within such mycelia usually do not form adhesive
sufficient storage of nutrients necessary for subsequent aggregates nor hardened structures; however upon
development to ascocarps. Conditions are then adjusted reaching maturity they do contain nutrient reserves
appropriate to induce the nutrient-primed mycelia to sufficient to supply substantially the entire nutrient re
enter the sexual growth cycle. Substantial care is taken 25 quirements for subsequent development of fruitbodies.
during development from primordia appearance to as The use of nutrient-primed mycelia as spawn has
cocarp maturation to maintain conditions that ensure several advantages with respect to the efficient produc
that the developing ascocarps do not abort. In particu tion of morels. In addition to growing at a rate commen
lar, conditions of soil moisture, humidity and air Surate with serving as a steady source of inoculum,
exchange are maintained as necessary to promote asco 30 nutrient-primed mycelia, particularly sclerotia, may be
carp development and minimize disease. preserved for extended periods of time. It is possible
The first step of morel production is the development that, in nature, sclerotia remain dormant for extended
of nutrient-primed mycelia spawn. The use of nutrient periods of time, such as over the winter months, until
primed mycelia as spawn represents an important aspect conditions become favorable for initiation of growth.
of the invention with regards to efficient production of 35 Storage at about 5' C. is found to be satisfactory for
morels. Although cultivation of morels could be ef. long-term preservation.
fected starting with spores, production would be much Mature nutrient-primed mycelia are used as spawn to
slower and, thus, impractical for commercial cultiva inoculate appropriate substratum. Two variations on
tion. In addition, the traditional use of grain spawn the method of the present invention may be followed. In
would be inappropriate because the spawn would lead 40 the first variation (Method I), nutrient-primed mycelia,
to cultures that are highly contaminated with other preferably in the form of sclerotia, are divided into
fungi and bacteria. pieces which are used to inoculate a substratum. Upon
One method is to culture sclerotia for use as inoculum addition of nutrients, additional nutrient-primed myce
spawn. A common method of culturing sclerotia is to iia are formed within the substratum before induction to
fill a container with wheat or other vegetative material 45 the sexual cycle. In the second variation (Method II),
to between about 40 to about 80 percent of its volume. nutrient-primed mycelia either sclerotia or aqueous
The wheat is then covered with a perforated liner, typi developed mycelia are directly inoculated into a sub
cally plastic film or metal foil, although other materials stratum, and the nutrient-primed mycelia and any addi
can be used, and the remaining 20 to 60 percent of the tional mycelia which growtherefrom are induced to the
container volume is then nearly filled with moist soil. 50 sexual cycle, without adding nutrients.
The volume of the container may range from about 50 An important aspect of the invention is induction or
ml to multiple liters, but is typically about 500 ml. The triggering of the fungus to enter the sexual growth
wheat berries or other vegetative material may be sup cycle in which ascocarps are produced. One important
plemented with additional nutrients consisting of both contributing factor in induction is deprivation of avail
organic and inorganic nitrogen sources, other minerals, 55 able exogenous nutrients to the fungus so that assimila
vitamins and carbohydrates which help to promote tion and storage of nutrients by the fungus ceases or
storage of the nutrients that are required during subse significantly slows. Accordingly, the environment of
quent ascocarp development. The container is covered the fungus is altered from a nutrient-rich environment
and autoclaved to kill possible contaminating organ to a nutrient-poor environment. For purposes of this
isms. The soil layer of the sterilized container is inocu invention, a "nutrient poor' environment is an environ
lated with ascospores, with vegetative hyphae or with ment lacking readily available nutrients for supplying
small pieces of sclerotia, and the jar is again sealed. The developing ascocarps; thereby, such developing asco
container is maintained at a temperature of between carps utilize the nutrients which have been stored, as in
about 10 C. and about 30° C. and preferably between the nutrient-primed mycelia.
about 18" C. and about 22 C. 65 Another important factor which appears to contrib
Hyphae from the inoculum grow through the soil ute to induction is exposure of the fungus to high quanti
layer and colonize the grain. After about one week, a ties of water in the substratum in which the fungus is
loosely compacted mass of hyphae appear in the soil growing. Typically, the substratum is hydrated substan
 5 4,757,640
6
tially to saturation for the purpose of promoting induc completely colonize the tray in about one week. As the
tion to the sexual cycle. By substantially saturated is mycelia develop, no further water is added, thereby
meant at least about 90% of the capacity of the substra allowing the substratum to dry, preferably to a substra
tum to hold water, but preferably approaching 100% tum moisture content of below about 75%. Drying of
capacity Preferably, during exposure to high quantities the substratum prior to feeding is considered to be an
of water, there is a continuous exchange of water. This important factor in inhibiting growth of bacteria and
may be accomplished, for example, by percolating other fungi which would harm or compete with the
water through the substratum in which the fungus is developing morels.
growing. Although Applicants are not bound to any Morels, being fungi, do not produce their own food
theory as to why the high level of water seems to pro 10 as do photosynthesizing plants, but rather obtain their
mote induction, the water may provide a triggering total nutrient supply from external sources. When a
"shock” to the system, e.g., by change in osmotic pres nutrient-poor substratum is deliberately provided, the
St.
In Method I, nutrient-primed mycelia in the form of site nutrients, andsometime
morel tissue must be provided with the requi
in this variation, nutrients are fed to
sclerotia are divided into pieces between about 0.5 and 15 the mycelia growing from the inoculum. The additional
about 4 cubic centimeters in size and inoculated into a nutrient-primed mycelia in the form of sclerotia that
thin layer of substratum which is typically between develop from the vegetative growth after such nutrient
about 1 and about 4 cm. deep. Good results occur when addition should contain, in stored form, substantially all
there are about 6 to about 30 cc. of divided sclerotia per of the nutrients that are needed for efficient fruitbody
square meter of substratum surface. Additional mycelial 20 development.
growth from sclerotial inoculum is enhanced by soaking Nutrients are provided to the mycelial growth mate
the sclerotial pieces in water just prior to inoculating rial in a manner so that the nutrients may be later with
them into the substrate. drawn to leave the substratum again nutrient-poor.
Preferred support substratum is nutrient-poor, per Removal of nutrients promotes differentiation of nutri
mitting the availability of nutrients to be controlled 25 ent-primed mycelia into the sexual cycle and decreases
through application and subsequent removal of an ex the incidence of contamination.
ternal nutrient source to the substratum. Suitable sub As a convenient means of providing a removable
stratum includes any standard bark, soil or sawdust source of nutrients, a nutrient-rich medium is placed
compost or potter's soil with or without added minerals onto the substratum, into which source hyphae can
known to those skilled in the art. For example, Super 30 grow and from which source the hyphae can distribute
soil (R) (R. McL. Co., San Francisco) has been used nutrients throughout the mycelia colony. As one means
successfully either directly from the commercially sold of providing such a source, jars are prepared similar to
bag or leached two times with two equal (v/v) volumes those used to culture the sclerotia. Typically jars are
of water. The substratum should allow adequate drain nearly filled with organic material; a perforated heat
age, should provide buffering capacity, should have 35 resistant liner (usually metal foil) is placed over the
good water-retaining capabilities, and should provide organic material; and the liner is covered with soil to :
adequate aeration to allow proper gasous exchange. the top of the jar. The jar is again covered with another
The substratum that is now being used is about 25% layer of perforated foil, further sealed with a sheet of
sand and about 75% organic material. A small portion metal foil and then sterilized.
of lime is also added. The organic portion of the soil is The nutrient source with which the jar is nearly filled
primarily ground fir bark (85%) and also contains 10% provides the organic material. The organic material is
sphagnum and 5% redwood bark. The soil mixture has metabolized and eventually is stored in the sclerotia as
an available water content of 55% and an air capacity of carbohydrates and lipids. The stored material is eventu
25%. It is expected, however, that a more optimal sub ally utilized for ascocarp formation. The nutrient source
stratum may be developed. 45 most commonly used in the development of this cultiva
The substratum is steam-pasteurized or hot water tion method is wheatberries; however, other vegetative
pasteurized or autoclaved. Pasteurized substratum is material, including mixed compost, is suitable. If wheat
then typically mixed with water to produce a workable berries are the nutrient source, they should be provided
slurry. The slurry is poured into a tray that has holes in at a ratio of about 1000 grams to about 8000 grams (dry
its bottom for drainage. After the slurry is added to the 50 wt.) per square meter of substratum. However, this ratio
desired depth in the tray, it is allowed to drain until the may vary significantly and is considered only as a gen
soil is void of gravitational water; i.e., is below field eral approximation.
capacity, allowing for maximum air spaces. This is ad It is desirable that as much sclerotia be produced
vantageous in at least two ways. First, it allows for within the substratum as is possible during this stage
increased sclerotia production, and more specifically, 55 because there appears to be a direct relationship be
sclerotia are formed throughout the substratum. Se tween the amount of sclerotia in the substratum and the
condly, removal of standing water helps to minimize total weight of ascocarps that develop per unit area of
later microbial contamination problems. Also, as an the substratum. Growth of sclerotia in substratum paral
alternative approach for tray preparation, trays may lels growth of sclerotia in jars, and the same nutrient
first be filled with the substratum, as above, and then factors which enhance growth in the jars enhance
pasteurized. growth in the substratum. Accordingly, the organic
After the poured substratum is inoculated with scle material may be supplemented with vitamins, minerals,
rotial pieces, the temperature around the tray is main additional protein and other substances.
tained between about 10 C. and about 22 C., the rela In this example of Method I, the top layer of foil is
tive humidity is maintained between about 75 and about 65 removed from the cooled sterilized jars, and the jars are
95 percent, and the water content of the substratum is inverted onto the surface of the substratum. Hyphae
maintained between about 50% and about 75%. Soon grow upward through the holes in the second layer of
after inoculation, hyphae grow from the sclerotia and foil, gather nutrients and distribute the nutrients to the
 4,757,640 8
7
mycelial colonies. During feeding, the soil moisture is wetted at about the time of inoculation to provide the
maintained at a level of between about 45% and about high amount of water which promotes induction. Better
70%, the relative humidity is maintained at between results, however, are obtained if the nutrient-primed
about 85% and about 95% and the temperature is main mycelia are maintained in the substratum and additional
tained between about 10 C. and about 22 C. Feeding mycelial growth is allowed to colonize the substratum
continues for a period of between about 7 and about 40 for about seven days under conditions similar to condi
days, typically about 16 days. At the end of the feeding tions during that period in Method I when the sclerotia
period, both conidia and sclerotia may be observed in are maintained in the nutrient-poor substratum but be
substantial numbers on the surface of the substratum. fore water is percolated through the substratum. Next,
Having provided the mycelial growth and newly O in a similar manner to the first variation, water is perco
formed attendant sclerotia with substantially all of the lated through the substratum, promoting initiation of
nutrients needed for subsequent ascocarp formation, the primordia from the nutrient-primed mycelia.
nutrient source is removed. Removal of the nutrients is There are several advantages to Method II relative to
a necessary step for cultivation because the sexual cycle the Method I. One of the more notable advantages of
will not commence to any appreciable extent in the 15 Method II is the permissible depth of the substratum.
presence of excess nutrients that are external to the For this method, the substratum can be considerably
mycelia. The use of an inverted jar or the like contain deeper, typically between about 6 and about 16 cm.
ing nutrient material permits the immediate removal of Cultures with a thicker substratum can contain more
most of the available nutrients, leaving the mycelia in a nutrient-primed mycelia and thus eventually support
nutrient-poor substratum. 20 more ascocarps per unit area of substratum surface than
Subsequent to removal of the nutrient source, a small can a thinner substratum layer.
amount of additional moisture is added to the substra However, Method I may be preferred because it is
tum, e.g., about 1 liter per square meter of substratum more closely analogous to processes used to cultivate
surface, and vegetative growth is allowed to continue other types of fungi and, therefore, may be more adapt
for a period of about ten days. During this period, the 25 able to cultivation in existing facilities or with available
substratum moisture content is maintained at between apparatus.
about 45% and about 70%, the relative humidity is Following hydration in either method, the substra
maintained at between about 85% and about 95% per tum is allowed to drain, and the cultures may be aspi
cent, and the temperature is maintained at between rated to further remove water. The relative humidity is
about 10 C. and about 22 C. After this period, the 30 maintained at between about 85% and about 95%, and
sclerotia have matured. the temperature is maintained at between about 10 C.
The mature sclerotia and associated mycelia, rich in and about 22 C. The substratum moisture content is
stored nutrients but deprived of exogenous nutrients, maintained at between about 55% and about 65% dur
are now ready for exposure to high amounts of water, ing this period.
which contribute to induction to the sexual cycle. Pref 35 At the end of this period, i.e., approximately 1-3 days
erably the substratum and morel mycelium are hydrated after hydration, morel primordia start to form. Primor
by a slow percolation of water through the substratum dia are spherical hyphal aggregates which are about one
for a period of between about 12 and about 36 hours. millimeter in diameter. Within a few days, the primor
Water is added to the substratum at a rate of between dia form protuberances which represent the first sign of
about 250 and about 1000 ml per hour per square meter 40 ascocarp fundament formation.
of substratum surface area. The substratum and the A growth period extending from the initial appear
percolating water are maintained at a temperature of ance of primordia until the morel ascocarp reaches a
between about 10 C. and about 22 C. height of about thirty millimeters represents an impor
In Method II, either the mature nutrient-primed scle tant period for ascocarp development. During this per
rotia which are produced in the jars or the nutrient 45 iod, the temperature is maintained at between about 10
primed mature aqueous-developed mycelia are inocu and about 22 C. and preferably about 18 C., the rela
lated into a wetted, nutrient-poor substratum at a much tive humidity at between about 85 and about 95 percent
higher rate, e.g., typically between about 1500 and 4000 and the substratum moisture content at between about
grams (fresh weight) per square meter of substratum 50 and about 60 percent. Unless very favorable growth
surface. These nutrient-primed mycelia contain all the 50 conditions are maintained, immature ascocarps are
stored nutrients that are necessary for hyphal prolifera prone to abort.
tion and subsequent fruitbody development. The nutri It has been found that maximum yields of ascocarps
ent-primed mycelia may be inoculated into the substra are obtained when the air flow near the substratum is
tum whole or divided. Sclerotia may be inoculated maintained at a substantially steady rate of between
directly from the jars described hereinbefore or wetted 55 about 20 and about 40 cm per minute.
with water first, e.g., typically an 18 to 24 hour immer After the morel ascocarp reaches the height of thirty
sion. Aqueous-developed mycelia are simply removed millimeters, conditions are maintained that are favor
from the aqueous nutrient medium and carefully able to continued development and maturation. The
washed with water. Inoculation into the nutrient-poor temperature during this part of the maturation may
substrate represents an additional deprivation of exoge 60 range from about 10 C. to about 27 C., the relative
nous nutrients to the nutrient-primed mycelia, one of humidity may range from about 80% to about 95%
the factors found to contribute to induction to the sex percent, and the soil moisture may range from about
ual cycle of growth. 30% to about 55%. As the ascocarps continue to de
The other factor found to contribute importantly to velop, they may turn a dark grey. The ascocarp color
induction, i.e., exposure to high amounts of substratum 65 then changes from grey to a golden-brown, at which
water, may commence contemporaneously with inocu point the morels are mature. After the first crop of
lation into the substrate or a relatively short period of ascocarps are harvested, the cultures may be reinduced
time thereafter. The substratum may be thoroughly to produce a subsequent crop(s).
 9 4,757,640
10
Using the method as described above with Morchella inducing said nutrient-primed mycelia to enter the
esculenta, yields of 25 to 500 ascocarps per square meter sexual reproductive cycle of such species, and
have been obtained. maintaining conditions appropriate for development
Although most of the development of the method has of ascocarps of such species.
concerned isolates of Morchella esculenta, the methods 5 2. A method according to claim 1 wherein said nutri
of the invention are generally applicable to other spe ent-primed mycelia are generated in potato dextrose
cies within the genus Morchella. For example, success broth.
with the species tentatively determined as Morchella 3. A method according to claim 1 wherein said myce
crassipes and Morchella costata have been obtained. lia are induced to the sexual reproductive cycle through
As an alternative to growing nutrient-rich hyphae in 10 the deprivation of exogenous nutrients and by exposure
a liquid nutrient, morel mycelia may be cultured in a to high amounts of water.
substatum which uses paper and urea as the respective
primary carbon and nitrogen sources and yields morel iod4.subsequent
A method according to claim 3 wherein for a per
to inoculation, said nutrient-primed my
vegetative hyphae which are seemingly intermediate celia are maintained in said nutrient-poor substratum at
between normal filamentous hyphae and sclerotial hy 15 a substratum moisture content of between about 45%
phae. These "modified', somewhat enlarged, irregu and about 70%, a relative humidity of between about
larly shaped hyphae are either found singlely or in small 85% and about 95% and a temperature of between
non-coalesced clusters and are dispersed throughout the about 10 C. and about 22 C., and subsequently, said
contents of the substratum. substratum is hydrated to expose said nutrient-primed
In this procedure, nutrient-rich hyphae are cultivated 20 mycelia to high amounts of water.
following the protocol outlined in Method I. Sclerotia 5. A method according to claim3 wherein subsequent
pieces are added to the substratum, and the fungus is to induction, the water content of said substratum is
allowed to grow for one week after which the sclerotia
pieces are removed. Following colonization, a mi adjusted relative
to between about 55% and about 65%, the
humidity is maintained at between about 85%
crowave-pasterized nutrient package is added to the 25 and about 95% and
surface of the substratum. This package is typically a between about 10 C.the and
temperature is maintained at
about 22 C. until primordia
plastic bag filled with moist paper, e.g., shredded 20 appear.
pound rag bond paper, the moisture content being less 6. A method according to claim 3 wherein from the
than 50% of the dry weight of the paper, preferably appearance of primordia until ascocarp development to
about 30-40%, and the underside of the package is 30 a height of about 30mm, the water content of said sub
perforated with small holes that provide entry points stratum is maintained at between about 50% and about
through which the fungus grows into the package. 60%, the relative humidity is maintained at between
After the substratum resident mycelia grow into and
occupy the paper packets, generally about 5-7 days, an about 85% and about 95% and the temperature is main
tained at between about 10 C. and about 22 C.
aqueous solution of less than about 1%, e.g., about 0.5 35 7. A method according to claim3 wherein during said
weight %, of urea is applied, approximately 40 ml per period from primordia appearance until ascocarp devel
square foot, to the substratum at 2-3 day intervals for an opment to a height of about 30 mm, the air flow near
additional two weeks. The packets are then removed, said substratum is maintained at between about 20 and
and the cultures are treated similarly using the method about 40 cm per minute.
outlined in Method I following nutrient removal. 8. A method according to claim 3 wherein, from a
While the invention has been described in terms of a period from ascocarp development at a height of about
particularly preferred embodiment, modifications obvi 30 mm to ascocarp maturity, the water content of said
ous to one with ordinary skill in the art may be made substratum is maintained at between about 30% and
without departing from the scope of the present inven about 55%, the relative humidity is maintained at be
tion. For example, conditions are described hereinabove 45 tween about 80% and about 95% and the temperature is
which are particularly favorable for promoting growth maintained at between about 10 C. and about 27 C.
of morels during various stages of their growth, such 9. A method according to claim 4 wherein said sub
factors, including substratum moisture, temperature, stratum is hydrated by percolating water through said
humidity, air flow etc. It is to be understood that substratum at rate of between about 250 and about 1000
growth may well proceed, at a less favorable rate at 50 per m2 of substratum surface per hour.
conditions outside of the stated preferred conditions ml10.
and that short-term excursions from the preferred con water Aat method according to claim 9 wherein said
ditions may not seriously affect the growth rate of mo about 22 C. is percolatedofforbetween
a temperature
a period
about 10 C. and
of between about
rels. Thus, for example, whereas a lower temperature of 12 and about 36 hours.
a favorable temperature range is stated in respect to 55 11. A method for culturing ascocarps of species of the
several stages of growth of the ascocarp, short term
temperature excursions to temperatures approaching genus Morchella comprising
cultivating asexual mycelial growth of such species in
the freezing point of water are consistent with the con the presence of a nutrient that provides both or
tinued survival of the ascocarps. ganic and inorganic nutrients for a period of time
Various features of the invention are set forth in the 60 sufficient for said mycelial growth to mature into
following claims. nutrient-primed mycelia in which are stored the
What is claimed is: nutrient supply needed for subsequent ascocarp
1. A method for culturing ascocarps of species of the development,
genus Morchella comprising
generating nutrient-primed mycelia in an aqueous 65 inducing growth
said nutrient-primed mycelia to the sexual
cycle of such species,
nutrient medium, and maintaining conditions appropriate for develop
providing a nutrient-poor substratum and inoculating ment and maturation of ascocarps of such species,
said nutrient-primed mycelia into said substratum, wherein said cultivation and induction are carried
 4,757,640 12
11
out by inoculating a nutrient-poor substratum with 12. A method according to claim 11 wherein said
pieces of nutrient-primed mycelia, wherein myce nutrient source includes an aqueous solution of urea
lial growth in said substratum is promoted from which is supplied by periodic applications.
said inoculum, wherein the environment of said 13. A method according to claim 12 wherein said
substratum is appropriately regulated to colonize
5 nutrient source includes paper having a moisture con
tent less than about 50%.
said substratum with hyphae, and wherein follow 14. A method according to claim 13 wherein an aque
ing said colonization a nutrient source, which in ous solution containing less than about 1% urea is ap
cludes cellulosic material plug a nitrogen source plied each 2 to 3 days in an amount of at least about 40
and which promotes the storage of essential nutri O ml per square foot. is k is k &
ents in said hyphae, is supplied to said hyphae.

15

20

25

30

35

45

50

55

65