c2) United States Patent Miller ‘US00690769182 US 6,907,691 B2 Jun, 21, 2005 (10) Patent No. (5) Date of Patent: (54) CULTIVATION OF MORCHELIA (75). Inventor: Stewart C. Miller, 1521 S. 9 St, Lafayette, IN (US) 47905-1859 (73) Assignee: Stewart C. Miller, Lafayette, IN (US) (*) Notice: Subject to any disclaimer, the tem ofthis patent is extended or adjusted under 35 US.C. 1544b) by 173 days. (21) Appl. Nos 10/180,690 (22) Filed: Jun, 26, 2002 6) Prior Publication Data Us 20080000000 AL Fan. 1, 2004 (1) In. cL AIG 1/04 (2) US.Ch aml (58) Field of Seareh aL 66) References Cited US. PATENT DOCUMENTS. 3982969 A S197 Como Jeeta 11979 Pinckard 1082 ama isa “isa R 5/1986, tensa ATSSLR 11986, Ower et cc 3 “11986, Mgnier etal ISTH A TIVES. Ower,daceased et al 4810501 31989) Schindler 48663878 A 971989 Omer, dacesed eta Shwagas A S901 Satie 5125208 A 611992 Hiromoto SUI8682 A 1/1093 Jaerete 53427592 A 6/1995 Romaine ota 5980223 A 6/1098 Carson ta SOMI2 A 81900 Holi eta. FOREIGN PATENT DOCUMENTS @ aaust 51987 @ 37338 61990, FR 2942262 AL * 8/1990 we Souris A= 1/1981 w az A + 4/1002 ‘AOLGILO4 BP os559 A + 211904 AOIGiL04 we 6261631 A * 9/1994 ‘01670 w 1011307051998 we Hoaise7s R998 we 1a301766 A+ 1/1908 ANIGILON ra 2ooens2083 4/2000 we aanno7sses 32001 (OTHER PUBLICATIONS WO 92/11752: Janette, Inoculum from ectomycorbizal fungi forming endomycorshizal infection with herbaceous plants 23.0792" Derwent~Ace-No. 1994-021852, 1998 -408888.* 1992-111630,1992-2800169, Website information fom wowirulletree.com for New World Trullieres, Ine. “Trule Cultivation Specialists” Copyright 2003, “Phosphate Activity of External Hyphae of Two Arbuscular -Mycotthizal Fung!” Joner et; Mycological Research 104, No. 1 (2000); pp Si-86. “Endophytic Fungi Associated with Palms", Frolic etal Mycological Research 104, No. 10 (2000); pp. 1202-1212 “The Mushroom Handbook", Louis C.C. Krieger, Dober Publications, Jun, 1967; pp. 20-21,32-89, 56-57, Plates 26 & 27. “Answorth & Bishy's Dictionary ofthe Pung etal; CAB International Publishing “Mushrooms of Northesstern North America”, Bessette et als Print Net (1997); pp. 476-477. “Allelic Vesiation and Segregation in morchella Deliciosa and M, Esculeata", Gessner otal; Mycologia, vol. 79, No 5 (1987) pp. 683-087, A rapid Staining Method for Detection of Endophytic Fungi in Turf and Forage Grasses”, Saha etal; The Ameri- ‘ean Phytopathological Society, vol. 78, No. 2, (1988); pp. 37-239, Hawksworth “Dissibution and Molecular Characterization of the Root Endophyte Phislocephals Fortnii Along an Environmental iadient in the Boreal Forwst of Alberts", Addy et al; “Mycological Rescarch 104, No, 10 (2000); pp. 1213-1 * cited by examiner Primary Examiner—Tesi Pham Law “Assistant Esaminer—Francis . Palo (74) Auorney, Agent, or Firm Baker & Daniels on ABSTRACT A process for cultivating Morchella ascocarps using, myce- lium and a tee seedling, the tree seedling having 4 root system and a shoot system, The process includes the steps of inoculating the root system with the mycelium 1 produce an inoculated tree seedling; stimulating the mycelium to form slerotiaby severing the shoot system Irom the root system; and inducing the selerota to produce ascocarps by providing conditions conducive to the formation of ascocarps. Ako, the growth of tres may be accelerated by inoculating the root system with the mycelium. 37 Claims, $ Drawing Sheets @ of § Drawing Sheets) Filed in Color)U.S. Patent Jun. 21, 2005 Sheet 1 of 5 US 6,907,691 B2 d < 2 7 % o cz S ist es oO o a 2 3 “ os Jd ire FiG. LoU.S. Patent Jun. 21, 2005 Sheet 2 of 5 US 6,907,691 B2 3a Fig.U.S. Patent Jun. 21, 2005 Sheet 3 of 5 US 6,907,691 B2 FIG 4U.S. Patent Jun. 21, 2005 Sheet 4 of 5 US 6,907,691 B2 FIGsUS 6,907,691 B2 Sheet 5 of 5 Jun. 21, 2005 U.S. Patent suomipuo ayy Suds oprrong ‘wojionpolg dres0asy Bu}onpuj woudojoaag epojag 004] paiemnoouy moun} %e [ woseag Sumo) S204], pareInDoU mop, %"O\h sSuypoag o0ay oveynoouy wotdswes Bumpans 2211 so| waisdg 100g arejnaeuy suoneinoouy 10US 6,907,691 B2 1 CULTIVATION OF MORCHELLA FIELD OF THE INVENTION ‘This invention relates tothe cultivation of any ofthe fungi species ofthe Family Morchellaceae and the genera Morch- ela BACKGROUND OF THE INVENTION ‘The edible fungi species of the genera Morchella are known by their ascocarp o¢ fruiting body. The ascocarp represents the mature embodiment of the sexval seproduc- tion eyele of the morel and is lined with tiny, mieroseopie elongated sacs, each of which is called an ascus. Inside each fscus are microscopic spores lined up like small eggs, approximately eight spores per ascus. These spores escape from the lid of the ascus and take to the air marking the beginning of the lifecycle ofthe morel mushroom. When the spores land on aa appropriate food source, such as moist, ‘dead, rotting, or decaying plant lif, the spores will “hate” producing small hair-like structures called hyphae. The hyphae begin o spread throughout the food source produe- ing an interwoven mat or feeding network ealled myclium. ‘The byphae’s absorption of nutrients from the nom-living food source demonsiraes the saprophytic nature of the ‘morel. Under certain, unfavorable conditions, this mycelium ‘contracts to form hardened protective belies called scleno= tia, The selerota then lie dormant wat favorable conditions arise. During favorable conditions, the scleratia develop ascocarps, The ascocarps of the Family Morchellaceae, especially those of morchela esculenta, morchella deliciosa, morch- cella crassipes,elata, semi-libria, and morchella angusticeps “black” have been highly sought after fo many years and are considered to be the most delicious ofall mushrooms. Every spring thousands of morel hunters take t0 the woods in search of the everclusive mor, also known as the “sponge mushroom.” In fat, certain towns in Michigan hold mushroom festivals every spring to celebrate the opportunity to take to the woods and find this delicious. morsel Unfortunately, tbe fruiting ofthe moze occurs naturally only under select conditions, thereby limiting the availability of this highly sought aller delicacy. Processes centered around the saprophytic nature of the movel have been suggested for commercially cultivating ‘morels. Such processes include that described in U.S. Pat. No, 4,757,640. However, these processes have not proven to be successful in the commercial production of these mushrooms, eiter inthe fekl, or in environmentally con- troled conditions. SUMMARY OF THE INVENTION “The present invention provides a process for cultivating Morchellaceae ascocarps using mycelium and a tree seedling, the tre seedling having a root system an! a shoot system, The process involves inoctlating the root system ‘with mycelium to produce an inoculated tree seedling, ‘stimulating the mycelium to form sleroia, and inducing the sclerlia to produce ascocarps. The present invention furher provides a process for aceclerting the growth of trees by inoculating the rool system of the tree with mycelium. Tn one embodiment, the processes ofthe present invention ‘may include cultivating the mycelium by introducing spores ‘onto a culture medium and incubating the spores nil the ‘pores produce mycelium. % o 2 a another embodiment, the processes may include inocu lating the root system by introducing the root system of the ‘woe seedling iato the eulture medium and iaeubating the culture medium until the mycelium has infected the root system, Alternatively, the processes of the present iavention may. include inoculating the root system by adling a layer of| planting medium on top of the culture medium, placing @ {ree seed on top af the planting medium, germinating the tree sce ilo a tree seeding, and growing the tre sedling until the root system is infected withthe mycelium, In till another embodiment, the processes of the inven ‘ion include cultivating mycelium by introducing plurality fof spores into a container of culture medium, placing the conliner of culture medium in a planting medium, covering the culture medium andthe planting medium wilh germi- ‘ating medium, and incubating the spores until the spores produce the mycelium, Tn this embodiment, the root system may be inoculated with mycelium by placing at least one tee seed in the germinating medium, germinating the tre seed into the tree ‘ceding, and growing the ice seedling uni the rot system is infected with the mycelium, Alternatively, the root system injecting the mycelium into the tee. In yet another embodiment, the root system is inoculated by grafting a portion of an infected tree onto an uninfected tree seedling In another embodiment, the root system is inoculated! by’ growing an uninfected tre in close proximity to an infected Tn another embodiment, the process of euivating Moreh ellaceae may include growing the inoculated tree seedling {or at letst one growing season, and then stimulating the ycelium to form selerota by distessing the ire seedling, ‘In another embodiment, the process of cultivating Moreh cllacene includes inducing the sclerotia to produce asco- ‘caps by providing conditions conducive tothe formation of sscocarps vay be inoculated by DESCRIPTION OF THE FIGURES ‘The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings ‘will be provided by the Patent and Trademark Office upon request and payment of the necessary fee "The above-mentioned and other features and objects of this invention, and the manner of attaining them, will become more apparent and the invention itself wil be bet tnderstood by reference to the following description of embodiments of the iavention taken in conjunction with the scompanying drawings, wherein FIGS. 1a-Ie iluszate the inoculation step of the present invention according Io one embodiment. FIGS, 24-26 illustrate the inoculation step of the present invention according to another embodiment FIGS, 34-2 illustrate the inoculation step of the present invention according to yet another embodiment FIG. 4 is « photograph illustrating mycelium. FIG. $ is a photograph illustrating an inoculated root FIG. 6 is a flow char of the process of the present wention aecording 10 one embodiment Corresponding reference characters indicate correspond ing pars throughout the several views. Although the draw-US 6,907,691 B2 7 ings represent embodiments of the preseat inveation, the ‘drawings are not necessarily to scale and certain features ‘may be exaggerated ia order to better illusteate and explain the present invention. The exemplificaions set out herein ‘Mfustrate ombadiments of the invention and such exempli fications are not to be consid as limiting the scope of the invention in any manner DESCRIPTION OF THE INVENTION ‘The embodiments disclosed below are not intended fo be ‘exhaustive oF limit the invention to the precise forms dis ‘closed in the following detailed description. Rather, the ‘embodiments ae chosen and deseribed o that others skilled jn the art may utilize the teachings. The present inventor has discovered tht saprophytism is ‘only one of the characteristics exhibited during the growth ‘eyele of the morel fungus. During its life eyele, the morel also demonstrates mutualism, For example, i he ease ofthe apple ie, the spores ofthe more! hatch on a rotting apple ‘The mycelium, acting as saprobe, absorbs nutrients from the cotting apple. Next, the hyphae f the mycelium spread from the rotting apple tothe root at the apple tee seed. The root of the apple Inve then begins poking its way into the round taking the hyphae with it ‘The developing fungus begins it's mutualistic behavior, also refered to as myconhizal phase, when the hyphae of the mycelium invade the root of the tree, working its way Ino the cells ofthe oot system. The mutualistic sssocistion between the mycelium and the tee root can be referred 10 ‘generally as mycorthiza. Once the mycelium has completely ‘entered the root cells ofthe tree the mutualistic association Js then referred to as endomycorriza, or an endophyte ‘Through the mycelial inoculation of the rots of the tee a symbiotic mycorrhizal or endomycorthizal infection devel- ‘ops overtime. The fungus and tree coexist together to form 8 mutualisti relationship, ot symbiosis. The tee provides the mycortiza fungus with a food source from the uit of ‘sp. The fungus, in turn, promotes healthy growth ofthe ree by transferring water and nutrients from the sol, which the tree would otherwise have difficulty obtaining. “The preseat inventor bas further discovered that both the twee and the morel fungus coexist inthis mutualistic manner ‘unt the relationship is disrupted, Such disuption ean be the result of many things including: te te losing its sap or the ‘death of the lee, such as by the infection of Dutch-Eim ddiscase, Whether the disruption & natural or initiated, the mycorthizas recognize the loss of their food source’ and respond defensively by retracting the mycelium into a sclerotic nodule. The mycelium retraction initiates the ‘development of the selerotia, which ultimately produces the ascocitp or fruiting body when the time and conditions are right ‘This discovery has lead to one embovliment of the present invention, which provides a process for cultivating Morch- lla ascocarps using tree seedling having 8 root system and a shoot system, and Morchella mycelium. The process ‘generally involves inoculating the root system with Moreh ella mycelium to produce an inoculated tree seeing, stnms- lating the mycelium to form scleolia, and inducing the scleotia to produce ascocarps, The inoculation step generally calls for inoculating the root system of a tre seedling with mycelium. The mycelium tsed in the inoculation step may be obtained using several Ulfferent cultivation methods In one embodiment, the mycelium is cultivated by into ‘ducing one or more Spores onto 2 culture medium and 4 incubating the spores unl the spores produce mycelium. This cultivation of mycelium may be accomplished by phicing dried ascocarps of whole, unwashed, and freshly Picked morel mushrooms in paper bags. The paper bags prevent moisture from being trapped inside the ascocarps, thereby impeding rot o bacteria growth on the ripened and ‘ried spores. The spores may be saved for short periods of time, without any adverse affect on spore geemination, by placing the paper Bags ina relrigerator. ‘An acceptable culture medium is then prepared. Accept able culture media include standard mycological culture media sold by laboratory supply houses. Such mycological culture medium is composed primarily of simple earbaby- ‘rates and gelatin, and is easily attainable and extremely reliable by viene of its nutrient consistency and its sterility ‘Alternatively, the culture medium ean consist simply of natural eashohydeates or decaying plant material “The culture medium is then inieluced into containers, such as Peri dishes, jars, and plastic, foam, or peat eups. To promote efficiency and eliminate waste, biodegradable seed ontiners may also be used as culture medium containers. ‘These containers typically consist of a paper-based, ‘dooar-ike material that decomposes in the ground ater lime. These biodegradable containers promote ellicency because the container and the inoculated tree seedling that ultimately is produced in the container (as described infra paragraph 0034) can be planted directly into the ground. To keep out competing fungi and bacteri, its benelcial to use containers that have lds. Alternatively, the containers can be tighlly stacked to preveat invading competition ‘As shown in FIG, 1a, the ascocarps 2 are removed from the paper bag and shaken over the top ofthe eulture medium ‘ike a slishaker. The spores 3 land densely over the culture ‘medium 4 and are spread over or stirred into the culture ‘medium 4 using a sterile fork. Alternatively, tiny pieces bout the sizeof a pencil hed may be cu fom the ascocarps. And placed on top of, of inside of, the culture medium. The spore-containing culture medium is then allowed to incubate at a temperature in the range of about appro ately 0° C=378° C. (32° F100” F), 0 allow the spores 3 to develop a network of mycelium 5, 36 shown in FIGS. 1 & 4, Altough the mycelium can develop at any tem- perature Within this range, the mycelium development is ‘optimal when the spores are incubated at a temperature Within the range of 72° C-239° C. (45* B~15° F). Typically in this temperature range, the growth cate of the mycelium in the medium during incubation is dlircetly ‘raportional to the temperature. That, the mycelium grows ‘ore rapidly when incubated at temperatures in the higher end of this range. Mycelium can develop alter as litle as 24 ‘hours of incubation. However, 1 insure a substantial supply ‘of mycelium, the mycelium is incubated for one to 60 weeks. As shosin in FIG, 4 confirmation of mycelium cultivation may be observed by viewing under the micro- scope at 10%. ‘The tree seedlings used inthe present invention ean be either germinated from seeds, or can be purchased from users. Any compatible tree seedling may be used. Such compatible ee seedlings include, but are not limited 10, elm, ash, oak, peach, poplar, douglas fr, ebetry and apple ‘White many different types of tre scedlings ean be used, the elm tree is inexpensive, expendable, produces a voluminous amount of seed, and is easy to germinate. The elm tee is considered expendable because Dutch-Elm disease has reciuced the elms life expectancy to a minimum, Some od surviving elms sil exist, which produce vast amounts ofUS 6,907,691 B2 5 seeds. The expendable and plentiful elm seeds provide a ood conduit for the sbecessful production of the morel Imushroom, Because it is the ultimate death of the tee that Simulates the formation of sclerotia, and ultimately the production af the ascocarp ‘Several methods ean be used to inoculate the tee seed lings. For instance, in one embodiment shown in FIG. Le, he root system 7 ofthe tee seedling 6 is introduced inte the ‘mycelium $-containing culture medium 4, andthe resulting culture medium 4 containing the mycelium § and the root system 7 is incubated until the mycelium § has infected the root sysiom 7. More specifically, the tee seedlings 6 are placed in the conttiners 8 of incubating mycelium 5 50 that the roots 7 ate covered with the cultivating medium 4. The roots of the tre seedings 6 may be bent or curled at the ‘same time to insure that they are substantially enveloped by the culture medium 4, The seedlings 6 and mycelium § are Jncubated inthe culture medium 4 until the root system 7 is adequately infected withthe mycelium 8, The seedings may be incubated at a temperature within the range of 0 2 C.-378° C. (32° F100" F), although adequate infection ‘occurs more predictably when incubated at a temperature Within the range of 7.2° C-23.9° C. (45° E-75" F). The seedlings are Watered sparingly and exeess water is drained away 10 prevent over watering. Adequate infection usually ‘occurs within about 1 10 20 days, and can be confirmed microscopically at 10-40 alter soaking the root segment ‘with a 1:1 (grim to mL) ratio of rose bengal and sodium hydroxide for about 5 to 20 minutes, Matening the root segment hetween two slides, and viewing the root segment through a green flter; an example of whieh is shown in 1G. 5.FIG, 3 is 40 microscopic photograph ofthe byphac of| a spore entering the root ofthe tee seeding. TInanather embodiment illustrated in FIGS. 2-2, a layer ‘of any acceptable planting medium 10s placed on top ofthe imyeclium S.containing culture medium 4 and at least one twee wed 12 is placed on top of, or within the layer of planting medium 10. Acceptable planting. medium is any ‘medium that fosters the germination of a tee seed and the growth of the tree seedling. Acceptable planting. media Jnclude, but are not limited to: soil, compost, manure, hums, peat moss, processed paper, pul, sphagnum, perlite, ‘synthetic material and mixtures thereof. The seed 13 is thes Jncubated for period of 1 to 30 days to allow the seed 12 to germinate into a tee seedling 6. The tee seedling 6 is grown until the root system 7 ofthe tree seeing 6 spreads Into the mycelium S-contaning culture medium 4 (FIG. 26) and becomes adequately infected with the mycelium 8. Adequate infection is illustrated in FIG. $ and in this ‘embodiment wsually occurs within about 1 to 30-day. ‘Adequate infection can he tested microscopically as deseibed above, Tn yet another embodiment shown in FIGS. 34-3f, a planting medium 10s pluced in a container or pot 14 having ocd! deuinage, such as a clay, plastic, or biodegradable pot, And w small hole 18 is scooped out ofthe planting medium as more particularly shown in FIG, 3a. As illustrated in FIG. 3b, 4 biodegradable cootainer 16 of spore 3-contaning, ‘eure medium 4 is placed in the bole of the planting ‘medium 10 so thatthe culture medium 4 is substantially level with the top of the planting medium 10, The culture medium 4 and planting medium 10 are then covered with @ Jayer, approximately one-half inch deep, of an acceptable _germinating medium 18, as shown in FIG. e. An acceptable perminating medium 18 i any medium in which a tree Sccdling can germinate, Such germinating media include, but are not limited to: sol, compost, humus, peat moss, % o 6 ‘compressed peat moss dises, processed paper, perlite, syn- thetic material and any mixture thereof. Itmay be beneficial fo use a sterile germinating medium 18 to deter invading bacteria and fungi. The spores 3 are then incubated until the spores 3 produce mycelium 8 (see FIG. 3d); about 110 30 ays a mately 0° C temperature FTF). Atleast one tree seed 12s then placed on top of o within the germinating medium 18 (see FIG. 32), and the seed 12 js incubated until the seed 12 germinates into a tee seedling 6 as shown in FIG. 3f. The seed 12 and resulting tree seedling 6 are watered, although relatively sparingly and excess Water should be drained away’ to prevent over Water- ing. As shown in FIG. 3f the tree seedling 6 is grown vat the root system 7 spreads inwo the mycelium S-contaning culture medium 4 and root system 7 becomes adequately fected with the mycelium 5. Adequate infection in this, embodiment usually occurs within about 1 to 30 days, and is illusated in FIG, § Alternatively, the myvelium and the ve seedling may be cultivated simultaneously. In this embodiment, the spores fare inlroduced onto a culture medium and a iree seed is ‘noice into «planting medium. The seed and the spores fare simultancously incubated until the spores produce the ‘mycelium and the tree sced germinatcs into a tee seedling. ‘Weally, the seeds will germinate in approximately the same period of time, approximately 1 t0 30 days, asthe spores. ‘Then, the twee Seeding i placed in the culture medium and the culture medium is incubated until the mycelium has adequately infected the root system. Adequate infection in {his embodiment usualy occurs within about one to 30 days. ler the root contacts the myeelivm culture Other methous of inoculating tre seedings include inject- ing hyphae or mycelium directly into the roots of the tree scealing. Alternatively, tte seedlings may be inoculated by gtalling or notching a portion of am infected root ot shoot ‘oto an uninfected tee seeding, Tee seedlings may also be Jnoctlated by growing the lee ceding in close proimity to an infected te. Tn one embodiment, after inoculation ofthe tee seeding and before the stimulation of selerota, the inoculated tree seedlings are planted in prepared fields or in containers and allowed 1 grow for at ast ane growing season. An appro- priate prepared field should have good water drainage 10 prevent standing water or puddles. Ideally, the soil of the prepared field should be finely tilled and have a datk, rch, sandy, loam quality, Ifthe nutrients in the soil seem to be Jacking, additional nutrients may be added by sprinkling @ Ientlzer over the soil and tilling the fertlize into the soi. may be helpfal to leave narrow grassy lanes between the rows of clusered seedlings (0 allow the maneuvering of equipment for mulching, watering and other purposes. Mulching by mowing the grass and letting moderate amounts of clippings accumulate around the trees may assist maintaining a cool and damp environment for the 001s, and may help 10 eliminate competing grasses and weeds In addition, shading (providing shade) the seedlings may also {sist in maintaining a cool and damp environment for the ‘The seedings ae allowed to grow in the prepared fle or indoors frat least one growing season, A growing season {equates to approximately one year. Although the seedlings ‘may be planted any time before the frst frost, September is an ideal time to plant the seedlings, because this allows the 32° F-95° F), with a. prefered the sange of about 7.2° C.-23.9° C. (45°