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Cultivation and analaysis of psilocybe species and an investigation of Galrina Steglichi.

Jochen Gartz

Originally published in Annali Museo Civico di Rovereto, vol. 10, pp. 297-306, 1995

Abstract:

Cultivation and analysis of Psilocybe species and an investigation of Galerina steglichi. Cultivation and formation of sclerotia of Psilocybe mexicana could be demonstrated on various grain substrates. Analysis of sclerotia from wet rice grain revealed the presence of psilocybin, in most cases psilocin and always low concentrations of baeocystin. Psilocybin, psilocin and baeocystin levels varied in the blueing fruit bodies of the new species Psilocybe natalensis from South Africa. The highest concentrations of these alkaloids were found in naturally grown and cultivated fruit bodies of Psilocybe azurenscens which is an indigenous species of the Pacific Northwest, U.S.A. The relative alkaloidal content of psilocybin, psilocin and baeocystin found in Galerina steglichii from Germany was similar to that measured in Psilocybe nataliensis. Psilocybin was also found in the cultured bleuing mycelium of these species.

Key words: Cultivation, Analysis, Psilocybe mexicana, Psilocybe natalensis, Psilocybe azurenscens, Galerina steglichii.

Recent ethnomycological and chemical investigations confirm earlier results that psychoactive species of various genera are growing wild in Europe, North- and South America, Australia and Asia (GARTZ, 1991, 1993, 1995; GUZMAN, 1983).

We described some ethobotanical facts and cultivation results of a new fungus from Thailand, Psilocybe semuiensis GUZMAN, BANDALA & ALLENin comparision with Psilocybe tampanensis GUZMAN & POLLACK from Florida and Psilocybe semilanceata (FR.) KUMM. fr om Europe (GARTZ et al., 1994).

In January 1994 we also found the first indigenous blueing Psilocybe species from South Africa on a field trip in the province Natal. This overall whitish and large species without a ring or even a velum grows on cow pastures and is named Psilocybe natalensis npm. prv. here. (GARTZ et al., 1995).

Since 1979 an interesting and large Psilocybe species was collected along the Columbia River network in the costal regions of the Pacific Northwest, U.S.A. These mushrooms turn also blueish after bruising and live in soils enriched with deciduous wood-debris Until. Now we describe these fungi as Psilocybe azurescens nom. prov. (STAMETS & GARTZ, 1995).

In continuation of these studies, in this paper some analysis and cultivation experiments of the species from America and South Africa including of some results of the "classic" fungus Psilocybe mexicana HEIM and a new Galerina (BESL, 1993) are described.

EXPERIMENTAL:

The strain of Psilocybe mexicana was obtained from the "underground mushroom movement" of the U.S.A. A successful fruiting experiment on rye grass seed (Lolium sp.) water mixture by using a casing layer (POLLOCK, 1977; STAMETS & CHILTON, 1983) and the subsequent microscopic examination of the few fruit bodies (GUZMAN, 1983) confirmed the identity of the species. Mycelium was kept as a stock culture on 4% malt agar.

Cotton - plugged 500 ml Erlenmeyer flasks were filled with 100 g grain and 180 ml water, sterlised by autoclaving, cooled, inoculated from stock cultures and incubated at 23 C in darkness to promote the formation of sclerotia. Rye grain, sfot rice grain and rye grass seed were used, respectively. Fruit bodies of Psilocybe natalensis (leg. O'Neill's cottage, Natal-South Africa, Jan. 22, 1994) were dried at 20-40 C.

Possible present residual water was removed from the mushrooms by freeze drying. Mycelium obtained from spore prints (STAMETS & CHILTON, 1983) was also kept as a stock culture on 4% malt agar. The mycelium from a cultivation on 100 ml agar was analysed after 4 weeks of cultivation.

Mycelium from Psilocybe azurescens (STAMETS & GARTZ, 1995) on 4% malt agar was used to inoculate a rye grain/water mixture identically to the cultivation of Psilocybe mexicana. After 3 weeks cultivation 500 g of sawdust soaked with water in plastic bag was inoculated with the mycelia on grain (STAMETS & CHILTON, 1983). The duration of the spawn run was 4 weeks.

In March commercial garden mulch in a shady outdoor bed was spawned with the mycelia on sawdust. A weekly water to keep the moisture content high. In September of the same year about 200 mushrooms appeared. Some were dried for anaylsis in the same way as Psilocybe natalensis.

Dried fruit bodies of Galerina steglichii from (BESL, 1993) were also analysed. It was possible to isolate a strain from spores on 6% malt agar. The extraction procedures of mushrooms and mycelia as well as the analysis by using HPLC and TLC are described elsewhere (GARTZ, 1989, 1991b).

RESULTS:

It was found Psilocybe mexicana soon formed yellowish to brownish sclerotia on rye grass seed/water. STAMETS & CHILTON (1983) repported similar growth pattern of sclerotia after 3 weeks on the same substrate. Observations on the formation of sclerotia in the highly similar species Psilocybe tampanensis after a cultivation of 4-8 weeks were reported by STAMETS & CHILTON (1983) and by GARTZ et al. (1994). In contrast, Psilocybe natalensis and Psilocybe azurescens under any cultivation only formed whitish mycelium throughout various substrates but no sclerotia.

The dry weights of the slightly blueing sclerotia of Psilocybe mexicana varied significantly during cultivation on rye grass seed/water. It seems that the tendency to lose its moisture is the reason for such a variation.

In addition, the cultivation on soft rice grain/water also soon fromed sclerotia after only 2 weeks. It was found that total darkness is not necessary for the formation of sclerotia on this substrate. Incubation of Psilocybe mexicana in diffuse daylight but without direct sunlight also promote this vegetative form.

Sclerotia formation on rice grain (100g)/water after 2 months is 15-20 grams dry weight. The alkaloidal levels obtained from these sclerotia were relatively high but varied even in the same batch (Table 1). Larger sclerotia contained more psilocin than smaller agglomerations. It seems that during the growing process of the sclerotia a signficant enzymatic decomposition of psilocybin to psilocin (BOCKS, 1968) occured (Table 1). In own investigation the same reaction was found in old fruit bodies of Psilocybe cubensis (EARLE) SING. (GARTZ, 1989). Only a few sclerotia were obtained on rye grain/water mixture after prolonged incubation (up to 12 weeks). It seems that the substrate based on soft rice grain was the best to promote a regular formation of sclerotia in Psilocybe mexicana.

Biological Efficiency Study

Abstract The effects of various combinations of wheat bran, rye and millet (at 20% and 30% of total dry sub-strate wt) on crop cycle time, biological efficiency (BE)
and mushroom quality were evaluated for a commercial-ly used isolate of Grifola frondosa (maitake). Supple-ments were combined with a basal ingredient of mixed
oak (primarily red oak) sawdust, and the resulting mix-ture was pasteurized, cooled, inoculated and bagged with an autoclaving mixer. Times to mushroom primordial
formation and mushroom harvest were recorded, and mushroom quality was rated on a scale of 1–4, where 1 was the highest quality and 4 was the lowest quality. The
combinations of 10% wheat bran, 10% millet and 10% rye (BE 47.1%, quality 1.8 and crop cycle 12 weeks) and 10% wheat bran plus 20% rye (BE 44%, quality 1.7 and
crop cycle 10 weeks) gave the most consistent yields and best basidiome quality over time. Introduction
Strong consumer demand has stimulated increased world-wide production of maitake (Grifola frondosa). The in-creased demand for maitake is due to the unique culinary
and medicinal properties associated with this choice mushroom. Annual commercial production has increased 41-fold (to 33,100 t in 1997; Chang 1999) since 1981, the
year when commercial production of maitake first began in Japan (Takama et al. 1981). Maitake production and consumption is also increasing rapidly in the United
States (up 38% in 1999–2000; USDA 2000). Presently, most maitake is marketed as food. Powdered basidiomes also are used in the production of many health foods such
as maitake tea, whole powder, granules, drinks, and tab-lets (Royse 1997; Mizuno 1999).
Commercial production of most maitake is on synthetic substrate contained in polypropylene bags. A common substrate used for commercial production of
maitake is supplemented sawdust. Oak (Lee 1994; D.J. Royse, unpublished data) is the most popular choice in the United States and Japan, while beech (Kirchhoff
1996; Yoshizawa et al. 1997) and larch (Stamets 2000) are also preferred to a lesser extent in Japan. In China, cottonseed hulls have been successfully used as a
substitute for sawdust (Zhao et al. 1983). Brans, derived from cereal grains, such as rice (Takama et al. 1981), wheat (Mayuzumi and Mizuno 1997), oats and corn,
are widely used as nutrient supplements. Other nutrient supplements used for maitake cultivation include millet (D.J. Royse, unpublished data), corn meal (Kirchhoff 1996), and soybean cake (Mizuno and Zhuang 1995). There are not many reference texts available for use in producing maitake. The techniques currently used to grow maitake are mostly adapted from those used to pro-duce other specialty mushrooms, such as shiitake.
Exten-sive research has been carried out on the most efficient methods, genotypes and nutritional formulation of spe-cialty mushrooms other than maitake (Diehle and Royse
1986; Royse and Bahler 1988; Royse et al. 1990; Stamets 2000). The rapid increase of maitake production in Japan and the United States has focused the need to
develop more efficient substrate formulas to improve yield and quality and to shorten the crop cycle. In this study, two experiments were conducted to determine the
effects of selected nutrient supplements at various levels on maitake crop cycle time, biological efficiency (BE), yield and quality. Significant differences among different
formulations were found and the best combinations of nutrient supplements among those tested were identified. For continued growth of the commercial industry, efforts
directed toward improving BE, yield, quality, and re-duced time to primordium formation and harvest are de-sirable. Q. Shen · D.J. Royse ( .)
Department of Plant Pathology,
The Pennsylvania State University, University Park, PA 16802,
USA
e-mail: djr4@psu.edu
Tel.: +814-8657322, Fax: +814 8637217
Q. Shen · D. J. Royse
Effects of nutrient supplements on biological efficiency,
quality and crop cycle time of maitake ( Grifola frondosa)
Received: 9 April 2001 / Received revision: 18 May 2001 / Accepted: 2 June 2001 / Published online: 11 August 2001
© Springer-Verlag 2001

Cleanliness Precautions
Inoculating your jars is the main step where contamination is possible, and thus must be done in as clean of an environment as possible. If the room you&8217;re working in is clean enough, you can get away with inoculating them in open air. The needle of the syringe, if not absolutely sterile, can carry bacteria and spores from other molds into your cake, contaminating and ruining the cake. Wash your hands and face with antibacterial soap. Wear clean clothes. Anything in the area of the syringe and jars could contaminate your cakes if it is not clean.

Glove Box (Optional)
If you&8217;re concerned about sterility, a good way to accomplish this is to buy a "glove box," an enclosed, semi-sealed box with holes for gloves to go through and a see-through top. Disinfect the inside of the box with Lysol spray disinfectant.

Inoculation: Cleanliness Simplified
begin carefully inoculating them with the syringe. It's a good idea to have a lighter handy as well to sterilize the needle as you go. Flame the needle until it gets very hot, then carefully squirt a little bit of spore solution (if you can spare it) to cool down the needle before sticking it in the cake. Putting a hot needle into the cake will get burnt-on rice flour all over the needle.

Birthing the Cakes
Once a cake is completely covered in white mycelium, wait at least 1-2 more days before taking the cake out of the jar and "dunking". Dunking is essentially submersion of the cake underwater for 12 to 24 hours. This is best done under refrigeration. Just don't overdo it, by 48 hours underwater they will be dead.

As for what kind of water to use, natural spring water is best but you can even use it straight from your faucet if need be. Temperature during the dunking should be cold, or as cool as possible and still above freezing. Time spent dunking should be not less than 6 hours for minimal benefits. 12 hours is about right for dunking in between flushes and at birth but 24 hours is the maximum for full rehydration of nearly spent cakes.
There are at least 2 options for how to dunk. My prefered method is to dunk each cake individually by placing it in a jar, filling with water, then screw on the lid to keep the cake submerged. This way is good for small batches, and has the advantage of keeping each cake isolated so no contaminants are spread from cake to cake. For larger batches, you can simply place several cakes in a large pan or bucket, cover with water and weigh down the cakes by placing a weighted down lid, plate, etc. on top of the cakes.

When dunking is complete you are ready, and in a fairly clean room, begin transferring the cakes from their jars into their fruiting chamber (described in the next step). Remove the lid of each jar. Then, put the lid back over the top of the jar. Slowly turn the jar upside down, so that the cake is resting on the jar lid. You may need to gently tap the jar to knock the cake loose. Take the jar off the top of the cake and then carefully pick up the cake and turn it over, so it is sitting right side up on the lid. Placing down a piece of foil, , put them it into the fruiting chamber in some damp perlite. Once all the cakes have been transferred, you&8217;re ready to induce fruiting.

Inducing Fruiting (Producing Mushrooms)

In order to initiate fruiting, three main conditions must be met for the cakes:
First, they need light. Only a dim light is needed. A fluorescent lamp or indirect sunlight is plenty of light. Mushrooms do not gain energy from the light like plants do, but in this particular species of mushroom light sends a signal to the mycelium that it is time to produce mushrooms.

A source with a wide spectrum of light, especially containing lots of blue light (daylight and fluorescent plant lights are very good examples of light with lots of blue) is best, but a low wattage (15 watts is plenty) incandescent light bulb will supply enough light.

Second, they need a fairly high humidity. 90-95% humidity is a good range for fruiting. The best and easiest way to do this is by lining the bottom of the fruiting chamber with damp perlite. A common mistake is to get the perlite too wet, and end up with a swamp of water and perlite that is very difficult to clean up, and will drown the cakes. Get enough perlite to make at least 1" (2.5 cm) thick layer on the bottom of the fruiting chamber, and put it into a colander, strainer, or cloth enclosure that it can&8217;t slip out of.

Wet it thoroughly with normal tap water, and let the water drain out. Then move the perlite into the fruiting chamber and smooth out the surface. You now have a layer of damp perlite that the cakes can be set directly on, and which will keep the humidity in the chamber high enough for the cakes to fruit. By the time your cakes have stopped producing mushrooms, the perlite might start getting a little bit skunky smelling.

If you want to reuse it, put it in a baking pan and cook it at 350 degrees in your oven until it is dry. Let it cool, and it&8217;s ready to be used again. You can also add some Hydrogen Peroxide to the wet perlite to help it stay clean a bit longer.

Lastly, it is a good idea to lower the temperature range a bit, to about 75-80 degrees F. Like the light, this signals the cakes to begin fruiting. However, most strains fruit so easily that lowering the temperature is not absolutely necessary.

Pinning, Fruiting, and Harvesting

For the first week or two, the cakes will generally not do anything. Then, very small bumps, called "pins," "pinheads," or "primordia" will begin to grow out of the surface of the cake. These are the beginnings of mushrooms. Many will never grow any larger. However, some will grow until they are full-grown mushrooms. A mushroom is ready to be picked when the edge of the cap tears away from the "stem" (the stem of a mushroom is called the stipe).

Often, there will be a thin veil between the cap and stipe. If this is present, you can wait until the veil tears before picking the mushroom. To pick a mushroom, grasp it near the base where it is joined to the cake, and gently twist it until it comes off. Immediately begin the process of preserving it, either by refrigerating it or by drying it, mushrooms will begin to rot immediately.

Each cake will produce about 1-3 waves or "flushes" of mushrooms, normally with 2-5 days of dormancy between flushes. After about a month or so of fruiting, most cakes will be spent, and will not produce any more mushrooms unless rehydrated by dunking underwater for 24 hours.

Basic Kit Setup and Preperation