CULTIVATION AND ANALYSIS OF PSILOCYBE SPECIES AND AN INVESTIGATION OF GALERINA 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.

For the first time, BRODIE (1935) observed a formation of sclerotia in a psilocybian mushroom which is now known as Panaeolus subbalteatus (BERK. & TZ, BR.) SACC. (GARTZ, 1993).

Psilocybin and its precursos baeocystin as well as psilocin were found in every fruit body of Psilocybe natalensis (Table 2). The highest concentrations wwere found in the smallest basidiocarps, as early was the case in Psilocybe cubensis (GARTZ, 198 9) and other species (GARTZ, 1995).

In Psilocybe natalensis psilocybin was found to be accompanied by only slight amounts of baeocystin. Other species like Inocybe aeruginascens BABOS (GARTZ, 1993) or Psilocybe semilanceata (GARTZ, 1991, a, b) contain much higher concentrations of this substance.

The alkaloidal concentrations in Psilocybe natalensis were very similar to the amounts in the subtropical and somewhat similar (GARTZ, 1989) Psilocybe cubensis (GARTZ, 1991b) and in Psilocybe samuiensis from Thailand (GARTZ et al., 1994).

Only psilocybin was found to be present in the cultured, even blueing mycelium grown on malt agar. Amounts of psilocybin, ranging from 0.13 to 0.28% dry weight were analysed in 4 different batches grown over a 4 week period.

Psilocybe azurescens is one of the most potent hallucinogenic mushrooms ever found. Very high concentration of psilocybin were detected in all extracts of this species (Table 3). Such substanial amounts of baeocystin have also been found in collection of Inocybe aeruginascens BABOS and Psilocybe semilanceata from Europe (GARTZ, 1991a, b: 1993).

Relative high levels of psilocin were even detected in every fruit of Psilocybe azurescens (Table 3). The mushrooms are characterised by a typical blue colouration of the stipe caused by handing of the mushrooms. Very old fruit bodies can possess blue flecks on the pileus and a blueish stipe too.

The alkaloidal levels ofthe 2 analysed fresh mushroom from the Pacific Northwest were in the same order of magnitude as that found earlier in dried mushrooms from the same area (STAMETS & GARTZ, 1995) and recently in "naturally" cltivated fruit bodies from Germany (Table 3). We also detected psilocybin and in some cases baeocystin and psilocin n the mycelium from agar depending on the concentration of malt extract (STAMETS & GARTZ, 1995). Psilocybe azurescens is a lignicolous species ultilzing a number of wood type and is able to grow on a wide variety of wastes including garden mulch, newspaper and cardboard. As a primary composer like Psilocybe bohemica SEBEK (GARTZ, 1993) Psilocybe azurescens is more robust and contains higher amounts of indole derivatives than this Czech species. Addiontally, Psilocybe azurescens shows a more aggressive growth of the mycelia in various substrates and a high fruiting potential than Psilocybe bohemcia and even it relative Psilocybe cyanescens WAKEFIELD (STAMETS & CHILTON, 1983; STAMETS 1993).

Recently, the qualitative detection of psilocybin, psilocin and baeocystin in extracts of Galerina Steglichii BESL has been described (BESL, 1993). this detection was the first evidence of the occurence of the alkaloids in the genus Galerina. these tiny blueing mushrooms were described as a new species from the botanical garden of Regensburg, Germany (BESL, 1993). In early 1993 they grew on an acidic substrate in a warm house there. The orgin of this species is unclear.

My own analysis revealed alkaloidal concenrations very similar to Psilocybe natalensis (Table 4). In the ushrooms extracts psilocybin and psilocin were found to be accompanied by only slight amounts of baeocystin. There was also a variation of the content of these indole derivatives in single mushroomsfrom the same location.

In contrast to earlier experiments (BESL, 1993) the own isolated mycelia grew as a fast pace on 6% malt agar. Psilocybin was also found to be present in the blueing mycelium of Galerina steglichii grown on agar over a 4 week period. Amounts of these alkaloids, ranging from 0.05% to 0.14% dry weight, were analysed in 5 different batches of mycelium.

Interestingly, no other indole derivatives were detected in the extracts of the in vitro gorwn myclium.

ACKNOWLEDGEMENTS:

The author thanks J. Allen, C. Andrew, P. Stamets and the mycologist from Regensburg, Germany who generously supplied valuable information, a mycelial culture and mushrooms for analysis. Appreciation is also extended to M.T. Smith for his help in search for Psilocybe natalensis during our field trips in Natal in January, 1994.

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