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Fructication and yield of the edible mushrooms Pleurotus pulmonarius and Stropharia rugosoannulata are clearly enhanced when

65 (2000). Some species grow best in fermented pasteurizedwet wheat straw, primordia of Pleurotus pulmonarius appeared two days earlier and primordia of Stropharia rugosoannulata

appeared 18 days earlier when compared to pure wheat straw substrate. This concentration of b-adenosine had no impact on themushroom yield of Pleurotus, but resulted in a 2.2 fold increase in yield for Stropharia. b-Adenosine at 25 mg per kg wet wheatstraw increased the yield of Pleurotus with 52% and the yield of Stropharia with 258%, but this concentration delayed primordialformation in Pleurotus.

# 2003 Elsevier Ltd. All rights reserved.

Keywords: Lolium perenne; Pleurotus pulmonarius; Stropharia rugosoannulata; Mushroom fruit body formation; Oyster mushroom; King Stropharia;

Rye grass

1. Introduction

Mushroom growth and fruit body formation are greatlyinuenced by various environmental factors. Abiotic andbiotic factors such as temperature, humidity, light, pH,gaseous exchange, nutrient status, minerals, sexualityand genetics, microbial interactions, hormones andenzymes can all have an inuence on mushroom devel-opment (Ganny and Gooding, 1987; Stamets, 1993).The mushroom substrate, which is the main source ofnutrients, is one of the crucial factors that greatly aectgrowth and fructication (Elliot, 1994; Moore, 1998).Dierent species of cultivated mushrooms have

dierent substrate requirements (Philippousis et al.,

substrates while other species grow best in sterilized,pasteurized or simply moistened substrates. Suitablesubstrates lead to fast colonization and high density ofmycelium, enhance primordial formation and fructi-cation and increase mushroom yield (Stamets, 1993).The nutrient availability from the substrates and myce-lium greatly contributes to the formation of the fruitbodies or carpophores (Chang and Miles, 1988; Ruitersand Wessels, 1989).The addition of organic supplements to wheat or rice

straw resulted in earlier fructication and increased theyield of Pleurotus mushrooms (Royse et al., 1991; Royseand Zaki, 1991; Krisnamoorthy, 1997; Chowdhury etal., 1998). Supplements can also inuence the chemicalearlier fructication of Pleurotus pulmonarius. Mushroom fruitwheat straw is supplemented with 30% Lolium perenne grass cha. The bioactive compound in the methanol extract of grass cha

was identied as b-adenosine. In vitro biological activity tests showed that 0.012 mg of b-adenosine per ml of medium stimulateding trials showed that when 12 mg b-adenosine was added to 1 kgb-Adenosine, a bioactive compmushroom

Denisa L. Domondona,1, Weidong HeJoseph

aLaboratory of Phytopathology, Department of Crop Protection, Fa

Coupure Links 653bDepartment of Organic Chemistry, Faculty of Agricultural and Applied B

Received 18 May 2003; received

Abstractund in grass cha stimulatingroduction

orbert De Kimpeb, Monica Hoftea,*,ppea

of Agricultural and Applied Biological Sciences, Ghent University,

0 Ghent, Belgium

ical Sciences, Ghent University, Coupure Links 653, 9000 Ghent, Belgium

vised form 11 November 2003

2004) 181187

www.elsevier.com/locate/phytochemPoppe and Sedeyn (1989) have performed production-stimulating tests with the king Stropharia, Strophariarugosoannulata on straw substrates. In general, the

0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.phytochem.2003.11.004

E-mail address: monica.hofte@ugent.be (M. Hofte).1 Present address: Department of Biology, Benguet State Uni-

versity, La Trinidad, 2601 Benguet, Philippines.composition and nutritional value of the Pleurotusmushroom (Tshinyangu, 1996; Krisnamoorthy, 1997).Furthermore, nitrogen-rich supplements inuenced cropyield and fruit body size of shiitake (Kalberer, 2000).* Corresponding author. Tel.: +32-9-264-6017; fax: +32-9-264-

6238.Phytochemistry

species used mainly for pasture, hay crop or groundcover (Adams et al., 1998). In Europe, grasslands

agar (56.3 mm). Also the mycelial growth was denser

ations of possible primmation was observed onP. pulmonarius 20 daysiny primordia were plen0:30 ratio of wheat straw

plates with boiled grass cha extract. Pinhead forma-tion was not observed, however, with Stropharia (datanot shown).

tochemistry 65 (2004) 181187occupy about 52 million hectares. Of these 52 million, atleast 12 million hectares use ryegrass cultivars for pas-ture (Prine, 1991). Maintenance of these pasture landsmeans that a continuous grass seed production is nee-ded. Processing of grass seeds generates grass cha thatis left, as a waste, in mountain heaps in most grass seed-processing centers. The abundant availability of thiswaste and the fact that it stimulates earlier fructica-tion makes it an interesting supplement to add tomushroom substrates. However, it is not known whygrass cha enhances fructication. In this study wedetected, isolated and identied one of the bioactivesubstances in the grass cha of Lolium perenne andproved that it enhanced fructication of Pleurotus andStropharia.

2. Results and discussion

2.1. In vitro trials with boiled grass cha extracts

Boiled extracts obtained from grass cha and wheatstraw were combined at dierent concentrations andwere tested for bioactivity with two mushroom species,Pleurotus pulmonarius and Stropharia rugosoannulata.The mean mycelial diameter of P. pulmonarius on theagar plates with dierent combinations of boiledextracts showed no signicant dierence between 100%wheat straw, 90:10 wheat straw/grass cha and 70:30wheat straw/grass cha extract (data not shown). ForS. rugosoannulata, the diameter of mycelial growth in70:30 wheat straw/grass cha boiled extract (74.7 mm)was signicantly dierent from the 90:10 wheat straw/grass cha (57.7 mm), 100% grass cha extract (71.0mm), 100% wheat straw extract (52.7 mm) and plainindoor culture of this mushroom on straw is verycapricious with late and low fructication. Poppe andSedeyn (1989) added dierent types of hay to wheatstraw and observed that ripe seed-setting Lolium grasshay, but not young Lolium grass hay, stimulated fruitbody production. The addition of pure Lolium grassseed also gave good results, but was too expensive.Finally, they found that the addition of cha fromLolium seeds even gave better results and resulted inearlier fructication and a very strong yield increase.Later on Domondon et al. (2000) showed that theaddition of grass cha to wheat straw also enhancedthe yield of various Pleurotus species and stimulatedearlier fructication. In addition, the carpophores har-vested from wheat straw supplemented with 30% grasscha had a signicantly higher protein content than thefruit bodies harvested from pure wheat straw substrates.Lolium perenne, or ryegrass, is an important forage

182 D.L. Domondon et al. / Phy2.2. Isolation of the bioactive compound

Grass cha of ryegrass, Lolium multiorum, wasextracted until exhaustion with hexane, dichloro-methane, ethyl acetate, methanol and water as shown inFig. 1. The crude extracts obtained were evaluated fortheir ecacy in stimulating mycelial growth and pri-mordial formation. Only the methanol extract added toagar resulted in denser mycelium and induced pri-mordial formation. Therefore, this extract was subjectedto RP vacuum layer chromatography (VLC). Eachfraction was tested for bioactivity using agar plate test(plain water agar for mycelial growth, fruiting mediumfor primordial formation). Fractions F5 and F6 thatstimulated primordial formation were then subjected tocentrifugal counter current partition chromatography(CPC) to obtain 12 fractions (Fig. 1). Crystallization ofthe most active fraction of this series aorded nally apure compound which was identied as b-adenosine 1by MS and NMR spectroscopy, including 1H NMR,13C NMR, COSY, HETCOR and DEPT spectra. Thebasic data are identical with those published for b-ade-nosine (Arpalahti et al., 1998; Trifonova et al., 1999)and with our data obtained from authentic b-adenosine.

Table 1

Primordia formation by P. pulmonarius on fruiting medium supple-

mented with boiled extracts from wheat straw (WS) and grass cha

(GC)

Substratesa Number of primordiab

100% WS 51.2 C90:10 WS/GC 100.3 B70:30 WS/GC 202.3 A100% GC 81.4 B

a Substrates: WS=wheat straw, GC=grass cha.b Mean of four replicates. Primordia formation was observed for a

period of 30 days. Values followed by the same letter are not sig-

nicantly dierent for P=0.05.extract as compared to the agar plates with extracts of100% wheat straw. This indicates that the bioactivecompound in the grass cha is thermo stable as shownby the development of more primordia on the agarplates with 7 /boiled grass chalated with(Table 1). Tafter inoculationtiful on the agarPinhead for the plates inocu-

ther observ ordial formation.

Plates wer onditions for fur-with the 70:30 wheat straw/grass cha extract whencompared with the other treatments.

e maintained in fruiting c

lata grown on wheat straw supplemented with grass primordial formation (primordia appeared after 37

tocheD.L. Domondon et al. / Phy mistry 65 (2004) 181187 1832.3. In vitro trials with -adenosine

The isolated b-adenosine 1 was further tested for sti-mulation of primordial formation in comparison withcommercial b-adenosine in agar plate tests. The resultsshowed that the puried compound and b-adenosine ata concentration of 0.012 mg/ml induced primordial for-mation 5 days after exposure to fruiting conditions.These results coincide with our previous experimental invivo trials where wheat straw substrates supplementedwith grass cha also showed P. pulmonarius primordiaformation after 5 days. Interestingly, plates supple-mented with b-adenosine or the isolated compound 1showed a prominent formation of rhizomorphs. Whenisolated compound 1 or commercial b-adenosine wasadded at concentrations of 0.025 or 0.05 mg per ml ofmedium however, primordia only appeared after 10days.

2.4. Fruiting trials with -adenosine

Table 2 shows the number of days to primordial for-mation, the harvest period and the mean of total yieldfor Pleurotus pulmonarius and Stropharia rugosoannu-

cha or b-adenosine. Full mycelial colonization took 14days for P. pulmonarius and 21 days for S. rugosoannu-lata irrespective of the type of substrate. When the fullycolonized substrates were opened and exposed to fruit-ing conditions, the rst primordium of Pleurotusappeared after 5 days on the substrates supplementedwith 30% grass cha. Primordia appeared 2 days earlieron wheat straw substrates supplemented with 12 mgb-adenosine kg1 wet substrate when compared to purewheat straw substrate. This concentration of b-adeno-sine however, had no eect on mushroom yield. b-Ade-nosine at 25 mg/kg wet substrate delayed primordialformation, but increased Pleurotus mushroom yieldwith 52%. In the fruiting tests carried out by Domon-don et al. (2000) primordial formation was delayedwhen pure grass cha was used as a substrate in com-parison with pure wheat straw or wheat straw supple-mented with 10 or 30% grass cha. It is possible thatthe abundance of adenosine in pure grass cha is thefactor that delays fruit body formation.Stropharia primordial formation occurred after 24

days on wheat straw supplemented with 30% grass chaand after 27 days on wheat stray with 12 mg b-adeno-sine. Here, however, also 25 mg b-adenosine enhanced

Fig. 1. Isolation scheme for b-adenosine 1.

is one of the rst recognized secondary messengers,which modulates various eukaryotic cellular processes(Laychock, 1989). In fungi, it was postulated to be

ing kewi P ated m th of nteader 1989 pos-eady of a rasshe s cycli con-uces rma ncesuctio

HPLC analysis of the nucleoside proles of medicinal

b-adenosine, that enhance fructication and mushroomyield. Accordingly, it can be concluded that fructica-tion is not caused by a single factor or substance but it is

Gen imen

he 1H EPT MRined M-E R apn). ectra red

sting

3.3 software. Melting points were determined on a

d of

Harv

Pleur

25

44

30

30

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each

iation. Treatment means were compared by Duncans Multiple Range Test.

ntly

tochedays) when compared to pure wheat straw substrate(primordia appeared after 45 days), but this concen-tration was less eective when compared with 12 mgb-adenosine. For Stropharia, both concentrations ofb-adenosine signicantly increased the total mushroomyield with 220 to 258%.Ohga (1989) showed that adenosine, adenine and

adenosine triphosphate were the most importantgrowth-activating substances for shiitake in hot waterextracts of Welsh onion. In addition, Yoshikawa andOoga (1989) conducted an in vitro study on adenine andits derivatives as mushroom growth enhancers. Theirresults showed that addition of adenine or its derivativessignicantly increased the weight of Agrocybe aegeritacarpophores. Addition of 15 mg/l of adenine, adenosineor adenosine monophosphate to substrates producedfruit bodies with a total weight of 13.96 g/dish versus9.3 g/dish for the control.Adenosine is a precursor of cyclic-adenosine mono-

phosphate (cylic-AMP), synthesized in the presence ofthe enzyme, adenyl cyclase (Grin, 1993). Cyclic-AMP

Table 2

Number of days to primordia formation, harvest period and total yiel

supplemented with grass cha (GC) or b-adenosine (b-adn)

Substrate Days to primordia formationa

Pleurotus Stropharia

100% WS 9 45

WS/30% GC 5 24

WS+12 mg b-adn 7 27WS+25 mg b-adn 14 37

a The spawn run for full mycelial colonization of the inoculated bag

deviations for days to primordia formation and harvest period were forb Values are the means of three replicates. SD indicated standard dev

Within each column, values followed by the same letter are not signica

184 D.L. Domondon et al. / Phymushrooms showed that Ganoderma lucidum, Lentinusedodes and Cordyceps sinensis contained large amountsof adenosines in their fruiting bodies (Shiao et al.,1994). These previous ndings of adenosine abundancein mushroom fruit bodies may further give light to thepresent results that b-adenosine enhances in vitro pri-mordial formation in P. pulmonarius. Apparently, theavailability of adenosine in the grass cha, which prob-ably served as a precursor for cyclic-AMP, enhancescellular processes, such as morphogenesis, resulting inan earlier primordial formation and enhanced fruitbody production. Likewise, there is a ready source forthe synthesis of adenosine nucleosides for cellularsynthesis. Therefore, it can be concluded that the avail-ability of adenosine in substrates could be an importantfactor in mushroom fructication.However, primordial formation occurred earlier and

mushroom yield was much higher in wheat straw sup-plemented with 30% grass cha compared to wheatstraw supplemented with b-adenosine. This indicatesthat grass cha contains other components, aside from

dierent from each other for P=0.05.P. pulmonarius and S. rugoso-annulata on wheat straw (WS) substrate

est period (d)a Total yieldSD (g fresh weight)b

otus Stropharia Pleurotus Stropharia

40 21010 C 3415 C60 3959 A 20510 A40 23014 C 10912 B45 32017 B 12217 B

e 14 and 21 days respectively, for Pleurotus and Stropharia. Standard

value smaller than 1 day.

mistry 65 (2004) 181187Moreover, Lou and Montag (1994) showed that thedominant fractions of nucleosides in mushrooms areadenosine, guanosine and uridine. A reversed phase-

and an orthogonal accelerated time-of-ight MS analy-zer using a Z-spray interface and operated in positiveion electrospray mode and controlled with Mass lynxfruit body prod n. (Micromass) consi of an Alliance 2690 LC (Waters)cha induces tsequentially indynthesis ofprimordial foc-AMP andtion and enhaobtaJapawith a JNMass spX270 FT NMwere measuparatus (Jeol,with a LCTsible that the r availability denosine in g T , 13C, D and COSY N spectra were

by 24% (Alexan and Lippert, ). Thus, it is

200 mg/l enhanc ycelial grow Calvatia giga 3.1. eral exper tal procedures

fruit body-induc substance. Li se, cyclic AMinvolved in nutrition, reproduction and morphogenesisand dimorphism (Pall, 1981; Grin, 1993). In Coprinuscomatus, the level of cyclic-AMP was increased onlyduring fruiting, hence, it was surmised that it acts as a

interplay of several compounds and several factors.

3. Experimental

Hz, OH-5 ), 5.88 (1H, d, J=6.33 Hz, H-1 ), 8.14 (1H, s, tration of 0.05 mg/ml and added to hand-warm fruiting

tocheBuchi 535 apparatus (Buchi, Switzerland). CPC wascarried out with a CCC-1000 High Speed Counter-current Chromatograph (Pharma-Tech Research Corp.USA), equipped with a SSI 300 pump, a PharmaciaLKB Uvicord S II detector (254 and 280 nm) and aRetriever II fraction collector. All precoated TLC plates(2002000.25 mm) for both normal (Silica gel 60 F254)and reversed phase (RP-18 F254s), and the solvents usedfor isolation and purication, were from Merck,Germany.Grass cha (500 g) was successively extracted until

exhaustion with n-hexane (n-C6H12), dichloromethane(CH2Cl2), ethyl acetate (EtOAc), methanol (MeOH)and water (H2O). The extracts were ltered over a lterpaper (Scheiler & Schuell, 595/2 folded lters, 1 240mm) and concentrated or dried in vacuo in a rotava-por at a temperature of 40 C. The residues of thecrude extracts were tested for their ecacy in stimu-lating mycelial growth and primordial formation ofPleurotus in agar plates. The MeOH extract (30.81 g),which showed the best mycelial growth and stimulatedearlier primordial formation, was selected for furtherstudy.Twenty-two grams of the methanol extract were

adsorbed by Lichroprep C18 and fractionated with RPVLC (P4 glass-sintered funnel packed with 30 g ofLichroprep C18, 4063 mm, Merck). The elution wasperformed under vacuum by stepwise gradient startingwith 500 ml each of water (two times), water/methanol97.5:2.5, 95:5, 92.5:7.5, 90:10, 85:15, 80:20, 75:25, 70:30,100% methanol, and then THF. These ltrates weremonitored by RP-TLC, of which, 12 fractions (F1-F12)were obtained. Active fractions (F5 and F6, 320 mg)were combined and subjected to CPC with the solventsystem CH2Cl2/MeOH/H2O (7:13:8): the lower phase(LP) as the stationary phase and the upper phase asmobile phase (MP); elution conguration, tail to head;sample dissolved in nine ml UP and nine ml LP, lteredwith glass ber lter (Acridose, 0.45 mm, GelmanScience) and injected into the coiled columns (350 ml)with a sample loop of 14 ml; ow rate, 1 ml/min; frac-tions collected in 14 ml; revolution speed, 1000 rpm witha pressure at 140 psi; equilibrium, 90 ml; run time, 340min; push out with a ow rate of four ml/min, andfraction in 8 ml; detection, 254 and 280 nm. Based onthe TLC monitoring, 12 fractions were obtained (F1-F12). The most active fraction F2 (39 mg) was puriedby crystallization with MeOH to obtain the pure com-pound 1 (20 mg) as a ne white solid (MeOH): mp234.4235.4 C [lit. Mp 234235 C (Budavari et al.,1989)]; 1H NMR (270 MHz, DMSO-d6) 3.47 (2H, s,NH2), 3.59 (2H, m, H-5

0), 3.97 (1H, q, J=3.3 Hz, H-40),4.15 (1H, dd, J=5.0, J=3.3 Hz, H-30), 4.62 (1H, dd,J=5.9, J=6.3 Hz, H-20), 5.24 (1H, d, J=4.6 Hz, OH-30), 5.49 (1H, d, J=3.0 Hz, OH-20), 5.51 (1H, d, J=4.0

0 0

D.L. Domondon et al. / PhyH-2), 8.36 (1H, s, H-8) ppm; 13C NMR (67.5 MHz,DMSO-d6) 61.8 (C-5

0), 70.8 (C-30), 73.6 (C-20), 86.1 (C-40), 88.1 (C-10), 119.4 (C-5), 140.0 (C-8), 149.1 (C-4),152.5 (C-2), 156.2 (C-6) ppm; ES+MS (LCMS): m/z 268(M+1, 5%), 137 (7), 136 (100), 108 (20).

3.2. Microorganisms and plant material

Two mushroom species, Pleurotus pulmonarius (Phy-topathology Laboratory collection, Ghent University)and Stropharia rugosoannulata (cap tissue isolate fromMerelbeke, Belgium) were used to test the ecacy ofgrass cha as a stimulant for fructication or primordialformation. Mother cultures were prepared in poly-styrene Petri plates with malt extract agar and were usedas sources of mycelial discs for bioassays. Mushroomspawn and mushroom substrate (chopped and ham-mermilled wheat straw) were obtained from the mush-room spawn laboratory Mycelia (St. Amandsberg,Ghent, Belgium). Lolium perenne grass cha was col-lected at the grass seed-production center in Merelbeke,Belgium.

3.3. In vitro trials

3.3.1. Boiled extractsWheat straw and grass cha, 200 g each, were boiled

separately for 15 min in 2 l each of water. The followingcombinations were prepared to determine the eect ofboiled grass cha extract as a supplement: (a) 100 ml ofboiled wheat straw extract, (b) 90 ml of boiled wheatstraw extract+10 ml of boiled grass cha extract, (c) 70ml of boiled wheat straw extract +30 ml of boiled grasscha extract, (d) 100 ml of boiled grass cha extract and(e) 100 ml of distilled water. Each of the treatments wasplaced in asks and 1.5 g of agar was added.

3.3.2. Crude grass cha extractsThe dierent crude grass cha extracts were dissolved

in acetone at a concentration of 160 mg/ml and addedto hand-warm water agar at a nal concentration of 1.6mg of crude extract per ml of water agar.

3.3.3. FractionsThe dierent fractions obtained during isolation were

dissolved in methanol at a concentration of 50 mg/mland added to hand-warm fruiting medium (Chiu andTo, 1993) at a nal concentration of 0.05 mg and 0.1 mgper ml of medium.

3.3.4. Pure compoundsThe isolated compound 1 was compared with a-ade-

nosine (Sigma-Aldrich) and b-adenosine (Sigma-Aldrich) for primordial stimulation. These compoundswere dissolved in water-methanol (70:30) at a concen-

mistry 65 (2004) 181187 185

For Pleurotus pulmonarius trials, the inoculated bags

hybrid mushrooms. Mushroom Journal 78, 305311.

Laychock, S.G., 1989. Coordinate interactions of cyclic nucleotide and

phospholipid metabolizing pathways in calcium dependent cellular

Oei, P., 1991. Manual on Mushroom Cultivation: Techniques, Species

tochemistry 65 (2004) 181187medium at a nal concentration of 0.05, 0.025 and 0.012mg per ml of medium.

3.3.5. Inoculation and incubationAll media were dispensed in polystyrene Petri plates

at a volume of 20 ml/plate. After solidication, agarplates were inoculated centrally with 9 mm mycelialdiscs of P. pulmonarius or S. rugosoannulata. Theinoculated plates were incubated in dark conditions at24 C. After 7 days, mycelial diameter was measuredwith a vernier calliper. Growth density was photo-graphed, rated and recorded with a plus sign. Plateswere subsequently maintained in fruiting conditions(20 C with a photoperiod of 12/12 h (day/night) anda light intensity of 1000 lux) for possible primordialformation. The primordia were recorded regularly for aperiod of 30 days.

3.4. Fruiting trial

The basal substrates for the growing tests were chop-ped and hammermilled wheat straw supplemented withcha of perennial ryegrass, Lolium perenne. Wheatstraw and grass cha were pasteurized separately byimmersion in water at 65 C for 1 min. The pasteurizedsubstrates were cooled at room temperature anddrained in a sieve up to 70% moisture (Oei, 1991).When the substrates were cooled, the following treat-ments were prepared: (a) 100% wheat straw, (b) 100%wheat straw+12 mg b-adenosine kg1 wet straw, (c)100% wheat straw+25 mg b-adenosine kg1 wet strawand (d) 70% wheat straw+30% grass cha. The com-bined wet weight of wheat straw and grass chasupplement per replicate was 1 kg. Each treatmentwas carried out in three repetitions. Each replicate wasinoculated with 50 grams of mushroom spawn. Pre-pared oat grain spawn was used for inoculation. Fiftygrams of oat grain spawn were inoculated alternatelywhile lling the polyethylene bags (2540 cm) withsubstrates layer by layer. The topmost layer of the sub-strate was inoculated with more spawn grains to hastenfaster colonization and prevent growth of competitormicroorganisms. b-Adenosine dissolved in 50 ml of waterwas added in droplets in the bags of inoculated wheatstraw. Bags were closed with adhesive tape and the basalcorners of the bag were cut to provide aeration and fordrainage of excess water. For Stropharia trials, the inocu-lated bags were incubated in dark condition for 3 weeks ata temperature of 25 C. After full mycelial colonization,the bags with were opened and a casing layer composedof 50% deep garden soil and 50% black peat wasapplied at a depth of 3 cm. The fully colonized bagswith casing layer were transferred in a fruiting roomprovided with articial light of 1000 lux with a photo-period of 12 h/12 h and at a temperature of 20 C.

186 D.L. Domondon et al. / Phyprocesses. Current Topics in Cellular Regulation 30, 203242.

Lou, S.N., Montag, A., 1994. Changes in the nucleostatus of mush-

rooms during storage and thermal processing. Deutsche Lebens-

mittel-Rundschau 90, 278284.

Moore, D., 1998. Fungal Morphogenesis. Cambridge University

Press, Cambridge.Grin, D.H., 1993. Fungal Physiology, second ed.. John Wiley and

Sons, New York.

Kalberer, P., 2000. Inuence of urea and ammonium chloride on crop

yield and fruit body size of shiitake (Lentinus edodes). Mushroom

Science 15, 361366.

Krisnamoorthy, A.S., 1997. Inuence of organic supplements on yield

and protein content of oyster mushroom. Madras Agricultural

Journal 84, 604606.were incubated in dark conditions for 2 weeks and thenexposed to light conditions like the bags of Stropharia.Casing layer was not, however, necessary for Pleurotuscultivation. The mean yield of carpophores was deter-mined for both species at the end of the fruiting trials.

Acknowledgements

This research was funded by the Joint Japan/WorldBank Graduate Scholarship Program and by the Facultyof Agricultural and Applied Biological Sciences, GhentUniversity, Belgium.

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beta-Adenosine, a bioactive compound in grass chaff stimulating mushroom productionIntroductionResults and discussionIn vitro trials with boiled grass chaff extractsIsolation of the bioactive compoundIn vitro trials with beta-adenosineFruiting trials with beta-adenosine

ExperimentalGeneral experimental proceduresMicroorganisms and plant materialIn vitro trialsBoiled extractsCrude grass chaff extractsFractionsPure compoundsInoculation and incubation

Fruiting trial

AcknowledgementsReferences