flammulina velutipes the culinary medicinal winter mushroom

Flammulina velutipes,The Culinary Medicinal

Winter Mushroom

V.P. SharmaSatish KumarR.P. Tewari

Directorate of Mushroom Research(Indian Council of Agricultural Research)

Chambaghat, Solan-173 213 (HP)

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Printed: 2009, 500 Copies

Published by:DirectorDirectorate of Mushroom Research (ICAR)Chambaghat, Solan – 173 213 (HP), INDIAPhone: 01792-230451; Fax: 01792-231207E-mail: [email protected]; [email protected]: www. nrcmushroom.org

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CONTENTS

Page No.

Foreward v

1. Introduction 1

2. Morphological and molecular characterization 2

3. History of cultivation 4

4. World production 4

5. Nutritional value 6

6. Medicinal attributes and their properties 7

7. Physiological requirements 10

8. Genetics and breeding 11

9. Preservation of cultures 14

10. Cultivation in India 15

11. Cultivation in other countries 21

12. Effect of environmental factors on the production 30

13. Post harvest management 32

14. Insect-pests and diseases 34

15. Spent mushroom substrate management 37

16. References 37

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FOREWORD

The land resources in the world for raising food crops are limited andthere is no possibility for its further increase. This warrants a wise use ofland with due regards to its sustainability for long term productivity.Keeping in view this limitation mushroom cultivation is a wonderful activitywhich fits very well in the present scenario and also alleviates environmentalpollution besides producing a highly valuable protein rich product. Indiais blessed with varied agro climate, abundance of agricultural waste andmanpower making it the most suitable for the cultivation of all types ofmushrooms. Our Centre is giving greater emphasis on the diversification inmushroom cultivation. Farmers are being motivated to cultivate these newmushrooms for better returns. Diversification will provide opportunity tothe seasonal mushroom growers for round the year cultivation and utilizationof different cultivation substrates. I appreciate the efforts and labour put inby the authors in compiling and editing the Bulletin for its use by themushroom growers and researchers.

Manjit SinghDirectorDirectorate of Mushroom Research,Chambaghat, Solan- 173 213 (H.P.)

Flammulina velutipes, The Culinary Medicinal Winter Mushroom

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Introduction

Varied agroclimatic conditionsand availability of agricultural andindustrial wastes in India offer greatopportunities for cultivatingdifferent mushrooms on commercialscale. Introduction of newmushrooms is important to meet outthe increasing public appetite for newand different foods. Total worldproduction of different mushroomsespecially Lentinula, Pleurotus,Auricularia and Flammulina has goneup many folds in the last few years.Occasionally the glut of a particularcommodity during bumperproduction period also fetches poormarket price to the growers whichhappened in the early 1990 and madeseveral growers search for alternativecrops to supplement their income.Different mushrooms while addingvariety, flavour and eye appeal to ourfood, are also nutritious because oftheir proteins, fiber, vitamin andmineral content. A wide variation inthe chemical composition of aparticular mushroom has beenobserved by various workers whichmay be due to the environmentalconditions, stage of sampling andmethod of analysis. Majority of the

Indian population being vegetarian,consumption of mushrooms wouldcertainly augument their diet whichis deficient in proteins and minerals.Mushroom consumption can thusprove a boon to the growing childrenas well as breast feeding mothers.Some mushrooms have specialsignificance for medicinal purposes.Specialty mushrooms have beentraditionally used in China and Japanfor medicinal and tonic purposessince time immemorial. Mushroomconsumption has proved beneficialfor the patients suffering fromhypertension, high sugar and heartproblems. Beneficial effects ofshiitake consumption are known asenhanced vigour, energy, potencyand diminished ageing. Therefore,the cultivation and consumption ofmushrooms can contribute towardssound health.

Among new mushrooms,Flammulina velutipes is importantmushroom and ranks fifth in term ofglobal mushroom production.Species of Flammulina have beenreported to occur naturally onvarious deciduous tree speciesnamely, poplar (Populus spp),willows (Salix spp), elms (Ulmus spp)

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plum (Prunus spp), maple (Acer spp)and birch (Betula spp) as a parasiteand later as a saprophyte growing onthe trunks or stumps of these broad-leaved trees from the end of autumnto early spring. Infection occurs onlyon wounded or weakened trees.Fruit bodies appear after a fewmonths at temperature between -2 to14oC, under low intensity winterlight (Poppe, 1974, Zadrazil 1999).Under in vitro conditions fruit bodieswere produced by culturing onpotato dextrose agar in darkness at30o C for 7 days and subsequenttransfer to 5oC (Chandra andPurkayastha, 1972). Flammulinavelutipes was cultivated as early as800AD in China and today it is beingcultivated all over the worldespecially in China, Siberia, AsiaMinor, Europe, Africa, NorthAmerica, Australia, Taiwan andJapan. This mushroom is particularlyknown for its taste and preventive aswell as curative properties for liverdiseases and gastroenteric ulcers. Inaddition, winter mushroom has alsobeen reported to containimmunodomodulatory, antitumorand antibiotic substances

Common names: Enokitake, Enoki,golden needle mushroom, wintermushrooms, velvet foot, or velvetstem.

Taxonomic Position:

Kingdom: Fungi; Division:Basidiomycota; Class: Homobasidio-mycetes; Order: Agaricales; Family:Tricholomataceae; Genus: Flammulina;Species: callistosporioides, elastica,fennae, ferrugineolutea, mediterranea,mexicana, ononidis, populicola, rossica,similes, stratosa, velutipes.

Morphological and Molecularcharacterization

Flammulina velutipes is long thinwhite mushroom used in the Cuisineof Japan and China. The mushroomnaturally grows on the stumps of theChinese hackberry tree, called enokiin Japanese, but also on some othertrees like mulberry and persimmon.There is a significant difference inappearance between the wild and thecultivated mushrooms. Cultivatedmushrooms are not exposed to lightresulting in a white color, whereaswild mushrooms usually have adark brown color. The cultivatedmushrooms are also grown toproduce long thin stems, whereaswild mushrooms produce a muchshorter and thicker stem. Its fruitbodies are small but delicious. Thepileus is 2-3 cm in diameter andhemispheric or convex in early stages


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of its development, graduallyopening to a plane as it grows. Thesurface of the pileus is viscid whenmoist and is yellowish brown or darkbrown, usually with a light brownmargin. The flesh is almost white.The gills are white or light-creamcolored, and they are adnexed ordecurrent to the stipe. The stipe isstiff, 2-9 cm long and 2-8 mm indiameter. The lower part of the stipeis dark brown, while the upper partis light brown and sometimes almostwhite. The spores are white, with aflat surface, cylinderically oval, and5-7 X 3-4 µm in size.

Neustroeva (1984) studied themorphology of 30 strains of Collybia(Flammulina) velutipes collected from11 tree host species. The strains fellinto 2 distinct groups: the 1st group,consisting of isolates collected fromlinden (Tilia cordata) and elder(Sambucus nigra]) was characterizedby more rapid growth. Three strainsselected from each group producedfruit bodies within 23-25 days (group1) and 40-50 days (group 2) afterinoculation on a solid substrate.

Molecular characterization (Konget al.,1997), revealed that variousisolates of F. velutipes collectedworldwide have two differently

sized IGR (intergenic region) of 1.61and 1.58 kb. Based on restrictionanalysis of the IGR and ITS (internaltranscribed spacer) rDNA using 6different 4-bp-recognizing enzymes,22 isolates were classified into 4groups with 92% similarity. Inrandom amplification polymorphicDNA (RAPD) analysis with 20primers, 68 collected isolates wereclassified into 7 genotypic classeswith 87% similarity. Interestingly, 30isolates producing white fruit bodieswere classified into one group.Nucleotide sequence andpolymerase chain reaction (PCR)-restriction fragment lengthpolymorphism (RFLP) analysis of theribosomal RNA gene (rDNA) regionscontaining the internal transcribedspacers (ITSs) and the 5.8S rRNAcoding sequence was used todifferentiate 7 typical Flammulinastrains (MH09201, MH09208,MH09210, MH09234, MH09286,MH09289 and MH09297). Thesenucleotide sequences revealed thepresence of strain-specific deletions,insertions and substitutions(Palapala et al.,2002). RFLP patternsproduced using restrictionendonucleases Dra I, Fok I, Hae II, MboII, and Nla IV, enabled identificationof specific Flammulina strains. PCR-RFLP analysis of the ITS regions

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appears to be a useful tool for theidentification of Flammulina strains.Digestion of F. velutipes collectionswith two restriction enzymes, Bgl Iand Bst UI, gave variable restrictionfragment patterns and revealedbiogeographically identifiablepatterns. Three haplotypes of F.velutipes were identified withgeographical distributions chiefly inAsia, Europe and North America.(Methven et al., 2000). A cDNA librarywas constructed using mRNA from7-day (7d)-old cultures and alsoscreened for genes that may beinvolved in fruiting bodydifferentiation. One cDNA clone,FDS (F. velutipes differentiationspecific), was isolated by differentialscreening (Azuma et al., 1996).

History of cultivation

It is reported that (Yang,1986:Wang, 1995), F. velutipes firstcultivated in China during the 8th

century. In 1928, it was cultivated onsawdust and rice bran in Japan(Nakamura, 1981). This mushroomhas been cultivated in Japan for over300 years, initially on wood, and laterin the bottles During the 1960s, itscultivation revolutionized in Japan,which became its largest producer inthe world and enjoyed this position

till the 1980s. Since the early 90’s,China has occupied the first place inits production. It was estimated thatin the Mainland China its productionwas about 200,000 tons during 1995(Meiging, 1997) which rose to 600,000tons in 2006 (Chang and Bushwell,2008). Production data from othercountries too indicated a fastergrowth rate in terms of its totalproduction. In the United States theproduction of Flammulina increasedat an estimated rate of 25% or moreduring 1991 - 1995 (Royse, 1995).Production of Flammulina is based onsynthetic substrate contained inpolypropylene bottles or bags. Thesubstrates most utilised areagricultural residues, such ascorncobs, cottonseed husk,sugarcane bagasse, etc., besidessawdust (Chang, 1989; Yang, 1986;Fan et al., 1990; Wang, 1995; Royse,1995).

World Production

Diversification of the mushroomindustry, in terms of number andquantity of species cultivated, hasaccelerated worldwide during the1980s and 1990s. Twenty years ago,70% of the world’s mushroom supplywas Agaricus bisporus, but by the mid-1990s, this had fallen to only 37%.


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(Royse, 1997). Total mushroomproduction worldwide has increasedmore than 36-fold in the last 37 years,from about 350,000 metric tons in 1965to about 6,160,800 metric tons in 1997and 12,250,000 in 2002 (Table 1). Thebulk of this increase has occurredduring the last 15 years. Aconsiderable shift has occurred in thecomposite of genera that constitutethe mushroom supply. During the1979 production year, the buttonmushroom, Agaricus bisporus,accounted for over 70 per cent of theworld’s supply. By 1997, only 32 percent of world production was A.bisporus. The People’s Republic of

China is the major producer of ediblemushrooms, producing about 14million tons (Table 2) in 2006 (Changand Bushwell, 2008).

Flammulina ranks at fifth place inthe category of edible mushroomsfor production and consumption.During 1990, its production wasestimated to be approximately143,000 tons, which increased to230,000 tons in 1994, showing aremarkable jump of 61% (Chang1996) which further increased to285,000t in 1997 (Chang 1999). Japanis the main producer of wintermushroom (Furukawa 1987). In 1986,

able 1. World production of cultivated edible mushrooms in 1986 and 1997.

Fresh weight (x 1,000 t) Increase

Species 1986 1997 (%)

Agaricus bisporus 1,227 (56.2%) 1,956 (31.8%) 59.4

Lentinula edodes 314 (14.4%) 1,564 (25.4%) 398.1

Pleurotus spp. 169 (7.7%) 876 (14.2%) 418.3

Auricularia spp. 119 (5.5%) 485 (7.9%) 307.6

Volvariella volvacea 178 (8.2%) 181 (3.0%) 1.7

Flammulina velutipes 100 (4.6%) 285 (4.6%) 130.0

Tremella fuciformis 40 (1.8%) 130 (2.1%) 225.0

Hypsizygus marmoreus — — 74 (1.2%) —

Pholiota nameko 25 (1.1%) 56 (0.9%) 124.0

Grifola frondosa — — 33 (0.5%) —

Others 10 (0.5%) 518 (8.4%) 5,080.0

Total 2,182 (100.0%) 6,158 (100.0%) 182.2

Source: Chang (1999)

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Japan produced 74,387tonnes; by1991, production rose to 95,123 tonesand, by 1997, Japan produced 174,100tones -an increase of about 45% in sixyears. From these data, it is evidentthat other countries are enjoying afaster growth rate, in terms of totalproduction. In the United States, alsowinter mushroom production hasincreased at an estimated rate of 25%or more per year for the last fouryears.

Lee and Park (1994) investigatedthe profitability of bottle mushroomcultivation in the Korea Republic andsuggested that for a production levelof 2000 bottles per day, it requires atotal expenditure of worth 5 millionand obtains an annual net income of

worth 110 million whereas, for aproduction level of 1000 bottles perday, Pleurotus ostreatus requires a totalexpenditure of worth 30 million andprovides an annual net income ofworth 300 million.

Nutritional value

Flammulina velutipes is a popularedible mushroom in Japan where atender cultivated form of the wildversion is called “enokitake.” It iscommonly used in Japanese cookingand increasingly can be found insalads in gourmet restaurants. F.velutipes contains 31.2 per centprotein, 5.8 per cent fat, 3.3 per centfiber and 7.6 per cent ash on the basisdry material.

Table 2. World production of cultivated edible mushrooms in 1978 to 2002.

Year Fresh weight (x 1,000 t)

1978 10,60

1983 14,51

1986 21,76

1990 37,63

1994 49,09

1997 61,68

2002 122,50

2006 (China alone) 140,00

Source: Chang (2002), Chang and Bushwell (2008)


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Medicinal attributes and theirproperties

Flammulina velutipes, a deliciousmushroom is rich in peroxidase,superoxide dismutase, and othersand can prevent some severediseases like cancer and coronaryheart disease. It is beneficial forphysical and intelligence of children.Winter mushroom containscompounds that prevent as well ascure liver disease and gastroentericulcers provided it is taken on aregular basis (Ying et al., 1987;Yoshioka et al., 1973). In addition,like many other specialtymushrooms, winter mushroomcontains immunodomodulatory,antitumor, tumor inhibiting, and anti-biotic substances. Both mycelium andfruitbody of F. velutipes could berecommended for formulatingantioxidative dietary supplements.

Winter mushroom containsseveral types of amino acidsincluding valine, which inhibits thegrowth of Ehrlich ascities tumourand sarcoma 180 in mice. It is alsoreported to increase body height andweight (Ying et al., 1987). Wintermushroom contains a cardiotoxicprotein (flammutoxin) that isrendered harmless when subjected

to heat (1000C) for 20 minutes. Thedose is 8-9 g/day.

Purification of lysine andflammutoxin revealed that it is asingle polypeptide chain(Bernheimer and Oppenheim,1987).It contains large amounts oftryptophan, serine and glycine, andfew or none of the sulphur-containingamino acids. The toxin appears notto be a phospholipase and it was notinhibitable by any of a variety oflipids. Flammutoxin caused efflux ofpotassium ions from humanerythrocytes and swelling of the cellsbefore haemolysis. Flammutoxin didnot lyse human erythrocytes in thepresence of non-electrolytes withhydrodynamic diameters of >5.0 nm,although it caused leakage ofpotassium ions and swelling of thecells under the same conditions(Tomita et al., 1998). Flammutoxin,assemble into a pore-forming annularoligomer with outer and innerdiameters of 10 and 5 nm on thetarget cells. The electrophysiologicalproperties of flammutoxin channelsusing planar lipid bilayer techniquewere studied, and found that thelarger-conductance channel offlammutoxin corresponds to thehaemolytic pore complex, while thesmaller-conductance channel may

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reflect the intermediate state(s) of theassembling toxin(Gulnora et al.,2000).

Cao et al. (1989) reported thatPA3DE shown to have a MW of 5.4 x106 which contained D-glucose, D-mannose and L-fucose in the molarratio 22.31:1.46:1.00, and had β -glycosidic linkage. It showedinhibitory activity against theSarcoma 180 tumor in mice.

A new fungal immunomo-dulatory protein (FIP-fve), havingmolecular mass of FIP-fvedetermined by SDS-PAGE agreedwell with the value of 12 704 Dacalculated from its amino acidcomposition and sequence wasisolated and purified from the ediblegolden needle mushroom. FIP-fveconsisted of 114 amino acidsresidues with an acetylated aminoend and lacked methionine, half-cystine and histidine residues. FIP-fve was able to haemagglutinatehuman red blood cells. Theimmunomodulatory activity of FIP-fve was demonstrated by itsstimulatory activity toward humanperipheral blood lymphocytes andits suppression of systemicanaphylaxis reactions and local

swelling of mouse footpads. FIP-fvewas found to enhance thetranscriptional expression ofinterleukin-2 and interferon- gamma(Ko-Jiunn, 1995).

Tomita et al., (2004) cloned andexpressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that ofFlammutoxin (FTX), a 31-kDa pore-forming cytolysin from F. velutipes.Ng and Wang (2004) investigated aprotein designated as flammin andexhibiting a molecular mass of 30kDa, and another protein designatedas velin and possessing a molecularmass of 19 kDa, from the fruitingbodies of F. velutipes. Flammin andvelin inhibited the translation in arabbit reticulocyte lysate system withan IC50 of 1.4 and 2.5 nM,respectively. Flammulin, anantitumour protein, was purifiedfrom the aqueous extract ofbasidiomes of Flammulina velutipes toelectrophoretic homogeneity, itsmolecular weight was 24 kDa,determined by SDS polyacrylamidegel electrophoresis (Zhou et al.,2003).Zhou et al. (2003) extracted mRNAof F. velutipes using Quick PrepmRNA Purification Kit forflammulin.


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Flammulina velutipes showedinhibitory effects on HP urease ( Kim,1996). A new protein that decreasestransepithelial electrical resistance(TEER) in the human intestinal Caco-2 cell monolayer was found in awater-soluble fraction of themushroom F. velutipes. This protein,termed TEER-decreasing protein(TDP), is not cytotoxic and does notinduce cell detachment, but rapidlyincreases the tight junctionalpermeability for water-solublemarker substances such as LuciferYellow CH (Mr 457) through theparacellular pathway (Watanabe etal.,1999). The anti-complementaryactivity (immunostimulatingactivity) of endo-polymer was foundto be 77% in F.velutipes (Song etal.,1998). Paaventhan et al. (2003)reported that Fve, a major fruitingbody protein from F. velutipes, amushroom possessing immunomo-dulatory activity, stimulateslymphocyte mitogenesis, suppressessystemic anaphylaxis reactions andoedema, enhances transcription of IL-2, IFN- gamma and TNF- alpha, andhemaggluti-nates red blood cells. Itappears to be a lectin with specificityfor complex cell-surfacecarbohydrates. Fve is a non-covalently linked homodimer

containing no Cys, His or Metresidues.

Zhang (2005) observed that somepolysaccharides from F. velutipesinhibited tobacco mosaic virus andcucumber mosaic virus infectingChenopodium amaranticolor. Anantiviral protein (Zb) was isolatedand purified from F. velutipes bydifferent chromatographic methods.SDS-PAGE showed that the proteinhas a molecular weight of ~30 ku (Fuet al.,2003). Flammulina velutipessignificantly inhibited mycelialgrowth of the four phytopathogenicfungi (Cochliobolus sativus, Fusariumculmorum, Gaeumannomyces graminisvar. tritici, and Rhizoctonia cerealis),responsible for foot and root diseasesof winter cereals (Badalyan et al.,2002).

Ishikawa et al. (2001) isolated twonew cuparene-type sesquiterpenes,enokipodins C (1) and D (2), fromculture medium of F. velutipes, alongwith enokipodins A (3) and B (4). Allthe metabolites showedantimicrobial activity against afungus, Cladosporium herbarum, andGram-positive bacteria, Bacillussubtilis and Staphylococcus aureus.Wang and TziBun (2001) isolated a

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single-chained ribosome inactivatingprotein (RIP) with a molecular weightof 13.8 kDa from the fruiting bodiesof the F. velutipes. The protein wasnovel in that it possessed a molecularweight lower than those ofpreviously reported RIPs and that itwas capable of inhibiting humanimmunodeficiency virus (HIV-1)reverse transcriptase â-glucosidaseand â-glucuronidase.

Mitchell et al. (2007) demonstratedthe anti-complementary activity forthe exopolysaccharide of F. velutipes.Two polysaccharides were isolatedfrom the basidiomycete F. velutipes,via successive hot extraction withwater, 2% and 25% aq. KOH, and thensubmitted to freeze-drying (Smiderleet al., 2006). A hemagglutinin with amolecular mass of 12 kDa wasisolated from the fruiting bodies ofthe mushroom Flammulina velutipes.Its molecular mass was similar to thatof the fungal immunomodulatoryprotein isolated from F. velutipes (FIP-fve) with ice-cold 5% acetic acid and50 mM 2-mercaptoethanol asextraction medium and to that of thelarger 12 kDa subunit of F. velutipeslectin isolated with phosphate bufferas extraction medium (Ng et al.,2006).Transgenic lettuce lines containingthe decarboxylase gene (oxdc)

isolated from a Flammulina sp. wereproduced by Agrobacterium-mediatedtransformation (Dias et al.,2006)

Physiological requirements

In nature, the main substrates formycelium growth are woodycompounds, which are cellulose,lignin and monosaccharides. F.velutipes grows and fruits onrelatively simple natural media suchas malt agar and potato-dextroseagar as well as on synthetic media.Ammonium compounds and aminoacids are suitable nitrogen sourcesfor the mycelial growth and fruitbody formation. Inorganic nutrientssuch as magnesium and phosphateare effective for the growth ofmycelium and for initiation of fruitbody formation. The phosphate ionsare reported to be indispensable forfruiting. The effect of various traceelements such as Fe, Zn, Mn, Cu, Co,Mo and Ca are also recognized.Among vitamins, thiamine has beenreported to effect the mycelial growthand fruit body formation of F.velutipes.

Furlan et al. (1997) reported thatmycelial growth rates were higher onWDA (wheat dextrose agar) mediumthan on PDA (potato dextrose agar)


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or MPA (malt soya peptone agar)media .Best growth of F. velutipes wasobserved in a medium containing 4%glucose as the C source, 0.2%peptone as the N source, 0.15%KH2PO4 and 0.05% MgSO4, and a pHof 6.0. The highest yield of myceliawas obtained when Zajiao 13 wascultured on this medium at 25oC for8 days (Su et al., 2001).

F. velutipes grew in the wastewater from the starch industry assubmerged culture and growthfurther improved with modificationsto the nutrient composition of themedium (Zhu et al., 1997).Boyle(1998) reported that lignindegradation is important for growthand observed that N-availabilitylimits growth rates of F. velutipes onwood while availability of simplecarbohydrates, micronutrients orvitamins does not. Wang (2000)recorded that F. velutipes grew wellin medium with soyabean powder,peptone, beef cream and yeastpowder as N sources but could notgrow well with nitrate or aminenitrogen as the N source. Yatohgo etal. (1988) purified a lectin which wasdimeric acidic protein consisting of2 identical subunits with an apparentmolecular mass of 11 kD, with a pHvalue of 5.4, and devoid of cysteine,

methionine, and histidine as aminoacid constituents.

Genetics and breeding

Morphological analysis, matingstudies and analysis of ribosomal ITSsequences were used to determinethat a number of new species werehidden within the epithet F. velutipesincluding F. populicola (growing onPopulus roots), F. rossica and F. elastica)For true F. velutipes, data suggest thatthe center of origin was Asia,spreading to Pacific North Americavia the Bering land bridge and toEurope and Eastern North America,possibly via the North Atlantic landbridge. Hybridization was suspectedin this group based on morphologyand the ability of some species tointerbreed in vitro, and wasconfirmed by finding a hybrid ITSsequence in F. velutipes.

The life cycle of F.velutipes is littledifferent from other basidiomycetessince monokaryotic fruit bodies areformed and dedikaryotization isdisplayed. It can produce bothmonokaryotic as well as dikaryoticfruit bodies, however , haploid fruitbodies are smaller than dikaryoticfruit bodies with poorly developedpilei and few basidiospores.

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F.velutipes is tetrapolar and has fourmating types. Dikaryotization occurswhere both A and B alleles differ. Allcombinations of monosporousisolates are dikaryotized amongstrains having differentincompatibility factors. Dikaryoticmycelia possessing differentphysiological and morphologicalcharcterstics from their parentaldikaryons can be obtained bycrossing many monosporous isolates.From these dikaryotic myceliasuitable strains for commercialcultivation can be obtained.

Tissue Culture

Zheng et al. (2001) inferred thatthere was significant genetic stabilityin culture characteristics between theparental strain and isolated strains,and among the isolated strains fromtissues of different parts of fruitbody. Contrary to this Zhang-Gonget al. (2001) reported that he tissuesfrom middle stipe of F. velutipes fruitbodies cultured on three media (I.potato extract 20% (w/w), glucose2%, MgSO4 0.015% and KH2PO4

0.02%, II. potato extract 10%,mushroom stipe extract 10%, sugar2% and agar 2%, and III.maltose 2%,peptone 1%, yeast juice 1% and agar2%) media had hyphal growth

potential) or hyphal growth velocityvalues higher than those fromjuncture of pileus and stipe, whereasthe tissues from basal stipe on IIImedia didn’t grow. The tissues fromany part of F. velutipes on medium IIIhad hyphal growth velocity orhyphal growth potential valueshigher than those on I and II. Magaeet al. (2005) developed a simplecolorimetric method to detectdegenerated strains by using a liquidmedium supplemented withbromothymol blue and lactose. Theability of a strain to develop normalmushrooms could be determined bythe colour of the medium.

Protoplast fussion

Yamada et al. (1983) usedprotoplast fussion to develop newstrains and observed that themycelium age (2 to 24 day-oldcultures) greatly influencedprotoplast isolation. The highestyields of protoplasts (6.7 x 10.7 cells/ml) were obtained from 2 to 3 day-old mycelial cultures. Protoplastregeneration was most rapid on OS(onion juice + soy sauce) medium.Zhao and Chang (1993) discussedthe applications of proplast fussionin comparison with othermonokaryotization methods for the


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genetic improvement and observedthat protoplasting is rapid, simpleand effective.

Breeding through Oidia

Yu et al.(1998) determined theoccurrence, germination, nuclearbehaviour and polarities of sexualityof oidia from different isolates of F.velutipes. It was observed that bothmonokaryons and dikaryons couldgive rise to oidia. Most oidia werecylindrical or oval in shape, and onlya few were round or Y-shaped. Oidiagerminated easily, and the germ tubewas usually wider than the oidia.Oidia produced by monokaryonswere all uninucleate and had thesame sexuality as the parentalmycelium. Oidia from dikaryonswere also uninucleate. Binucleate ormultinucleate oidia were notobserved. A portion of the oidiaproduced by dikaryons had the samemating type as one of the parentalmycelia, while the other portion hadthe same mating type as the otherparent. Zhang et al.(1990) usedoidiospores for making crossesbetween varieties. No significantdifferences in hyphal growth rate,fruiting capacity, yield and qualitywere found between crossesinvolving oidiospores and those

involving basidiospores. The use ofoidia was simpler and more rapidsince they could be obtained moreeasily than basidiospores.

Markers

Isoenzyme markers of hybridsfrom 4 crosses between differentstrains of F. velutipes were analysedby PAGE (Hu et al.,1997) . All thehybrids resulting from these crosseshad zymograms different from thoseof their parents. The 2 biochemicalmarkers EST (esterase) and PRO(soluble protein) showedcomplementary effects that resultedin the presence of new bands inhybrids. Hybrid heterosis couldtherefore, be predicted at an earlystage using zymograms combinedwith the number of markedheterozygous loci.

Yun (1996) reported that brownand white strains respond similarlyto the various pretreatments appliedalternating temperature (the besttreatments were 5-10 and 5-15o C),ultraviolet radiation for 30-50s,electric current for 5s, thiaminehydrochloride (0.05-0.1%), urea andIAA. The brown strain had a largerpileus and longer stipes than theyellow strain, while the yellow strain

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produced more biomass and agreater number of fruit bodies. Xie etal. (2004) identified the allele for thewhite colour through, a pair ofsequence-characterized amplifiedregion (SCAR) primers. The SCARprimers amplified the uniquefragment for all strains that carry theallele for the white colour of thefruiting body of F. velutipes. Theproducts of polymerase chainreaction analysis of the SCAR primercould be detected directly under anultraviolet lamp; electrophoresis wasnot always necessary. Zhu et al. (2007)evaluated the germplasm resourcesof F. velutipes using sequence-relatedamplified polymorphism (SRAP) tostudy the genetic difference betweenyellow and white strains. Thirtytested strains were classified into 14categories. SRAP analysis wasefficient, stable and repeatable, andcould be used in the construction ofthe genetic linkage map,identification of strains andmolecular marker-assisted breeding.Esterase isozymes from F. velutipesshowed many bands and variationsamong the different stocks on the gel.The stocks of F. velutipes in Japan wereclassified into three groups (A, B, andC) according to the cluster analysisof esterase isozymes. Group C was

characterized by a larger spore size,slower spawn running, and a palerpileus color than groups A and B.Furthermore, group B showed asmaller spore size, slower spawnrunning, and paler pileus color thangroup A (Nishizawa et al., 2003).

Radiation

Jin et al. (2000) developed a highyielding strain using 60 Co- gammaray treatments indicating thatprotoplast radiation induction is aneffective method for F. velutipesbreeding.

Preservation of cultures

Kitamoto et al. (2002)demonstrated that the sawdust-freezing method using acryoprotectant is a reliable and easypreservation method for fungusstock cultures of F. velutipes. Fivedisks of F. velutipes kept in a vialcontaining 1 of 3 cryoprotectants:10% glycerol, 5% DMSO or 10%polyethylene glycol and stored for 7years at -20oC, -85 oC or liquidnitrogen temperature revealed thatfour mycelial disks preserved at -20oC showed higher yields than thosepreserved at the other temperatures(Ohmasa et al.,1996). Among the


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cultures derived from strain FMC224,the control cultures preserved bysubculture showed the lowest yield.

When F. velutipes was cultured onordinary SCM, xylanase and CMC,amylase activity peaked on day 6, 4and 3, respectively. When F. velutipeswas cultured on carbon-source-changed SCM, amylase activitypeaked twice, on day 3 and 5, andxylanase and CMC activity peakedon day 5. Protein and RS contentschanged over time on both mediaand were related to enzyme activities.Protein and RS contents and the totalactivity of the 3 enzymes were higheron ordinary SCM than on the carbon-source-changed SCM (Li et al., 2002).

Spawn preparation

Saw dust spawn: The sawdust ofEucalyptus sp. (80%) and rice bran(20%) are used for the spawnpreparation. The mixture is adjustedat the moisture of 60% (Yang, 1986)and then filled in the glass jar of 500ml capacity. After autoclaving at121oC for 1 h, the spawn medium isinoculated with bits (one disc of onecm diameter) of mycelia of straingrowing vigorously in PDA slantsand then incubated at 24oC in dark.The spawn in the jars becomes ready

for inoculation to the substrate after20 days growth when the mixtureturned totally white.

Grain spawn: The boiled wheatgrains mixed with CaSO4 and CaCO3

are filled into half litre glucose/milkglass bottles upto 3/4th of thecapacity. Bottles are plugged withnon-absorbent cotton and aresterilized at 22 lbs p.s.i. (1260C) for1.5-2 hours. Sterilized bottles aretaken out from the autoclave whilestill hot and are shaken to avoidclumping of the grains. These bottlesare allowed to cool down overnightto room temperature. Polypropylenebags (12.5x27.5 cm ) accommodatingabout 250-260 g of spawn in each bagcan also be used for the sake ofconvenience in transportation. About40-50 bags are inoculated from onemaster culture bottle. The bags afterinoculation are incubated at 25±1°C.In two weeks after inoculation,spawn bags become ready for use.

Cultivation in India

Sawdust and rice bran / wheatbran are commonly used assubstrates for cultivation. Saw dustof broad tree leaves is required forthe cultivation of this mushroom.Sawdust media have oxygen and

V.P. Sharma et al.

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water which is necessary for mycelialgrowth. Rice bran/wheat is used asa supplement which provides manyof the essential nutrients. Much of thelignin, cellulose, and monosacc-harides are provided by the sawdust.Saw is wetted thoroughly with waterfor 16-18 hrs. Generally, equalquantity of saw dust is poured inequal quantity of water in 100ltscapacity tubs. After wetting 5 per centwheat bran is added in the saw dustand mixed thoroughly.

Containers: This mushroom canbe grown in a variety of containers

like Polypropylene bags, plasticbottle, vinyl bag, filter bag andjars (Fig.1).

Filling: Polypropylene bags (2000 g)are generally used for the cultivation.Two kg substrate (soaked) is filledin each bag. The bags are pluggedwith non absorbent cotton byinserting a ring in the mouth of thebag.

Sterlization:The filled bags aresterilized in the autoclaves for 1½hour at 15 Ibs pressure per squareinch.

Fig.1: Different containers used for the cultivation of winter mushroom


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Inoculation: After the bags have beensterilized and cooled down to roomtemperature, they are inoculatedwith wheat grain based spawn @ 2-3%. Saw dust spawn can also be usedwhich growers may purchase fromspecialist spawn makers. Sawdustspawn is prepared by mixing tenparts of saw-dust with one part o frice bran and enough water toprovide a certain degree ofhumidity.

Incubation (Spawn run): The Bagsare placed/ arranged in incubationrooms where mycelia can growfavorably. The optimum temperature

for the mycelial growth is between22 and 25°C, so the temperature ofincubation room is kept between20°C and 23°C under the normalcommercial cultivation conditions.Mycelia spread over the whole bagafter 20-25 days (Fig.2).

Fruiting induction: When mycelialspread to 90% of the bag space, theplug is pulled off, the neck of the bagis unfolded and the surface of themedia is made smooth for fruiting(Fig.3). Bags are then placed in thedark at a temperature of 10 -14 °C andthe humidity is maintained at 80-85%. A moisture level in the bags is

Fig.2: Spawn run bags

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important to fruiting. Good fruitbodies are encouraged to form byadjusting the humidity in the roomand by maintaining the correctmoisture content of the substrate.Primordia are formed in 10-14 daysafter reducing the temperature (Fig.4).

Controlling: At 10 -12°C, the fruitbodies grow rapidly, but they areslender, long, and of poor quality(Fig. 5). For this reason, the growthof fruit bodies is controlled bylowering the temperature to 3-5°Cand providing air movement (3-5m/sec) as well as by providing the

rolled paper to hold the fruit bodies.This control is continued for 5-7 days,from the period when the cap’sdifferentiation is observed with thenaked eye to the period when thelength of the stem reaches 2 cm.

Cropping: When the fruit bodies are13-14 cm long, the rolled paper isremoved and fruit bodies are pulledup from the bottle and packed. Ittakes about 50-60 days from theinitial sowing to the crop. The firstcrop usually amounts to 200-280 g/bag and the second crop from 120-160 g in the same bag (Fig. 6). Bottle/Jar culture is not recommended for

Fig.3: Bags ready for fruit bodied induction


19

Fig.4: Initiation (left) and developing (right) primordia

Fig.5: Slender, long fruit bodies

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Fig. 6: Cultivation in polypropylene bags

commercial cultivation due toinconveience in handling (Fig. 7).Kachroo (1991) obtained increasedyield by dipping the bags in asolution of copper (0.04mg) and Fe(0.02mg) after spawn run. Sharma(2004) cultivated winter mushroomby filling 2 Kg wet substrate (mixedsaw dust) in Polypropylene bags,

sterilized at 1210C for 2 hrs,inoculated and incubated at 250C.After the completion of spawn runtemperature was lowered to 150C.After lowering the temperature, fruitbodies appeared in 18 days whichbecame ready to harvest in the 6days. A biological efficiency of 45 percent was achieved.


21

Management: The initiation of fruitbodies starts in dark but light isnecessary for further development.At the time of fruit body formation,temperature of cropping roomshould be lowered to 8-120C withrelative humidity from 80-85 percent.

Harvesting: When the fruit bodiesare 14-18 cm long, the fruit bodies areharvested (Fig 8). They are packedin PP bags or can be sun dried. After

harvesting second flush appears inabout 15 days. Only two flushes areharvested. About 360-400 g freshmushrooms can be harvested per bagcontaining 2 Kg wet substratethereby giving 36-40% B.E.

Cultivation in other countries

Substrates: Various substrates andcultivation procedures tried forwinter mushroom cultivation inother countries are as under:

Fig. 7:Cultivation in jars

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The mushroom is cultivated in aplastic bottle or a vinyl bag orpolypropyene bags at 20°C and 70%humidity, on a substrate of saw dustor corn cobs, and a number ofadditional ingredients for 30 days.Afterwards, the mushroom is grownfor another 30 days in a slightly coolerbut more humid environment. Thegrowth is constricted to force themushroom to grow long and thin.The mushroom available in thesupermarket often still shows theimpression of the bottle around thebase of the mushroom.

F. velutipes has been cultivated onwood logs or on saw dust medium.Since white, stiff, and durablesporocarps are preferred, now-aday’s cultivation on sawdust iscommon. The quality of the F.

velutipes cultivated on wood logs wasinferior hence presently itscultivation is mainly done on sawdust.

Production of most enokitake isbased on synthetic substratecontained in polypropylene bottles.Substrates (primarily sawdust andrice bran; 4 : 1 ratio) are mechanicallymixed and filled into heat resistantbottles with a capacity of 800 to 1,000ml. Sawdust consisting primarily ofCryptomeria japonica, Chamaecyparisobtusa or (aged 9 to 12 months) Pinusspp. appears to offer the best yields.In United States, a bran-supplemented medium, consistingprimarily of corn cobs, serves as theprimary medium. After filling intobottles, the substrate is sterilized (4h at 95°C and 1 h at 120°C),

Fig. 8: Crop ready to harvest


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mechanically inoculated andincubated at 18° to 20°C for 20 to 25days. When the substrate is fullycolonized, the original inoculum isremoved mechanically from thesurface of the substrate and thebottles may be placed upside downfor a few days. At the time of originalinoculum removal, the airtemperature is lowered to 10° to 12°Cfor 10 to 14 days.

To further improve qualityduring fruiting, temperature islowered to 3° to 8°C until harvest. Asthe mushrooms begin to elongateabove the lip of the bottle, a plasticcollar is placed around the neck andsecured with a Velcroreg strip. Thiscollar serves to hold the mushroomsin place so that they are long andstraight. When the mushrooms are 13to 14 cm long, the collars are removedand the mushrooms are pulled as abunch from the substrate. Themushrooms then are vacuum packedand placed into boxes for shipmentto market.

San-Antonio and Hanners, (1983)cultivated F. velutipes successfully onwood logs using grain-spawn diskinoculum. Miller (1998) also usedlogs and sawdust, for the cultivation

of F. velutipes mainly in China andJapan.

Gavrilova and Lysenkova (1988)tried various substrates consisting ofPopulus tremula sawdust + wheatstraw (10:1), sawdust + wheat straw+ spent malt (10:1:1), and sawdust +spent malt (10:1), produced yieldsequal to 15.5, 19.8 and 20.9% of theweight of the substrate, respectively.Cellulose, lignin and carbonutilization from the substrates duringgrowth and fruiting revealed that theinitial 0.17-0.41% N concentration ofthe substrates rose to 0.38-0.80%.Schmidt, (1985) also describedoptimum culture temperatures, pH,nutrient media and nitrogen sourcesfor F. velutipes.

Dang-Jian et al. (2001) conductedtests to investigate the effects offermentation period, culture material(CM), height and carbendazimconcentration in the medium on thegrowth and physiology of F. velutipescultivar (F801 strain). In test I, cottonseed hull medium wassupplemented with carbendazim at0, 0.1, 0.15, 0.2 and 0.25% to preparemedia (1), (2), (3), (4) and (5),respectively, of which medium (1)was sterilized, but the others were

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not. When F801 was inoculated onmedia 1-5, the carbendazim inhibitedhyphal growth, and this inhibitionincreased with increase incarbendazim concentration. In test II,medium 1 was fermented at 12oC,and sampled after 48, 72, 96 or 120 h.The highest fresh mushroom yieldwas obtained on medium sampledafter 96 h of fermentation. In test III,the medium fermented for 96 h wasbagged to heights of 10, 8, 6 or 4 cm,then inoculated with F801 strain. Thehighest biological conversionefficiency 112% was found on the CMwith a height of 8 cm.

Payapanon and Tontyaporn(1999) studied the effects of heattreatments (100oC for 4 h (A), 100oCfor 3 h/121 oC for 1 h (B) or 121o C for1 h (C)), used to sterilize substratemedia (rubber sawdust alone or incombination with rice bran, cornflour or water, 800 g inpolypropylene bags), oncontamination of F. velutipes cultures.Two flushes were harvested fromeach treatment. For the first flush,contamination was in the range 0-4,15-25 and 50-80% for heat treatmentsA, B and C, respectively. For thesecond flush, low contamination andhigh production rates were observedin treatments in which the substrate

media were heat treated at 100o C for4 h.

Ding et al.(1990) reported that theuse of plant growth regulatorsincluding alkylal 30, gibberellin,alkylal 30 + gibberellin, ethylene,kinetin, and methyl α -naphthylacetate during different growthperiods of Flammulina velutipesshortened the time for the appearanceof fruiting bodies, and increased thenumber, yield and quality of thefruiting bodies. Alkylal 30 +gibberellin gave the best results.

The fruit-bodies were muchheavier in flowing air, mainlybecause of greater pileus size. Youngfruit-bodies left singly on amycelium grew larger than those leftin groups; they attained 60-80% of thetotal weights of groups of 3-7 fruit-bodies each in still air and 54-64% ofthe weight of 5 fruit-bodies in theflowing air. Fruit-bodies of the samerank weighed more in smaller thanin larger groups, but the totalweights of groups of 3-7 fruit-bodieseach did not differ significantly.There was also competition betweenfruit-bodies in their late phase ofrapid elongation. Pileus/stipeweight ratios increased withdecreasing numbers of fruit-bodies


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on a mycelium and were greater inflowing than in still air. The resultsindicate that fruit-bodies in groupsinhibit each other’s growth bycompeting for materials, required forgrowth, which are supplied inlimited amounts from the myceliumand distributed among individualsproportionately to their ranks (Gruen,1983).

Zadrazil (1980) describeddifferent containers suitable forgrowing edible fungi and observedthat plastic container is the best forF. velutipes. Growth of fruiting bodieswas better on the sides of a cylindricalcontainer than on a flat surface.Various types of apparatus forconstructing cylinders and blocks ofsubstrate, and for providing themwith aeration and warmth while thecrops are produced. A new methodof bag cultivation for F. velutipes,regarded as regeneration cultivation,was introduced by Cai (1989). Parket al. (1978) recorded optimum yieldsof fresh material of F. velutipes in 800ml-flasks containing 660 g poplarsawdust to which rice bran wasadded at 40% by volume andadjusted to 70% moisture content,with a growing temperature of 6oC.F. velutipes was grown in glass jars on

50 g damp wheat straw alone orenriched with 20 g soaked crushedbean, pea or wheat seeds, or on 100 gdamp wood shavings alone orsimilarly enriched. It fruited betteron supplemented wheat straw(Hubsch, 1983).

Gramss (1981) grew F. velutipesusing substrates containing beechsawdust, wheat straw, wheat flour,wheat bran and pea haulm. Thefungus needs a substrate with highcarbohydrate and N contents, each inan easily available form. Acaciamearnsii sawdust proved to be a goodsubstrate for growing F. velutipes (Lin,1991). Chu-Chou (1983) cultivatedwinter mushroom on sawdust fromNew Zealand timbers. Gramss,(1977)cultivated F. velutipes on blocks madeof mixtures of sawdust, clover, hay,dried pea haulm, dried lucerne,wheat flour, sugar and othermaterials. Pawlak and Siwulski(2001) evaluated yield of two F.velutipes cultivars (F-01 and F-04) onsawdust cultivation mediaconsisting of sterilized pine or beechsawdust as well as a mixture of bothtypes. They observed that cultivationmedia influenced the yield of F.velutipes.

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Yoshizawa(1998) reported thatsawdust prepared from smoke-heated logs of sugi and karamatsu,the hot-water, 1% sodium hydroxide,and ethanol-benzene extractscontamination were reduced incomparison with non-treatedsawdust. Poppe and Heungens(1991) evaluated ten woods (Acer,Alnus, Betula, Carpinus, Fagus,Fraxinus, Picea, Pinus, Populus, Salix)as substrates for F. velutipes.

Tang (1988) described thecultivation of F. velutipes in plasticsheds, either in bags or in boxes on asubstrate consisting of 95% cottonseed hulls. Boxes gave better yields.A high humidity of 85-95% should bemaintained, it is recommended thatthe sheds should be kept open for 2h daily. Early harvesting, when thefruiting bodies reach a length of 13-15 cm should be done for betterquality fruiting bodies. Miao( 1988)cultivated F. velutipes in beds ofcotton seed hulls and highlyinconsistent results were obtainedthe three crops he raised. Zhao (1989)described the cultivation of F.velutipes underground in jars on asubstrate of 93% cotton seed hulls, 1%sucrose, 1% gypsum and 5% groundmaize. Illumination was provided by15-25 W incandescent lamps spaced

25 m apart. A temperature of 13-15oCand a high humidity of 80-90%favoured growth. With this systemthe average yield was 0.197 kg/jar.

Xiong et al. (1999) compared thegrowth of F. velutipes on mediacontaining 88% cotton seed hulls, 1%gypsum and 1% refined sugar with10% quail droppings (aged for 0-40days). It was observed that growthrate increased with aging ofdroppings up to 30 days, and thendecreased slightly with aging for 40days. Average yields were 178 g/jarwith fresh droppings, increasing to219 g with droppings aged for 30days and 214 g with droppings agedfor 40 days. In another combinationconsisting of 78-93% cotton seedhulls, 5-20% quail droppings, 1%gypsum and 1% refined sugar thegrowth was most rapid with 10%droppings and slowest with 20%droppings. Average yields were 193,213, 199 and 158 g/jar with 5, 10, 15and 20% droppings, respectively.

Application of vitamins to thegrowing medium increased hyphalgrowth and productivity of F.velutipe. Chen-Hai et al.,(1995)obtained high productivity and goodquality mushrooms using a substrateof cotton hull locules (50%), rice bran


27

(20%), wheat bran (15%), cornpowder (10%) and 5% bean cake,whereas, Wang (1989) observed thatthe best culture medium for thecultivation was the mediumcomprising of cotton seed husks(88%), wheat bran (10%), vitamin B1(0.2%), vitamin B2 (0.3%), MgSO4

(0.5%) and CaSO4 (1.0%). Li (1989)grew four strains of F. velutipes onsubstrates consisting mainly of cottonseed hulls or unsterilized maize cobs.He observed that the period betweeninoculation and appearance of thefungus was longer on maize cobsubstrate than on cotton seed hulls,but the yield was from 2.9 to 9.6 timeshigher on maize cob substrate thanon cotton seed hulls, depending onthe strain of the fungus. Yang-Xiao(2001) used three types of media withdifferent carbon sources and revealedthat there was no significantdifference in the yield of F. velutipesbetween the medium of fir sawdustmixed with crust of cotton seed andthe medium of sole cotton seed crust.However, the medium of fir sawdustwas more practical due to its its lowcost.

Lu et al.(1989) cultured F. velutipeson 7 media: 1. 80% distillers’ barley+ 20% cotton seed husk; 2. 40%

distillers’ barley + 60% cotton seedhusk; 3. 88% cotton seed husk + 10%broiler concentrates + 1% sugar + 1%lime; 4. 100% cotton seed husk(control); 5. 89% cotton seed husk +10% broiler concentrates + 1% lime;6. 98% cotton seed husk + 1% sugar+ 0.5% urea + 0.5% lime; and 7. 89%rice straw + 10% concentrates + 1%lime. The highest mycelial growthrate, 39 cm/day, was achieved on thelast medium (7) and the lowest rate,24 cm/day, on the first medium (1).The harvest times for the first crop ofmushrooms were approximately thesame, but they varied significantlyfor the 2nd harvest (31-37 days). Thebiological efficiency was the highest,(98.6%), in medium 3 and lowest(50.9%), in medium 7, with the order(3) > (2) > (6) > (1) > (5) > (4) > (7).

Ji-Hong et al.( 2001) inoculated F.velutipes on 4 media, namely, (I) 70%maize straw (MS), 10% cottonseedhulls, 15% wheat bran (WB) and 5%maize flour (MF), (II) 88% MS, 5%WB, 5% MF, 1% sucrose and 1% lime,(III) 90% MS, 5% WB and 5% MF, and(IV) 79% MS, 5% WB, 5% MF, 10%sawdust and 1% lime. The bestmedia formulae for hyphal growth ofF. velutipes were II and IV,respectively. Jinzha No. 19 a cultivar

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of F.velutipes was the best strain withbiological efficacy of 73% on mediumII.

Tang-Xiang et al.(2001) evaluatedfour media comprising of flourformulae, (1) 80% rice straw, 10% ricebran, 7% corn flour, 2% plaster, 1%sucrose; (2) 80% P. notatum, 40%cotton seed hulls; (3) 92% cottonseedhulls, 5% rice bran, 1% calciumsuperphosphate, 1% plaster and 1%urea, and (4) 90% P. notatum, 6% ricebran, 2% calcium superphosphate,1% plaster and 1% sucrose. Thesemedia were sterilized for 1.5 h at 126o

C and inoculated with F. velutipes.The hyphal growth rate on the 4media was ranked 4<2<1<3. Thehighest biological conversion ratewas recorded on formula 3 (106.68%),next highest on formula 2 (90.61%),the third highest on formula 4(80.76%), and the lowest on 1(76.73%). The input output ratios offormulae 1, 2, 3 and 4 were 1:1.86,1:2.17, 1:2.08 and 1:2.03, respectively.

Thielke (1989) cultivated F.velutipes successfully on coffeegrounds, a waste material producedin the manufacture of coffee powder.Arai et al. (2003) reported hot water-soluble fraction (HWSF) from cornfiber (CNF) an abundant by-product

of the wet corn milling process, canbe used as a promotive substance forcultivating F. velutipes. Gao et al.(1992) gave preliminary report on theuse of feather meal in the cultivationof the winter mushroom.

Commercially, winter mushroom isgrown in jars The mushroom hasbeen cultivated in Japan initially onwood, and later in the bottles.

Solid state cultivation (SSC)F.velutipes is cultivated on coffeespent-ground or coffee husk whichis also known as solid statecultivation (SSC). SSC is carried outusing substrates filled in plastic bags(20x35 cm) filling100 g substrate ineach bag on dry wt basis (Fan etal.,2001). These substrates aremoistened with water (60% and 55%for coffee husk and spent-ground,respectively) generally 4-5 h beforeautoclaving and are autoclaved at121oC for 1.5 h. On cooling these bagsare inoculated with spawn @10% andmixed thoroughly to facilitate rapidand uniform mycelial growth. Higherspawn rates viz. 25% did not givesignificant higher yields. The mouthof bags are sealed using a cotton plugor thread. Then these bags areincubated in the dark at 24oC. Coffeehusk and spent-ground without any


29

nutrients supplementation has beenused for cultivation of F. velutipes LPB01 in solid state cultures. Syntheticsubstrate consisting of primarilysawdust and rice bran in the ratio of4 : 1 is being also used for thecultivation of winter mushroom inJapan. Sawdust consisting primarilyof Cryptomeria japonica, Chamaecyparisobtusa or aged (9 to 12 months) Pinusspp. appears to offer the best yields.

On coffee husk substrate, firstfructification occurred after 25 daysof inoculation and the biologicalefficiency reached about 56% withtwo flushes after 40 days. On spent-ground as substrate, firstfructification occurred 21 days afterinoculation and the biologicalefficiency reached about 78% in 40days. There was decrease in thecaffeine and tannins contents (10.2and 20.4%, respectively) in coffeehusk after 40 days. In coffee spent-ground, the tannin contentsdecreased by 28% after 40 days. Thisdecrease was attributed to thedegradation of caffeine or tannins bythe culture because these were notadsorbed in the fungal mycelia.Results showed the feasibility ofusing coffee husk and coffee spent-ground as substrate without any

nutritional supplementation forcultivation of edible fungus in SSC.Spent ground appeared better thancoffee husk.

Xing et al., (2007) studied theeffects of adding As, Hg, Pb and Cdto compost on the yield and heavymetal enrichment of fruit bodies ofF. velutipes. The results showed thatthe As, Hg, Pb and Cd contents of fruitbodies of F. velutipes increased withan increase in heavy metalconcentrations of compost. Hgdecreased the biological efficiency,but Cd slightly improved thebiological efficiency. The regressionanalysis showed that there was asignificant linear correlation betweenthe heavy metal concentrations ofcompost and the correspondingcontents of the fruit bodies.According to the safe limits of heavymetals of the national agriculturalstandard ‘Green Food: Edible Fungi’(NY/T 749-2003). It was concludedthat the safe limits of As, Hg, Pb andCd in compost of F. velutipes is lowerthan 1.0, 0.1, 10.0 and 1.0 mg/kg,respectively. Zou, et al. (2005)revealed that growth-promotingagents accelerated hyphal growth ofthe fungus and increased its yield. Anoptimum combination was also

V.P. Sharma et al.

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identified, which shortenedvegetative growth period by 4.6 daysand increased the biologicalefficiency by 55.08%.

In United States, a bran-supplemented medium, consistingprimarily of corn cobs, serves as theprimary medium. After filling intobottles, the substrate is sterilized for4 h at 95°C and 1 h at 120°C,inoculated mechanically andincubated at 18° to 20°C for 20 to 25days. When the substrate is fullycolonized, the original inoculum isremoved mechanically from thesurface of the substrate and thebottles are placed upside down for afew days. At the time of originalinoculum removal, the airtemperature is lowered to 10° to 12°Cfor 10 to 14 days (Royse, 1996).

Effect of moisture and spawn rate:Substrates were prepared withdifferent moisture levels namely 45,50, 55, 60, 65, and 70% for SSC.Similarly, different spawn rates weretested, which included 2, 5, 10, 15, 20,and 25%. After 20 days fermentation,the protein and fibre contents in thesubstrate were measured. Moisturecontents 55-60% and spawn rate 10%were the best.

Effect of environmental factors onthe production

Age of the mycelial culture is veryimportant in determining thefructification. It is difficult to formfruit bodies if the culture is tooyoung (less than 5 days) or too old(older than 2-3 weeks duration).Theoptimum temperature for themycelial growth is between 22oC and26 oC. The mycelium grows slowly,but does not die, when exposed to atemperature of 3 oC – 4 oC. On theother hand, at around 34 oC growthceases and over 34 oC the myceliumis killed for a short time. Thetemperature necessary forprimordial formation is between 10and 20 oC. The fructification inducedmore quickly at a temperature of 15oC than 5 or 10 oC. Optimumtemperature for fruit body growth isalmost the same as that for primordialformation.

Moisture, humidity andtemperature are indispensablefactors for growth of mycelia andfruit bodies formation. It was foundthat mycelia grow most quickly inthe presence of 60-65% water.Flammulina velutipes is aerobic and,accordingly, must be supplied with


31

sufficient oxygen. It has beenobserved that pileus diameterdecreased with the increasingconcentration of CO2. Stipeelongation has been found to be lesssensitive to CO2 than pileusexpansion and stipe elongation aswell as pileus expansion are bothprevented by high concentration ofCO2.

Primordia of F. velutipes areinduced in the dark condition, butlight is essential for the maturationof the fruit body. Without light, onlyrudiments of a fruit body are formed.

Sakamoto et al. (2002) investigatedthe influences of temperature on thefruit body of F. velutipes and revealedthat the fruit body could be inducedafter reducing of the ambienttemperature under completedarkness, but could not be inducedafter light irradiation withouttemperature reduction. Fruit bodiesformed under complete darknesselongated without pileus formation(pinhead fruit body). Pihakaski (1977)grew F. velutipes on liquid nutrientmedium, first in darkness at 25 oC for7 days and thereafter in continuouslight at 15 oC . Fruit body primordiawere visible after 10-11 days from thestart of the light period. Experiments

on the commercial cultivation of F.velutipes in beds showed that areasonably high CO2 concentrationwas a key factor in the control of capopening and growth promotion, andthat low light conditions inducedelongation towards the light. Theresponse of F. velutipes to light underCO2 concentrations of 0.114-0.152%was used to improve the yield andquality of fruiting bodies(Zhao,1990). Kaneko and Sagara(2001) revealed that fruit-bodies ofsome lignicolous agarics grewstraight downward. F.velutipes fruit-bodies showed almost the samebehaviour as the ‘Non-CoprinusType’. Based on these results,gravitational responses inhymenomycetes are overviewed.

Sakamoto et al. (2004) showed thatfruit bodies of F. velutipes could beinduced in complete darkness aftera sharp temperature reduction from23o to 16oC. However, the fruit bodiesthat form in complete darkness havea long stipe with an undevelopedpileus on the top (pinhead fruitbodies) and are thinner and whiterthan the normal fruit bodies whichare formed in the light. This findingsuggests that F. velutipes fruit bodiescannot mature in complete darkness.Sakamoto et al. (2007) identified a cell

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wall-associated protein (PSH) thatwas specifically induced in thepileus, but not in the stipe, followinglight treatment of the pinheadfruiting body. Cloning and sequenceanalysis of the gene encoding PSH(psh) revealed a motif in the C-terminal region of the predictedamino acid sequence that was similarto hydrophobin. The level oftranscription of psh was low in thestipe, but it was expressed at a highlevel in the pileus of the normalfruiting body. Transcription was alsolow in pinhead fruiting bodies, butincreased after light treatment. Theseresults indicate that psh isspecifically expressed during pileusdifferentiation induced by lightstimulation.

Post harvest management

The mushroom are sold as freshor canned, however, the freshmushroom are prefered. Cut off theroot system (approximately 4 cm)and wash briefly before use. They aretraditionally used for soups, but canalso be used for salads and otherdishes. They have a fruity flavor anda crisp texture. The mushroom canbe refrigerated for about one week.

Shelf life of F. velutipes isapproximately 14-20 days at 1oC, 10

days at 6 oC and 2-3 days at 20 oC .Browning of the pilei, gills and stipesand polyphenoloxidase activitymarkedly increased during storageat 20 oC. Total free amino acid contentalso greatly increased at 20 oC(Minamide et al., 1980). Fluctuatingtemperature had a particularlymarked effect on the quality of thismushrooms (Ito and Nakamura,1985).

The effect of packing materials(polyvinylidene chloride coated,oriented nylon, antifogging, wrap orvacuum packing film) on the keepingfreshness of F. velutipes at 25 or 2 oCwere investigated. The besttreatment for maintaining quality waspacking in antifogging film; F.velutipes could be stored for 28 daysat 2 oC (Chi ,1998).Low-densitypolyethylene (LDPE) film embeddedwith 30 micro m (AC30) or 60 microm (AC60) of silver-coated ceramicand used as packaging film for enokimushrooms revealed that, AC30 hada storage life of 14 days comparedwith 10 days when packaged withLDPE without ceramic at either 5 or20 oC (Eun et al.,1997). The effects oftemperature (0 or 6 oC) and packing(sealed in polyethylene or sealed inpolyethylene with a hole, alone or incombination with corrugated


33

cardboard) on the shelf life of F.velutipes revealed that the besttreatment was packing in sealedpolyethylene and storing at thelower temperature; shelf life wasextended to 15 days (Chi et al.,1996).

Wang and Xu (1992) designedan experimental drier used for thinlayer drying studies of F. velutipes(Sing). Data were obtained for 8drying conditions involving windspeed of 1-3 ms-1,temperature of 40-70 oC and RH of 16-30%. Twomathematical models, a diffusionmodel and the Page equation, wereused. The Page equation was foundto be more suitable for describing thedrying characteristics of a singlelayer of F. velutipes. From an analysisof the drying rate, energyconsumption and product quality, atemperature of 48oC and wind speedof 2 ms-1 were found to be theoptimum conditions.

F. velutipes stored in air at 5, 10,20, 30 or 35 oC for 60 h, and exposedto ethylene at 100 p.p.m. for a 24-hperiod starting 12 h after storageshowed no response to ethylene atany temperature (Inaba et al.,1989).The quality of mushroom, as affectedby vacuum packaging, packagingunit and shelf temperature, was

investigated during simulatedshipping and marketing. Vacuumpackaging of a 200-g-unit mushroomseemed to have higher potential forquality maintenance until the 14thday of shipping, while no significantquality changes were observedbetween vacuum- and non-vacuumpackaging after 21 days ofshipping(Park et al.,2003).

Fresh mushrooms (100 g) werepackaged under various conditionsand stored at 10 oC for 14 days. Thehalf-vacuum package was best interms of quality preservation of thefresh mushrooms. A polyolefin filmwith respective gas permeabilities toO2 and CO2 established anequilibrated atmosphere of 1.7-2.4%O2 and 4.1-5.6% CO2 inside thepackage at 10 oC. This polyolefin filmwas shown to contribute topreserving the freshness of themushrooms. Temperature fluctuationsbetween 5 and 15 oC did not induce aharmful atmosphere inside thepolyolefin package, though hightemperatures accelerated the qualityloss (Kang et al. 2001).

Jasinghe et al. (2006) investigatedthat ultraviolet (UV) radiation-induced conversion of ergosterol tovitamin D2 in fresh mushroom of

V.P. Sharma et al.

34

enokitake mushrooms. A variety ofchromatographic techniques wereused to study the chemicalproperties and composition of totallipids in fresh and freeze-driedsamples of the edible fungus F.velutipes. The total lipid content(average of 5 samples) was 0.43% FW(3.58% DM) in fresh samples, and3.43% FW (3.53% DM) in freeze-driedsamples. In fresh samples, thephospholipid and neutral lipidfractions each constituted over 40%of the total lipid content whileglycolipids accounted for 14.8%. Thepredominant lipids in these fractionswere triacylglycerol (64.4% of theneutral lipid fraction), cerebrosideand acylsterylglucoside (35.8 and28.5%, respectively, of the glycolipidfraction), and phosphatidyl cholineand phosphatidyl ethanolamine (48.0and 42.2%, respectively, of thephospholipid fraction). Freezedrying only affected these valuesslightly (Takenaga et al.,1995).

Insect-Pests and Diseases

Various competitors, diseasesand insect-pests assocated withmushrooms are described below.

Moulds: Green mould is the mostimportant competitor associated

with winter mushroom. Addition ofwood (Quercus acutissima) vinegar tothe sawdust and potato dextrose agarmedia used for the cultivation of F.velutipes @ 10% not only completelyinhibited mycelial growth ofTrichoderma harzianum but alsopromote the growth F.velutipes(Chang et al., 1995) . Jiang ( 2001)studied the the effect of 5 fungicideson the hyphal growth of F. velutipesand contaminated moulds.Chlorothalonil and iprodione provedto be the most effective in managingthe moulds associated with wintermushroom. Both of their averageinhibitory rates were higher than 90%.Copper hydroxide and chlorothalonilexhibited the strongest inhibition oncontaminated mould. The suitableconcentration of copper hydroxidewas from 0.7-0.8 g/litre. Ogawa et al.(1975) reported that it is necessary toadd benomyl to the medium beforesterilization @ 40-60 ppm to avoidcontamination.

Bacterial rot: Pseudomonas tolaasii isthe main bacterium associated withF.velutipes. It results into brown orblack lesions on the fruit bodies(Shirata et al.,1995; Suyama and Fujii,1993). The bacterium produced 8toxins, tolaasin I, the main which


35

plays an important role in thedevelopment of symptoms on fruitbodies (Shirata, 1996).

Soft rot : P. agaricicola causes soft rotand produced pitting on the fruitbodies of winter mushroom. SEMstudies demonstrated the sequentialremoval of hyphal wall layers,thereby exposing the chitin skeletalmatrix, which was then degraded. Asecond type of damage typified bycollapsed, shrivelled, and in somecases lysed hyphal cells was alsoobserved. Culture plate assaysrevealed that P. agaricicola producedchitinase. This, together with earlierevidence of a β -glucanase enzyme,accounted for the degradative abilityof the pathogen (Gill and Tsuneda,1997). Okamoto et al. (1999) identifiedthe bacterium as P. agaricicola sub sp.carotovora causing soft rot of wintermushroom.

Virus-like particles: Presence ofvirus-like particles of ca. 50 nmdiameter associated with the dsRNAsconfirmed by electron microscopyhas been also reported in wintermushroom (Magae and Hayashi,1999).

Nematodes: Nematodes likeAphelenchoides composticola,

Ditylenchus myceliophagus,Paraphelenchus myceliophthorus andRhabditis species are associated withthis mushroom (Fang and Luo,1992;Tan et al.,1992). Tylopharynx foetidusexhibited strong pathogenicity to themycelia and fruit body of F. velutipesand also found to be associated withsoft rot disease caused byCladobotryum varium of mushrooms(Cai et al., 1999).

Mites: The pygmephorid Luciaphorusauriculariae, is regarded as adestructive pest of F. velutipes (Zouet al., 1993).

Okabe (1993) reared individualsof Histiogaster sp. separately oncolonies of F. velutipes anddevelopment period, fertility and thelongevity of females were observedat various temperatures. The mitesdeveloped faster when they werereared at temperatures of 20, 25 or 30oC.

Flies: Lycoriella spp are associatedwith winter mushroom. Atemperature of 20 oC is the mostcongenial for the development ofthese flies and they lay eggs whichare oval 0.17 mm in diameter and 0.27mm in length. The average eggperiod is 4 days. The body length of

V.P. Sharma et al.

36

Flow Chart of Flammulina production

Sawdust +Wheat/Rice bran

wheat grain based

Pasteurization22 p.s.i. for 1½ hr

Spawning

@ 4% dry wt. basis

Incubation

(22-25o C, high CO2, dark)

Wetting - 65 %

Substrate

Spawn

Pinning

(12-15o C, RH 85%, 1500ppmCO2, light > 800 lux)

Maturation

Harvesting

Marketing / Sun drying

Packing


37

each instar from 1st to 3rd was 0.7,1.5 and 4.5 mm, respectively (Choi etal., 1997).

Spent mushroom substratemanagement

Substrates used for the cultivationof winter mushroom can be used inthe production of vegetable cropslike tomato (Lee et al., 2005).

Marketable yields of tomatoplants grown in 4 l of RSE on non-recycled hydroponics was 6% higherthan those in perlite medium onrecycled hydroponics. Total porosityand container capacity of RSE werehigher. The amounts of P, K, Mg andCa in RSE after tomato cultivationalso increased.

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flammulina velutipes the culinary medicinal winter mushroom

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