On the Mycelial Growth of the Lorel Or False Morel, Gyromitra Esculenta (Pers.) Fr
Karatenia 18(auppL.) 1978 On the mycelial growth of the lorel or false morel, Gyromitra esculenta (Pers.) Fr.
ILMA ROPONEN and MATTI KREULA
ROPONEN, I . A KREULA, M. 1978: On the mycelial growth of the torel or false morel, Gyromitra escultenta (Pers.) Fr. - Karstenia 18 (suppl.).
The growth factors of lore I mycelia were investigated in solid and I iquid media. The mycelial strains used in the experiments were isolated by germinating spores of lore Is in Petri dishes on nutr i ant agar. The strains i so I a ted were stored in the co I d in test tubes as pure cultures, and propagated vegetatively by Inoculation. The results showed that the production of spawn is pass i b I e in axen 1c substrates. This method of cuI t i vat ion needs a ·high degree of sanitation . Spawn growth is at a maxi mum after 3-4 weeks, when the sub strate i s fully covered with the mycelium. The pH range 4-7 was observed to be fabourable for the growth of the myce l ia tested . The mycelia grew vigorously when formic acid was added to a concentration of 0.01-0.1 per cent in the substrate, but growth was inhibited when the concentratIon was 0. 2 per cen. or more. The effect of temperature on mycelial growth was tested at +5°, 10°, 15°, 20° and 30° C; optimum growth was obtained at 10-25° C. The mycelia were very sensitive to elev at =~b;~fa~~~centratlons, and effective aeration and shaking were needed for growth in 1 iquid
Waste materials from the prepared pulp industry containing hemicel tutose and t ignin appear to be su itab le for the cultivation of lore I mycelia after the addition of a small amount of urea.
I. Rap
Introduction vaara 1947, Korhonen 1973). In Finland, studies on The composition of mushrooms and the factors influ the effects of the breaking of the soil surface and encing their growth have been studied at the Bio the removal of peat on the growth of the lorel (Jal chemical Institute during the last few years . The kanen 1975) as well as other pssobilities for its cultivation of lorels (Gyromitra escuLenta) has been cultivation (Suhonen 1974) are in progress . very difficult. Some information about G.escuLenta .Another variety of Gyromitra~ G. gigas, which and factors that have an effect on its growth is is larger and lighter in colour than the ordinary presented here. lorel, is met to some extent in groves of southern and central Finla nd. Occurrence, yieLd and use of the LoreL In Finland the lorel is one of the 30 commer G. esculenta, like MorcheLLa, belongs to the sub- class cial mushrooms (Pohjola et al. 1975) picked by trained of Discomycetes and _to the group of Pezizales. collectors. The yield varies cons iderably in different The .colour of the cap of the lorel varies from years. As an example of the variation in yield Fig. 1 yellowish-brown to dark-red-brown. It is densely shows the quantities of lorels received yearly in folded and its brim is arched inwards. The layer pro 1972-76 (total 114 tons) by Valia Finnish Cooperative ducing spores is on the surface of the dried cap as Dairies' Association for marketing . a white powder. Harmaja (1969 a, b, 1973 a, b) has studied These quantities do not comprise the whole the systematics of Gyromitra Fr. yield of lorels collected in Finland during the above The lorels a ppea r early in the spring after ·years, but they illustra te the variations in the yield the snow has melted. In southern Finland the fruit in different years. -bodies of the lorel appear as early as at the end of Fresh lorels are poisonous . Pyysa1o (1975) has March, and in Lapland they can be found a s late as studied by gas chromatography-mass s pectrometry the in July. The species is common over the whole country, contents of toxins in lorels, i . e . gyromi trin and i t~ particularly on pine-growing moors. They are usually homologues, and their removal from the mushroomr . In found in places where the surface layer of the soil our laboratory we have a l so studied the t ox i c affec t s is broken, that is, bes ide ditches, roads, patways of gyromitrin in animal e xperiments. A ca reful 10 and gravel-pits, and in forest-fire and log-felling minute s' heating and rinsing or long-te rm drying re areas wh ere the mossy cover is thin or tota lly lackin~ moves the toxin, and the mushrooms can then be used and in former woodpile and barf.: ing stands (Rauta- for food . These results show that the old methode Karstenia l8(suppl. ) 1978 59 found by experience for destroying the toxin and a pure culture of the mycelium and the culture flasks making the lorel edible are sound. Due to their good were s haken after inoculation and thereaft~r once a fla vour the lorels have always been popular. week . Under f avourable conditions the mycelia com pletely covered the substrate i n 3-~ weeks. In the Philippines the mycelium of Volvariella has been Fig. 1 . The quantities of Gyromitra esculenta de grown in moi s tened chopped straw (Ho 1972).
livered to the Val i a Finnish Cooperative In the plant dep~rtment of the Biochemical Dairie: ' Association in 1972-76. Re s earch Institute the cultivation of certain native edible mushrooms, suc h a G Pleurotus os treatus, Flammulina velutipee , Xt4 echneromyces mutabilis and Naematoloma eapnoides has been studi ed. This work
ha~ included aloe a s tudy of mycelial growth of the lorel (Roponen 1975). In the present study the :uita bility of c ertain wood waste materials as sub strate for the mycelium of the lorel was tested. The ttl effect of acidity, temperature, light and aeration +Jc on the mycelial growth was also examined . QJ Materials and me thods -' ::J u The mycelia l strains of the lorel used in the exper Ul iment s , Klx3 and K9, were isol ~ ted from single sporo QJ phores obtained from s outhern and central Finland b·y ttl growing their spores on nutrient agar in Petri dis h L.. +J es. The isolated strains were stored as slant surfa ce cultures in test tubes on a 2% malt-agar or modified E Hagem-agar in a refrigerator (+4° C). The vegetative 0 L.. inoculation of t he mycelia wa s performed a s eptically. >. Strains a vigorous a s possible were selected from (.!) the mycelial strains cultivated from s pores (Zamenhof ..... et al. 1967, Willo 1968 , Peng 1972) . 0 The tests were conducted in test tubes , Petri Ul QJ dishes and flaaks. The liquid media used contained c 1% hemicellulose ~ nd Ca - lignosulfonate (both dried c waste wood materials). The uo lid ~ ubstrates, in ~ flasks , were a-s follows: fresh and old (kept for a year outdoors) birch and pine bark residue; undecom posed and decomposed pine litter; sawdust - wheat bran (3:1) . Urea, 75-320 mg per litre distilled water, was added as the nitrogen source, ~nd the control me dia contained no added urea. 1972 -73 -71. -75 The substrates were autoclaved at 120° C for 30 min. The pH was adjusted with KOH or HCl before sterilization. After inoculation the cultures were incubated for 4 weeks. Nitrogen contents were deter Methode f o r growing mycelia mined by Kj eldahl ana lyses. Formic a cid (98-100% It has not yet been possible to cultivate the sporo pro analy.si) was obtained from Merck. phore: of the lorel (G. e ~ cu L enta) and of its The incuba tion of cultures was carried out at relative Morch,, zza under controlled conditions . the constantly regulated temperatures of +5 to 30° However, much experie nce has been obtai~ed in the C or in l greenhouse in which the air temperature cu ltivation of the mycelium of Morchella (D'Yvoire was 18-24° C during the day and 8-14° C at night. In 1889, Heim 1936, Brock 1951, Gilbert 1960, Litch addition to daylight the cultures were given artifical field e t al . 1963, M~kinen 1976), whereas only light (Philips HDK 251, 400 W) for 16 h per day (from l ittle is known about the factors effecting the 5 a.m. to 9 p.m . ) in winter . mycelial growth of G. esculenta (Raudaskoski et a l. The relative rate of mycelial growth was ob 1976. 1977). tained from the ma ximal rate cf extension of the Solid (Sinden 1937) and liquid (Laniece 1966) colony diameter i n mm or from the weight of mycelium E ub s trate ~ have been used for the production of mush dry matter in mg on a given medium. On solid substra room mycelia. In the method of Sinden for Agaricus tes of bark residue and litter the estimation of specieE, c ookod grQin to which calcium sulfate ha d growth wa n made by naked eye and under a Reichert boen adde4 wa ~ used. The substrate wa s inoculated with micros cope. 60 Karsten ia 18(suppl.) 1978
Preliminary t ests were performed in aerated acid test was performed since it has been observed that liqu id culture in f las ks and in aerated plastic film in its natural habitat the lorel grows close to a nt tubes . The poor growth observed in these tests shows - paths. The object wa s to t e s t whether the formic acid that further detai led studies are necessary. excreted by ants has an effect on the growth of the mushrooms , or whe ther the a ssociation i s due to 3pores Results and discussion being t r ansported by ants. The e t showed that f ormic Effect of acidity on the growth of myce lia acid promotes mycelial growth . Certain scientists ha ve tried to elucidate the beneficial effect of ants on Fig . shows that mycelial strain Klx3 of the lorel the growth of mushrooms (Weber 1966, Martin 1970 , Sohmid grew well at pH 4-7. The mo st suitable acidity for 1971). most mushrooms is pH 5-7 (Smith et al. 1975).
Effect of temperature on mycelial growth Effect of light o n mycelial growth Fig . 3 shows the results of a series of t e sts accord Th e action of light on the growth of mushrooms varies. ing to which mycelial strain Klx 3 grew well at Champignons grow and develop fruit-bodies in the dark. temperatures from +10° to +25 ' C. At +5° C the growth The studi es have shown that some s pec ies of Ba sidio was about half of that at the optimum temperature, mycetes need light for fruiting initiation, for in while a t +30 ° C it wa s particularly poor. Also in the stance Pleurotus strains. When the mycelia of the experimen s of Treschow (1944) the growth of white lorel were grown in light in the greenhouse their mycelial strains increased fairly evenly up to +24° growth became s lower and the white mycelia began C, after which it decreased s haprly. to tur n brown and form dense patches. Rauda skoski et al. ( 1976) have obtained similar results with the Effect of formic acid ·an mycelial. g rowth mycelia of G. esoulenta and Mlkinen (1976) with the mycelia of Morahella e sculenta. When 0.01- 0.1% f or mic acid was added to the substrate the growth of myc elia increased considerably. Addition of 0. 2% formic acid to the substrate prevented entirely the growth of mycelial strain K9 (Table 1). The formic
Fig . 2. The effect of pH on the growth of the mycel ium of Gyromitra esculenta (KlX3).
HEM 1% UREA 320 mg /L E' 12 u • ~10 -A? A- .....~ A >. c A 0 ...J 8 0 u cu ..c. 6 +J
0 - I. ..c. +J ~ 0 L... 2 (!) -l o, 2 3 5 6 7 pH Karstenia 18(suppl.) 1978 61
Fig. 3. The effect of temperature on the growth of the mycelium of Gyromitra esculenta (Klx3).
g 10 '& ..,.,. 1;. >. B ~ c __,0 0 u 6 1\ ..c.CLJ +-' 7- 0 I. \ -..c. +-' ~ 0 L.. 2 \ (.!) ~ 0 1 5 10 15 20 25
Table 1 . The effect of formic acid on the mycelial growth of the lorel (Strain K9) on a 1% hemicellulose and 0.032% urea substrate. The growth period was four weeks. Each test was made in triplicate . The initial acidity was adjusted with KOH to pH 4 . 2.
HCOOH, ml/1 in substrate Fresh weiV>t of Dry weight of Dry matter of N content of Total N of mycelit.nn, rrg rnycelit.nn, rrg mycelitnn, % myce litnn, % reyceliurn, mg of dry wt. 0 684 135 20 4.7 6.3 0.1 765 237 31 4. 4 8.6 0.5 1212 224 18 4. 4 10.0 1.0 3188 197 6 4.7 11.2 2.0 0 0 0 0 0 5.0 0 0 0 0 0
Mycelial g rowth o f the .lorel in waste materials In wheat grain moistened with a hemicellulose from the c ellulose industry -urea solution and on sawdust-wheat bran substrate Hemicellulose a nd lignosulfonate are waste materials mycelial strain Kh3 grew well. In a hemicellulose o f cellulos e production and could be used a s sub -urea liquid culture the mycelium grew only on the stra tes for mushrooms, with urea as the source of surface of the liquid. The liquid layer had to be shal nitroge n. In our experiments the mycelium of the low (below 5 em) . lorel did not grow on media conta ining solely hemi In fresh waste bark from birch and pine mois cellulose or calcium lignosulfonate in distilled tened with a hemicellulose-urea solution and in fresh wat e ~. In contrast , on medi conta ining hemicellulose pine litter the mycelia of the lorel did not grow and urea , or hemicellulose, urea and lignosulfonate, at all, or at most very poorly. The mycelia grew only the mycelium grew we ll ; in fact better than on a 1% on decomposed wa s te material. malt extract (Fig. 4). 62 Karstenia 18(suppl.) 1978
Fig. 4. The growth (0 em) of the mycelium of Gyro mitra esculenta (strain K9) in Petri dishes in pure liquid cultures. The substrates con tained: (1) 1% hemicellulose, 1% calcium lignosulfonate and 0.032% urea, (2) 1% hemi cellulose and 0.032% urea, (3) 1% malt ex tra ct , (4) 1% hemicellulose nd (5) 1 % calc ium lignosulfonate.
E 1, _ u 10 & I >. V/-o2 c 8 0 -' 0 !/~~1 u 6 Q) y•y 3 ..c +' ~-· I. Iii. ~~0 ~ ~-~ 0 -..c &?..J....•'~,_. +' ~ 2 0 """"" L.. (!) ..~~I I I """- ~ ~ ~~ 0 2 6 8 10 12 14 16 Days
References Laniece, A. 1966: Production and use of liquid mu~hroom Brock, T.C. 1951: Studies of the nutrition of t'lorchella spawn. U.S. Patent Office 3,286,399 . esculenta Fr. - Mycologia 43: 402-422 . Litchfield, J.H., Overbeck , R.C. & Davidson, R. S.l963: Gilbert, F.A. 1960: The submerged culture of Morchella Factors affecting the growth of morel mushroom - Mycologia 52: 201-209. mycelium in submerged culture. - Agric. Food Harmaja, H. '1969a: A wider and more natural concept of Chern. 11: 158-162. the genus Gyromitra Fr. -Ka rstenia 9: 9- 12. Martin, M.M. 1970: The biochemical basis of the fungus attine ant symbiosis. - Science 169: 16- 25. -''- 1969b: A neglected species, Gyromitra ambigua (K rst.) Harmaja, n. comb., and G. infula s. str. M&kinen, Y. 1976: Huhtasienen viljelykokeista. ( ulti - vation experiments with morel.) - Luonnontutkija in Fennos kandia. - Karstenia 9: 13-19. 80: 33-40. -"- 1973a: Piispanhiippa k&s itt&&kin kaksi eri lajia, Peng, J.-T. & Wu, L.-C. 1972: Variations in the c ulti joista toinen ehkli myrkyllinen. - Sienilehti vated mushrooms. -Mus hroom Science 8: 103-113. 25 (3): 10-12. Pohjola, K., Hintikka, V., Makinen, Y. & Layout 1973b: Amendments of the limits of the genera -"- Christiansson , I. 1975: Mets&s ienineuvojen opas. Gyromitra and Ps eudorhizina, with the descrip - Ammattikasvatushallitus, Metsliopetuksen OS·1Sto. tion of a new species, Gyromitra montana. - 75 pp. Karstenia 13: 48-58. Pyy s alo, H. 1975a: Gyromitriini korvasieniruoissa . - Heim, R. 1936 : La culture des morilles. - Rev. Mycol. Sienilehti 27 (2): 39 . 1 (suppl. 1): 10-11, (suppl.2): 19-25. _u_ 1975b: Studies on the volatile compounds in mush- Ho, M.-S. 1972: Straw mushroom cultivation in plastic houses. - Mushroom Science 8: 257-263. rooms. - Dr. Ph. Thesis 139 pp. - Technical Jalkanen, E. 1975: Luonnossa tapahtuvien korvas ienen Research Centre o f Finland, Helsinki. viljelykokeiden ongelmia. - Sienitalousseminaa Raudaskoski, M., Poh jola , K. & SaQrvanto, I. 197 6:
ri 7-8.4. 1975, Jyv ~ skyl&n yliopisto, Biolo Effect of temperature and light on t he mYcelial gian laitos, Tiedonantoja 1: 13-14. Jyv&skyl&. growth of Gyromitra escule nta in pure culture. Korhonen, M. 1973: 100 sient&. - 168 pp. Helsinki. - Karsteni a 16: 1 -5 . Karstenia l8( suppl.) 1978
11 - - 1977: L~mp b til a n ja valon vaikutus korvas ienen tuvasta tutkimuksesta Suornessa. - Kuopio, Kehi rihma ston kasvuun puhdasviljelmlssl. - Sieni- tysalueraha sto Oy . lehti 29:15. Treschow, C. 1944: Nutrition of cultivated mushroom. - Rautavaara , T . 1947: uomen sienisato. - 534 pp. Porvoo. Dansk Bot. Arkiv 11: l-180. Roponen, I. 1975: Korvasienten kasvatuksesta labora to Weber, N.A. 1966: Fungus -growing ants . - Science 153: rio-olosuhteiss a . - Sienitalouss eminaari 7-8.4. 587 -604 .
1975, Jyv~skyl&n yliopisto, Biologian laitos, Wills, C. 1968: Yeaot partial dominance: effect of Tiedonantoja 1: 12-13. J yvaskyla. environment and background genotype . - Science Schmid, R. 1971: Biochemis che Grundlage der Symbiose 160 : 549-550. zwischen Ameisen und Pilzen. - Naturw. Rdsch. D' Yvoire, B. 1889: La morille. Procecte de culture 24: 482-485. potagere applicable a taus les jardins. - Bull. Sinden , J.W. 1936: U.S. Patent 2,044,861. Soc. d'Acclimation 36: 18 - 26. Sm i th, J.E. & Berg, D.R. 1975 : The filamentous fungi.I. Zamenhof, S. & Eichorn, H.H. 1967: Study of microbia l Indus trial Mycology. - 34 0 pp. London. evolution through loss of biosynthetic functions: Su honen, I. 1974 : Selv itys syOtlviin sieniin kohdis- Establishment o f 11 defective 11 mutants. - Nature (London)216: 456 - 458.