Mushroom Spawn Production and Infrastructure Requirements R.C. Upadhyay, $.K. Singh and R.P, Tewari National Research Centre for Mushroom VMOU eR Ae Page Ice arene) ‘hambaghat, Solan-173 213 (HP)Technical Bulletin Mushroom Spawn Production and Infrastructure Requirements R.C. Upadhyay, $.K. Singh and R.P. Tewari Ww ICAR National Research Centre for Mushroom (Indian Council of Agricultural Research) Chambaghat, Solan-173 213 (HP)ie: ogs, 210,50 Copies Publish by: Director ‘National Research Centre for Mushroom (Chang, S328) ND Phone: 01792-28051, 290167 ONRCM, 2008 Asis sve No po tiscali may eee any a ey ay easing pl coping edi a noi tr eta sem with pi psn in ing fo compe abi Desa & Paid: ‘Namal Vijay rien, 140/,Nnina Inara Ave, Phas, Now Dei 108 ‘TL; SHES, 26148, 25054, Moe: 05817 {nivoduetion ‘story of gpm production Raking of pr cues 4) Prpanton of cute medion 8) olton fom fi boy 3) Tiswecutue a) Spore cate 6) Subeuuing ‘Maintenance atd conservation of stock cultures Spam Preduton © Sabte 4 Substrate preparation 4 Mote maser saa prepara Comme pawn preparation * ‘Spawn sorage and transport Spawn production aa gnce ‘Recent advances in spawn production eshnalogy Precautions in spawn preparation UO Magen of emai UL, estan rong cos 12, Livof we sae bri ia 18, Seed eee 8INTRODUCTION {n nature, mushroom spores help in survival of a species from one generation to the next. These spores are extremely small microscopic propagules and therefore difficult to handle as seed. Spores need time and specific conditions to germinate and during this time competitor fungi might germinate and grow faster to exhaust the available substrate. Therefore, a pure culture of the desired mushroom mycelium is first raised on a convenient artificial culture medium and then added to the substrate to give it an advantage. The term spawn has been defined as the vegetative mycelium from a selected mushroom grown on a convenient medium (Klingman, 1950). The spawn comprises mycelium of the mushroom and a supporting medium, which provides nutrition to the fungus during its growth. Spawn is used as inoculum or “seed” for the substrate in mushroom cultivation. Right kind and quality of spawn is very important for mushroom cultivation. ‘The rapid rate of development of mushroom production technology from a primitive cave culture in France to a high-tech industry during the last three centuries is a success story which has kept pace with the ever increasing demand for this commodity and there is every reason to be optimistic about its further growth in the years to come (Rai and Verma, 1997). The world mushroom production has registered a 3-fold increase from two million tonnes in 1986 to ‘about six million tonnes in 1997. Five major mushroom species Agaricus bisporus, Pleurotus spp., Volvariella volvacea, Lentinula edodes and Auricularia spp. accounted for 82 per cent of the total world mushroom production (Chang, 1999), Not all mushrooms are edible, but some are highly poisonous, while edible fleshy fungi are called mushrooms, poisonous ones are termed ‘toadstools’. It has been estimated that out of 10,000 species of fleshy fungi (Kendrick, 1985) about half of them are edible (Chang, 1993) and as many as 100 species are highly poisonous. Therefore, utmost care should be taken in procuring pure cultures, Preferably, to start with, pure desired mushroom cultures should be procured from an authentic mushroom culture bank. Agaricus bisporus, popularly known as the white button mushroom, has the widest acceptability in India and its present production level is estimated between 50,000 to 60,000 tonnes per annum. The second most popular oyster mushroom species (dhingri) accounts for approximately 7000-8000 tonnes per year. ‘The isolation, purification and maintenance of mushroom cultures require technical expertise and aseptic high-tech laboratory facilities. Therefore, small mushroom growers can not maintain their pure culture and or spawn. They have to rely entirely on commercial spawn producers, reliable governmental and non-governmental organizations that play a vital role in supplying reliable spawn of a desired mushroom strain or variety. ‘This technical bulletin shall provide technical guidance and information to mushroom growers, entrepreneurs and young individuals those who are = >ie in taking up papi as sore of iacne, bss or tof scala ay, HISTORY OF SPAWN PRODUCTION Culpeper in 1652 deste pam as myelin of mushroom from 1652 {0 184 AD speva ws ater rom the wil ater than made Fey, ‘Wee the abet of gaia pv, een kinds of pa erent ‘iin svn (fom the asus and mead), ake pa eg of ‘beds hough whic ushroom myetiom has run), Mil wack sawn (ich ed and mae fm mite of re dung, cow dug and om it) and sunue svn (on sed hase mau compe mague) Han and Co, (UR) were the fist to podace pawn spas o vs fe from onan cca in 1885 and soda bicep it UK and capored to Ac, Gemy and USA, ‘Theft pure cafes was produced by Costantin in Fase (1894) om horse manu compos, In 198 Dagger reared pure cure from sushoom tu, Later on Myc cle was use nora ove manure in bots (1915, The proces of mang pa cn gain was induced bythe Pesan Ste University which tw putes of Ths ues were asignd oh ney bye incor Po LW Sinden in 982 Lites under the patent were aval o any aban guid make the grin sawn. The gain sawn was fuer pede by Stoller in 194. Since the proces forthe pation of pie sam, dames ‘vent chang, You sil aed rer cule, cere gi, the gain is srl, ced an the pot go ot Simple Ye ey "No his ‘ere hal anjone can mae svn jaa ano can pow mskcons, RAISING OF PURE CULTURES 2) Peat fata media ses infasracr, muta and equipment, ey weet ‘yl cue ing to prc guy so The eet ye ' nied ort coin ctu mda. vay of cule mada canbe sed to gon vitae mlm i not ost sds ‘o.grow tmstrooms. These cute media are als used as substrate for olation, ‘ultilicatiog, maintenance and pesenaton of masiroom cultures For the comico te patil ses 2 fo reins of ice: eda are fiva belo whch canes rang sans oe des Molt Exiract Agar (MEA) Milentat + OM Agaragar powder Ms al Wer ‘ite fl 0 ‘Wats i bold, ied wth malt extract andthe ingredients ave dsoed ag den as by tring aero pom. The pf he smeium i adjusted between 65 to 7.) before autoclaving, ‘Wheat Estas Ager (WEA) Wheat gas ok Agar power a Water ‘ie iH 0 Wea ais ar Del in ator 14 hous ad are ered ooh tbemui/ ces cot adc The volume of gin ext aso Hine with at and hen agape by iin conti and pH isadjuge, ato Deine Agu (PDA) Feand ded Pam; ty Deatase Mg Aga oder Me 1 10 tes ae pele we, cut it sol ps (om) and bed in ‘ate fr M25 mites, The oo enacts eed hgh hese eth and poo cays cade. The volume ofthis enact sae to ne ire. ‘Dente and agua poner isthe mil by ings pl 8 adie. Gre potatoes sold ob wed ashy conan aig aad wich ‘maybe aml o strom aye, Compa Exact Agar Paseurad and conditioned compost 130g es weight) Aageaga poder Ng ‘Wace tie # io ‘Pascurizd composts bole in 1-2 ive of wate fl the volume of "treed bl his eet hgh ceca ad te te oe Compo cnr mai. Agape ise siigandpaused Streptomycin sale @SOmg/ ire may be added afer autoclaving to eliminate bacterial contamination, (Out Meal Agar Oat mel fakes iy Agar powder my Wate lite # 10 (at meal aks are bie in sufcet water or? hous, The volume of he ‘operant aise lo one. Agragar poder is mined by sircing and pH is adj ‘Rice Bren Decotion Mei Rice ran My Agaragar powder Me Water Hine pH 10 ‘Rie rans bo in water for 1-20, ered trough cheese Chand then agar agar powders mined by sing, pH is ated If hep ofthe medium slow 5, N/, NGOH isa ro by opto raeitto 70 Wheres, ifthe His sboe 70 canbe brought down by adding Ni HCL The piso be aus 7 Oboe serizaton hasbeen observed tut the pH dens appromaty 05 upon acing Afr acing the Hlcoms down 6 ee, wich 'smast suitable fr masroom mecum o sow the cates are wo be raed in Pet plates then the medium sist serait il coasts 120ml hkevam tered culture medium is poured in ach presteriized Pa plat ft culture are oe mii in testubs then fe mediums poured in ge umber of ctu bis @ Sil/ tube, Te cute rubs or cial ass are then pla wit absent coton and autoled under moist seam a 18 youd pr sun inch (psi) pressure for 25.30 mings, A psu at 15 psi eliminates all mies, which mayer compet with eet mycelium of desired msc. Afr stazaton cate tubes replace in slating poston io pride mor surface ara fo te vegetative eel growth fa ushoom. The cute medium s allowed os in culture oes or in a pats or ev hours bef they anc use fer nocalaton or sbalung. The Pe pte ae steiize in an «vena 18PC or 2: hous and illowe 1 cxl down room empeatue efi they are sed pling media 1) Isolation from fruit body ‘We ned lal cue meu bth in ets and coe aes, scale, inclton nc, wie moth est tubes spit amp, sss, aces, match, 1 ethanol (ected spit, lamia ow, fest asics (ri bod nan noedation room Inouaon rom shoul te proed with a Aouble doe ety or ar uri atthe ene pint to cut cc and foe iyo iin he oom, Mos ofthe contamination ents though diet air rents entering ino inneltion rom, The laminar fow othe cea beach has mic ites tht help in inating microbes and ds prices pean the az, When the aminar fw is pot on, all dust pais and microbial contamination, mor than 4 ions in ize are end withthe filter and clan aiisbown ou inthe wortngarea. Uta viet (UV) igh shul be pt cn fr abot al an hore woking inthe anna ow Al atria excepe the living cules andro shul be exposed to UV dh co eizate contamination present 0 the sure of ature tubes, Pt plates, sso, fps. The ula vie igh shuld te sich bie wing ania fw because eos to UY High is harmful to al ving onanism ncuing tuman beings and can cause maton, skin diseases or ven cance. Separate foot ware and apron (co) soul be worm before entering into inaction room and these wars shoud nore sed outside pawn ah Before saing he iol, the surface area of ncn tbl sl be swab Withcton dipped in ecified pt. Aer aig of UV Highs hand shoul be waked With rectified spit (10% ethanol) and aiid boeing spit amp to avoid acids Pore cltures of fsb fungi/mushooms can be prepared ther by sve ‘alu matispre cats Tissue culture Incase of Ain sei, aban tage young frit body shuld be fshly hares and bought tothe bboratoy fr isu cule. The it hay shoud be ceaned and adered casing oil may be Fenoved withthe hlp of ntonswab, Te rat ay i ed wit the ep of cape inclaton nee and ip in. eres chore HCl) oT ethanol fo 3 seconds for a wile on spit Lp ame o Kl mcoes preset ons sure. The scalpel is surace serie by dipping iin 70 thao and then heating ito ame Afr cong Scale thefts spt longa and tise bits of appl 3mm ae cut fom cu 54, coe as Sie juncion cola region The is ar aseptcall)vng ae meintd tafe oto resteried Prats conning ue med eas alconvenient cukure medium withthe help of a steriied forceps. These Petr plasare raped within adic in BOD inabtrat21C+11C for 6 days The actly growing melon fom ede alongwith uae smdium of about Sum sz shoud be trate toa numer of est tubes (Fig 1), These est rubes are then ncubted ina BOD incubator a 25 41°C for 2 weeks Tsp clean thenbewse anol or pan reparation. Incas of Poss, te ise cols fm he junction files and tpn, Res ofthe proved issame In case of tropical mushrooms Ake ans and Calybspecis the Pe lats and te tubes comaining bis of tbody ae incubated at 3032 instead of 29 Spore etre ‘There exist diference in the sexuality pattem in culated mushrooms Agaric bus, Lata eds, Planta sp. and Aico sp, ae heterotic whereas Artisan Vr sondary and primary bonita, spt Single pore cultures may no evel su in fruiting in heterothalic spaces, Therefor, in most ofthe spawn labs, the pare myc clues reise! fom tise clre. Neel, mulispore culrres canbe raised fom any nshtooe species for spawa peeparation. It has its advantages, for asing a pre culture, mas of poe now spore pit 's required. The spore print can be obtained from rut body on toa steniized Petr plate or plain paper In cae of avin a healthy fut body whose vei is sil nats haves and land wth action svab to emove the casing sol Bel, Pets plas and eakers are seize nan oven at IC fr 24 ours ef takng a spre prt The rit bodys mounted ona coi and kept inn open Pett pate, covered wit beaker and enclosed ina Bel jar for 48 hours, Mion of prs shed inside Pr lor ona plain pape. This spar pit cia be toed at dy place ulus Incase of Fan, spore int can obtained b simply plc te mate vit body wil facing om pper si, na Per pate oron rized plain oper within 8 hows Pare cues from pecan be raed in two ays, Mai poe ate Muti spore cules abo se fo ing pre cure I red fom a mas of germinated basidiospores. From a por print, a aop fl of spore mass ispcked sng seized inoculation ned, suspended in 10m! ele did Water in 18150 mum st tbe, and mined thoroughly. One ml of hs suspension isin in inl tere ware. ain, ne lofi pe suspension i spread wth he blp of glass mon toa Pet plat conning 2m of ‘vee ure medium. These Pe platesare incubated in BOD incobtor 2125CH'C or one week. The fa going mul spore colons ar picked up vith pice of agar aga and rane esl repre comeriet cute ‘medium in tet tubes unde asec conditions, Tes test ubes are incubated for 23 wees in a BOD incubator, Muli sore cule is then ready for ‘noewation inthe wheat grain substie for spawn preparation, Single syrecule Insecosar homotalic mushroom pei like Agra hg, nly 65% ofits spre afte. In such cass, ele spores can beefed and canbe ‘sed forcvation purpose, wheres speci of Pts Lin urnaria anv cus eter and singh pres ae notre. Sige spon it suck cast are no wed for preparation of culture and sawn sey may not eventually rt intuit, Nevers, singe spores ae of immense Amporanceit developing new vars, hybrids and singe poe selections using pmoernbreding techniques. The proceso aig singe sore uur he sameasthar of multi spore cube. In single spore isolation, the spore suspension irr cuted to obtain a concentration of 20-90 spore oe spreading them in Pa plates. stead of picking up a growing ma spore cloy, nly arming single spore myeeivm is carefully marked, picked and transfered ‘to sterile test rubes. These test tubes are incubated for 23 weeks in a BOD incu. The singe pore cut colony wl ke vey slow rowing and canbe identi visually ©) Subuturing AS a result of isolation and junction, a pure vegetative mycetial «altar i established. These cultures ne maicaied and mii orbit sein inoculation of grain substrate for lng scale production of spawn and vse in ure fora vey of purposes Pare cate of Agia bps and ews jaa ae shown in ges ‘Land 3, especie, Sub-okring is Fig. 2 Pee car of ris hn lone by tranecing a sll pict of powingpure cular alog with eve medium ona stale medium, The ogostions of most comma wed mods hae aeady been described, In onder to maintain vigour of the cel he culture media may be changed in subsequent sub-cfuring. Tessa that els ae kept in Fig 3. Pe cate of Pas gon —~<@— —2 —=efigeator at °C for 23 months and again subculrred n fescue tubes or use in making spawn MAINTENANCE AND CONSERVATION OF STOCK CULTURES ‘Thereare varios metodo maintenance and conseration of mushroom, cole anda goo culture colecton cen adots more than on eid to ‘preserve them. The mushrooms might be of academic industrial, medicinal ‘or of hortcutural importance. When a new genus ara species is discovered and dese, it i genely deposed ina csi gemplasm bank, Tis ensures avaity of the oganism fr ws in fire In ton, mushroom sais having indutia importance are pateted and preserved although aay of sch sais became etic (Jong and Biringha, 191), Since there sno sastny method to check and vate the quality of span by api one spr xamination aretha of preserving ted strains teed and prove desis of primary moran I no depenerate changes teal during te preparation or manterancef mason cals andof sav heath preserva wold as ben eae simple oie proces Unfors tte Degestion of euro spava reesio ths of died ats adn to slow deveapen, port of suri and Jove of prod (Chang and Mis 198, Sladelnana, 1984) Spore of ketal or seandaryhomudlc pci are produced through asexual proces wil bn gent dienes (Pee, 195). Spores from primary homie spies weld be expected to be genetical sim Ofte ciated fungi, oly Flr basispors ae primary homathlic but il xb varatons (Chang a, 198). 1f sgl poe cute ae ruil, mating ts fr ete pcs woud be egied rote, aswellsts fo iting aii, hie. poe ane maintained for bomatalic spit tou be eesarytocek ey ofthe cults by rela futng tests, Consequently in practi, vepative mycelia of only Known ogi are stored (nl, 188) Aer pure mye caus re baited a wide arity of methods ae aval forthe conservation of mushroom cues suitable fra purer reed eg, preservation relatively shorter peri, ora lag pero. The choice of preston metnd depends won man fats bth vaily of recessry equipments and unis commny a deeruning act in sach decison. Frequent subculturing Under recommended temperature and pH conn, most masbroom eelium continues to grow unl hematin fa suitable cre medium are exhausted. Therefor, bese clues remain vale ol fo few mons ‘depending upon the growth rate, substrate and method of storage etc. Using a K§— =—————— sytem of pride anf a esol ital, ck ues ae fen ‘maintained ina actively growing sate unde optimum lborory condtons. “Aer bing simon mye growth,mtroom cuts te stored unl beng become neces. Fo storage pups, clues ae repre on agains in cle oes nd ott hs. Theseus este in teks at om tenprates fr ot ew wel. The prods beween sub- aug can beextended opto M6 nots by sage a Cin areata ar cold soo. In oratory, the ele mushroom sais are subcultured on ugh contre maa, Veils su and Cl inane naked at 303°C fo I and 1 ay, epetey, The cher mshrom sais ae incubated at 273 for 23 weeks tl th slans a aly over wth myetum, nce fll gown cafe of has as een oti, they arf be Kept at rm rempratue. Vous shoud be subled been 68 wes. Stas of Lens, Pees aA species can be Kein aera 2 4, and they shold be subculture very 34 months. ‘Deviation fom the orginal characteris ofthe cures can be detected with mew clues. The mas commen degenerate symptoms are 1s of sow growth, mycelium that tha and wea in apeurance, or mate or ff bot as aomal goth ate. A sow growing myeium nes more time for colonization and tends to cary vias parle, A fu meeium causes the ayainto sk together tis harder to spread in compos tan normal rai I tendo frm oma an it gieslower yl. Moet ofthese pes shold be discarded (Chang and Miles, 1989), Cultre abs of Votan sp. fas clamespors, hich ae brow in colour, Culture tues showing more >San Antoni 1978) eprted at clue vit and mushroom pdcion ‘er ao alee by yogic storage for 8 yeas. Eliot and Challe (1979) stored )2 cores of Aoi hp and elated species for 3 yeas in tbe Ghsshose Crops Rese so, Lileumpo UX and pri 8% secovery rate, Jodon af, (1982) ao reported tha gh cures of the comercial misuoom 4 amen (A Bon), wer preserved i guid ston fo 10 yeas ith ape chage in morplagil or pbyslogial barter: Chal a Eli (196 found that 1 guns gh soliton ‘sed as cryoprotectant was good in preserving the cultures of guia sp., pins, Leip, etsy, comma, Tena asp, and bar omic bt ot sate aoa whe Howeies thy found tata aqueous DMSO (dnt snide solo sare constant and ell rei of ar. Chen 198) pore hat cares ofall 122 sino pres of Bsidomyetsinduingimprtant edi mushrooms survive ater with sw cong (Cini) tan rapid ‘ein, Slow feng and rapid having poe gave the hight viiliy coun, Sigh 204) preserved musioom mycelium moped on wheat spins nde of myelin iui nitrogen and eed suv, yield and gent stabi of 1 ele mushrom stock cates afer sever yas. The ‘modified cyoprsenation reek ae expen demonaion of ec sabiltyof stock cultures, and validated the us of lgidairogen ‘ropestraton fr longer presenti of mshoom atures ‘New Granular structure medium A ew gram sructure cute media deloed by Kiang (19) an te wed asaneoconicsusinte forthe raional eral gin dium wed inspvn manufacture nds medium ft presto of mason tas ‘The ingens speed are sods oid stew powder (724), wheat your (21s), soybean ower (3), compen adits (2) and aces (05%) The medion cane prepared a serie in 500 mj The meal ‘iy an the eemomie ropes of mushroom stains canbe etn fo at east ve yas ifthe myc spree at 14 on ganar structure sedi, When the sans prepare fr cokvaion by spew met, xs inocaum acesoberemoved each ine fom te spec 0 mac Removed ingcaumis thm rnsfered and epucedin aw. actin incl istakenithasto be done ata temperate of ina see option, Ung tis mtd, a 50 ml aro he reserved sin can pie thera and ‘at, inoculum for long ime. Alternately fesh granular medium is replaced ‘nto the arand mye grows upwards fo hej btn oop wi ep 120 toca te aoa, When th meal oth reaches be suas, thers immediately read to 24'C, This yl can be conned fr many yeas. This method was climed tobe pracily superior and els in venation of mycelium othe pressed mustroom sri. (Cryopreservation in ‘mechanical freezer ‘Because viability of stored cls increases dramatically with lower tempat. ralo tempt mechani feces ar ee desi fy eading itn compas operat een t-14VCor-180 Cel maybe sored indent at sucenty low temperatures bw - 150 that is las wonton temperature of wate, Blow this temperature enayme act is completely suspended and thermally driven ection cannot ecu, The eure ae prepared in he same way a for guid itogen vation and placed fst at~20°C and then at 70°C and nally in freecers tmaaied tlw -130C. Tecate peserton by this matbo s 35 good as ink itogen. Ics tive 2 compared othe at of per ite drs! uid irs, as ing oeating expenses. Nevers ta dow temperature ezes are run on clei and tere arent wry sucess in developing countries where elec suply i erate and on the spot eas ae inacesble ‘The hice of neta depend o the reieme of he callcton, the epment and fait vase, ible | compas detent method of preservation with regard css of materi, tour, longeity and genetic Say, Is eeommened that each mushroom stin/soae shouldbe rained bya ast wo diferent methods, In genera, strage in iid tito an nina oi preservation cigs are es se forpsertion ‘ables Comparison of mushroom cote preservation methads Meta of prseration Cost angsty Genisoy ‘Nate Labour sowels Vale Sonpatmm = Law High Aenpertre Songein erigertor Medaum High 6 mos arte Sageondcro Low Low/meim 4Syean Moderate Sage inwaer Low Law/tmedm 2-3yeas Moder Storage in dep fezer Nevum Low/medium 45x Modeat (ie) Freezedrying of! igh —‘Initally hgh M0yeas—Good/medim coh il ir High «Lowder God ‘Ultadow mechanic High Low 0 8 frees) = Nota uae with mush cure tak When iow temperate eszers anew. Longviy & gn sali ye tobe sper o ober etd of preservation,of edible mushrooms, The handing techigus, fezng protools, yo servation and thang ates canbe ptinzd fora para sain abn ‘maximum survival, Onc te strom a ben ses fenn ad ore inkiquiitogen, beste perin ageusobeindfnte becuse no ceil and orpyscal changes can ocur at sch low temperatures (Grout and Monts, 1987), Mashroo resis ‘The maintenance and producto ofthe real pre cutue save wth desable qualities ike oration andthe ist etic sage athe ses of amuse cto, Maso cule rept banks pay vit role isopply of pure and authentic cule to mest of the mushroom spann roduing unt. One can oan pore clues or making pawn fom any of ‘he National or Interaional postr, Some of he are sed ow: Nain! i) National Research Cente fr Masioom (CAR), Chamght Solin (HP). i) Divsonof Mycology and Pat Patoogy IAI, isa, New Dei 1002, i) Indian Ist of Hoictre Reach (ICAR), Banglore, Karta, i) insite of Miri Technology TMTECHH, Ser 3D, Chanda ¥) Department of Micbidloyy Puja Agscultual Univesity, Labia, Puja, vi) Deparment of Pt Ptolog, Mata Rana Pratap Rajan Agra Univesity, Up, Raat. Jwersational i) American Type Colee Colleton (ATCC), Rockvile, Marland, USA. i) Intnl Meoogcltnsie, Kv, Sure, UK ii) Nation Regonal Reseach Laboratory (NRRL), USDA, Peto, ini, USA, i) Femenatoa Reach Insite (FD) apn 4) Canadian Collin of Fungus Culture (CCFC), Canad, 1) Coleg of Apical Sines, Panna Stare Unie, USA. vi) Dutch Mustroom Experimental Station. the Neth. vi) Deuche Samming von Mikoranismen Brunch, Germany ——o—__— SPAWN PRODUCTION: Substrate oe gts Mstoom pan canbe prepared onan kindof cereal gis ke wh, amine ba (ar ile) jar soghun), ore. The age rains arya gear reserve of food material er grain ssi thence of mshroon amesiu ut iti esablsbd and frdng onthe compat, they may be snr eft noo conpot or adverse conditons, Wheres esa gains pride mae pois of naa per gram of sun sia the gins of ot types ow equal wel te small ons wil penta the compos sooner ‘Mashcoom — Cultivation Spawn substrate Grain colonization species method period Agoriis spp. Trays, plastic Cereal grains 2025 days ‘ags/shetf system Avia sp, Wood logs! Sawis/gnin 1820 days sys bags |i sp Wontop/ Sonate 2025s synths bags gin/woed sticks | Pas spp. Synthetic bags Cereal grins 10415 days Volar spp. Outdoor! ‘Used tea leaes/ 10-12 days indoor in cages pip kanes! paddy straw + sa dete gninseton waste Other substrates Although cereal rains are suitable for making spewn of any mushroom “ate bt due to poi cos. arity of agricul wastes econ cobs, wooden ick ice stay, sant and ede ees thea een se. A varity of subtrates for diferent mushrooms hae bee suggsted, ‘Sawadust of red wood tree species is preferred for making spawn, Wheat tran ranging from 10 to 20% mined wth saat to increase moisture ena Used a ees washed and ed bef osing then for spa ‘pip eves ae mixed with saw dustin the ratio 31 and wed asSubstrate preparation Spawn ubstate shoud ne following stale harass should nt consi any ike compounds desinle mushroom species + Largesurface area of substrate shoul be vale fr fungal colonization + Itshould provide ese autres required by mashroom mycelium to row, + Ceca rains should be fe fom das. * Coral gins shoul ot eon, oor damaged by ines, Spawn-preparation asing cereal grains as substrate is described in deals The cre rein are thoroughly washed in suffice: water 34 ims o remove soil debris, straw particles and uncesbl ses and grass washed grains are then soaked in safc water for 2040 minus {0 se energy. These gins are taken in a wie mouth comainr and boiled in suficient water for 20-25 minutes Fig 1), Normal, {or soaking and bolng 20 ig of wheat grain, 35 ies of wars requted, After boiling, grains should aba 38-40% moigure Excess water from the boiled gins istemovedby spreading on sive made of fie wie mesh or rmslin cloth, The grains ate allowed a eae such fo afew Fig. igo wht ais fous ota the wate on the surface is evaporated (Fig. 8). Now the gains are nixed with Gypsum (calcium sulfate, C280) and hall power (Cau carbonate CaCO) so that the pH of the grains is around 710, they do ‘not form lumps (Fig 9), Diltet pope hire gen diferent rats for mixing ‘Bypsum and calcium Fig. 8. Deng wae ed pias carbonate. The best results have been chee by using 20g sypumand Sgcalcum carbonate for 10 kg grains, These calcu laonsareon dy weight bass of grin wed for sparen Fst gypsum and calcium catbonate are mixed separately and then the mixture of both is thorougily mixed with gains, Ths mining shoud bedone on asmoah ufc er wearing esto aod contamination, ip 9. Nino CSO, and 2CO, wt od when gins Mother aster spawn preparation About 30 g prepared sbsate fied cereal gins coated with gypum and calcium carbonate) ile in glucose /mik bots upto 23 volume and pluged with no-bsrbent coon, These bates are then autocad at 2 b pac preset 126°C for 2 hours Fig. 10). Tes aula tes are in ‘ig 10 Horizontal atc fr sein of sum suhat, the oom for 24 hous so that hey are cooled o ambient temperature and the ces moisture accumulated inside the botle wal is evaporated. These autoclaved bottles containing sterilized rains are then exposed to UW ight inside heinocltion chamber rbot 3 mites ice of growing mcm is —S EEEaseptically transfered 10 these tls and inoue bots are netted at 25 Inoculated bortes are geily shaked on 5! and 10 day after inoculation, Fully colonized mother spawa totes can be used for inoculating. commercial spawn bags after twot three wees Inoculated bites are incubated at 2.2510 for Agari, Pleas, Letinala ad udari species bt at HOI2C for species of Volrarielle and. Calecye Uninocuated and ready master spawn in bottles is shown vide Figur 11 Commercial spawn preparation Commercial spawn can be prepared in heat es: stant polypropylene bags. Normally for bal and one kg spawn, the bags shouldbe of 88x17 Sem and ‘hem size, respectively, Plpropylene tags shoud have double sing at the bottom and afer filling the grains they are plugged withthe help of a polypropylene neck and nonabsoreat xn, The polypropylene bags of 150 gauge shoul be wed, The gs artes seized at 2b pi. pesue for LS to 2 ows in an autoclave. Autoclave bags are shaked well before inoculation co thatthe water droplets accumulated inside the baa ar ebsohe by th gyn. Thesterfized bags ar kept inh laminar Now under UV igo about 30 minutes. Tent fieen prams of grains rom master span bones inoculated pet bag under aseptic conditions (Fig 12). One botle of maser spawn is cafe for iocltng 25 o 30 commercial sawn bags of half ig capacity Inoculated gs are aguas ota te inca swe mie wi oer agains. However, ede theme period fr saws preparation, the quantity of inocu maybe increased. Then th bags ae kept in nebation oom or in BOD neuatr for myeetiumspread a 28°C Fig. 1Sand 4), During incabation the bags ore ely examined for mould infevaton,comtaniatedhas should be immediately removed and atocled before discarding the bags to avoid build-up of contamination inthe vicinity, Normally it takes 20-25 das for complete spread of mec on the gains for Apri specs. Ready 0 use commercal spawn in polypropylene bags is shown in Figure 15, Gi Fig, Unnorulate and dy mse gun ints Fig 3, Poe cuts, maser pawn and coermercal spawn in icubation rom at 25°C aig. 15 Ready tw commenti spuvn in pps Fig Ih pure cates and poe in BOD incr ‘SPAWN STORAGE AND TRANSPORT IF possible eslypepre pawn hou ewe while the mycelium sin thestato active roth. These bugs afer completion of log rot phase ‘an be mine upto 34 montis tC Th commercial pawn should be «jt packed in vetted Bags or cardboard bores. The spa seuld be cad in reget vans to lage dstnes. erative it can also be anspor to lng tances during ight by pbb transport or pate vehi sothtthe emperatareof planter pando noe rsebeyend 30.32. The spawn of Void and Cola should not be stoned treated temperatures as thee mshroomsare esi low temperatures OM spa bas Aira may ao ooze brownish Kuda cooler tespraturstat ay tes in crop loses SPAWN PRODUCTION AT A GLANCE Preparation of Mother / Master Spawa Step 1 Healthy and cleaned cereal grains 4 Siep2: il gain in ater 225min) 4 Step: Remove acess water tough seve 1 Sip Dry grins in shade (44 bs) 4 Sep: Mix CaCO, (0.5) and CaSO, (2h) Siep6 —FUL30g grains in each sacs bo Sep; Pig coton and auolae at 22 psi for2 hy Shep: Inoculte mycelium of desired stain wing lamina ow Siep% _Inoulate in BOD at 25°C for 2025 days (Tropical mushrooms a2 C) Sept Mother/ Mater spn sey Preparation of Commercial spawn Step: Use polypropylene gs instead of bates for grin ling Siep2) Upto atovig Step To 7 are same as of master pave Step: Inoclte about 10 gm fom moter span to polpropyleas ay conning rains t Sep: Shake bags afer 7-8 days t Sep Incubate at 25% in incbation room or BOD (Tropical aushiooms a 0.32 C) t Stepll: Commercial spawn s ready in 2025 days. —@- ane >RECENT ADVANCES IN SPAWN PRODUCTION ‘TECHNOLOGY Séors of improvement have ren repel in varios ses inl in sqm pructon chology. ater commercial su was prepared in ik argue bots, wich soe ficult carom ore place oot Heatiesstartplpoplne gs hare elton thee indsy. igh tech spawn labs now use polypropylene microfilm windows for aeration, Honevespolropleetanscat otesof 10 capacity ae ako used in Euope and USA for spawn producion, but it has not ben inroced in India due to high es ofthe mater Shite (Ltn eds) cuted continuously in liquid medium and has been used as liquid spawn. Shtake mushroom was culated n steeds substae and th technique i sil patie in deloping counts, Norma fru odes were hares fom the colin substrate bck afer 120 dys nuttin with soli paw The incubation tne was ect 90 ay withthe ue of lig spam, Ligd cure can be 2 wef frre of his mshrom PRECAUTIONS IN SPAWN PREPARATION Abou mun of rations ave ben ug in evant hate, sill he mzjoe precautions ae sted below; Si ypine shoul be manned tronghout ight to ang of pre ‘ale osrage and argo of spam, Exes of vito to inoeulaton cm shoud be dni Jno oon sol be dine eu by exit formalin or by insling noe enero imate chances of cotamiation. Dutngincutton of span, tes ands huldbe ingested equny in emove contaminate bigs, ‘To avoid spread of contamination in the vicinity, the contaminated bags ‘must be autoclaved and buried in the si Before spawning note subst tested sp under reigned conditions should be brought to ambient termperatures. Tei mecessary foreach rowing eee to star witha fesh spawa, ‘Regular floor cleaning with surface disinfectant lke dete, lysol ec, should be dove vice a ec. Cts shouldbe wrapped with almium ito prevent conintion of cates during storage unde eigate contons 10. Cokuresand spawn shoud nverbe sored a sub ero temperate in deep fears Howeve cultures can epee a ura low temperatures sng cry poets flowing proper proaolsalcady described separate, —___@—_____ (el 1, Over along of eure medium beyond the prescribed presse and time stoald be ave ait may cus caramalation of sup in he sium MANAGEMENT OF CONTAMINATION ‘Te mos cmon conaminaisecoureed during pawn prepartion are species of Aegis, Pow, Trckerna, Capo, Chara, Alrnara, Mucor, Ricopws, Farin and Deckew (Mazumdar and Rathaiah, ‘00 Oi, 1991, Thape ct 1956). Grains are the main our of eontaminatin ‘The fungal conanination can be usualy emgnized with he typi clos of thet mycelium, pores or conti. Armes a dstinctive one canbe opined 15 lesion between inocled mushroom mycelium and contamination. f the ‘onaminars ar allowed to grow, thy may spi age numberof spawn tgs If such comainated bags are not timely removed and disinfected it may trcome a perenial source of contamination Section of gu quality grains, oper auclavingand strict gene in spawn ab can eee contamination foageatertent. th problem continu, cirbendazin: or thiophanate-methyl (@005g/4g of bed grains can el in redoing te loses caused y furgal contamina Bacterial contamination is moe del to detect. Some bacteria give a greasy appearance and emit a pungent or foul order sme Ifthe bacteria contamination no detected in mast spawn bos al he comme pave (x compost prepared fm them wl became uses and may sina os of spn. ocr. Wet pe dea cnsed by Bali spcies ia common bacterial contamination problem in mushroom spawn (Ablawat «al, 1999), ‘The dieae canbe managed by maininng pH of spawn substrate nea 6.0 ‘and by incubating it between 20.25°C, If the problem continues, antibacterial compounds like neomycin, stepomycin or sepoccin @ 1080 yp/gam of Spawn be added fe bing of wheat rains to manage he dese, INFRASTRUCTURE AND RUNNING COST Proposed Inslaton Capacity : 000 kg/annum 20 tonnes/annam) [Now a dys theresa ot of awareness about mushroom culation in India ‘nd pple are coming forward to ak wp commercial culation bth as self ‘employment venture and subsidiary source of ncome. The quality of mushroom ‘sedorspavn det afesthe mshoom production ach mushroom species sis pice, Oly fu pecs ame Arn gs, ams,‘ale 2: Sunn requirement for growing feat musroom species in nia (200s 205) ‘Mushoom Species Ansual Spawn at pe tone Annual spawn Prodaction of prepared substrate/ requirement (Tones) compost (kg) (Toenes) | _Agaricas bisporus. 50000, 9 im Pearotas 999. ‘A00) 5 1000, Caboeyte inca 5000 vv 0 ‘Volvariefla vafsaces 1000 B 135 Toad 2 i ‘Theft anual mushroom production in nda by the end of 206 as teen mated around 00 toanes ad fo meting ut ths demand abot 5200 onnes of mushroom spa wl eee, In Ii, pan is poduced and supplied by Research Isiues and Agricukare Universes and private spawn laboratories Mos of th export one unis he hei own sawn roducton flit. The cs of te spn isthe majreemamic fat for cathaton of ecaymusrooms ie lwetsand Clay ind. hse is prepare by the producer hse he he pot willbe more and one is assured of the quay mshroons Selection of site and Layout of a pawn production unit Uisalvays desl the sawn unisbood be ated fom comps yard and growing rooms. sepurate block for pawn unit canbe conc above the ofc room, The lan eit fir 366 bags 50 gm saw pet smo ae as flins: Siore rom ‘Storage room is equited for rae of wheat grins, cheials,P bags PP necks, cotton bundles et, There should be minimum 45 shelves for smarimum pce ulin, The oom shold be 444m sz. Boing ond ing roms Tre room oul be 4m sett canbe ued fo washng, boing nd flag rns wi sti vate supply and damage Bing cal oes shld be placed nea the wae dainage One orto fins and evs sould al be provided so tha gris coud be died oui. A plato made of marble or cement shuld be om the opps se ofthe Boing cate. The plato is wd fr ming chemicals with ras, ing and png bags The parm shoul be 1.10 m($)x09(6) 4). ——————— ee i Astolaving wom ‘Te basalteiling are rough for serizaton, Avdlv of 780 min X 58mm sie shouldbe aed or serlzton, The bags faking should teept or 6 hours so hat water droplets onthe PP bags are absorbed before inoculation, There should be small window berveen autoclaving oom and ‘nocalation chamber. Sterized bags are to be transported ealy through this window ciel tothe inoculation room, The autoclaving room shoud be SxdxAminsze, nocuaton room ‘The sterlizd bags before inoculation are to be kept inthe amiar flow ina double door com ote between atolving and nebation oo. The bags ‘before inoculation should be kept for minimum 15 to 30 mines under UV ight befor inoculation. A small BOD (6 cf) and eigen is so reuired fon storing master spun and culture toes Te inoeaation rom should be 4x3x3x4min size Tneubation room “nubation room shouldbe environmentally contol for ncbation of ‘movulted bags at desired temperature, Iron racks with 5 tiers at a distance of ‘Mem between wo ter, 37.5 cm wie and 15m long could aeommodate 72 tas ba ig spam in each er On.asngl rack about 350 to 40 bags could ‘ekipt An incubation om of 4x3x 4 msize could accommodate 15 racks of ‘the above size. The size ad number of incubation room canbe increased as pet the requirement of the paw oratory. Cold 00m ‘Thee wom of 3134 suse fo string uly colonized pan bug, ‘Croom shouldbe Fl insulted andthe temperature sou be itn between Ato 6. The col oom shoul have one door fr aking prepared ‘Spam Out of the cold rom. One ai curtain shoul be fted awe the door Coridors _ Alive Corridor of 6x24 m should ea te main emry of the spew ‘ui. An ar curtain at che main etry shoud als be provided jocaupments are roped or commercial sawn produce: (6 quintal capacity 6 Nos)‘Astalaes (10m depth $50 mm di) one laminae fow -6sne- One OD incubator (909090 on one Refit (210 ie capacity) - One Racks for keeping g(x 15° wih te = 15 No. Toles or tansporting bags oe No Ethos fins Two 10, Areata thee Nos. Minor insramerts Spi lamps 2 Nos) notion eles {Nos bigs sieves moni on oa rae (x4), (6 Ns) and sting ol 2 No) Consumables Consumables should alvays be purchased in bulk andthe tll quaity rei front math hold kp in tests, Consumables rue are heat gain rected spit polypropylene bps (1x11, FP nels Apron seen, cos bts perpaescolue tub aluminon foi goes and non absorbent coon et Marpoer regret ‘Two sons are equi for preparing 140 bags of $0 g each sing 80h wheat grin dl Economics of spawn production Unit (20 tonnes pr annum) ‘an nd ung (Ta rea 90.) Relity Unit Sie (om) Tol area, m) Coiorcom oe Ome a bh ‘Storage oom One aa 6 Ruilng and fling om One KE 6 Attain om One HKG 8 Tnoaton oom Ore NG 2 Inctuion oom Ore NEG 6 Colt om One SG 9 ~~ * ‘Cost of building, equipment, consumables and manpower A, Non-Recurring expenditures 4) Lan ingot ee Area) Co, mt. Rs) Tok Co (| (Coa of and and ‘x9 Rs. 2000 180000 delopst Cotofminatanot 2 8.4500 4c fic cum coor ‘Cost of storage rocen m R450 noo) Coitafeiing mom NL RAs) co Conafancnerom 313 RAS stn Coif sued mu 5000 x00 intro con Cos of old om x3 Ha 5000 5000 ‘including insolation Toul 5.0.58 b) Eeuipments ‘Machinery Equipe Nocof Unit Price Tolle waits Rs) (Bs) ung nd sokig prs “ 0 2,00 (lr cpuciy) Disi/Kemsene operated S000 5. tune LPG contin ‘Sie (Mie on a) 400 ‘wonder fame 1 |) (rz a 15000 sho ida 0m At, 152s Small autoclaes o 5.000 00 0t2Sites cnn) ita Ait Dow O eat 0 60 60am sie) ‘near sto) st ee)Reiger ene a (20 ies cai) nae: a Ext fine @ ts Arcata ® so Sol Cn | Abeta set o 5000 5.000 Minor egies Cn Refigeation in cld rom, Oh 2800 asym Toul 5950 2B Recurring Ependues 4. Consumables: Accomm firing 20 ss year par (40,0 tos X50 we veto pode nna $40 asf Sarto ‘be inoculated to avoid 10% contamination alo, ten Roof UsirPace Thal ant ais 9 9 ‘Whe! in 10g. RGSS, 310 Non born cation 0G Relig. 09 PP bag 150 age Ce a) ties fr 0g saw) PP els AONONeS —RS6S/IO0Nos 8040 | Gps We ReSMig m0 (G2 yy en Calcium carbonate O15 Rs Tet 35 GaOSKe/at dy wheat) Meta it White Rs, 2c “ Die keneteorLPG ——STUliesor RF om) Opsedl eciy cages "00 si BA) sto 0 {ois ssa) —— —_, Anche p00 procution Rs SON per moni =e | ean abe Rs 20/ pron (2 No =n) ai. Total 129000 Irs ond depreciation [ont Con a, & Dee Shines 00 3 Onbuiting | ‘Shepton 34830 es | Shite In On Mutiny 1 dein 538100 950) 19h ines 2, Manpower Tol cos of Production Rov mils 2786 iy ed gs zw ee deprection M80 Says 1, Mtonnes spawn year 2 Sep = ny 4 lem io te l= 19900 of pout fr ky spa = B20LIST OF SOME SPAWN LABORATORIES IN INDIA A) Anda Pradesh 4) Departmen of Horticulture, Plc Gardens, Hydra 50 00 ii), Department of Horticukare, Visakhapatnam, AP 3B) Armachal Pradesh |) Directorate of Horticulture, Gor. of Arunachal Pradesb, tranagar, Arunachal Pradesh ©) Assam ') Assistant Horticulruns., Deparment of Hortulure Got of Assam, Gealpara, Assam D) Bihar i) Deparment of Mirobilogy, Musuoom Cente, RAU, Pos. -B8 15, Bitar i) Mastuoom Spawn Cee DRDA Dua, Bia )Chhatisarh i) Mushroom Project, Deparment of Plant Pathology, Indira Gandhi Krishi Vishwa Vidyalaya, Rar -92 012, Chatigar, F) Delhi i} Division of Plant Patol, IARI, Post «110012, New Det. ii) National Horticulture Research and Development Foundation, Bageani Bhavan, 47 Parka Road, nsituonal Area, Janakpur, New Debi- 110 058. G) Haryana i) Mosioom Spm Laboratory, Deparment of Plant Patio, ee (GCS Haryana Apriclural Univesity, His, Haryana fi) Haryana Ago Indus Corporation, ‘Marth! Ro, Sonepat, Haran. 1) Himachal Pradesh 4) Director, ‘National Research Centre for Mushroom, ‘Chamboghat, Solan 173.213, HP fi) Department of Past Pathology, De YS Pamar Uniesy of Horticulture and Fores, avn, Solan (HP 173290. Jemma and Kashi 4) Spawn Podocin Labora, ‘Tolab Tilo, Jammu, 18K, 4) Spawn Prducton Labwaty, Govt of Ja and Kash, ‘alma, Srinagar, Kashi, J&K 2 ton _ i ue and Agnes Research : Pada, Ranch £34010, , ) Karnataka §) Mastroom Spawa Laboratory, Jndan Inet of HoiclalRescarc, ‘Hessarghatta Lake Post, Banglore, Ai) Dept. of Horticulture, Lah, Bogle, L) Kena 8) Depart of Pat Patho (AICP Collegeof Apicutue, Kerala Aysctual Univesity, Va Tiron faa 6550 8) Dino, re Ril Gs Rea i, ‘Thirwananttapwar, Kerala (8552,AN) Meghalaya 3) Divison of Plant Poly, ICAR Research Complex for NEH Region, ‘Uo’ Roa, Barapa, Mealy - 798 10, 0) Mizoan i) Dinero of Hortcutue, ‘zw, Mizoram, P) Nagaland ’) Spawa Laboratory, Directorate of Horticulture, Kohima, Nagaland, 0) Orisa §} Spam Production Laboratory, Osea Ag Indust Corporation, Route, Oris ii) Spawn Production Unit, Site Horécltue Departmen, Bhutaneshwar A) Posjab i) Deporte of Mirobinog (AICMIP), Punjab Ags Univesity, Ludhiana (Punjab), 8) Rajasthan ') Departmen of Plant Pathology (AICMIP), Maharana Pratap Rajatan Agra! Unvesty ‘Udaipur -313 001 Rajasthan, 1) Sikkim |) Joint Secretar, Science and Technology, Govt. of Sikkim, Gangtok, Sikkim, U) Shillong i) Asisant Horicutuis, Mushroom Development Centre, Sillong- 793. ¥) Tamil Nada 1) Department of Plat Pathology, ‘Tam Nad April Unies, Comore 64 ON, Tai Nadu W) Tripura Misivoo Laboratory, -—Gammagar, Agel, Tripura 1: 1X) Uiraatal |) Mashoon Rec Cet, ‘Deparment of Plant Pathology, GE Pant Unive of Agi. Ad Technology, Panga -263 HS, Urantl Y) Uttar Pradesh 4) Depunmet of Pam Puhlogy (AICMID, ND Unies of Agate and Techaoogy, ‘Kumarganj, Gavia - 224 229 (UP). i) done Dieter, Industal Tring Cee, Sabana, UP aa toe! 1) Departmen of Plant Pathology, ‘Noth ry Canis, _" Pundibari, Coochbchar, WB. of Plan Pathology, an , SELECTED REFERENCES OP, Rai, RD. and Verma, RN. 1999, Bacterial contaminants in “ti the mshom, ics hrs (Lang Sing Msioom Ru, 111976. Storage of ngs cultures in water, Tinto of Si 61S‘Challen, M.P. and Ello, J. 1986. Polypropylene straw ampoules forthe storage of mirooranisms in Liquid nitrogen. Journal of Miata Mads 5:11 (Chang, 7,198. Mustroom bly he inpact on mushroom proaction and mushroom products. In: Chang, $.7, Buswell, LA, and Chice, 5, (eds) “Masham Boy an Motos Fad. Hong Kong, Chinese Univesity Pres, p10, Chang, $7 1999. World production of cultivate edible and medicinal mushrooms with emphasis on Lenin dads (Berk) Sing. in Chia, ‘emia Jena of Madina Maho, 129130, (Chang $7. and Miles, RG, 1989. ile msmoms andor enon, CBS Delhi, India, Chang, ST Miles, PG. and Wal, C.C, 1981, A study of monosporous ioltes of Yoav vues Mucho See Hart 2) 63 (Chen, YY. 1987 The preservation of basiiomyetes culture dy uta tow femeraturefeevng dc My Sra 62; 10 10, Culpeper, N. 1652. Compe Hel, Fousam 1952 epi, London 11, Dog, BM. 198. 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Manual of Mushroom Cultivation, Toot Foundation, Amterdam, 77, Petersen, LH, 1995, There's oreo mushroom than meets the eve, Mating Studies inthe Apaicales. Mpeg $7 (I) 1-12, 2% Rai RD.and Verma, RN 1997, Vision-2000- Peps Plan, NCMRT, Solan, NCMRT pg, 2 San Antonin. 1979, Sablty of spawn socks of the calivated ‘mushrooms stored for nin yeas in iguidnizogen 16°C) to (196°C), Mason Science (Part 1 103, 4H Sate, AN. and Dighe 8.1987, ‘A simple and economic method fr long. {erm peseraion of muskroom cultures, Ci See 40). 485, SI. Sinden, 10,195, Gain spawn, is nae advantages and we. 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Snell, J.J.S. 1984. General introduction to maintenance methods. In maintenance of Microorganisms. (Eds, Krisop, B.E. and Snell, J.J.S.), Academic Press, London. Stadelmann, R. 1986. Preservation and degeneration of mushroom strains, Mushroom Journal 158: 41 Stroller, B.B. 1962. Some practical aspects of mushroom spawn making, Mushroom Science, 5: 170-184, Thapa, C.D. Kumar, $. and Seth, P.K. 1976. Competitors of spawn and their control. Indian Journal of Mushrooms 2): 58. ‘Tan, C.S., Van, I.C.W.,Stalpers, L.A., Van, I.C.W. and Griensven, L.J.D. 1991. Freeze drying of fungal hyphae and stability of the product. Genetics and breeding of Agaricus. Proceeding of the first International Seminar on Mushroom Sciences, Mushroom Experimental Station, Horst, the Netherlands. Xiang, Y. 1991. A new granular structure medium for spawn manufacture and the preservation of strains. Jn Science and Cultivation of Edible Fungi. (ed. Maher) Balkema, Rotterdam,