Chiang Mai J. Sci. 2013; 40(2) 161

Chiang Mai J. Sci. 2013; 40(2) : 161-172http://it.science.cmu.ac.th/ejournal/Contributed Paper

Development of an Edible Wild Strain of Thai OysterMushroom for Economic Mushroom ProductionJaturong Kumla, Nakarin Suwannarach, Amornrat Jaiyasen, Boonsom Bussabanand Saisamorn Lumyong*Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.*Author for correspondence; e-mail: saisamorn.l@cmu.ac.th

Received: 10 March 2012Accepted: 5 July 2012

ABSTRACTThe edible oyster mushroom genus Pleurotus are collected and cultivated in

many countries. In 2011, fruiting body of Pleurotus giganteus was collected from northernThailand and pure culture was isolated. The fungal identification based on morphologyand molecular characteristics. In this paper we report the optimum in vitro cultureconditions and the fruiting bodies production. Among ten culture media tested, potatodextrose agar was the best for the mycelia growth. The fungus was able to grow attemperatures ranging from 15-35oC, with an optimal growth temperature of 25oC.The optimum pH for mycelia growth was 7.0. In addition, the fruiting bodies inductionconditions of this fungus were examined in pure culture. The primordia were formedunder lowered temperature, high humidity and a 12 h light photoperiod conditions anddeveloped to mature fruit-body. For the spawn production, sorghum grain was thesuitable solid substrates for spawn production of P. giganteus. Pleurotus giganteus couldgrow on sawdust and produced the mature fruiting bodies as well as casing with soillayer induced the primordial production. This report provides valuable informationfor the first time concerning the possibility to cultivation P. giganteus in Thailand.

Keywords: agaricomycetous mushroom, cultivation, Pleurotus giganteus

1. INTRODUCTIONThe cultivation of edible mushrooms has

become an attractive economic alternativeover past few years, mainly due to increasein its demand and market value [1,2].Mushrooms are delicious food and are richin proteins, vitamins, and minerals whilecontaining little fat [3]. Several species ofmushrooms are important because of theirmedicinal properties such as; they are activeagainst human pathogen, cancer, diabetes,hypertension, hypercholesterolemia condition

and turmor [4, 5]. Cultivation methods foredible mushrooms varied considerablyaround the world. Depending on locationof production, many specialty mushroomscan be grown using ether indoor and outdoormethod. In most countries, the consumersaccept the cultivated mushroom such asAgaricus bisporus, Auricularia spp., Lentinusedodes, Pleurotus spp. and Volvariellavolvacea for consumption [6]. In 2002,world production of cultivated mushroom

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was estimated to be 12,250 thousand tonsand China’s mushroom production wasover 14 million tons in 2006 which nowChina has become a leading mushroomproducer and consumer in the world [1].In Thailand, mushroom production in2007 was estimated at 10,000 tons [7].An edible mushroom such as, A. auricula,Coprinus comatus, L. edodes, L. sarjov-coju,P. cysidiosus, P. ostreatus, Schizophyllumcommune, Trimella fuciformis andV. volvacea were grown on a commercialscale [8,9]. However, many types of wildmushrooms are also collected for sale andconsumption.

The most well known species of Pleurotusare the largest group of cultivated in theworld and they were found wide intemperate forests and some species intropical forests [10,11]. In addition,morphological differences among membersof this group are few; therefore, theirtaxonomic assignment has been in the pastan issue of debate. Recently, variousmolecular analyses have been introducedfor the verification of them, such as RFLP,RAPD, SSU rDNA and ITS sequenceanalyses [2,12,13]. Many wild species of thisgenus such as, P. cornucopie, P. cystidiosis,P. eryngii, P. flabellatus, P. florida andP. ostreatus were selected for cultivation inthe commercial scale [18]. Oyster mushroomcan be cultivated within a wide range oftemperature on different natural resourcesand agricultural wastes [14]. However, thespecific conditions of nutrition, humidity,carbon dioxide levels and temperature willaffect mycelial growth and fruiting bodiesproduction of mushroom [15-18]. Thisstudy focused on the identification offungus base on morphology and phylogeny.In addition, the culture conditions formycelial growth and fruiting bodiesproduction in pure culture were studied.

Moreover, the evaluation of suitable solidmedia for the best spawn production wasalso investigated.

2. MATERIALS AND METHODS2.1 Fungal Strain

Fruiting body of P. giganteus CMU54-1were collected from Medicinal Plant Garden(18o48’22"N, 98o54’52"E and altitudes1,042 m), Doi Suthep-Pui National Park,Chiang Mai Province, Thailand in 15 May2011 and kept at the Research Laboratoryfor Excellence in Sustainable Developmentof Biological Resources, Faculty of Science,Chiang Mai University. The mycelia wereisolated from fruiting body by asepticallyremoving a small piece of mycelium frominside and transferring it to potato dextroseagar (PDA). The plates were incubated at30oC in the dark. The mycelia emergingfrom tissue were picked and transferred toPDA. The pure cultures were kept on PDAslants for further use. Same cultures werekept in both sterile distilled water at 4oCand 20% glycerol at -20oC for long-termpreservation.

2.2 IdentificationThe fruiting body was identified based

on morphological characteristics [19,20]and a molecular analysis of the DNA of thefruit-body extracted through a modifiedCTAB method. Fruiting body and mycelia(1 g) were frozen in liquid nitrogen andground into a fine powder with a mortarand pestle. The powder was placed in a1.5 ml Eppendorf tube and 500 μl CTABextraction buffer (2% CTAB, 100 mMTris-HCl, 1.2 M NaCl, 20 mM EDTA,pH 8.0) was added and mixed with thematerial. Samples were incubated at 65oCfor 60 min in a water bath and occasionallymixed. An equal volume (500 μl) ofchloroform: isoamyl alcohol (24:1) was

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added to the samples and briefly mixedby vortexing. After centrifugation for15 min at 13,000 rpm, the upper aqueouslayer was transferred to a new sterileEppendorf tube. The extraction wasrepeated until the interface was clear. Thesupernatant was removed to a newEppendorf tube, containing 2 volumes ofcold 100% ethanol. The DNA was pre-cipitated at -20oC overnight and centrifuged15 min at 14,000 rpm, 4°C. Then the pelletwas washed with 70% ethanol and dried atroom temperature. It was resuspended in100 ml of 0.002% RNase (5 mg/ml) inTE buffer and incubated for one hour at37oC. The suspension was stored at -20oCpending use for amplification.

2.3 Fungal ITS and LSU RegionsSequencing and Phylogenetic Analysis

The internal transcribed spacer (ITS)regions of nuclear rDNA were amplified bypolymerase chain reaction (PCR) with primersITS4 and ITS5 under the following thermalconditions: 94oC for 2 min, 35 revolutioncycles of 95oC for 30 s, 50oC for 30 s,72oC for 1 min, and 72oC for 10 min.In addition, the 28S ribosomal RNAgene was also amplified with primerLROR and LRO5 under the followingthermal conditions: 94oC for 2 min, 30 cyclesof 95oC for 30 s, 52oC for 30 s, 72oC for1 min, and 72oC for 10 min. The PCRproducts were checked on 1% agarose gelsstained with ethidium bromide under UVlight. PCR products were purified by usingPCR clean up Gel extraction NucleoSpinR

Extract II purification Kit (Macherey-Nagel,Germany Catalog no. 740 609.50) followingthe manufacturer’s protocol. The purifiedPCR products were directly sequenced.Sequencing reactions were preformedand the sequences were automaticallydetermined in the genetic analyzer

(1ST Base, Malaysia) using PCR primersmentioned above. Sequences were used toquery GenBank via BLAST (http://blast.ddbj.nig.ac.jp/top-e.html) and a multiplesequence alignment was carried out usingthe subroutines in Clustal X [21]. Aphylogenetic tree was constructed using thePUAP beta 10 software version 4.0 [22].

2.4 Fungal CultivationTwenty five milliliters of culture media

for each experiment were poured into Petridishes (9.0 cm, in diam) after autoclavingat 121oC for 15 min. A sterilized cellophanedisc (9.0 cm, in diam) was placed on thesurfaces of the test media. Mycelial inoculawere prepared by growing on PDA at30oC in darkness for one week. Mycelialplugs (5 mm diam) from the periphery ofthe growing colony were then inoculatedcentrally of the media. All plates were sealedwith parafilm after inoculation. Colonydiameters at one week after inoculationwere measured. The covered cellophanediscs were removed, died at 105oC overnight and maintained in desiccators for20 min before weighing. Mycelium dryweights were calculated. Four replicationswere made for each treatment.

2.5 Effect of Media on Mycelial GrowthThe following 10 different media were

used in this experiment: corn meal agar(corn 20 g, glucose 10 g), Czapek Dox agar(NaNO3 2 g, KH2PO4 1 g; MgSO4.7H2O0.5 g, KCl 0.5 g, FeSO4.7H2O 0.01 g, sucrose30 g), glucose yeast extract agar (glucose10 g, yeast extract 2 g), malt extract agar(malt extract 20 g), oatmeal agar (oat 20 g,glucose 10 g), potato dextrose agar (potato200 g, glucose 20 g), soybean meal agar(soybean 20 g, glucose 10 g), V8 medium(V8 juice 200 ml, CaCO3 2 g), wheat agar(wheat 20 g, glucose 10 g) and yeast extract

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peptone glucose agar (yeast extract 10 g,peptone 20 g, glucose 20 g). All media wereadded with agar 20 g and adjusted volumeto 1,000 ml with distilled water. Final pHwas adjusted to 6.5 by using 1 N HCl and1 N NaOH. After inoculated culture mediawere incubated in the darkness at 30oC forone week.

2.6 Effect of Temperature and pH onMycelial Growth

In this experiment, cultures of P.giganteus CMU54-1 on PDA were incubatedin darkness at 15, 20, 25, 30, 35, 40 and45oC. The pH of the medium was adjustedto from 2.0-9.0 with 1N HCl or 1NNaOH before autoclaving. After inocula-tion, cultures were incubated at 25oC inthe darkness.

2.7 Fruiting Bodies InductionPotato dextrose agar was chosen for

this test. The medium was autoclaved andtransferred to Petri dishes. After inoculationwith a 5 mm diam piece of mycelial diskcut from the growing edge of a 2 week-oldculture, each Petri dish was sealed withParafilm. The cultures were incubated at25oC in the dark. After ten days ofincubation, Parafilm was removed and thecultures were transferred to 20±2oC,80±10% RH and illuminated for 12 h withfluorescent lamps at about 1,500 lux.

2.8 Determination of Suitable SpawnProduction

Nine solid substrates such as, brownrice grain (Oryza sativa), corn grain (Zeamays), job’s tear grain (Coix lacryma-job),black kidney bean (Phasecolus vulgaris),mung bean (Phaseolus aureus), rye grain(Secale cereale), sorghum grain (Sorghumbicolor), soy bean (Glycine max) and wheatgrain (Triticum aestivum) were used in this

experiment. Each grain was prepared byboiling for 10-15 min. The grains wereplaced in 18×180 mm test tube toapproximately 10 cm depth and autoclavedat 121oC for 30 min. After cooling, eachtube was inoculated with a mycelial plugcut from the periphery of the growingcolony on PDA and incubated at 25oC fortwo weeks in darkness with five replicatesof each experiment. Linear growth of themycelium was measured and growth ratewas determined.

2.9 Cultivation for Fruiting Body Pro-duction

Sawdust of Albiza saman was used asmain substrate for cultivation. Substratewas mixed with 6% rice bran, 2% CaCO3,0.2% Na2SO4 and 0.2% MgSO4 on dryweight basis of substrate. The mixedsubstrate had reached a humidity of 70-75%with water and 300 g of mixed substratewas filled in plastic bag (12.5 cm wide and18.5 cm long). The bags were sealed usingcotton plugged polyvinyl chloride pipe ringand covered by piece of paper. The bagswere autoclaved at 121oC for 30 min andthe sterilized bags were cooled for 24 h.The bags were immediately inoculatedwith 5 g of master spawn (three week-oldof P. giganteus CMU54-1 on sorghumgrain) and bags were kept at roomtemperature (25±2oC). After 45 days, themycelia covered the substrate and the bagswere shift to 20±2oC, relative humidityabove 75% and illuminated for 12 h. Inaddition, three different test casings includinga control, scratched surface and addedsaturated soil with water were investigatedthe fruiting body production and the entireprocess was repeated twice with tenreplicates. The number of primordia andfruiting body per bag was recorded.

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2.10 Statistical AnalysisAll data were analyzed using SPSS

V16.0 for one-way analysis of variance(ANOVA) and Duncan’s multiple rangetest was used for significant differences(P<0.05) between treatments.

3. RESULTS AND DISCUSSION3.1 Identification

The morphological identificationindicated that the fungus isolate CMU54-1was Pleurotus giganteus (Berk.) Karunarathna& Hyde (sym. Lentinus giganteus Berk.and Panus giganteus (Berk.) Corner) [23]which a popular edible mushroom inSri Lanka [24]. Pileus was 40-118 mm broad,plano-convex becoming centrally depressedat maturity, grayish brown to dark brown,margin wavy to lobed, inrolled whenyoung, incurved at maturity, surfacesmooth and small flattened scales in age.Lamellae was deeply decurrent, anastomosingridges on the stipe, subdistant to crowded,3.5-5.0 mm broad, cream to white. Stipewas 162-175 mm long and 5-12 mm thick,central, roughly, tapering to narrowed

base, attachment laterally, solid, the samecolor as the pileus (Figure. 1A, 1B). Sporeprint white. Basidiospore 4.0-9.0×7.0-10.0μm, Q = 2.1, elongate to cylindrical,smooth, hyaline, inamyloid, thin-walled(Figure. 1C). Basidia 8.0-10.5×20.5-40.0 μm,clvate, bearing 4 spores (Figure. 1D).Sterigma 5.8-11.2 μm long, Pleurocysidiaabsent. Cheilocystidia 6.0-12.2×12.0-44.0μm, narrowly clavate, some with a taperingapex, 0.3-0.5 μm diam, hyaline, thin-walled(Figure. 1E). Pileipellis 4.0-6.0 μm diam,with hyphae arranged parallel to the pileusbut somewhat irregularly entangled andclamped. Hyphal was system dimitic.Hyphae of context 3.8-4.5 μm diam,branched, with clamped. Hyphae of stipe5.0-6.5 μm diam, scantily branched, withclamped. This study reported that thenew collection site of P. giganteus fromMedicinal Plant Garden, Doi Suthep-PuiNational Park, Thailand. However,Karunarathna et al [31] reported the threecollection site of this mushroom inThailand such as, Mushroom ResearchCentre (Ban Pha Deng, Mae Taeng District),

Figure 1. Pleurotus giganteus. A-B, Fruiting bodies at natural habitat, C, Basidiospore,D, Basidia and E, Cheilocystidia. Bar A-B = 5.0 cm; C-E = 10.0 μm.

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Sangasabhasri Lane to Huai Kok Ma village(Doi Suthep-Pui National Park) andhighway No.110 to Mae Sai, Doi Tung(Chaing Rai).

The ITS and LSU gene regions weredeposited in GenBank as JQ724360 andJQ724361, respectively. The maximum-parsimony (MP) analysis of the ITS gene of28 sequences resulted in 759 characters, ofwhich 316 characters were constant,78 variable characters were parsimonyuninformative, and 365 characters wereparsimony informative. Heuristic searchesresulted in a tree length=1,136 steps,CI=0.6391, RI=0.7892, RC=0.5044 andHI=0.3909. While, The MP analysis ofLSU data of 25 sequences results in 938characters of which 728 characters wereconstant, 48 variable characters wereparsimony uninformative, and 162characters were parsimony informative.Heuristic searches resulted in a tree

length=359 steps, CI=0.6825, RI=0.8331,RC=0.5685 and HI=0.3175. Thephylogenetic studied of ITS and LSU generegions of CMU54-1 were shown inFigure. 3 and 4, receptively. Both phylo-genetic analyses indicated that Inocybaceae,Pluteaceae and Pleurotaceae are the familylevel of order Agaricales with 100%bootstrap support. The analysis confirmedthat the ITS and LSU sequence of CMU54-1were placed in the family Pleurotaceae, orderAgaricales which related to the Pleurotusspecies. The result was supported thatP. giganteus was transferred from L. giganteusto Pleurotus which the new combinationmushroom [23].

3.2 Effect of Media on Mycelial GrowthMycelial growth of P. giganteus on ten

different growth media was presented inTable 1. After one week of incubation, thefungus grew well on malt extract agar,

Figure 2. Maximum-parsimonious trees inferred from a heuristic search of the internaltranscribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 sequencealignments of 28 isolates. Lentinus tigrinus and L. polychrous were used to root the tree. Brancheswith bootstrap values ≥ 50% are shown at each branch and bar represents 10 showedsubstitutions per nucleotide position.

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Figure 3. Maximum-parsimonious trees inferred from a heuristic search of the 28Sribosomal RNA gene alignments of 25 isolates. Lentinus tigrinus and L. squarroslus wereused to root the tree. Branches with bootstrap values ≥ 50% are shown at each branchand bar represents 10 showed substitutions per nucleotide position.

Figure 4. A-B, Fruiting bodies formation of Pleurotus giganteus in induction conditions,C-D, Cultivation of Pleurotus giganteus, C, Primodia formation, D, Fruiting bodiesformation. Bar A-B = 1.0 mm; C-D = 5.0 cm.

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potato dextrose agar and V8 medium. Thehighest mycelial growth rate (17.85±0.30mm/day) and biomass yield (82.65±3.63mg/plate) were observed on potatodextrose agar. The smallest of the mycelialgrowth rate and biomass yield werefound on oatmeal agar and V8 medium,respectively. These results agreed with theresult of Gibriel et al [14] and Rawte andDiwan [16], who reported that themaximum biomass yield of P. columbinus,P. florida and P. flabellatus were observedon potato dextrose liquid and solid media.However, malt extract agar was the bestmedia for mycelial growth of P. eryngii [18],P. ostreatus [25] and P. pulmonarius [26].Furthermore, Khandakar et al. [27] reportedthat corn extract agar was a suitablemedium for the mycelail growth ofP. citrinopileatus.

3.3 Effect of Temperature and pH onMycelial Growth

The mycelial growth and biomassproduction were responded on the differenttemperature treatment. P. giganteus grewat temperatures ranging from 15-35oC

(Table 2). The statistical analysis of the datashowed that 25oC was the best temperaturefor mycelial growth and biomass productionof this fungus. The results were accordingto the previous studies that the optimumtemperature for mycelial growth of oystermushrooms was range from 25-30oC[17,27]. After one week of incubation at40 and 45oC were stunted and failed toresume growth after additional one weekincubation at 25oC.

The effects of pH variations onP. giganteus mycelia growth are shown inTable 3. The fungus had the ability to growat pH 4.0-9.0. The pH 7.0 was the optimumpH value which produced the highestmycelial growth rate (18.00 mm/day) andbiomass yield (163.10±4.40 mg/plate). Thisresult showed different growth respond topH when compared with other Pleurotusspp. For examples, P. citrinopileatus waspreferred alkaline pH values which pH 8.0was the optimum pH value [27]. While, P.eous, P. eryngii, P. flabellatus and P. floridashowed the maximum mycelial growth inacid condition [16, 28].

Table 1. Growth of Pleurotus giganteus on different media.

The results are mean and standard deviation of four replicated. Data with differentletters with in the same column indicated the significant difference at P<0.05 accordingto Duncan’s multiple range test.

Growth Media Growth rate (mm/day) Dry weight (mg/plate)

Corn meal agarCzapek Dox agarGlucose yeast extract agarMalt extract agarOatmeal agarPotato dextrose agarSoybean meal agarV8 mediumWheat agarYeast extract peptoneglucose agar

14.15±0.44c12.30±0.42d13.80±0.37c17.60±0.49a11.35±0.82e17.85±0.30a12.30±0.77d17.75±0.50a13.65±0.90c16.70±0.53b

23.68±0.91f18.93±0.62g38.95±4.67e57.65±3.54c22.05±1.50fg82.65±3.63a22.40±2.01fg38.43±1.50e43.93±3.77d69.55±3.82b

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Table 2. Growth of Pleurotus giganteus on potato dextrose agar at different temperature.

The results are mean and standard deviation of four replicated. Data with different letterswith in the same column indicated the significant difference at P<0.05 according to Duncan’smultiple range test.

Table 3. Growth of Pleurotus giganteus on potato dextrose agar at different pH.

Temperature (oC) Growth rate (mm/day) Dry weight (mg/plate)1520253035

6.85±1.39c10.80±0.49b17.90±0.20a17.75±0.50a2.75±0.70d

20.63±5.00d42.13±5.18c116.15±9.55a89.20±7.15b14.30±1.02d

pH value Growth rate (mm/day) Dry weight (mg/plate)

456789

9.45±1.05d15.55±1.06c17.95±0.20a18.00±0.00a17.75±0.30ab16.90±0.53b

91.20±7.53e108.10±6.51cd112.90±5.92c163.10±4.40a141.90±2.69b103.88±4.89d

3.4 Fruiting Bodies InductionThe white primordia of P. giganteus

were found after two weeks of incubationin the low temperature, high humidityand illuminated conditions. The primordialwere developed to mature fruit-bodieswithin 10-15 days. The mature fruitingbodies were colleted and descriptions ofthese fruit-bodies were recorded. Pileuswas 14.0-17.2 mm broad and stipe was62.0-75.3 mm long and 5-8 mm thick. Theother macroscopic and microscopiccharacteristics were similar the naturalfruiting body (Figure 4A, 4B). The resultssimilar the previous studies that loweringthe temperature and light during incubationwas necessary to induce the pure cultureprimordia formation and mature fruitingbody development of Pleurotus spp.[29,30].

3.5 Determination of Suitable SpawnProduction

The mycelium growth rates of P.giganteus in the test tube culture on varioussubstrates were investigated and showed inTable 4. After two weeks of incubation,sorghum grain and corn grain had thethickest mycelial growth in comparisonwith other media by visually assess. Thefungus significantly grew faster on sorghumgrain (9.86±0.22 mm/day), followed bycorn grain (9.12±0.19 mm/day) and jobstear grain (9.28±0.29 mm/day). The resultsshowed that sorghum grain was a suitablesubstrate of spawn production of P.giganteus based on the low cost. This agreedwith Thulasi et al [28] who reported thatthat sorghum grain was the best substratefor spawn production of P. eous andP. florida. In addition, the previous studies

The results are mean and standard deviation of four replicated. Data with differentletters with in the same column indicated the significant difference at P<0.05 accordingto Duncan’s multiple range test.

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Table 4. Growth rate of Pleurotus giganteuson solid media after two weeks incubatedin test tube.

range of different natural resources andagricultural wastes such as, log, sawdust,straw dust and weed [14, 31]. Furthermore,previous reports indicated that the casingoverlay with soil could improve the oystermushroom production [32, 33].

4. CONCLUSIONSPleurotus giganteus could grow best on

PDA and sorghum grain which proved to besuitable medium for mycelail growth andsolid substrate for spawn production,respectively. The optimum temperatureand pH values for the mycelial growthwere 25oC and 7, respectively. Interestingly,the fungus was able to form the maturefruiting bodies in terms of mushroomproductivity which provided valuable forcultivation. Future studies will attempt theselection of suitable techniques for a largescale commercial production of thismushroom in Thailand.

ACKNOWLEDGEMENTThis work was supported by grants

from The Royal Golden Jubilee Ph.D.program (PHD/0309/2550), GraduatedSchool of Chiang Mai University and TheOffice of Higher Education Commission,Thailand under National Research University(A1) program.

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3.6 Cultivation for Fruiting Body Pro-duction

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