Technical Bulletin Diseases and Competitor Moulds of ... · PDF fileDiseases and Competitor Moulds of Mushrooms and their Management Technical Bulletin S.R. Sharma Satish Kumar V.P

Diseases and Competitor Moulds ofMushrooms and their Management

Technical Bulletin

S.R. SharmaSatish Kumar

V.P. Sharma

National Research Centre for Mushroom(Indian Council of Agricultural Research)

Chambaghat, Solan-173 213 (HP)

Printed : 2007, 1000 Copies

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

N.R.C.M. 2007All rights reserved. No part of this technical bulletin may be reproduced inany form or by any means without prior permission in writing from thecompetent authority.

Designed & Printed at:Yugantar Prakashan Pvt. Ltd.WH-23, Mayapuri Indl. Area, New Delhi-64Ph.: 011-28115949, 28116018

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CONTENTS

Page No.

Foreword v

1. Introduction 1

2. Fungal Diseases and Competitor Moulds 2

A. Button Mushroom 2

B. Oyster Mushroom 36

C. Paddy Straw Mushroom 38

D. Other Mushrooms 39

3. Viral Diseases 44

4. Abiotic Disorders 65

5. Bacterial Diseases 70

6. References 77

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FOREWORD

The cultivation of Mushrooms is a carefully controlled biological system,however contamination with microorganisms, which are in ways, isinevitable. In India majority of the mushroom holdings are lacking adequatecompost preparation, pasteurization and proper environmental controlfacilities, which lead to the development of various diseases and pestssufficiently to a level to cause considerable yield loss. It is therefore veryimportant for the mushroom growers that they should know the importanceof diseases and competitors and should understand the importance ofhygiene to grow mushrooms successfully and profitably. I would like toadvise the mushroom growers to pay maximum attention to preparecompost/ substrate of optimum quality and maintain highest level of hygieneto avoid these problems. I appreciate the efforts and labour put in by theauthors in compiling and editing the bulletin for its use by the mushroomgrowers and researchers.

Rajendra Prasad TewariDirectorNational Research Centre forMushroom, Solan – 173 213 (HP)

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I. INTRODUCTION

Like all other crops, mushroomsare also affected adversely by a largenumber of biotic and abiotic agents/factors. Among the biotic agents,fungi, bacteria, viruses, nematodes,insects and mites cause damage tomushrooms directly or indirectly. Anumber of harmful fungi areencountered in compost and casingsoil during the cultivation of whitebutton mushroom. Many of theseact as competitor moulds therebyadversely affecting spawn runwhereas others attack the fruitbodies at various stages of cropgrowth producing distinct diseasesymptoms. At times there iscomplete crop failure dependingupon the stage of infection, qualityof compost and environmentalconditions. General distribution ofvarious competitor moulds andpathogenic fungi is as follows:

I. Those occurring mainly incompost include: Olive greenmould (Chaetomium olivaceumand other spp.), Ink caps(Coprinus spp.) Green moulds(Aspergillus spp. Penicillium spp.and Trichoderma spp.), Blackmoulds (Mucor spp., Rhizopus

spp.) and other (Myriococcumpraecox, Sporotrichum sp.,Sepedonium sp., Fusarium spp.,Cephalosporium spp., Gliocaldiumspp., and Papulospora spp.).

II. Fungi occurring in compost and incasing soil: White plaster mould(Scopulariopsis fimicola): Brownplaster mould (Papulosporabyssina), Lipstick mould(Sporendonema purpurescens),False truffle (Diehliomycesmicrosporus) and green moulds.

III.Fungi occurring on and in casing soiland/or on the growing mushrooms:Cinnamon mould (Pezizaostracoderma), wet bubble(Mycogone perniciosa), Dry bubble(Verticillium fungicola), Cobweb(Cladobotryum dendroides), Pinkmould (Trichothecium roseum) andgreen moulds.

IV. Fungi attacking the fruit bodies only:Fusarial rot (Fusarium spp.).

At any phase of growth anundesirable growth or developmentof certain moulds can occur and canadversely affect the final mushroomyield.

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II. FUNGAL DISEASES AND COMPETITORMOULDS

A. WHITE BUTTON MUSHROOM(Agaricus bisporus, A.bitorquis)

a. Diseases

1. DRY BUBBLE

Pathogen : Verticillium fungicola

Common Name : Verticilliumdisease, brown spot, fungus spot, drybubble, La mole.

This is the most common andserious fungal disease of mushroomcrop. If it is left uncontrolled, diseasecan totally destroy a crop in 2-3 weeks(Fletcher et al. 1986). Verticilliumfungicola was major pathogenresponsible for considerable yieldlosses of cultivated mushrooms inManchuela area provinces of Cuencaand Albacete, Spain (Gela, 1993). Ina disease survey of commercialmushroom houses, V.malthousei wasisolated from 11.3% of mushroomsampled (Foree et al. 1974). FromIndia the first report of the heavyincidence of dry bubble disease wasfrom mushroom farms located atChail and Taradevi (Seth et al. 1973).

The pathogen has been invariablyisolated from the compost and casingsamples collected from mushroomfarms in Haryana, HP and Punjab(Sharma, 1992). Thapa and Jandaik(1984-85) have recorded theincidence of dry bubble from 25-50%at Solan and Kasauli and upto 15%at Shimla and Chail during 1980-81.Artificial inoculation with thepathogen at the time of spawningand at different loads of inoculumhad delayed pinhead formation by 5days and reduced the number andweight of fruit bodies by 2.26-47.2%and 2.19-38.01%, respectively(Sharma and Vijay, 1993).

Symptomatology : Whitishmycelial growth is initially noticedon the casing soil which has atendency to turn greyish yellow. Ifinfection takes place in an earlystage, typical onion shapedmushrooms are produced.Sometimes they appear as small-undifferentiated masses of tissueupto 2cm in diameter. When affectedat later stage, crooked and deformedmushrooms with distorted stipes

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and with tilted cap can be seen.When a part of the cap is affectedharelip symptom is noticed. Affectedmushrooms are greyish in colour. Ifthe infection occurs at later stage,grey mouldy fuzz can be seen on themushrooms. Sometimes littlepustules or lumps appear on the cap.On fully developed sporophores, itproduces localized light browndepressed spots. Adjacent spotscoalesce and form irregular brownblotches. Diseased caps shrink inblotched area, turn leathery, dry andshow cracks. Infected fruit bodiesare malformed, onion shaped andbecome irregular and swollen massof dry leathry tissue (Sharma, 1994).In A.bitorquis, the dark brownblotches caused by V.fungicola varaleophilum are sometimes coveredwith a layer of grey colouredmycelium particularly in the centre.In A.bisporus it causes minorspotting though in variety Les Miz-60 it causes fruit body deformation.An isolate of V.psalliote fromA.bitorquis causes more confluentbrown spots on A.bitorquis but couldnot infect A.bisporus (Zaayan andGams, 1982).

Causal Organism : Verticilliumfungicola

The fungus produces numerousone celled thin walled, oblong to

cylindrical, hyaline conidia, 3.5-15.9x 1.5 - 5u on lateral or terminal,verticillately branchedconidiophores (200-800 x 1.5-5.0 u).Conidiophores are relatively slenderand tall. Conidia accumulate inclusters surrounded by stickymucilage. The fungus abounds insoil.

Epidemiology : Verticillium iscarried on to the farm by infectedcasing soil. Spread is carried out byinfected equipments, hands andclothing. Phorid and sciarid flies arealso known to transmit this disease(Renker and Bloom, 1984). Underlaboratory conditions sciarids andphorids were found to transmit 84-100% and 76-100% of V. fungicolarespectively, into two different media(Kumar & Sharma, 1998). Mites arealso known to transmit the diseasefrom infected to healthy mushroom(Fikete, 1967). The fungus is soilborne and spores can survive in themoist soil for one year. It alsoperpetuates through restingmycelium from dried bulbills and inspent compost. The optimumtemperature for diseasedevelopment is 20°C. The periodfrom infection to symptomexpression is 10 days for thedistortion symptoms and 3-4 days forcap spotting at 20°C. The pathogengrows best at 24°C. However,

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Diseases and Competitor Moulds of Mushrooms and their Management

V.fungicola var aleophilum andV.psalliotae grow best at highertemperature (27°C) (Fletcher et al.1986). High humidity, lack of properair circulation, delayed picking andtemperature above 16°C favour itsdevelopment and spread (Sohi,1988). It becomes more commonwhen cropping is extended beyond61 days. A number of wild growingfleshy fungi also serve as source ofinoculum. Air borne dust is alsomajor source of primary infectionand may enter houses throughexhaust vents. If infection occursearly, it causes more severemalformation of fruit bodies.Holmes (1971) reported thatinoculum introduced before 21st daycaused low mushroom yield and highdisease incidence. However,inoculum introduced after 14 daysof casing caused the highest diseaseincidence. According to Nair andMacaulley (1987) when crops ofA.bisporus and A.bitorquis wereinfected at casing with V.fungicolavar fungicola and V.fungicola varaleophilum respectively, a relativelyhigh incidence of disease wasobserved but disease was less in thecrops infected at spawning or aftersecond flush. Reduction oftemperature from 20C to 14C andRH from 90% to 80% for 5 days couldnot reduce the severity of the disease.

All the commercial strains aresusceptible (Sharma 1994).However, Poppe (1967) in pot trialsfound brown strain from Francemost resistant to dry bubble disease.

Management

a) Physical methods : Use ofsterilized casing soil, properdisposal of spent compost andproper hygiene and sanitation areessential to avoid primaryinfection (Sharma, 1994). Wuestand Moore (1972) reported thattreating mineral soil with aeratedsteam at 54.4°C for 15 minuteseliminated V.malthousi that hadbeen experimently establishedfor 17 days in axenic soil culture.Further in 1973, Moore andWuest reported that thirtyminute treatment with aeratedsteam at 60°C and 82°C, hinderedspore germination and soilcolonization by V.malthouseimore than similar treatment at98°C. Heat treatment of infectedcasing layer at 63°C for one hourcompletely prevented sporegermination (Poppe, 1967).

b) Biological method : Accordingto Trogoff and Ricard (1976)spraying casing soil with 100x106

Trichoderma propagules/litre/m2

controlled V.malthousei in

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several trials on naturallyinfected mushroom holdingswhere dry bubble disease wasendemic. Under laboratoryconditions, leaf extracts ofCallistemon lanceolatus,Cannabis sativus, Citrus sp.,Euclyptus sp., Dhatura sp.,Urtica dioica, Solanumkhasianum and Thooja compactacaused 27.77%, 13.05%, 16.66%,22.22%, 5.55%, 6.66%, 22.77%and 27.77% inhibition,respectively of V.fungicola(Sharma and Kumar 1998-99)Bhat and Singh (2000) reported5 bacterial isolates effectiveagainst V.fungicola.

c) Chemical methods : Inlaboratory trials V.malthouseiwas controlled by Zineb on a largescale, Bercema - Zineb 80 used at0.1 - 1.2% controlled the diseasewhen used before and betweenthe flushes (Philipp, 1963).V.malthousei was controlled by 3sprays with Dithane Z-78 at 0.25or 0.50% or Hexathane at 0.30%given at the time of casing, atpinhead formation and afterflushes of crop (Seth et al. 1973).Application of chlorothalonil as adrench reduced the incidence ofV.fungicola tolerant to certainbenzimidazole fungicides.

However, incorporation ofchlorothalonil into the casinglayer caused toxicity to crop anddepressed the yield (Gandy andSpencer, 1976). However, Zaayenand Rutjens (1978) obtained goodcontrol with 2 application ofDaconil 2787 (chlorothalonil) at3g/m2 without any adverse effecton yield. Treatment should beapplied directly after casing andagain 2 weeks later. According toGeijn (1977) disease can becontrolled by spraying withcarbendazim, benomyl orthiophenate methyl at 100, 150and 200g/100m2, respectively in100-150 litres of waterimmediately after casing. Casedbeds can also be treated with 0.5%formalin or 100g carbendazim,150g benomyl or 200gthiophenate methyl in 100-150litres of water per m2 of bed.Zaayen (1979) obtained highestyield with chlorothalonil at 3g/litre water /m2 applied directlyafter casing and again 2 weekslater. Good control of V.fungicolawas achieved by spraying withprochloraz manganese at 60g/100m2 within 7 days of casing andsubsequently at 2 weeksintervals (Fletcher and Hims,1981). Fungicides triadimefon(1g/m2), prochloraz (1g/m2),

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Delsene M (carbendazim +maneb) (8g/m2) andchlorothalonil (2g/m2) appliedafter casing increased the yieldfrom 39.9% in untreated controlsto 56.7, 56.3, 54.6 and 53.1%,respectively (Gandy and Spancer,1981). Zaayan and Adrichem(1982), Russell (1984), Eicker(1987) recommended theapplication prochloraz +manganese complex (Sportak50WP) at 1.5g a.i/m2, 9 days aftercasing. However, only partialcontrol has been achieved byintensive use of prochloraz inSpain (Gela, 1994). Eicker(1984) recommended theapplication of Tecto as drench(450g a.i thiabendazole/dm3) atdosages of 1.838g a.i/m2 aftercasing and 1.44g a.i/m2 betweeneach break. Application ofAmitrole T at 50g, paraquat at10g and diuron at 20-40g after 24hours of inoculation wereeffective against V.malthousei(Popple, 1972). Zaayan and Geijn(1979) suggested new possibilitesfor control of diseases whichadvocated application offormaldehyde (2 litre/100 litresof water/100m3) immediatelyafter casing for effectivemanagement of disease. Ifdisease reappears, replace

formaldehyde by benlate,bavistin, Topsin M throughoutone cultivation cycle. IfV.fungicola becomes resistant tothese fungicides, chlorothalonil(3g /m2) can be used immediatelyafter casing and again 14 dayslater or Curamil (pyrazophos) (at0.5ml/m2) can be applied aftercasing and thereafter at weeklyintervals. According to Flegg(1968) fumigation with methylbromide at a CTP of 600 oz/hr/1000 cu.ft or more can provide asatisfactory alternative to cookout with live steam.

2. WET BUBBLE

Pathogen : Mycogone perniciosa

Common Name : Wet bubble, Lamole, white mould, bubble,Mycogone disease

Wet bubble in white buttonmushroom incited by Mycogoneperniciosa Magn. has been reportedas one of the serious diseases fromalmost all the major mushroomgrowing countries of the world.Bubbles or mole (M. perniciosa),first described from Paris in 1888, isstated to be responsible for theheaviest losses in mushroom beds inFrance, England and United States(Nielson, 1932). The disease has also

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been reported to assume seriousproportions in other majormushroom growing countries of theworld such as United Kingdom,Netherlands, USA, China, Taiwan,South Africa, Brazil, Hungary,Australia and Poland from time totime. In India, this disease wasreported for the first time in 1978from some mushroom farms inJammu and Kashmir (Kaul et al.,1978). Later, this disease has beenreported from the States of HimachalPradesh, Haryana and Maharashtra(Sharma, 1994, Sharma and Kumar,2000, Bhatt and Singh, 2000).

Symptomatology : Many workershave described Symptoms of wetbubble at different stages ofmushroom development. Smith(1924) recognised two mainsymptom types, infected sporophoresand sclerodermoid masses, which heconsidered to be the result ofinfection by M. perniciosa atdifferent stages in the developmentof the sporophores. Thus, wheninfection took place before thedifferentiation of stipe and pileus theselerodermoid form resulted,whereas, infection afterdifferetration resulted in theproduction of thickned stipe withdeformation of the gills (Fletcher andGanney, 1968). Garcha (1978)

described the symptoms in the formof white mouldy growth on themushrooms, leading to theirputrifaction (giving foul odour) witha golden brown liquid exudate. Hsuand Han (1981) reported that theinfected sporophores may berecognised by two symptoms, one istumorous form, infected frompinheads, and other is malformation,infected at later stage. Both types ofinfections may exude water drops onthe surface of infected sporophores.These water drops later change intoamber colour. Tu and Liao (1989)observed that when young pin headsare infected they develop monstrousshapes which often do not resemblemushrooms. Fletcher and Ganney(1969) have reported about 31%infection at the base of the stipe inapparently healthy sporophores inthe form of black streaks. Sharmaand Kumar (2000) described thesymptoms as short, curly, pure whitefluffy mouldy growth of the pathogenon malformed mushrooms, whichcan be easily observed by nackedeyes. Cross section of deformedsporophores without cottony growthshowed black circular area justbeneath the upper layer. Umar etal., (2000) described dramaticcytological changes as a result ofinfection when young (up to 6mm)pin heads were infected. Large, very

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irregular, nodular and tumorousfungal masses are formed and nodifferentiation or organogenesis ofthe cell mass takes place.Mycopathogen grew on the surfaceas fluffy mycelium but was absentdeep on the lesions. TransmissionEM revealed two kinds of cell wallreactions, either focal swelling likecushion at the site of adhesion of M.perniciosa or focal lytic changes withswollen mitochondria.

Nielson (1932) stated that wetbubble caused heaviest losses among

all diseases in mushroom beds inFrance, England and United States.In USA, M. perniciosa was isolatedfrom 3.7 per cent samples collectedfrom various mushroom farms. Forerand his associates (1974) whileestimating the qualitative andquantitative losses caused by wetbubble and dry bubble inPennsylvania (USA), reported thatthese two diseases induced 2.2million lbs. as quanlitative and 19.7million lbs as quantitative loss ofmushrooms. Nair (1977) conducteda survey of 24 mushroom farms in

Symptoms of wet bubble disease

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New South Wales during 1975-76and observed that the mosteconomically important diseases inthese farms include wet bubble.Sharma and Kumar (2000) reportedthat the natural incidence of wetbubble disease of button mushroomranged from 1 to 100 per cent innorthern India. Loss in yield in A.bisporus (S-11) due to this diseaseunder artificial inoculationconditions has been reported to varyfrom 15.72 to 80.13 per cent. Bhattand Singh (2000) have reported theyield loss up to 100 per cent as aresult of artificial inoculation of M.perniciosa.

Etiology : The disease, wet bubble,is caused by Mycogone perniciosaMagn. and the perfect stage isHypomyces perniciosa. Mycelium ofthe pathogen is white, compact, felt-like. Hyphae branched interwoven,septate, hyaline, 3.5m broad.Conidiophores short, slender,branched, hyaline measuring 200 x3-5m and having sub-verticillate toverticillate brances which bear thinwalled, one-celled conidia measuring5-10 x 4-5m. Large two-celledchlamydospores present; upper cellwarty, thick walled, globose, brightcoloured measuring 15-30 x 10-20m,lower cell hyaline, smooth andmeasure 5-10 x 4-5m.

Host Range : Mycogone perniciosa,though a major pathogen of Agaricusbisporus, is also capable of infectingother mushroom species. Figueiredoand Mucci (1985) revealed that M.perniciosa can infect A. campestris.Sisto et al., (1997) have reportedPleurotus eryngii and P. nebrodensissusceptible to M. perniciosa. Sharmaand Kumar (2000) reported all thestrains of A. bisporus (U-3, S-11,791, S-910) and A. bitorquis (NCB-6, NCB-13) susceptible to M.perniciosa under in vivo conditions.

Spread : Spread of M. perniciosaoccurs primarily through casing soilbut the introduction of pathogenthrough other agencies, like spentcompost and infected trash, is notruled out. The infection can be air-borne, water borne or may bemechanically carried by mites andflies (Garcha, 1978). Hsu and Han(1981) reported water splash as animportant factor for wet bubblespread on the beds. Bech et al.,(1982) reported that spread throughcontact occurred readily duringwatering and especially harvesting.They also observed thatcontaminated containers can be asource of spread over greaterdistances. Contrary to other reportsit was also suggested that spores ofM. perniciosa can also be spread by

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air current (Tu and Liao, 1989).Kumar and Sharma (1998) reportedthat transmission percentage of M.perniciosa under in vitro conditions,by sciarid and phorid flies was 100per cent on MEA medium and 4-12per cent on compost.Chlamydospores have been reportedto survive for a long time (upto 3years) in casing soil and may serveas the primary source of inoculum.The aleurospores produced on thesurface of monestrous structures areprobably responsible for secondaryinfection.

Biology / Physiology : Lambert(1930) revealed that Mycogoneperniciosa is quite sensitive toprolonged exposure to moderatelyhigh temperature. The cardinaltemperatures for growth of theorganism on Thaxter’s agar are 8°C,24°C and 32°C. He also reported thatin agar cultures M. perniciosa waskilled by exposures to temperaturesof 42°C (106°F) or higher for 6 hr. ormore. According to Zaayen andRutigens (1981) thermal death pointfor M. perniciosa is 48°C. Bech andKovacs (1981) reported that aqueoussuspension of Mycogone spores canwithstand 42°C and 36°C for 10minutes and 1 hr, respectively. Hsuand Han (1981) reported thatoptimum temperature for mycelialgrowth, sporulation and conidial

germination was 25°C. He alsorecorded pH 6.0 as optimum forconidial germination. According toLiao (1981) chlamydospore failed togerminate on various media in vitroeven after heat (40-70°C) treatmentor application of chemicals andsolvent. However, germinationoccurred on potato dextrose agar(PDA) medium exposed to the gasproduced by mushroom mycelia incompost for 36 hrs at 24°C. In anthorstudy Bech and Kovacs (1981) foundthat aleurospores are unable togerminate in water, Richard’ssolution, pressed mushroom juice orin PDA but verticilloid sporesshowed a certain degree ofgermination in diluted mushroomjuice and on PDA. As reported by Tuand Liao (1989) the pathogen istolerant to a wide pH range in acidside and able to grow at pH 4.4,however, the growth becomesweaker or rather restricted at pH 8.4.Holland and Cooke (1991) reportedthat in malt extract agar medium M.perniciosa formed abundant thinwalled, hyaline phialo conidia andthick walled pigmented verrucoseconidia. During nutrient deplectionother propagules appeared, namely,lateral smooth conidia, infectedinterculary cells, chlamydosporesand arthro conidia. Singh andSharma (2000) have reported themaximum growth of M. perniciosa

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on PDA. Optimum temperature andpH for growth were reported to be25°C and 6.0, respectively. Mannoseand asparagine have been reportedas best sources of carbon andnitrogen, respectively. Sharma andKumar (2000) observed compostextract agar medium as the best forthe mycelial growth and malt extractpeptone dextrose agar medium forspore production. A pH range 5-6was found optimal for the mycelialgrowth.

Management : As the pathogeninflicts serious damage to the crop,various attempts have been made tomanage the disease through variousmeans.

a) Physical : Wuest and Moore(1972) suggested that aeratedsteam at 54.4°C for 15 minutescan eliminate M. perniciosa fromcasing soil. Munns (1975)suggested the use of plastic potsto cover mushroom showing wetbubble symptoms during thecropping season to prevent spreadof disease. Tu and Liao (1989)while working to find out anintegrated approach for themanagement of wet bubbledisease revealed that the use ofclean compost, pasteurization orsterilization of casing soil, goodpeak heating and fumigation of

mushroom house and use ofbenomyl or Mertect 40 per centwere effective in managing M.perniciosa. Zhang (1990)suggested three methods ofprevention of wet bubble diseasewhich include steam sterilizationof mushroom beds, formaldehydefumigation and fungicidalapplication. Another method likescreening and selection of diseaseresistant strains should also beexploited.

b) Biological : Jhune et al., (1990)screened 12 isolates of bacteriaand 71 isolates of actinomyectesisolated from mushroom compostand casing mixture and observedAJ-117, AJ-136 and AJ-139 aspromising bioagents. Though,almost negligible attempts havebeen made to control M.perniciosa through botanicals butthe inhibition of fungal growth byplant extracts is not uncommonand has been reported earlier bya number of workers (Flierman,1973; Michal and Judith, 1975).Gandy (1979) made aninteresting observation thatAcremonium strictum produces aheat stable antibiotic compoundpossibly a cephalosporin, whichis inhibitory to M. perniciosa butno attempts have been made toexplore this approach as both

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fungi are pathogenic tomushrooms.

c) Chemicals : Benomyl spray at0.5-4g/m2 immediately aftercasing has been reported veryeffective for protecting the crop(Gandy, 1974; Stanek andVojtechovska, 1972). Fletcher(1975) advised that adequatecontrol of wet bubble wasobtained by benomyl orThiophanate methyl at 10g a.i. atcasing while TBZ was lessaffective. Kim (1975) recordedsatisfactory control of wet bubbleby spraying benomyle @ 0.5g a.i/m2 , 3 days after casing. Geijn(1977) suggested the control ofwet bubble disease by sprayingthe crop with carbendazim,benomyl or thiophanate methylat 100-150 litre waterimmediately after casing.Basamid (Dazomet) and Vapam(Metham sodium) applied @100ppm to casing has also beenreported very effective (Kim etal.,1978). Application ofcarbendazim, benamyl,chlorothalonil, TBZ, prochlorazmanganese complex (Sportak 50WP) into casing mixture havebeen reported very effective forthe management of wet bubble byseveral workers (Hsu and Han,1981, Zaayen and Adrichem,

1982; Fletcher, 1983; Zaayel etal., 1983; Eicker, 1984; Jhune etal., 1991; Sharma and Kumar,2000). It was reported that ifcasing is contaminated controlcan be achieved by treating itwith 1 per cent formalin.Alternatively, a spray of 0.8 percent formalin on to casingsurface, immediately after casing,can be effective. However, thisconcentration can be injurious ifused at a later stage in cropdevelopment. Sharma et al.,(1999) have reported 62.5-100per cent inhibition of M.perniciosa in culture wheninoculum discs were drenched in0.5-2% formalin solution for 5seconds. Exposure of M.perniciosa cultures to vapours of1-4 per cent formalin for 6-24 hrsalso resulted on 100 per centinhibition of fungal growth onsub-culturing.

3. COBWEB

Pathogen : Cladobotryumdendroides

Common Name : Mildew, Softdecay, Hypomyces mildew disease,Dactylium disease.

This disease renders extensivedamage either by causing soft rot or

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decay of fruiting body. Merat (1821)described this disease as Botrytisdendroides and transferred it in tothe genus Cladobotryum by makinga combination C.dendroides (Bull :Merat) W.Gams et Hoozem. Salmanand Ware (1933) were the first toreport D.dendroides being parasiticto mushrooms. According toFletcher and Atkinson (1977)mushroom of any age of developmentwould be attacked by this fungus.This disease causes great damage tomushroom houses where humidityis high (Bozhkor, 1975). Forer et al.(1974) isolated C.dendroides from0.6% of mushroom sampled fromcommercial mushroom houses inPennsylvania. In India it was firstrecorded in Chail and Shimla (HP)(Seth, 1977) and later from Solanand Kasauli with natural incidenceranging from 8.17 - 18.83% in 1982and 1.93-25.63% (Seth and Dar,

1989). Under artificial inoculationconditions with different levels ofinocula, the loss in marketablemushrooms has been estimated at66.6% (Sharma and Vijay, 1996) and21.95 - 48.95% at differenttemperatures (Seth and Dar, 1989).Sharma et al. (1992) recordedC.verticillium a new pathogen ofA.bitorquis in Himachal Pradesh.

Symptomatology : Cobwebappears first as small white patcheson the casing soil which then spreadsto the nearest mushroom by a finegrey white mycelium. A floccosewhite mycelium covers the stipe,pileus and gills, eventually resultingin decomposition of entire fruit body.As the infection develops, myceliumbecomes pigmented eventuallyturning a delicate pink cover (Laneet al. 1991). In severe attacks, adense white mould develops over

Symptoms of cobweb

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casing and mushrooms change froma fluffy cobweb to a dense mat ofmycelium. The white colour canturn pink or even red with age. Onesymptom which can appear andwhich is generally not associatedwith the disease is cap spotting. Thespots can be brown or pinkish brown(Sharma, 1994). On inoculated fruitbodies, characteristic symptomsappeared within 24 hours ofinoculation when mycelial + sporesuspension were applied, symptomsappeared 4-12 days after infestation.Younger mushrooms are moresusceptible than fully developedones. Tuffs of conidiophers developon all sides of the web and growth ofengulfed mushroom is arrested. Onremoval of mycelial felt from affectedmushroom, drops of dark browncoloured fluids exudes emittingbitter foul smell (Seth and Dar,1989).

Causal organism : Cladobotryumdendroides (Dactylium dendroides)imperfect state of Hypomycesrosellus. Sterile hyphae form a turfand are prostrate, branched, septateand hyaline with approximatelyopposite branches, which divideabove into usually three pointedbranchlets. Conidiophers are erect,similar or branched in many whorls.Conidia single, elongate pointed atthe base, 2-3 septate, slightly

constricted at the septa and measure20-30 x 10-12.5 u. It produces sexualstage belonging to Hypomycesrosellus, which has been observed ondecaying dried fruit bodies of wildmushrooms in HP.

Epidemiology : High relativehumidity and temperatureencourage the disease. Spread ismainly by conidia. The pathogen isa soil inhabiting fungus and isnormally introduced into the crop bysoil contamination, spores,mycelium on crop debris or by farmworkers. Spores are easily spread byair movement, workers hands, toolsand clothing and by water splash(Sharma, 1994). Under laboratoryconditions, sciarids and phorid flieswere found to transmit 4-100% ofthe disease in to two different media(Kumar and Sharma, 1998). A highRH and temperature range of 19-22°C and 12-15°C resulted inmaximum loss in yield (Seth andDar 1989). Optimum temperaturefor growth is 20°C and for sporegermination is 25°C. C.dendroideshas been isolated from woodland soil(Canada) moss (Polytrichum sp.)(UK), a bracket fungus Stereum sp.(UK), dead wood (Pinus sp.) andmushroom farms (Lane et al. 1991).On the other hand disease caused byC. verticillatum on A. bitorquis wasfavoured by RH 90% and

14


temperature of 25-30°C (Sharma etal., 1992).

Management

Physical : Through disinfection ofcasing soil with live steam orsterilization of casing mixture at 50Cfor 4 hours effectively eliminates thepathogen. Regular cleaning, removalof cut mushroom stems and younghalf dead mushrooms after eachbreak and controlling temperatureand humidity helps in controllingthe disease (Sharma, 1994).

Biological : Under laboratoryconditions, leaf extract of Cannabissativus, Ricinus cummunis,Callistemon lanceolatus, Citrus sp.,Euclyptus sp., Dhatura sp. andUrtica dioica were found to cause5.55%, 10.55%, 18.55%, 26.11%,34%, 19.07% and 23.33% inhibitionof C.dendroides (Sharma andKumar, 1988).

Chemical : Terraclor(pentachloronitrobenzene) caneradicate Dactylium mildew evenafter the well establishment of thedisease (Stoller et al., 1956).Bozhkor (1975) suggested annualdisinfection of houses andsurrounding areas with 2%bordeaux mixture or with 5%formation solution at 0.5-1.0 l/m2 or

fumigation with 2.0-2.5 l formationands 0.5-1.0 kg chlorinated lime/100m3 for controlling disease. He furthersuggested that immediate spray aftercasing with benomyl at 1g in 0.5 -1.0l water/m2 also controls the disease.According to Russell (1984) singleapplication of prochloraz manganesecomplex (sporogon) at 1.5g a.i./m2 ofbed 9 days after casing givessatisfactory control of the diseases.Seth and Dar (1989) obtained bestcontrol of disease by applyingbavistin + TMTD at 0.9 and 0.6g/m2

followed by TBZ and benlate (0.9g/m2). Effective control of C.verticillatum was obtained byspraying with 0.05% carbendazim atspawning followed by 0.25%mancozeb at casing and carbendazimagain 15 days later (Sharma et al.1992).

4. GREEN MOULD

Pathogen : Trichoderma viride, T.hamatum, T. harzianum, T. koningii,Penicillium cyclopium, Aspergillusspp.

Common names : Trichodermaspot, Trichoderma blotch,Trichoderma mildew, Green mould

One of the most common anddestructive diseases in mushroomcultivation is the green mould which

15


is mainly caused by different speciesof Trichoderma, Penicillium andAspergillus. Among these moulds,Trichoderma spp. induce significantquantitative and qualitative losses inthe yield of Agaricus bisporus,Pleurotus spp., Auricularia,Calocybe indica and Lentinulaedodes. Kligman (1950) was the firstto report the presence ofTrichoderma in mushroom compost.Different species of Trichodermawhich have been reported ascompetitors and / or pathogenic onbutton mushroom include, T. viride,T. koningii, T. hamatum, T.harzianum, T. atroviride, T.pseudokoningii, T. logibrachiatum.Among all these species, T.harzianum is recognised as causingthe most severe problems (Morris etal., 1991; Seaby, 1996). Seaby (1989)recorded 9 distinct groups ofTrichoderma species and strainswhich had almost similar sporebearing structures as described byRafai (1969). These included, T.viride, T. harzianum (Th-1, Th-2,Th-3), T. koningii, T. pseudokoningiiand T. longibrachiatum. Geneticallydistinct biotype Th-4 of T.harazianum has been responsiblefor serious outbreak in USA. InIndia, T. viride was first reported byThapa and Seth (1977) on A.bisporus, and T. hamatum and T.

harzianum by Seth and Bhardwaj(1986-87).

Economic Importance : Greenmoulds caused by Trichodermaspecies were once recognised asindicators of poor compost qualityand were of minor significance. Thedevastating nature of T. harzianumwas undocumented in mushroomindustry until 1985 when it was firstobserved in Ireland and resulted inlosses estimated at 3-4 millionpounds to the U.K. and Irishmushroom industries. The secondwide spread epidemic occurred inearly 1990’s in Ireland (Seaby, 1996).Green mould epidemics have beenreported from the USA, Canada,South America, Asia, Australia andEuropean countries. T. harzianumbiotype Th-2 was responsible forsevere epidemics in Europe andbiotype Th-4 in America (Mamounet. al., 2000). Crop losses to greenmould are variable, however, sincethe onset of the disease inPennsylvania crop losses have beenestimated in excess of $30 million(Anderson et al., 2000). Yield lossesin first flush of A. bisporus byartificial inoculations have been upto 8% for T. pseudokoningii and 26%for T. atro viride (Grogan et al.,2000). Sharma and Vijay (1996)have reported yield loss in A.

16


bisporus from 12.5-80.8% by artificialinoculation of T.viride at differentinoculum loads and at differentstages of crop growth. Jandaik andGuleria (1999) reported 5-46.87%and 6.25-50.0% yield losses due to T.viride and T. harzianum,respectively under artificialinoculation conditions. Anderson etal., (2000) recorded significantdifferences among hybrid mushroomstrains in response to T. harzianumbiotype 4 (Th-4) infestation. Hybridwhite strains were the least resistantto green mould, sustaining yieldlosses upto 96%. Hybrid off-whitestrains exhibited intermediate

susceptibility with yield losses of 56-73%. Brown strains weresignificantly resistant to greenmould, sustaining yield losses of only8-14%.

Symptomatology : Differentspecies of Trichoderma have beenreported to be associated with greenmould symptoms in compost, oncasing soil, in the spawn bottles andon grains after spawning. A dense,pure white growth of mycelium mayappear on casing surface or incompost which resembles tomushroom mycelium. Later onmycelial mat turns to green colour

Symptoms of green mould

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because of heavy sporulation ofcausal agent which is a characteristicsymptom of the disease. Thereafter,the mould creeps to surface of casinglayer and infects the new parts anddeveloping newly borne primordia.Mushrooms developing in or nearthis mycelium are brown, may crackand distort, and the stipe peels in asimilar way to mushrooms attackedby Verticillium fungicola causing drybubble disease. Some species inducebrownish lesions / spots on capswhich may cover the entire capsurface under congenial conditions.

Causal organism : Several speciesof Trichoderma are associated withgreen mould disease complex of A.bisporus. The taxonomy of thisgenus has caused confusion and asuccession of mycologists haveinvestigated it since the turn of thecentury. In 1939, A. R. Bisbyreviewed the literature andconcluded that although there weredifferences in morphology betweentypes these were not consistent anddistinct. He, therefore, classified alltypes as T. viride whilst recognizingthat considerable but inconsistentvariability existed. In 1969, Rifairevised the genus and proposed ninespecies aggregates. His classificationis now the one that is generallyaccepted. The species recorded inmushroom culture include T. viride,

T. harzianum and T. koningi. T.viride is said to be weed mould, T.koningi a pathogen whilst T.harzianum has been ascribed to avariety of roles including pathogenand agent for biological control. Fourbiotypes namely, Th-1. Th-2, Th-3,and Th-4 have been furthercharacterized in T. harzianum onthe basis of occurrence, symptoms,morphological characters andphysiological requirements (Seaby,1989).

T. viride (T. lignorum) : This iswidespread in soil. Spore are ovoid,rough walled, green and measure2.8-5x2.8-4. The colony emitscoconut odour. This fungus grewslowly at 27°C but faster at 20°C.

T. koningi : This is commoninhabitant of soil. Spores are smoothwalled, cylindrical, green andmeasure 3-4.8x1.9-2.8m. Colonyemits no odour. Spores germinatefaster than other species and growthrate is 1-1.2mm/hr.

T. harzianum : Common in soils.Colonies growing rapidly, mostisolates 7-9cm in diameter after 3-4days, aerial mycelium floccose, whiteto greyish. Conidiation on MEAinitially as compact and produce aflat postule often concentric that isgreen whitish and later turn to dark

18


green colour. Chlamydospores fairlyabundant, intercalary or terminal,solitary, smooth walled, mostly 6-12mm. in diameter. Macronematousconidiophores highly branched.Conidia smooth walled, ovoid, greenin colour and measure 2.4-3.2x2.2-2.8m. Four biotypes, Th-1, Th-2, Th-3 and Th-4 have been furthercharacterized in T. harzianum onthe basis of morphological, cultural,physiological and geneticalvariations.

Epidemiology : Green mouldgenerally appears in compost rich incarbohydrates and deficient innitrogen. If the compost is tampledtoo hard in the beds, or the fillingweight is too high, this can make thepeak heating difficult. This iscertainly the case with compostwhich has a short texture and whichmight also have too high moisturecontent, resulting in improperpasteurization and conditioning ofcompost. Frequent use of formalinalso tends to promote thedevelopment of green moulds(Sharma et al., 1999). Differentsources of primary inoculum ofTrichoderma spp. could be dustparticles, contaminated clothings,animal vectors especially the mite,Pygmephorus mesembrinae, miceand sciarid flies, air-borne infection,infected spawn, surface spawning,

contamination of compost byhandling and machinery andequipments at the mushroom farm(Seaby, 1987). Spore concentrationless than 1x102 was unable to causeinfection (Grogan et al., 2000).Benomyl treated grain spawn orcompost spawn in normal composthad less T. harzianum. High relativehumidity accompanied by a low pHin the casing soil also promotes thedevelopment of Trichoderma spp.(Sharma and Jandaik, 1999).Chlamydospores produced by T.harzianum, T. viride, T.longibrachiatum and T.pseudokoningii survived theexposure of 9 hours at 60°C (Morriset al., 2000). T. harzianum inducedsignificant yield reductions at 30°Cthan at 20°C (Seaby, 1986).

Control : Green moulds can beprevented by

a) Very good hygiene

b) Proper pasteurization andconditioning of compost.

c) Sterilizing the supplementsbefore use and mixing themthroughly preferably afterspawning.

d) Using the correct concentrationof formalin (maximum 2%)

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e) Weekly sprays of mancozeb(0.2%)or bavistin (0.1%) TBZ(0.2%) ortreatment with zineb dust orCalcium hypochlorite (15%) havegiven effective control of thedisease.

b. Competitor moulds

5. FALSE TRUFFLE

Pathogen : Diehliomyces microsporus

Common name : Truffle disease

This is the most dreadedcompetitor in mushroom beds. It wasfirst reported by Lambert (1930)from Ohio, USA during 1929 anddescribed by Diehl and Lambert(1930) as Pseudobalsamiamicrospora. Glasscock and Ware(1941) observed it in UK and studiedits invasion in mushroom beds.False truffle incidence in theNetherlands was reported by Bels-Koning and Bels (1958) but itsserious incidence was noticed in thecrops of Agaricus bitorquis, grown athigher temperature (Zaayen and Pol-Luiten, 1979). Gilkey (1954)reclassified the fungus fromTuberales to the Eurotiales andnamed the genus Diehliomyces. InIndia, Sohi et al. (1965) observedfalse truffle causing serious losses tomushroom crops when the compost

temperature in the trays reachedbeyond 22-24C. The naturalincidence of false truffle in A.bisporus grown under naturalclimatic conditions has been reportedfrom 1-80% in the States ofHimachal Pradesh, Haryana, Punjaband Uttar Pradesh (Sharma andVijay, 1996). False truffle is a limitingfactor in the production of A.bitorquis in India because of itshigher temperature requirements.The disease is of common occuranceduring February or early March inA. bisporus in the plains of theNorthern India and during summermonths in A. bisporus and A.bitorquis in hilly regions of thecountry. Sharma and Jandaik (1996)reported 66-88 per cent incidence ofthis competitor in Himachal Pradeshduring 1993-1996 resulting in 58-80% yield loss.

Symptoms : The coulour of thefluffy mycelium is white to start withand turns a creamy yellow at a laterstage. It appears as small wefts ofwhite cream coloured mycelium incompost and casing soil, usuallymore conspicuous in the layer wherecompost and casing mixture meetand also on casing. Gradually themycelial growth become thicker anddevelops into whitish, solid,wrinkled, rounded to irregularfungal masses resembling small

20


brains (ascocarps of the fungus),looking like peeled walnuts. Theyvary appreciably in size ranging from0.5 to 3cm in diameter. At maturitythey become pink, dry and reddishand finally disintegrating into apowdery mass emitting a chlorinelike odour. The fungus does notallow the mushroom mycelium togrow and compost turns dull brown.The spawn in affected patches turnssoggy and disappears.

Causal Organism : Diehliomycesmicrosporus (Diehl and Lambert)Gilkey, ascocarps are formed fromthe dense tangled hyphal knotssingly or several knots coalesce toform large ascocarp. Ascocarps arefleshy, at first white then brownishand finally reddish brown containingnumerous sac like asci which areoval, sub-spherical, short or long-stalked, with 3-8 ascospores, 19-27x10.5-15m. Ascospores are

spherical, sulphur coloured with onedistinct oil drop and measure 6.5min diameter. Chlamydospores may benoticed in the hyphal web ofascocarp.

Epidemiology : Ascosporesdevelop in the truffles in 3 to 6 weeksand are released when the truffledisintegrates. Ascopore productionis abundant at 25 and 30°C but notat 15 or 37°C (Wood and Fletcher,1991). Ascopore germination upto70% has been recorded at 27°C aftergiving heat stimulus at 40-50°C forhalf an hour. (Zaayen and Pol-Luiten; Sharma 1998). The majorsources of infection are casing soiland surviving ascospores/myceliumin wooden trays from the previouscrops. Ascopores can survive for aperiods of 5 years in soil and spentcompost and mycelium for 6 months(Sharma, 1998) and thus serve as themajor source of primary inoculum.

Symptoms of false truffle

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Stage of infection and temperatureare important factors fordetermining the severity of thedisease. Optimum growth of thefungus has been recorded at 26-28°C.False truffle seems to depend eitheron mushroom metabolities or ondepletion of inhibitory factor bymushroom mycelium. It is mainly adisease of A. bitorquis wherein cropis raised at 25±1°C but it alsodevelops very fast in A. bisporuswhen crop is taken under naturalclimatic conditions and temperaturerises above 20°C.

Control

1. Compost should be prepared ona concrete floor and never onuncovered soil. Because duringcomposting there is rise intemperature which activates theascospores present in the soil.

2. Pasteurization and conditioningof the compost should be carriedout carefully. Maszkiewiez andSzudyga (1999) observed thatpasteurization of compost underoptimum condition completelyeliminated the false truffleincolum in the compsot.

3. Temperature above 26-27°Cduring spawn run and aftercasing should be avoided. During

cropping, temperatures shouldbe kept below 18°C . Under suchconditions, it is practicallyimpossible to grow A. bitorquisbut disease can be managedeffectively in A. bisporus.

4. Casing soils known to harbourtraces of spores should not beused. Young truffles must bepicked and buried before the fruitbodies turn brown and spores areripe.

5. Woodwork, trays or side-boards ofshelf-beds should be treated witha solution of sodium-pentachlorophenolate at the endof the crop which was infectedwith the truffle disease. Air-drying of wood-work for 2-3months may also eradicate thepathogen.

6. Good cooking out (composttemperature 70°C for 12h.) atthe end of the crop should becarried out which will killmycelium and spores of thepathogen in the compost.Wooden trays should beseparately chemically sterilized.Thermal death point ofascospores and mycelium hasbeen reported to be 70°C for 1hr. and 45°C for 30 minutes,respectively (Sharma, 1998).

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7. Initial infection can be checkedby treating the affected patcheswith formaldehyde (2%) solution(Sohi, 1988).

6. OLIVE GREEN MOULD

Pathogen : Chaetomium olivaceum,C. globosum

The first evidence of theoccurrence of C. olivaceum in Indiawas provided by Gupta et al., (1975)at the mushroom farm at Chail,Kasauli and Taradevi. Anotherspecies, C. globosum, was laterreported from mushroom farms inHP, Delhi and Mussorie (Thapa et al.,

1979). Yield losses ranging from12.8-53.65% have been reported inA. bisporus (Sharma and Vijay,1996).

Symptoms : The earliest signs ofthe fungus consist of aninconspicuous greyish-white finemycelium in the compost or a fineaerial growth on the compost surface10 days after spawning. Frequentlyinitial spawn growth is delayed andreduced. By late spawn run, fruitingstructures that look like gray-greencockle-burns-1/16 inch in diameter,develop on straw in isolated spots ofthe affected compost. The compostwill have a musty odour. Compost

Symptoms of olive green mould

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not supporting spawn growthgenerally supports the growth ofChaetomium and other severalmoulds and hence olive green mouldis not the exclusive colonizer of blackcompost. Spawn usually grows intoareas occupied by Chaetomium,although normal spawn growth isdelayed. C. globosum is also noticedon spawn bottles.

Causal organism : Chaetomiumolivaceum Cooke and Ellis, C.globosum Kunze ex Steudel

The fungus consists of a grayishwhite mycelium which laterproduces perithecia. Perithecia of C.olivaceum are superficial, opaque,globose, thin, membranous with anapical tuft of dark bristles of setae.Asci clavate and evanescent.Ascospores dark brown, broadlyovoid, umbonate at both ends andmeasure 9-12.5x7-9.5m. Peritheciaof C. globosum are scattered orgregarious, broadly ovate or ellipsoid,often pointed at the base, thickly andevenly clothed with slender flexuoushairs. Asci oblong-clavate andevanescent. Ascospore dark, broadlyovoid, faintly apiculate at both endsand measure 8-9.5x6-8m.

Epidemiology : The infectionusually comes through air, compost

and casing soil. It appears due todefective composting in phase-IIbecause of improper pasteurizationaccompained by high temperaturesin the absence of adequate fresh air.Improper stacking of the composttrays in the pasteurization roomwhich do not allow proper circulationof the air or overfilling of the roomcauses intensive condensation whenwet steam is introduced, result innon-selective compost whichharbours Chaetomium and othermoulds. Spores are resistant to heatand are probably not killed easilyduring pasteurization. It is also wellknown that spores of Chaetomiumare already present in the compost(Munjal et al., 1977, Sharma, 1992)which are activated by bad peakheating control. When compost is toowet, penetration of air is less whichresults in the conversions ofnitrogenous compounds in wrongdirection. Unfavourable conversionsoften results in renewed productionof anhydrous ammonia whichprompts the growth of ammonia.Sometimes the temperature is toohigh in certain spots of a room, ormay be less of oxygen which oftenresults in olive-green mouldappearance. Ascospores are spreadby air flows, clothes and othermaterials used in mushroom farm.

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Control

1. The fermentation period of thecompost should not be too short.It is essential to provide an activecompost that is not too wet andhas a good structure.

2. Do not add nitrogen, ammoniumsulphate, urea, chicken manureor similar materials just beforefilling.

3. There should be sufficient timefor peak-heating and sufficientsupply of fresh air duringpasteurization. Highertemperatures (above 60°C) forlonger time should be avoided.

4. Large number of fungicidesincluding Benomyl, Thiophanatemethyl, TBZ, Vitavax, DithaneZ-78, Dithane M-45, Thiram andCaptan have been found effectiveunder in-vitro conditions (Thapaet al., 1979) and sprays ofDithane Z-78 (0.2%) have beenrecommended for checking thesecondary spread (Sohi, 1986).

8. BROWN PLASTER MOULD

Pathogen : Papulaspora byssinaHots.

Papulaspora byssina was firstreported on horse dung compost

from Missouri (Hotson, 1917).Charles and Lambert (1933) laterreported its occurrence onmushroom beds and recordeddelayed yields in the presence of thismould. This disease has also beenreported from India (Munjal andSeth, 1974) causing 90-92% yieldloss in A. bisporus. This mould hasalso been reported to cause completecrop failure in oyster mushrooms inKasuali, HP (Dar and Seth, 1981).This fungus now is frequently foundat almost all the mushroom farms inIndia appearing usually duringspawn run (Garcha et al. 1987; Kaulet . al. 1978; Sharma, 1992). Thismould has invariably been isolatedfrom different compost and casingsamples collected from mushroomfarms in northern India and theincidence of the disease has beenrecorded from 5 to 9%. (Sharma andVijay, 1996). Loss in number andweight of fruit bodies as a result ofartificial inculation of the mould hasbeen found 7.7-53.5% and 3.0-50.7%respectively (Sharma, 1990; Sharmaand Vijay, 1993).

Symptoms : It is first noticed aswhitish mycelial growth on theexposed surface of compost andcasing soil in trays as well as on sidesin bags due to moisturecondensation. This develops furtherinto large dense patches gradually

25


changing colour through shades oftan, light brown to cinnamon brown;ultimately becoming rust coloured.No mushroom mycelium grows onplaces where plaster mould occurs.

Causal Organism : Papulosporabyssina

The mycelium is brownish,septate; later produces clusters ofbrown coloured many celled,spherical bulbils measuring 60-130x30-190µ. These are inter-wovenwith a net work of hyphae, are setfree later with the death of themycelium.

Symptoms of brown plaster mould

Epidemiology : Primary infectioncomes through air-borne bulbils orcontainers, compost and casing soiland workers. Its development isfavoured by wet, soggy and wronglyprepared compost. Highertemperature during spawn run andcropping favours the diseasedevelopment. In wet, greasy compostwhich had not received enoughoxygen during fermentation andmany of amines, development of thedisease is greatly favoured. Additionof less quantity of gypsum and moregreasiness favour the diseasedevelopment.

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Control

Composting should be carriedout carefully, using sufficient gypsumand not too much water. Peak heatingshould be of sufficient duration andat proper temperatures. Thecompost should not be too wet beforeor after peak heating.Munjal andSeth (1974) recommended localizedtreatment of infected patches with2% formalin while Seth andShandilya (1978) recommended 4%formalin for its control.Largenumber of fungicides namely,benomyl, carbendazim, thiophanatemethyl, vitavax, daconil, MBC,dithane Z-78, dithane M-45, captan,thiram and copper fungicides havebeen screened under in vivo and invitro conditions by various workers(Thapa et al. 1979; Kaul et al.,1978; Dar and Seth, 1981). Sprayingof systemic fungicides at 0.1%concentration has also beenrecommended.

9. YELLOW MOULD : (Matdisease; Vert-de. gris)

Pathogen : Myceliophthora lutea,Chrysosporium luteum, C.sulphureum

All these fungi produce yellowmycelial growth in the compost.Constantin (1892) reported M. lutea

from French mushroom caves for thefirst time. Yellow mould inducingmat disease has been reported fromJammu & Kashmir (Kaul et al.,1978), Punjab (Garcha et al., 1987)and Himachal Pradesh (Thapa &Seth, 1986-87; Seth & Bhardwaj,1989) inducing 5-20% loss on theyield of button mushrooms undernatural conditions. Artificialinoculations with M. lutea atdifferent stages caused 27-89% lossin yield. The incidence of the diseasein HP has been reported from 20-60% during 1981-83 and 10-70%during 1985-86. Recently, thedisease has also been noticed inDistt. Sonepat in Haryana State.

Symptoms : The yellow mouldsmay develop in a layer below thecasing (Mat disease), form circularcolonies in the compost (confetti) orthey may be distributed throughoutthe compost (Vert-de-girs). In India,M. lutea has been reported to inducemat disease. This fungus forms ayellow brown corky mycelial layer atthe interphase of compost and casingwhich is difficult to detect during theimpregnation of casing layer by thespawn and even during the firstbreak. It becomes apparent when itdevelops its stroma like morphologyand mushroom production isseverely inhibited.

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Causal organism :Myceliophthora lutea

The mycelium is whitish at firstthen yellow to dark tan withrestricted growth and creamish ordull white sporulation. Hyphaeseptate, hyaline, branched. Itproduces three kinds of spors, (i)Smooth, ovoid terminal condia bornesingly, (ii) Smooth, thick walledchlamydospores, terminal orintercalary and (iii) thick walledspiny chlamydospores.

Epidemiology : The major sourcesof primary inoculum are air, chickenmanure, spent compost anddefectively sterilized wooden trays(Seth & Bhardwaj, 1989).Thesecondary spread is mainly throughmites followed by flies, watersplashes, picking and tools. The

fungus survives easily through thickwalled chlamydospores. Diseaseseverity is generally more at 70%moisture content of the compost and19-20°C temperature.

Control

1. Proper pasteurization of thecasing mixture is very essential.Fungus does not survive theexposure for 6 hrs. at 51°C or 4hrs at 54°C.

2. Benomyl (400-500ppm) andblitox (400ppm) sprays havebeen found effective to controlthe disease and increase the yield(Seth and Bhardwaj, 1989).Spraying with calciumhypochlorite solution (15%) iseffective for eradication of themould growth (Sohi, 1986).

Symptoms of yellow mould

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10. SEPEDONIUM YELLOWMOULD

Pathogen : Sepedonium spp.

Yellow mould disease induced bySepedonium has been reported inIndia by Thapa et al., (1991) and theincidence of the mould has beenreported by vary from 5-20% withinsignificant reduction in yieldexcept in extreme cases. One morespecies, S. maheshwarinum, has alsobeen reported by Vijay et al., (1993)with very high incidence causingsevere losses or even complete cropfailures. Bhatt and Singh (2000)have recorded 1.6 to 8% incidence ofyellow mould in Haryana and UPStates and 32 to 64% loss in yieldunder artificial inculations.

Symptoms : This mould is mainlyobserved in the compost and isinitially white in colour turning toyellow or tan at maturity. It isgenerally present in the lower layersof the compost or at bottom of thecropping bags. Various types ofdistortions in fruit bodies arecommonly observed, probably due tothe production of volatile substancesor toxins. These toxins inhibit thespawn and ultimately mushroommycelium disappears from thecompost.

Causal organism : Sepedoniumchrysosporium (Bull.) Fries., S.maheshwarianum Muker.(Hypomyces chrysosporium Tull.)

Mycelium is white initially,turns yellow to tan with age.Hyphae septate, brenched, hyaline,moderately thick, 3-5mm wide.Conidiophores erect, bear lateralsimple or botryose cluster ofbranches, 4-4.5mm wide, usuallyseptate, bearing spores singly andterminally on the branches. Twotypes of spores are produced inlarge numbers. Conidia arehyaline, thin walled, ellipsoid orpyriform, produced singly from thetips of the phialids. Second type ofspores are like chlamydosporeswhich are globse, warted, darkyellow, thick walled and 13-21mmin diameter.

Epidemology : Primary source ofinculum are probably, soil, spentcompost, air or improperly sterilizedwooden trays. The chlamydosporeare thick-walled and resistant to heatand in this spore form, the fungusmay survive peak-heat. Spores canbe spread to the compost by aircurrents prior to or during fillingoperation, during the spawningoperation or with unpasteurized orspent compost sticking to wooden

29


trays. Conditions favourable forbutton mushroom cultivation alsofavour the Sepedonium mould.Higher N content, especially in theform of chicken manure, have beenreported to favour the moulddevelopment (Vijay et al., 1993). Itsappearance in the lower layers of thecompsot has been linked with morewetness. Sharma and Sharma(2000) have reported very highpopulation of Sepedonium spp. in3-12 months old chicken manurewhich may serve as the primarysource of inoculum in long methodof compost.

Control

Strict temperature monitoringand control during compostpasteurization and an adequatepost-crop cooking out are essentialto eliminate the threat ofinfestation. Preventing the entry ofspores during spawning andspawn-running by installing high-efficiency air filters are essential.Incorporation of 0.5% carbendazimin compost and sterilizing thechicken manure (for long methodof composting) with 2% formalinand 0.5% carbendazim has givengood results (Vijay et al., 1993).

11. INK CAPS

Pathogen : Coprinus spp.

Common names : Ink weed, wildmushrooms

The appearance of inky capsduring spawn run is commonlyobserved on the mushroom beds innorthern India (Kaul et al., 1978;Garcha 1984; Sohi, 1986). Artificialinculations of C. fimetarius atdifferent loads of inoculum atspawning has resulted in 20.14-94.4% reduction in the number offruit bodies and 14.68 to 94.43%reduction in the weight of fruitbodies (Sharma 1992).

Symptoms

Ink caps appear in the compostduring spawn run or newly casedbeds and outside the manure pilesduring fermentation. They areslender, bell-shaped mushrooms.Cream coloured at first, blueish-black later and are usually coveredwith scales. This fungus sometimesgrows in clusters in beds and has along sturdy stem which oftenreaches deep into the compost layer.Several days after their appearanceink caps decay and form a blackishslimy mass due to autodigestion.

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Causal organism : Coprinusatramentarium, C. lagopus, C.commatus, C. fimetarious

Caps are 1.5-4cm wide, at firstelongated oval, later conical, thencompanulate; surface white andcovered with small white scales thatdisappear within a few hours,margin splitting as cap expands,turning into umbrella shape atdissolution. Gills 6-10cm long, upto1 mm wide, free; first white, soonturn black on liquifying stem 2-4"long, 2-3mm thick, white, shinning,hollow, fragile, tapering upwardswith a small bulb at the base. Spores

8-12x3-5mm, elliptic and black (Kaulet al., 1978).

Epidemiology

The infection generally comesthrough unpasteurized or partiallypasteurized compost or casing soil orair. Ink caps appear if the compostcontains too much N, so if too muchchicken manure is used, or if thepeak heating period is too short.These are therefore, genuineindicator moulds which arebenefited from insufficientlyconverted N containing constituentslike NH3. Ink caps can also develop

Symptoms of Ink caps

31


if insufficient gypsum is added to thecompost or if peak heating has takenplace at too low a temperature or ifthe compost is too wet and poor intexture. Ink caps can directly usefree NH4 + and can also decomposecellulose very well, in addition tolipids and lignin. They are genuinecoprophillic fungi which have anoptimum pH of around 8. The largemasses of spores released throughinking of the caps can very easilyinfect freshly prepared compost.

Control : Use properly pasteurizedcompost and casing soil. Avoidexcessive watering. Rogue out youngfruit bodies of the weed fungus toavoid its further spread.

12. CINNAMON MOULD

Pathogen : Chromelosporiumfulva, C. ollare

Common name : Cinnamon brownmould, brown mould

The occurrence of this could hasbeen reported in mushroom bedsfrom J&K (Kaul et al., 1978) Punjab(Garcha et al., 1987) and differentparts of HP (Sohi, 1988).

Symptoms : AlthoughChromelosporium fulva(Ostracoderma fulva) has been

called cinnamon brown mould, itscolour ranges from yellow gold togolden brown to cinnamon brown.The mould first appears as largecircular patches of white aerialmycelium on the compost or casing.Within few days the spores areformed and the colour changes fromwhite to light yellow or to lightgolden brown. As the spores mature,the colour changes to golden brownor cinnamon and the colony developsa granular appearance. The fungusproduces numerous cup-like fleshyfruit bodies on beds.

Causal organism :Chromlosporium fulva, C. ollare

Perfect statge : Pezizaostracoderma

Apothecium discoid, varying insize from a few mm when young to1-2cm wide when mature; cupshaped, margin wavy, often splitting,tapering to a stem like base. Stem5-9mm long. Asci cylindricalmeasuring 80-160x8-12. Ascospores8 in number, arranged in a singlerow, ellipsoid, hyaline 8-12x4-8m.Paraphyses present, hyaline.

Epidemiology : Soil, casing mixtureand damp wood are the sources ofprimary inoculum. Inoculum canblow through open doors or splash

32


from floor during cleaning. Thespores of the fungus are easily air-borne. Over pasteurized compost,over-heated patches during spawnrun, high moisture content of thecompost and excess of ammoniapresent in the compost favour thedisease development.

Control : Casing soil should not bemade completely sterile by steam orformaldehyde. Newly cased bedsshould be sprayed with dithane Z-78 and maintain proper moisturecontent in casing layer.

13. LIPSTICK MOULD

Pathogen : Sporendonemapurpurescens

Common name : Lipstick, Redlipstick

This disease has been reportedfrom mushroom farms in Punjab(Garcha et al.,1987) and HP (Sohi,1986, 1988).

Symptoms : The disease firstappears in spawned compost as awhite crystalline-like mould, rather

Symptoms of lipstick mould

33


nondiscernable from spawn. As thespore of the mould mature, thecolour changes from white to pink,to cherry red and then to dull orangeor buff. White mycelial growth ismore in loose areas of casing and cancolonize well conditioned compost. Incrops where there is a serious virusdisease, lipstick mould usuallyoccurs as a secondary disease.

Causal organism : Sporendonemapurpurescens

Mycelium whitish at first, oftentaking on a “frosty” appearance andthen forming whitish balls ofmycelium. Hyphae septate andbecome segmented into chains of l-celled, short cylindric spores withtruncate ends. Spores have reddishpigment which gives the whitishmould a cherry red colouration.

Epidemiology : Soil, casing mixtureand spent compost are the sourcesof primary inoculum. It is furtherdisseminated by water splashes orpickers. The mould is reported to beassociated with the use of chickenmanure in the compost formula; thelitter is said to carry the lipstickfungus.

Control : Good hygiene is essential.Good pasteurization andconditioning of the compost willeliminate the pathogen.

14. LILLIPUTIA MOULD

Pathogen : Lilliputia rufula (Berk& Br.) Hughes

This competitor mould has beenreported from HP and Delhi (Sethand Munjal, 1981) with an incidenceof 1-40% during 1975-1979,maximum being in Chail (HP). Itseriously restricts the spawn spreadin the compost resulting in pooryields. The sexual stage has beenidentified as Gliocladium prolificumBainer. Chicken manure, horsemanure as well as casing mixture arethe primary sources of infection.Mycelium is viable upto 3 months (at10°C) and cleistothecia upto 9months under room temperature.Use of dithane Z-78 at 20ppmconcentration has beenrecommended for the control of themould. (Seth and Munjal, 1981).

15. PINK MOULD

Pathogen : Cephalotheciumroseum Corda

This mould has been observed inJ&K (Kaul et al., 1970) and Chailand Solan in HP as a white growthon the casing soil which turns pinkin due course (Seth, 1977; Sohi,1986). Yield loss upto 90% or evencomplete crop failures have also

34


been recorded. Hyphae are septateand branched. Conidiophores erect,usually branched and slightlyswollen at the tip. Conidiaacrogenous, single, pear shaped, 2-celled, the apical cell being larger,hyaline to pink, 11-18x7.5-9.5m.Infection generally comes throughair. Mould can be checked byspraying twice thiram or captan(0.04%) on casing soil at 10 dayintervals (Guleria and Seth, 1977).

16. OEDOCEPHALUM MOULD

Pathogen : Oedocephalumfimetarium, Oedocephalum spp.

This is a common mouldobserved on mushroom beds in HPand incidence upto 60% has beenobserved in a farm at Solan during1991 (Sharma, 1991). Artificialinoculation of casing layer with O.fimetarium @ 5g inoculum per 10kgcompost bag has reduced thenumber and weight of fruitingbodies by 19.9% and 11.63%,respectively (Sharma, 1991; Sharmaand Vijay, 1993). The mould formsirregular, light silver gray patches onthe compost surface during cooldown before spawning. Afterspawning, the mould is light gray butchanges to dark tan or light brownas the spore mature. Similar growthis also recorded on casing layer.

Conidiophores of the fungus areerect with a spherical cluster of largespores at its tip end. Oedocephalumsp. in compost indicates thatammonia and amines were notcompletely eliminated duringpasteurization and conditioning.Spraying or swabbing locally with 2%formalin controls the mould.

17. WHITE PLASTER MOULD

Pathogen : Scopulariopsis fimicola

This disease has been reported tooccur commonly in different parts ofIndia by several workers (Garcha,1978; Kaul et al. 1978; Sohi, 1986;Bhardwaj et al. 1989) causing about37% loss in yield. The diseaseappears as white patches on thecompost or casing soil. Thesepatches or mycelial mats may bemore than 50cm under favourableconditions. The white growthchanges to light pink after a week ofthe formation of the spot. Spawn runis reduced significantly and undersevere conditions complete cropfailure are also recorded. Myceliumof the pathogen is septate,conidiophore short, branched, borneirregularly as lateral branches ofhyphae. Annellospores ovate,globose, round or showingtruncation, buff to avellaneous inmass, occur in chains or clusters,

35


measure 4.8-9 x 4.8 µm. Thepathogen is favoured by under orovercomposted compost which stillretains the smell of ammonia andhas high pH (more than 8). Propercomposting and addition of optimumquantities of water and gypsum arerecommended. Sprays of benomyl(0.1%) and local application offormalin (4%) after the removal of themat are helpful in controlling thedisease.

B. OYSTER MUSHROOM(Pleurotus spp.)

a. Diseases

There are four fungal diseasesreported on oyster mushroom fromIndia. Their causal agents,symptoms and control measures arepresented in Table 1.

b. Competitor moulds/weedmoulds

Compared to white buttonmushroom, the information ondiseases and competitor mouldsoccurring in or on oyster mushroomsis less. Several competitor mouldshave been reported occurring in thesubstrate used for oyster mushroomcultivation. Variations in thenumber and types of moulds aremainly due to the use of a variety of

substrates, different methods ofsubstrate preparation and theconditions and containers used forcultivation.

Competitor moulds: Differentfungi occurring in the substrate andcompeting with mushroommycelium for space and nutritionare: Arthrobotrys sp., Aspergillusniger, A.flavus, A.fumigatus,Alternaria alternata,Cephalosporium aspermum,C.acremonium, Chaetomiumglobosum, Cladosporiumcladosporoides, Coprinus retirugis,C.sterguilinus, Coprinus spp.,Cochliobolus specifer, Drechslerabicolor, Furarium moniliforme,f.moniliforme var. ferbolutinans,F.moniliforme var. subglutinans,F.graminearum, Momniellaechinata, Mucor sp., Penicillium sp.,Rhizopus oryzae, Rhizopus spp.,R.stolonifer, Stachybotryschartarum, Stilbum nanum,Stysanus medius, Sclerotium rolfsii,Sordaria fimicola, Oedocephalumgloberulosum, O.lineatum,Trichoderma viride, Trichotheciumroseum, Trichurus terrophilus andPhialospora sp. (Sharma andJandaik, 1980, 1981; Singh andSaxena, 1987; Doshi and Singh,1985; Vijay and Sohi, 1989; Das andSuharban 1991). Loss in yield in

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Table-1: Fungal diseases of oyster mushrooms in India

SN Casual organism Symptoms Control References

1. Cladobotrym apiculatum White cottony growth Spray bavistin Upadhyay etC.verticillatum on the substrate; small 50ppm al; 1987;SohiC.variospermum brown irregular sunken and

spots or fluffy growth on Upadhyayfruit bodies; soft rot and 1980;decay of sporophores Goltapeh etemitting foul smell. al. 1989

2. Gliocladium virens Fruit bodies covered by Spray 100ppm BhardwajG.deliguescens mycelium and green bavistin or et al. 1987;

spots; young pin-heads benomyl Sharma andbecome soft, brown, pale Jandaik,yellow and decay. 1983Mature fruit bodies showbrown spots enclosed byyellow halo.

3. Arthrobotrys pleuroti Fluffy growth on Spray 50ppm Ganeshan,substrate and fruit bavistin 1987bodies; infected tissuesturn yellow, waterlogged and rot.

4. Sibirina fungicola Powdery white growth Proper Sharma andon stipe, gills and the aeration and Jandaik,primordia; primordia RH essential; 1983,show brownish spray benomyl Jandaik anddiscolouration and soft twice Sharma,rot and mature fruit 1983.bodies turn fragile.

different Pleurotus spp. by thesecompetitor moulds has beenreported upto 70%. In addition tothese moulds being competitivesome have been shown to producemetabolites which directly inhibitthe growth of mushroom mycelium.However, detailed information about

these competitor moulds especiallyon their relative importance,epidemiology and management isnot yet available. Most of thecompetitor moulds have beenreported to be completely inhibitedunder in vitro and/or in vivoconditions by benomyl (50 ppm),

37


carbendazim + blitox (100ppm each)and Thiram (100ppm) (Bano et al.,1975, Doshi and Singh, 1985;Sharma and Jandaik, 1980).

C. PADDY STRAW (Volvariellaspp.)

Though paddy straw mushroom(Volvariella spp.) was the first to becultivated in India as early as 1943by Thomas and his associates atCoimbatore yet very littleinformation is available on thediseases of this mushroom. This isstill being cultivated outdoors inIndia following primitive productiontechnology with very low biologicalefficiency. Paddy straw mushroomsare subject to a number of destructivediseases/competitor moulds likeMycogone perniciosa, Scopulariopsisfimicola and Verticillium spp. inother countries. In India, largenumber of competitor moulds andfew diseases have been reported onthis mushroom. Chaetomium spp.,Alternaria sp. and Sordaria sp. havebeen commonly observed ascontaminants on wheat, kans, maize,barely and jowar beds but not onlypaddy straw bundles (Gupta et al.1970). A ‘button-rot’ disease causedby Sclerotium sp. has been reportedby Muthukrishnan (1971) andbacterial ‘button-rot’ by Kannaiyan

(1974). Combination of insecticide,fungicide and antibiotic (Malathion0.025% + dithane Z-78 or benomyl0.025% + tetracycline 0.025%) arerecommended for the managementof pests and diseases (Kannaiyanand Prasad, 1978). Several othercompetitor moulds namely, Coprinusaratus, C.cinereus, C.lacopus,Psathyrella sp., Penicillium spp.,Aspergillus spp., Rhizopus sp.,R.nigricans and Sclerotium spp.have been reported from thesubstrate (Munjal, 1975; Bahl, 1984;Purkayastha and Das, 1991,Rangaswami, 1978). Partialsterilization of the straw and sprayson the beds with captan and zineb(0.2%) have been recommended forreducing the damage. Bahl andChowdhry (1980) have reportedPodospora favrelii as a seriouscompetitor and inhibits the growthof mushroom mycelium completely.Bhavani Devi and Nair(1986) havealso recorded Rhizoctoria solani onthe substrate which reduces thesporophore formation and causesmalformation of fruiting primordia.A serious effort is urgently neededto investigate the diseases of paddystraw mushroom and recommendthe package of practices to befollowed to the growers to achievegood yields.

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D. OTHER MUSHROOMS

Sporadic attempts have beenmade to cultivate few othermushrooms like giant mushroom(Stropharia rugoso-annulata), blackear mushroom (Auriculariapolytricha), shiitake (Lentinulaedodes) and milky mushroom(Calocybe indica) in different partsof the country and the competitormoulds/diseases recorded on themare briefly mentioned below:

Sohi and Upadhyay (1989) havereported Mycogone roseaparasitizing S.rugoso-annulataunder natural conditions. The mainsymptoms are white cottony growthon gills, light brown spots on stipeand deformity of the sporophores.Cladobotryum verticillatum hasbeen reported on Auriculariapolytricha (Goltapeh et al. 1989)producing white fluffy growth onsubstrate and fruit bodies resultingin 9-96% yield loss. Sprayingcarbendazim (50ppm) has beenreported effective for controlling thedisease. Trichoderma viride,Trichoderma sp., Aspergillus spp.and Fusarium sp. have beencommonly recorded as competitors(Sharma and Thakur, unpublished)during the cultivation of winter earmushroom. During the cultivation ofC.indica, several competitor moulds

namely, Aspergillus niger, A.flavus,A.fumigatus, Rhizopus stolonifer,Mucor sp., S.rolfsii, T.viride,T.haematum, Fusarium spp. andCoprinus spp. have been isolatedfrom the substrate (Doshi et al.1991). In addition Sharma andThakur (unpublished) have alsorecorded very high incidence ofCladobotryum and Oedocephalumspp. from the casing mixture.Incidence of T.viride has beenrecorded from 15-25% in thesupplemented bags as compared to5-10% in unsupplemented ones inL.edodes cultivation (Thakur andSharma, 1992).

GENERAL GUIDELINES

In order to decide the mosteffective measures for controlling adisease in mushroom, it is necessaryto understand the size of the initialinoculum, density, the rate at whichthe disease develops and spreads andthe time when the infection takesplace.

Based on these, the followingpreventive and/or eradicative controlmeasures are necessary for themanagement of these diseases:

● Ecological-by manipulations ofenvironmental factors such astemperature, humidity andventilation.

39


● Biological-by use ofantagonistic organisms throughincorporation of biocontrolagents and organic amendments.

● Chemical-by use of safe andminimum doses of specificfungicides, antibiotic etc.

A close relationship existsbetween crop management practicesand some endemic disease problemslike dry bubble, brown blotch andtruffle. Biological agents are beingincreasingly tried throughout theworld but with a limited applicationon commercial scale. Sanitation andhygienic measures are mostessential to manage the diseaseparticularly under Indian conditionsalthough under certain situationsuse of chemicals is also inevitable.

Sanitation and hygiene

Hygiene covers all the measureswhich are necessary to allow as littlechance as possible to the pests andpathogens to survive, develop andspread. Thus hygiene and sanitationgo hand in hand at all stages ofgrowing mushrooms. Farm hygieneis the best defense a mushroomgrower has against mushroom pestsand diseases particularly during thepresent days, when use of chemicalson food crop is being discouraged.

After having gone through thedetails of different diseasesdiscussed earlier we know thatmushroom pathogens gain entry toa mushroom farm in a variety ofways. They can fly in, drift in on thewind and crawl in. Also they can becarried on people, on the vehiclesand in the raw materials. Whatmakes matter worse is that they areusually difficult or impossible to beseen with the naked eyes. Based onthe critical observations during allthe stages of mushroom production,the following steps have become aroutine practice for successfullycultivating mushroom.

● The location of mushroom unitshould be in such an area whereeffluents of chemical industriesdo not pollute the water and alsothe air is free from toxic fumes orgases.

● Floor for the preparation ofcompost should be cemented/tiled and covered with a roof.

● Substrates used for compostpreparation should be fresh,protected from rain and mixed inexact proportion.

● Pasteurization and conditioningof the compost should be foroptimum duration at right

40


temperatures as over/underpasteurization may not producequality compost and invite manydisease problems.

● Do not allow free access ofpersons working in compostingyards to spawning and othercleaner areas without changingthe dress and foot-dip. Similarly,all machinery including tractorsand fork-lift trucks should not bemoved to the cleaner areas. Afterfilling, all equipment andmachinery should be thoroughlycleaned.

● Spawn should be fresh and freefrom all the contaminants.

● All equipments used forspawning, floor and walls ofspawning area must be washedand disinfected.

● The fresh air should be filteredbefore it enters the growingrooms to exclude all particles of2 micron and above.

● Casing mixture should beproperly pasteurized (60-65C for5-6 hours).

● Casing mixture should be storedin a clean and disinfected place.

All the containers, equipmentsand machinery used for casingshould be thoroughly washedand disinfected. Keeping dust toa minimum and not to have dustyoperations going on at the sametime elsewhere on the farm isalso very helpful.

● The pickers should use cleanoveralls and gloves. Pickingshould start from new or cleanercrop towards older crops.

● Waste from picking, chogs, trash,stems, unsaleable mushroomsshould be carefully collected notallowing to fall on the floor, andbe disposed off carefully.

● Avoid surface condensation ofwater on developing mushrooms.

● Add bleaching powder (150ppm)at every watering to managebacterial disease.

● Remove heavily infected bagsfrom the cropping rooms or treatthe patches by spot application of2% formalin or 0.05% Bavistin.

● Maintain optimumenvironmental conditions in thecropping rooms to avoid abioticdisorders.

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● Control insect-pests well in timeto avoid the spread of pathogenby them.

● At the end of crop, cooking out at70C for 12 hours is very essentialto eliminate all pests andpathogens.

Use of Chemicals

It is advisable to manage thedisease in mushrooms throughhygienic measures listed above.There are only a limited number ofpesticides registered for use onmushrooms. This is becausemushrooms themselves are fungiand most of the pathogens are alsofungi thereby making the choice offungicides very difficult. Moreover,because of short cropping cycle,residual toxicity of differentchemicals is of great concern and itmust be kept below the tolerancelimit. Mushrooms are very sensitiveto fumes, toxic gases and severalchemicals. This also limits thefrequent use of chemicals inmushroom industry. Equallyimportant factor which limits theuse of fungicides for themanagement of diseases inmushrooms is the problem ofresistance. Repeated and regularapplications of the same chemicalgreatly increase the chance of

resistance. If equally effectivealternate fungicides are available theproblem of pesticide resistance canbe minimized. On the other handthere are, unfortunately only a fewpests or diseases that can becontrolled satisfactorily byenvironmental manipulation alone.Some of the most common fungicidesrecommended for the control ofmajor fungal pathogens ofA.bisporus (Fletcher et al. 1986) andused in mushroom industry are:

● Benomyl(Benlate 50wp)- Forcontrol of Dactylium, Mycogone,Trichoderma, Verticillium, mix240g/100m² with casing ordissolve in water at 240g/200litres/100m² during firstwatering.

● Carbendazim (Bavistin) same asfor benomyl.

● Chlorothalonil(Bravo or Repulse)- to control Mycogone andVerticillium. Apply as spray 2week after casing and repeat notless than 2 weeks later @ 200mlin 100-200litre water/100m².

● Prochloroz Manganese(Sporgon)- to control Mycogone,Verticillium, Dactylium, give asingle application of 300g/100litres/100m², 7-9 days after

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casing. For double application,use 113g/100litres/100m², 7-9days after casing and repeat againbetween second and thirdflushes. For triple application,use 57g/100litres/100m², 7-9 daysafter casing and after first andthird flushes.

● Thiabendazole(Tecto)- to controlDectylium, Mycogone,Verticillium, apply at the samerate as Benomyl.

● Zineb(Zineb Tritoftoral)- tocontrol Dactylium, Mycogone,Red Geotrichum and Verticillum,Use 7% dust, at 350g/100m²every week after casing or 140g/100m² before watering. Forwettable powder, 1kg/1000 litres@ 5 litre/100m² after casing andbetween flushes. For Tritoftoral,5kg/100m² at 4.5m² betweenflushes.

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III. VIRAL DISEASES

including mycoviruses, fungalviruses, mycophages, doublestranded RNA (dsRNA) plasmidsand virus-like particles (VLPs). Theterm mycophage is clearlyunsuitable since virus infection hasvery rarely been associated with lysisin fungi. Although mycoviruses mayshare some of the characteristics ofplasmids, their morphology,nucleoprotein composition and thepossession of virion-associated RNApolymerase activity are consistentwith a viral nature. The termplasmid has already been abused incurrent literature as pointed out byReanney (1976) and to denote theviruses of fungi as plasmids wouldnot find ready acceptance. The termVLPs and mycoviruses have beenused by some authors (Bozarth,1972; Saksena and Lemke, 1978),with the understanding that thefirst term applies to those particlesoccurring in fungi and having avirus-like appearance in electron-micrographs but which have notbeen isolated and characterised,whereas the second term denotesthose which have been isolated andshown to have the morphology andnucleoprotein composition generally

a) Button Mushroom

INTRODUCTION

In recent years, viruses haveincreasingly been found inassociation with fungi, an associationthat has taken one of the two forms.In the first, the fungus is the vectorof the virus and in the second,fungus is the host of the virus. Hereonly the second form of associationi.e. fungi, especially the mushrooms,as hosts of viruses will be consideredin detail which has been reviewedearlier by Raychaudhury (1978),Sharma (1991) and Sharma andKumar (2000). Although thepresence of viruses in fungi has longbeen suspected (Sinden and Hauser,1950) experimental evidence wasnot forthcoming until 1962 whenvirus particles were demonstrated indiseased mushroom (Gandy andHollings, 1962; Hollings, 1962). Todate viruses or virus-like particles(VLPs) have been reported to occurin over 100 species from 73 generaof fungi, but only a small number ofthem have been isolated andcharacterised. Several terms havebeen used for the viruses of fungi

44

attributed to viruses. Thisdistinction offers an operationalconvenience and has been widelyadopted. Since mycoviruses havenot conclusively proven to beinfectitious as purified particles,some workers prefer to apply theterm VLPs in all cases.

HISTORY AND GEOGRAPHICALDISTRIBUTION

In 1948 a very seriousinfectitious disease of white buttonmushroom (Agaricus bisporus(Lange) Sing.) was observed in theUnited States of America on a farmin Pennsylvania run by the LaFrance brothers, and thus becameknown as La France disease (Sindenand Hauser, 1950). In England, adisease inducing brown staining onthe stipe was named as ‘browndisease’ by Storey (1958). Gandy(1958) observed the most commonsymptom in the form of large waterlogged patches on stipes ofmushroom from diseased beds andproposed the name ‘watery stipe’. Asimilar, possibly the same diseasewas observed in the mushroomindustry throughout Pennsylvania.The cause of this disease wasunknown and no existingdescription appeared to fit thedisorder, hence the name ‘X-disease’was coined by Kneebone and co-

workers (1961). Since 1959 asimilar disease ‘mushroom-die-back’has been studied in Englandwherein degeneration of myceliumrather than the symptoms on fruitbodies were more predominent andhas been attributed to a complex ofatleast three different viruses(Gandy and Hollings, 1962).Schisler and co-workers (1967),reported that one of these viruseshad been isolated from a whiteisolate of La France. They advocatedthe name X-disease and die-back bedropped. In the Netherlands,disorders of this type were notreported until 1964 when a heavyoutbreak occurred causingsignificant yield losses (DielemanVan Zaayen and Temmink, 1968). InAustrailia, mushroom diseases ofviral nature probably dated back tothe early days of mushroom growingin open ridge and disused railwaytunnels in the 1930s butconfirmation of existence of die-backwas reported in 1968 (Paterson,1968). Virus disease in buttonmushroom has been reported fromIndia by Tewari and Singh (1984;1985) and have also been describedfrom New Zealand, Polland, GermanDemocratic Republic, China,Denmark, Sweden and Canada.More recently La France disease hasalso been reported from Spain.

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Viral particles have beenreported, recently in Pleurotusostreatus and P.sapidus from Chinaand in P.pulmonarius, P.ostreatusand P.columbinus from France(Table-2). A double stranded RNApolyhedral virus has also beenreported in Volvariella volvacea fromChina, Virions of different shapes

and sizes have been detected inLentinus edodes from Japan andUSA. In India, virus and virus likedisease have been on buttonmushroom (Tewari and Singh, 1984;1985; Gottapeh and Kapoor, 1990)and oyster mushroom (KrishnaReddy et al. 1993). Generalsymptoms, transmission and control

Table -2: Virus and VLPS reported in different mushrooms

S. No. Host/Disease Shape Size Country

I Agaricus bisporus Spherical 25nm Australia,La France, Watery stipe, 29nm England,X-Disease, Die-back, 35nm Holland,mushroom disease 40-50nm America, France

GDR, India

Bacilliform 18x50nm U.K.

Club shaped 60-70nm dia or France,120-170 long W. Germanywith a spherical S. Africabody of 40-50nm & acylindried tail20-30nm in dia

Rods of varing 19x9-90nm Polandlongth 19x35nm GDR

20x130nm China

II Pleurotus spp.P. colombinus Spherical 26+2nm FranceP. ostreatus IndiaP. pulmonarius Spherical 24nm ChinaP. sapidus ChinaP. florida Flexuousrods 40-600nmlong

III Volvariella volvacea Spherical 35nm China

IV L.edodes Spherical 20nm, 23nm China,36nm, 45nm Japan30nm

Stiff or 17x200x1200nm Japan15x700-900nm China18x1500nm15x16x200-300nm

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measures of button mushroomviruses are discussed below:

SYMPTOMS

The various early names coinedfor mushroom virus disease givesome indication of the diversity andvariation of symptoms caused byviral infection. The symptoms,which have frequently beendescribed may be expressedindividually or in variouscombinations and in a wide range ofseverity. The symptoms of virusdisease vary from reduced yield todistorted mushrooms. During thespawn run period, there is no visibleindication of the disease however,once casing is applied, distinctivesymptoms may be restorted whensymptomless or slightly affectedmycelial isolates are grown incompost and induced to fruit. Thefull range of symptoms that areencountered in non-hybridmushrooms are also seen in hybridmushrooms. In extreme cases allsporophore initiation is inhibitedand the vigour of the mycelium isseverely reduced while in other casesit is difficult to detect thesesymptoms. This variation dependsupon a number of factors whichinclude virus concentration, time ofinfection, strain of spawn used and

cultural conditions. The generalsymptoms observed are as below:

1. Mycelium does not permeate orhardly permeates the casinglayer or disappears after thenormal spread. Mushroomsappear only in dense clusters,maturing too early.

2. Mycelium isolated from diseasedsporophores on agar shows a slowand degenerated growth ascompared with healthymycelium.

3. The delayed appearance of thepinheads of the first flush can bean important indication of thedisease as well as the formationof fruiting primordia below thesurface of the casing layer. Assoon as these mushrooms appearabove the casing soil, their pileiare already opened.

4. Symptoms of sporophores arehighly variable. The followingabnormalilties can be foundseparately or in combination:

a) Slow mycelial growth,development of abnormalmushrooms.

b) Slow development ofpinheads, dwarfing.

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c) Delay in appearance ofsporophores, reduced yield.

d) Off-white colour of the capand early maturity.

e) Sporophores with elongatedstems and small caps.

f) Elongated slightly bent stipes,sometimes with small earlymaturing pileus.

g) Premature opening of veil.

h) Mushrooms are looselyattached to the substrate andat the slightest touch arepushed over.

i) Accelerated post-harvesteddeterioration.

j) Watery stipes, streaking inthe stipes

k) Stipes are spongy and quicklyturn brown on cutting andshow an abnormal structure.

l) Thickened barrel-shapedstipes; the veil is attached tothe thickest part of the stipe,thus lower than usually. Pileiare small and fat.

m) Brown, slimy, cap occur owingto a secondary bacterial rot,stipes are sometimes tapering

downwards during the firstflush, sometimes a few lightbrown caps can be observed.

n) Veils abnormal or absent,hard gills are common.

5) A specific musty smell can beperceived in a growing roominfested with the disease.Whereas in Pleurotus, virusinfection causes dwarfing orelogation of stipe. However, nodistinct symptoms are visible inVolvariella.

Severe and total crop losses havebeen reported due to club-shapedvirus reported in A.bisporus (Albouyet al. 1973). It has been shown to bevery difficult to produce spawn frommushroom infected with this club-shaped virus. The symptomsinduced by virus disease in L.edodesinclude dwarfing, early maturity,hardened gills and thickened,elongated or barrel shaped stipes(Deahl et al. 1986).

Causal organism

Several viruses of differentshapes and sizes have been reportedon different mushrooms. In India,virions measuring 29nm and 35 nmin diameter have been foundassociated with a virus disease of

48


button mushroom. Virus likeparticles measuring 29nm indiameter have also been reported inbutton mushroom as revealed byimmunosorbent electron-microscopy(Goltapeh and Kapoor, 1990).

CHARACTERIZATION OFVIRUSES AND VLPs

Morphology of Viruses

Experiments by Gandy andHollings (1962) and Hollings (1962)demonstrated the presence of threetypes of virus particles associatedwith the diseased mushroomshaving die-back symptoms. Two ofthe viruses had isometric particleswith a diameter of 25nm and 29nmwhile the third was bacilliform witha diameter of 18nm and a length of50nm. A fourth virus type was laterreported from England (Hollings etal., 1968) and Holland (Dieleman-van Zaayen and Temmink, 1968)having 35nm diameter. Anotherspherical virus having a diameter of40 to 50 nm was later reported fromEngland by Hollings and co-workers(1968). Thus, five different types ofviruses have been reported inEngland and the acceptednomenclature for these viruses inUK is as below:

MV-1 : Spherical particles,diameter 25nm.


MV-3 : Bacilliform particles,19x50nm.



In addition to these five viruses,two more types having club-shapedand rod-shaped particles have beenreported in A.bisporus from differentparts of the world.

Recently, spherical viral particlesof 24 to 26nm in diameter have beenshown to exist in Pleurotus ostreatus,P.sapidus, P.columbinus and P.floridafrom China and France (Liang et al.1987, 1990; Liu and Liang, 1986;Molin and Lapierre, 1989) andflexuous rods measuring 40-600 nmlong from China (Liang et al. 1990).From China, polyhedral virusmeasuring 34 nm in diameter hasbeen reported in Volvariella volvacea(Chen et al., 1988). Rods as well asspherical types of viruses have alsobeen reported in Lentinus edodes

49


from China, USA and Japan.Different viruses and VLPs reportedfrom different parts of the world havebeen summarised in Table-2. InIndia, viruses and VLPs have beenreported infecting A.bisporus(Tewari and Singh, 1984; 1985;Goltapeh and Kapoor, 1990) andP.florida (Krishna Reddy et al.,1993).

Physico-chemical Properties

As is evident from the reportsthat several viruses having variousshapes and sizes have been foundassociated with diseasedmushrooms. However, the role ofindividual virus or VLPs in inducingthe typical symptoms of the diseasehas proved inconclusive. Recentbiochemical studies havesignificantly advanced ourunderstanding of the viral nature ofthe diseases or VLPs. Further, thewidespread occurrence of VLPs inhealthy basidiocarps and mycelium(Passmore and Frost, 1974, 1979)has raised questions concerning theetiological role of viruses in disease(Frost and Passmore, 1980).Because mycoviruses typicallypossess double stranded RNA (dsRNA) genomes, the discovery ofdiscrete ds RNA molecules indiseased tissues constitutes the mostconvincing evidence for the viral

etiology of La France disease (Hicksand Haugton, 1986; Lomke, 1976;Marino et al., 1976; Ross et al., 1986;Wach, et al., 1987).

It was also reported that a viralcomplex (Sonnenberg andGriensven, 1991; Romaine andSchlagnhaufer, 1991) involving a ssRNA virus and unrelated ds RNAvirus (es) plays a role in etiology ofLa France disease. A.bisporus fruitbodies affected by La France diseasecontain the specific set of 9 ds RNAmolecules which is genome of 36nmisometric virus (Van der Lende et al.,1994; Revill et al., 1994;Zobalgogeazcoa et al., 1995; Goodinet al., 1992). The nucleotidesequence of dsRNAs M2 (1.3kb) andL3 (2.8 kb) is invariably associatedwith the disease. The average G+Ccontent of these ds RNAs was 43percent close to that of A.bisporusnuclear DNA. S3 ds-RNA (0.39 kb)is occasionally found in largeamounts in diseased mushrooms(Harmsen et al., 1991). Harmsenand Wessels (1991) reported that LaFrance disease was associated with10 differently sized dsRNAs, whichappeared to be encapsidated by virusparticles of 25 and 34 nm. One ofthese dsRNAs was also present inhealthy mushrooms. Recently, it hasalso been shown that dsRNAs L5and M2 are encapsidated by 34 nm

50


particle (Ven der Lende et al., 1994).Reverse transcription-polymerasechain reaction assay (RT-PCR)showed the diseased mushrooms tobe either singly infected by LaFrance isometric virus (LIV) ordoubly infected by La Franceisometric virus and mushroombacilliform virus (MBV). La Francedisease is associated with theinfection by two autonomouslyreplicating viruses in which LTV isthe primary causal agent and MBV,possibly pathogenic, capable ofmodulating symptoms, is notrequired for pathogenesis (Romaineand Schlagnhaufer, 1995). MBV wasfound to have a monopartite ssRNAgenome of positive sense. Theputative RNA-dependent RNApolymerase and coat proteindisplayed homology with proteinencoded by plant virusesparticularly luteoviruses andcarmoviruses.

Transmission of LIV duringbasidiosporogenesis together withspore-borne nature of causal agentplayes etiologic role of virus in LaFrance disease (Romaine et al.,1993). In two separate trials anaverage of 75 and 65 per cent of theviable basidiospores discharged fromdiseased basidiocarp were infectedby LIV. Basidiocarp showing thepresence of dsRNAs in the stipe

tissue produce LIV infectedbasidiospores. Double strandedRNA having molecular weight of3.2x10 dalton has beendemonstrated with 35nm virusparticles in Volvariella volvacea(Chen et al., 1988) and 0.85x10daltons with 24nm particles inP.sapidus and P.ostreatus (Liange etal., 1987). In A.bisporus, dsRNA hasbeen demonstrated with 25 and34nm particles (Hicks andHaughton, 1986; Romaine andSchlagnhaufer, 1989) whereas withbacilliform particles measuring19x50nm, single sRNA has beenreported (Molin and Lapierre, 1973;Tavanizis et al., 1980). In L.edodesdsRNA has been demonstrated with39nm spherical particles. Most ofthe spherical VLPs or viruses areisometric, with sizes between thelimits of 25 and 45nm diameter.Many have never been transmittedeven by hyphal anastomosis to ahealthy mycelium and nothing isknown about their sedimentationcharacteristics, number ofcomponents or the composition ofthe viral nucleic acid andpolypeptide moieties. They are stillonly the VLPs. The status of otherparticles is uncertain for differentreasons. These are regarded by someworkers as artifacts, fragments from19x50nm, MV-3 virions, seentransversely (Hollings et al., 1971).

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These particles, derived only frompreparations containing MV-3, hada UV absorption spectrum lackingthe 260nm peak of nucleoproteinvirions and could not betransmitted. However, someworkers regarded these as virions ofa distinct type.

From L.edodes isometricparticles 25, 30 and 39nm(Ushiyama and Nakai, 1975) and 30,36 and 45nm (Yamashita et al.,1975) have been recorded in Japan,but whether these refer to the samethree viruses or to four differentviruses, is not known. Too little isknown about the rods from L.edodesmeasuring 38x300nm in dippreparation and 15x200nm in thinsections to decide whether or notthese could be tobamovirus particles.

Purification Procedures

It has proved much difficult toobtain consistently goodpreparations of mushroom viruses.In repeated tests mycelium fromagar or from liquid cultures has beenreported wholly unsatisfactory as thesource of virus (Hollings and Stone,1971) for virus extraction. Most ofthe virus is lost during the differentsteps in purification and therefore,sporophores with higherconcentrations of the virus should

be taken for grinding. Mushroomscontain powerful polyphenolsoxidase system and often copiousamount of polyphenolic complexesin virus extraction. Several methodsof extraction, clarification andpurification of viruses or VLPs havebeen tried in A.bisporus with varyingdegree of success. Some of theseprocedures are:

1. Hollings and co-workers(1971)attempted extraction withphosphate buffer andprecipitation of virus with citricacid which gave best yield ofviruses MV-1, MV-3 and MV-4.Precipitation of virus byammonium sulphate or bysodium chloride pluspolyethylene glycol(PEG) gaveunsatisfactory results(Hollingsand Stone, 1971).

2. Extraction in borate or phosphatebuffer and clarification withbutanol gave preparation ofviruses MV-1, MV-2, MV-3 andMV-5 and proved very satisfactoryfor virus 2 but virtually destroyedMV-4(Hollings, 1962).

3. Extraction in phosphate buffer,clarification with ethoxy andbutoxy and butoxy-ethanols hasyielded MV-1, MV-3 and MV-4(Kitano et al., 1961)

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4. Dieleman-van Zaayen andTemmink(1968) used themethods of Hollings and co-workers (1965) and Kitano andco-workers (1961) with slightmodifications followed bydifferential centrifugation andobtained good yields of MV-1, MV-3 and MV-4.

5. In case of Pleurotus ostreatus andP. sapidus, Liang and co-workers(1990) used 0.03 M phosphatebuffer (pH 7.0) for extractionfollowed by low speedcentrifugation and densitygradient centrifugation and afairly high concentration of bothspherical as well as rod shapedvirions was obtained. Molin andLapierre (1973) have also used asimilar method for purifyingspherical virus from P.pulmonarius.

6. Chen and co-workers (1988) usedTris-HCl buffer (pH 7.6) forextracting the spherical virusfrom V.volvacea followed by threecentrifugation at 5000g each forclarification. PEG 6000 and 0.1Msodium chloride were used forprecipitation and pellets wereagain suspended in 0.05M Tris-HCl and 1M and NaCl buffer.Concentrated preparations of the

virus were obtained by furtherdifferential centrifugation.

It can not be disputed that verypure virus preparations are essentialfor chemical, physical andbiochemical studies and that manybiological investigations aredependent on the availability ofatleast partially purifiedpreparations. It must be stressedhere that no two viruses are exactlyalike and consequently there areabout as many purificationprocedures as there are viruseswhich have been purified. Toachieve a purified virus preparationone has to take into account severalfactors like selection of propagatinghost, conditions affecting virusmultiplication, selection of properhost tissue for extraction, extractingmedia (buffers, pH and molarity)method of extraction, clarificationprocedures and methods of isolation,concentration and furtherpurification.

EPIDEMIOLOGY

The wide variation in symptomsreflect the variation in the economicimpact of viruses on mushrooms. Itis possible to have yield losses soslight that they are masked by otherfactors and the growers remain

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unaware of them. Alternatively, theinfection may be so severe thatvirtually no marketable mushroomsare produced. The first appearanceof pinheads is delayed by severaldays and they remain as a small grey-fawn clump without further growth,although some may shed spores.Loss of crop varies from slight to 95per cent (Barton, 1985; Dieleman-van Zaayen, 1970; Hollings et al.,1963; Rasmussen et al., 1969;Schisler et al., 1967). Various factorslike time of infection, culturalconditions and the strains of thespawn used greatly affect the loss inyield. Dieleman-van Zaayen (1972)reported that when artificialinoculation was done from 0 to 12days after spawning, the extent ofloss due to dieback varied from 37.5to 95.6 per cent over uninoculatedcontrol. He also concluded that: a)the time of infection is much moreimportant than the amount ofinoculum; b) with early infection,the amount of inoculum is of noconsequence, and c) the amount ofinoculum has a slight negativeinfluence with later infection, which,by itself, causes a small loss in yield.A survey among more than 1000Dutch growers showed that in 1967and in the first half of 1968, one outof three mushroom farms was

contaminated and on these farmsaverage yield loss was 15 per cent.Thus in 1967, in the Netehrlands,4.5 per cent or about 7,90,000 kg ofmushroom were lost (Dieleman-vanZaayen, 1972a).

Detection Methods

Diagnosis of virus infection inmushrooms is not easy because oftwo reasons. The first reasons is thatmushroom being an anatomicallysimple organism, responds to a rangeof adverse stimuli in only a limitednumber of ways. For examplesystems like elongation of the stipe,water logging of stipe, general lossin yield and bare patches in themushroom beds may be induced byvirus infection as well as a variety ofother biotic and abiotic factors. Thesecond difficulty with diagnosis isgenerally the low virusconcentration. The differentapproaches adopted for the diagnosisof virus infection in mushroom are:

1. Symptoms on the bed.

2. Comparative growth rates ofmycelium on agar.

3. Direct electron-microscopicexamination (EM).

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4. Immunosorbent electron-microscopy (IEM or ISEM).

5. Polyacrylamide gelelectrophoresis (PAGE).

6. Enzyme-linked immunosorbentassay (ELISA).

7. Reverse transcription-polymerase chain reaction assay(RT-PCR).

Symptoms on the beds

Symptomatology has beendiscussed in detail earlier and theycertainly indicate that something iswrong. However, it is difficult toconclude with authenticity that aparticular abnormally in mushroomis only due to virus. Moreover,viruses have been detected by othermethods in apparently healthymushrooms.

Agar growth test

This was the first test to bedevised and depends upon the factsthat affected mushroom myceliumhas a slower growth rate thanotherwise identical healthymycelium. Tissue cultures ofhealthy mushrooms in 2.5 per centmalt agar at 25°C grow aggressivelyand achieve a diameter of 80 to100mm in 21 days. The periphery

of the colony will have bare denseaerial hyphae which are white orpale cream in colour and resemblecotton wool. In the centre of thecolony, the aerial hyphae largelydisappear as they amalgamate toform rhizomorphs which have theappearance of fine white threads onthe surface of the agar. Incomparison, virus-infectedmushrooms cultured in the samemanner will achieve a colonydiameter of 5 mm upto 80 to 100 mm.If the mushroom is severely infectedcolony will be flat and slightly waxyin appearance with few aerialhyphae. The colour is usually a deepcream or even light brown and cansometimes be dark in the Centre.Rhizomorphs do not generally formon diseased colonies and are oftenreplaced by very flat aggregates oftissue which give a speckled orpepper and salt appearance to thecentre of the colony (Gandy andHollings, 1962, Nair, 1973). Theadvantage of this system is that itdoes not require expensiveequipment. The disadvantages are,firstly, the long time needed toobtain a result and, secondly, therelative insensitivity of the test.

Electron microscopy

Gandy and Hollings (1962) firstobserved virus-like particles in

55


transmission electron microscopewhile examining purified andconcentrated sap from mushroomsexhibiting dieback symptoms.However, virus purification andconcentration was a complex andtime consuming process which wasnot suitable for large number ofdiagnostic tests and Hollings and co-workers (1965) found that virusparticles could be detected morequickly if the juice from diseasedmushrooms, disrupted by ultra-highfrequency sound waves, wasexamined under the electronmicroscope. Hollings and co-workers(1967) modified that detectionprocedure further wherein the juicefrom the suspected sporophore wassqueezed through a piece of finecloth. The juice thus expressed wasmixed with 2 per cent PTA (pH 7)and mounted on carbon coated gridsfor examining in electronmicroscope. Thereafter the use ofelectron-microscopy allowed severalviruses to be found in either purifiedpreparations or ultrathin sections ofdiseased mushrooms (Barton, 1985;Barton and Hollings, 1979; Chen etal., 1988; Dieleman-van Zaayen,1972b; Dieleman-van-Zaayen andIgesz, 1969; Dieleman-van Zaayenand Temmink, 1968; Hollings et al.,1968; Koons et al., 1983; Leistner,1980; Lesemann and Koenig, 1977;

Liang et al., 1990; Molin andLapierre, 1989; Mori and Mori,1974; Mori et al., 1978; Passmoreand Frost, 1979; Tavanizis et al.,1980; Tewari and Singh, 1984;Ushiyama, 1975). Virus particles(MV-1, MV-2 and MV-3) could bedetected with reasonable certainly inseverely affected mycelium in aslittle as 1mg (fresh weight) ofmycelium disrupted by sonication(Hollings et al., 1965). One of theadvantages of this technique is thespeed in the detection in sampleswhen large number of virus particlesare present. The disadvantage is itsuncertainty of detecting levels ofvirus too low to cause disease at thetime of examination but which mayindicate a potential problem.

Immunosorbent electronmicroscopy

ISEM, originally developed byDerrick (1973) is a rapid method ofdetection and cheap to perform.Although it is a serological method,monospecific sera are not necessaryits first use with mushrooms wasreported by Del Vecchio and co-workers (1978). In this procedure,electron microscope grids are coatedwith carbon which behaves muchlike activated charcoal. It stronglyabsorbs proteins and by floating

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these grids on antiserum dropletsthey become coated with antibodymolecules. This coating can thenselectively adsorb virus frommushroom extract and the antigen-antibody aggregates can be easilyseen in EM. ISEM is almost 5000times more sensitive than direct EM.

Polyacrylamide gel electrophoresis

This is a highly sensitive andspecific detection technique, and isused for detecting double strandedRNA (dsRNA) in diseasedmushrooms (Marino et al., 1976). Inorder to use this technique, the viralRNA must be extracted fromdiseased mushrooms. This can beidentified by applying thepreparation to an agar gel columnwhich is subjected to an electric field.By staining the column after apredetermined time, the RNA, ifpresent, can be identified. Thistechnique is about 20 times moresensitive than direct EM and canalso be used for detecting specificviruses. However, the drawback isthat it is dependent upon thestability of the virions during theextraction of the dsRNA. Thistechnique has been widely used indetecting dsRNA in virus infectedA.bisporus, especially Lentinusedodes (Ushiyama et al., 1977),

Pleurotus ostreatus, P.sapidus(Liang et al., 1990) and Volvariellavolvacea (Chen et al., 1988).

Enzyme-linked immunosorbentassay

Virus detection by ELISA (Volleret al., 1976) had become widespreadamong plant and animal virologistsand was a simple and fairly rapid (1-2 days) detection method. Itsdrawback was that a monospecific(perfectly pure) antivirus serum wasrequired.

Any antibody to normalmushroom constituents gave verystrong, nonspecific, false positivetests. Mushroom was detected inpreference to or as well as, virus. Thegreat difficulty in adequatelypurifying most of the mushroomviruses for antiserum productionresulted in limited use of thistechnique for detecting MV-3 and aspherical virus in P.pulmonarius(Barton, 1985; Liu and Liang, 1986).

Tests have shown that directelectron microscopy can detect MV-1 at a concentration of 1mg/ml(micro-gram per ml). A little betteris dsRNA at concentration 250mg/ml. Most ELISA tests with plant andanimal viruses can detect down to2ug/ml, an increase in sensitivity

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over direct electron microscopy of5,000 times (Barton, 1985).

Reverse transcription-polymerase chain reactionassay (RT-PCR)

Harmsen (1990) described RT-PCR detection method for thepresence of dsRNA in spawn runcompost. This is a sensitive andreliable test available for detectionof dieback disease virus at any stageof cultivation of A.bisporus. Thismethod, in principle, could beapplied to mycelium in the compostsince dsRNA L3 encodes for one ofthe coat protein of 34nm particle.However, it is especially importantto test atleast two dilutions of eachcompost extract in a rangeequivalent to 0.5-5 ug freeze driedcompost per RT-PCR reaction. Lowdilution of the samples inhibit theRT-PCR by the presence of inhibitorycompounds and high dilutions lowerthe concentration of dsRNAs beyondthe detection limit.

Transmission and spread

Through mycelium

It was revealed at an early stagethat viable mycelium could transmitthe ‘dieback’ disease (Gandy, 1960).Viable diseased mycelium would

remain behind in trays after a cropand after inadequate disinfection,would anastomose with healthymycelium in the following crop andthus transmit the virus.

This is the most common methodof transmission and has beenconfirmed by several workers(Dielman-van Zaayen, 1986;Hollings, 1962, 1972, 1982; Hollingset al., 1963). However, this ispossible only among the compatiblestrains and not in others. Forexample, A.bitorquis was not infectedafter exposure to diseased A.bisporusmycelium and spores (Van Zaayen,1976). Although A.bitorquis hasbeen regarded as highly resistant orimmuse to mushroom viruses, it mayescape infection by incompatibilitywith A.bisporus, for heterokaryosisdid not occur between the twospecies (Raper, 1976). Mycogoneperniciosa heterokaryosis did notoccur between the two species(Raper, 1976). Mycogone perniciosaand Verticillium fungicola, bothparasitize A.bisporus and theirmycelial strands penetrate theintercellular spaces in mushroomsporophores, the known viruses ofM.perniciosa and V.fungicola areserologically unrelated to any of theknown viruses of A.bisporus andthere is no evidence of any virus

58


transmission occurring betweenthem. Tubular virus particles werefound in Plicaria sp., a weed mouldgrowing among the mushrooms intrays, and in very low concentrationin the mushroom sporophores(Dielman-van Zaayen, 1967) butthere is no evidence to suggest thatany transfer of the virus took placebetween the two fungi.

Through spores

This was first demonstrated formushroom virus (MV-1) by Schislerand co-workers (1963, 1967) andsubsequently for mushroom viruses2,3 and 4 (Hollings et al., 1971).Mushroom virus 4 particles have alsobeen visualized in thin sections ofmushroom spores and germtubes(Dielman-van Zaayen, 1972b). Lastand co-workers (1967) isolated 25nmvirus particles from some ofSchisler’s spore-derived culturesand confirmed the transmission ofthe disease. Spores can infect thecompost at any stage before and/orafter spawning and aftergermination the myceliumanastomose with disease-freemycelium thereby resulting in virustransmission. Infected mushroomsusually mature too early and growerscan not pick them all before theyopen and release the spores. Spores

from diseased mushrooms oftengerminate better and faster thanuninfected spores (Dielman-vaanZaayen, 1970, Schisler et al., 1967).Over 40 per cent of spores frominfected mushrooms germinatewithin a week on agar mediumcompared with none of the healthyspores. The spore load within amushroom house fluctuates greatly;over 3 million per minute wererecorded in the exhaust air from amine shaft with an accumulation ofunpicked mushrooms (Schisler et al.,1967) under ordinary croppingconditions, 1000 to 10000 spores perm3 were estimated from cascadeimpactor and volumetric spore traps(Gandy, 1971). Spores weredetected 5cm away from exitventilators but further away nospores were trapped (Gandy, 1971)but Frost and Passmore (1979) havereported that daily meanconcentration of order of 10-10 werepresent in the compost yard atdistances of 10 to 20m from thenearest growing room exhaust.Mushroom spores have beendetected in fairly good concentrationin air samples taken from thepasteurized filtered air-zone of thefarm where peak heating, spawning,spawn run and casing were done(Frost and Passmore (1979). Gandy(1971) also detected basidiospores

59


upto 16 per m3 in a spawn run roomat GCRI where the air was filteredto exclude particles greater than 2um and detected similarconcentrations on a commercialmushroom farm with a similar air-filtration system. Since minimumdose of basidiospores necessary fortransmission of the disease laybetween 1 to 10 spores per tray(Schisler et al., 1967) the efficiencyof transmission of virus diseases inmushrooms through spores will bevery high. Thirty years or more isthe estimate given by Schisler andco-workers (1967) as the life ofhealthy spores although they did notspecify the conditions of storage. VanZaayen (1979) claimed a life of 14years of spores stored at 4C. Atkeyand Barton (1978) found that virus-infected spores stored under morestringent conditions of normal room-temperature (20C) on a window sillin full sunlight were viable and ableto transmit 25 nm and 35 nmviruses after 6.5 years although theefficiency of transmission haddeclined somewhat compared withthat of fresh spores. Nair (1976)observed that infected basidiosporeswere smaller and had thin walls butthis was not verified by otherworkers (Stalpers and Van Zaayen,1981). Isometric virus particlesmeasuring 25, 30 and 39 nm indiameter have also been transmitted

through basidiospores of Lentinusedodes (Ushiyama and Nakai, 1975).

Transmission through vectors

There is no any report about theinvolvement of any vector for thetransmission of mushroom viruses.However, a very low level oftransmission of MV-1 by mushroomphorid fly (Megaselia halterata:Diptera) was obtained whenaseptically reared insects wereallowed to feed first on purified virusfrom a sucrose density-gradient andthen on healthy mushroommycelium. There is no evidence,however, that M.halterata canacquire the virus from infectedmushroom mycelium. Hollings andGurney (1973) also failed to transmitMV-1 and MV-4 from sterile virus-infected mycelial cultures to healthyones, using aseptically rearedmites(Tarsonemus myceliophagus).However, both phorid flies and mitesdo carry the mushroom spores fromone place to another within a tray orfrom tray to tray, thereby resultingin introduction of virus inoculum.Such agencies may be importantcarriers but not vectors.

Mechanical transmission

Many workers have attempted toinfect mycelial cultures by applying

60


cell-free virus preparations but nonehas succeeded so far even when thecultures were abraded or shakenwith carborundum powder or glassblades. Very low transmission hasbeen obtained when purifiedpreparations of mushroom viruses 1,2 and 3 were hypodermicallyinjected into sporophore initials ofhealthy A.bisporus grown inscreened isolation chambers(Hollings, 1962; Holling and Stone,1971). The viruses were notsubsequently detected in theinjected sporophores but wereconfirmed in the mycelium growingbeneath them. This has beenconfirmed with MV-4 with very lowlevels of transmission (Dieleman-van Zaayen and Temmink, 1968).

Temperature and time ofinoculation had great effect onsymptom development ininoculations at casing the cream X-disease and the La France isolatesproduced more severe symptomswhen held at a cropping temperatureof 20 to 21°C than when held at 15to 16°C (Hager, 1968). Diseaseseverity was also observed to becorrelated with time of inoculation.Inoculations at spawning were moredamaging than inoculations at casing(Hager, 1968; Last et al., 1967;Schisler et al., 1967). Contaminationof trays or shelves with fragments of

mycelium provides a very importantmeans of spread of all the viruses tothe next crop causing maximumdamage. For detecting viralinfections and predicting percentageof yield losses on the basis of dsRNAbands, Batterley and Olson (1989)have standardized the samplingtechnique from the mushroom beds/cropping rooms. It is not unusual toget positive detection by PAGE orELISA tests for mushroom viruseswithout observing any virussymptoms in the sporophores. Butpositive detection results using agargrowth test and direct EM are almostalways associated with yieldreduction or symptoms of thedisease.

MANAGEMENT OFMUSHROOM VIRUSES

For adopting suitablemanagement strategies formushroom viruses, one has to keepin mind that the disease is spread byviable mycelium and spores ofdiseased mushrooms; early infectionis dangerous, especially an infectionsimultaneous with or shortly afterspawning. Upto the time of casing,the compost and mycelium must beprotected. Owing to the lack ofuseful resistance with the species,control of the disease is based largelyon the use of hygienic practices

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directed at the elimination ofdiseased mycelium andbasidiospores from the production(Schisler et al., 1967, Van Zaayen,1976). Dieleman/van Zaayen (1970,1986) has suggested variousapproaches to reduce the spread ofmushroom virus diseases which havebeen summarized below:

When the disease is not present

1. Steam the compost for 12 hoursat a temperature of 70°C. Atemptying, remove the compostquickly.

2. Spray the wood with 2 per centsodium pentachlorophenate towhich 0.5-1.0 per cent soda(sodium carbonate) has beenadded, after drying spray withwater.

3. Disinfect doors, little holes in thefloor, shutters, racks, floors andwalls with formaldehyde (notwith sodiumpentachlorophenate). Also cleanthe manure yard and adjacentpatches of ground withformaldehyde.

4. Before filling, fit spore filters,during growing time these sporefilters should be replaced once ortwice according to the amount of

dust in the air. Use a fan forextracting air.

5. Immediately after spawning, usea pesticide against flies and coverthe compost with paper. Keep thepaper moist. Wet the paper twicea week with a 2 per cent solutionof the 40 per cent commercialformaldehyde. Repeat till a fewdays before casing. Never usesodium pentachlorophenatehere. Moisten the paper beforeremoving it carefully.

6. Quickly remove cuttings andlitter and destroy.

7. The entire farm and itssurroundings should bemaintained very clean and stayso. In the working corridorformaldehyde should be sprayed.Machines, refrigerator and otherutilities should be disinfectedwith a formaldehyde solution.

8. At the first sight ofcontamination, the disease can becontrolled best by immediatelysteaming out the concernedroom.

When the disease is alreadypresent

1. Adopt practices 1,3 and 4mentioned under when thedisease is not present.

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2. Immerse the wood in a 4 per centsodium pentachlorophenatesolution to which 0.5-1 per centsodium carbonate has beenadded.

3. Pick the mushrooms when stillclosed.

4. Keep each room as a separateentity with separate clothes,shoes, steps, buckets, pickingknives, picking racks, fans etc.Kill off diseased patches with saltand cover with plastic, make thelimits of the patches rather big.First pick from the healthy partsthen from the diseased patches.Wash hands often.

5. Admit as few visitors in thediseased rooms as possible andkeep the door towards theworking corridors closed. Kill offpests in particular. Have a shortpicking period only (not morethan 4 weeks).

Heat Therapy

When infected cultures weregrown at 33C for 2 weeks, and hyphaltips then sub cultured and returnedto 25C, many of the latter showednormal growth and did not containvirus (Gandy and Hollings, 1962).However, these findings were notconclusively proved by Dieleman-

van Zaayen (1970). Rasmussen andco-workers(1972) also obtainedincreased sporophore yields whentissue and spore cultures derivedfrom symptomatic sporophores ofwhite and two cream strains wereincubated at 32C for 2 weeks. Wuestand Mataka (1989) have observedmore extensive spawn run on horsemanure compost with thesymptomatic spawn incubated at30C than the spawn incubated at 23or 27C.

Spawn Strains

Immunity to the virus disease ofthe cultivated mushroom, A.bisporushas been found in several strains ofthe white mushroom, A.bitorquis,collected from nature. Some strainsof A.bisporus do not show symptomsas markedly as others. These are thebrown, cream and off-white strains,or some smooth-white strains knownto anastomose less frequently withothers, or A.bitorquis can help toreduce the general virus inoculumand can enable economicallyworthwhile crops to be grown.Hybrid strains can anastomose withboth white and off-white strains andtherefore, their widespread culturemay reduce the effectiveness of strainalteration as a means of virus control(Fletcher et al., 1989; Romaine,1987).

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b) OYSTER MUSHROOM

A mycovirus affecting P.floridahas been detected byimmunodiffusion and ELISA testsand found related to P.ostreatus virus(Krishna Reddy et al. 1993). Butvarions measuring 26±2nm and21nm in diameter have beenreported associated with virusdisease of P.ostreatus and it is not

clear as to which virus is affecting P.florida in India.

Symptoms induced in P.floridainclude; pileus curling upwards,swollen stalks and greatly distortedbasidiocarps. Premature sporeshedding and elongation of stalk aretypical symptoms of the disease.Management practices are almostsame as described in white buttonmushroom.

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IV. ABIOTIC DISORDERS

In addition to biotic agent whichadversely affect the mushrooms,there are a large number of abioticagents which create unfavourableenvironment for the proper growthof mushrooms resulting in thequantitative as well as qualitativeloss. These abiotic agents include lowor high moisture in the substrate,pH, temperature, CO2 concentrationin the room, wind velocity, fumesand relative humidity. Many of theseagents make the substrate non-selective for mushroom myceliumand encourage other moulds andpests while some interfere with thenormal mushroom production.Management of environment is ofgreat significance in mushroomcultivation and any deviation fromthe optimum requirements may leadto various kinds of abnormalities.Since a major proportion of buttonmushrooms is being produced undernatural climatic conditions in India,the following abiotic disorders arequite frequently observed.

1. Storma

Common name : Storma, Sectors,Sectoring.

Stroma are noticeableagreegations of mushroom myceliumon surface of spawned compost or thecasing. Discrete aerial patches ofwhite mycelium form a dense tissuelayer on the substrate surface.Stroma can be easily peeled fromthe surface of compost or casing.Stroma form on the compost in smalllocalized areas and the smallerpatches can coalesce into largerareas. After casing, stroma may formon the casing above a patch ofcompost-borne stroma or on casingwhere stroma does not exist in thecompost. Stroma on casing developesin advance of pinning but rapidlyputrefies once watering begins.Mushrooms can develop on stroma,but this is somewhat unusual.

A sector is a portion of spawn thatis distinctive when compared to thegeneral appearance of spawn. Asector may be extra-white, extra-dense or extra-ordinary fluffy and isalways different from the normalspawn. Sectors appear on or in thecompost and on the casing, and tendto disappear as the crop ages.

Stroma and sectors are relatedto the genetic character of the

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spawn but are sometimes inducedif spawn is mishandled or exposedto harmful petroleum based fumesor chemicals or certain detergentsduring preparation, storage, transitor at the farm. Production practicesduring cropping also affect theappearance of these abnormalitiesbut specific relationship has notbeen elucidated. Excessive CO2 andprolonged spawn run period alsoresult in stroma formation. A fewsectors will not affect yieldadversely but the presence ofexcessive stroma may reduce yield.Large patches of stroma 8 to 12inches are often removed from thecompost or casing surfaces with thehope that next growth of spawn willbe normal and bear mushrooms.Removing patches of stroma doesnot ensure growth of mushrooms inthese areas, so removal of stroma isa matter for each farmer to decide.This disorder has been commonlyobserved in seasonal farms in HPwhere proper aeration is lacking.

2. Weepers

Common names : Strinkers,Leakers

Mushrooms described as being‘Weepers’ typically exudeconsiderable amount of water frommushroom cap. When small water

droplets exude from stem or cap,the mushrooms are called leakers.These water droplets may be fewin number and relatively isolatedfrom each other or may besufficiently numberous to cover themushrooms. The distinctionbetween a ‘leaker’ and ‘weeper’ isthat the water droplets remain asdroplets on the leaker mushroomswhile it actually falls or flows from aweeper.Weepers are usually noticedsince they are quite unusual. Aweeping mushroom can dissolve intoa white foam. Water collects on thecasing surface beneath a weeper andthe area developes a putrid odourbecoming a ‘stinker’.

Factors that induce a mushroomto become a weeper are not knownbut low-moisture compost-less than64% coupled with high moisturecasing is where weepers arefrequently seen. The combinationof these two conditions often fosterweeper mushrooms prior to andduring the first break.

Smooth white mushrooms seemsto have some sort of protectionagainst leakers and weepers. Othermajor types-off-white, cream, goldenwhite are susceptible to this malady.The disease has been recorded inthe seasonal farms in HimachalPradesh.

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3. Flock, Hard cap and Openveil

Common names : Flock, Hard cap,Open veil, Saggine socks.

Flock is a physiologicallyinduced malformation of themushroom’s cap and gill tissue. Thecap opens pre-maturely and the gillsof the affected mushrooms arerudimentary, poorly developed andhave little pigmentation. Theflocked mushrooms generallyappear in first flush and maydisappear in subsequent flushesbut in some cases it continuesincreasing in subsequent flushes.

The mechanism that causes themushrooms to be flocked is geneticand certain strains have a greatertendency to develop theabnormality. Environmentalconditions including diesel exhaust,oil-based point fumes and certainanticorrosive chemicals in steamboilers or certain diseases like die-back, brown plaster mould and falsetruffle induce flock symptoms. Hardcap is a variation of flock syndrome.With hardcap, cap and gills are asdescribed for flock and the captends to be disproportionately smallin relation to stem diameter. Hardcap mushrooms are restricted to alimited area on the casing but at

times 30% areas may producehardcaps. Hard cap means a loss ofharvestable mushrooms. Open veilis the premature opening of veilwith abnormal gill development.Open veil sometimes occurs whena period of water stress of 1 to 3 days- is followed by a generous watering.It also occurs when fumes of certainorganic chemicals drift into or arereleased in a growing room. Overall,if open veil appears, it is safe toconclude that the mushroom hadbeen under stress during itsdevelopment. This abnormality isof common occurrence in H.P. andHaryana, especially during thetermination of the crop or underhigh temperature conditions.

4. Hollow core and Brown pith

These two disorders seem toafflict cream strains much morethan other strains, although off-white strains can have hollow core.When the bottoms of the stemsare trimmed after harvesting, acircular gap is seen in the centre ofthe stem. This hole may extendthe length of the stipe or it may beshorter. When the hollow cut endportion is brown in colour the saleprice is considerably reduced. Thisabnormality seems to be related towatering and water stress.

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5. Purple stem

Common names : Purple stem,Black leg, Storage bum.

Cut stems of the mushroomsdevelop a deep purple colour withinfew hours of harvest or after beingin cold storage (36F) overnight. Attimes colour is closer to black thanpurple and it occurs in all strains-smooth white, off-white, cream andbrown. Generally mushrooms from3rd break to the end of the crop aremost susceptible. Polyphenoloxidase, an enzyme increases inlater-break mushrooms and thisenzyme influences pigmentformation. Conditions thatpredispose mushrooms to thisphenomenon are unknown but thefrequency and the amount of waterapplied before harvest seems toaffect its occurrence.

6. Rose Comb

Large lumps and swelling arevisible on the mushroom cap. Thegills often grow in the top of the captissue and even on the top of the cap.These mishappen gills make theswellings look spongy. Themushrooms can even burst or splitand then turn brown.

The abnormality is caused bygases or vapours coming fromsolvents, paint or oil products andpolluted casing soil.

7. Scales or crocodiles

Scales arise through the surfacetissue failing to grow while the capdevelops further. The main reasonfor scales being formed is poorclimate control, in particular toomuch drying out or too great airvelocities. Strong vapours offormaldehyde or pest-controlproducts in excess can also cause theouter layer of the skin of half-growmushrooms to tear off. As themushroom continues to grown, theskin bursts and so-called ‘crocodile’skin is formed. The off-white andcream mushroom strains are moresensitive to scalyness than whitemushrooms. This is the mostcommon and serious maladyaffecting button mushroom inseasonal farms in HP.

8. Long stemmed mushrooms

The presence of long stems incombination with a number of othersymtoms can indicate virus diseasesbut it is often the result of too highCO2 concentration so that the stems

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extend more (drumsticks). Withthe improvement of aeration suchconditions can be avoided.

9. Brown Discolouration

Browning of small pin heads orhalf grown mushrooms is verycommon on seasonal mushroomfarms. This may be caused by hightemperature, sprinkling at highwater pressure(maximum pressureis 0.4 atm), chlorinating with toohigh a chlorine rate [maximum rateis 500ml (10%) per 100 litre of waterper 100m²] or incorrect use of

formalin, e.g. by spraying themushrooms with a formalin solution.

10. Oyster Mushroom

As compared to white buttonmushroom, there are fewphysiological disorders recorded inoyster mushrooms. Reduced light inthe cropping room results in longerand thicker stipes and pileus ispartly reduced. Insufficientventilation (1-2% carbon dioxide)and low light exposure inducebunched growth regeneration.

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V. BACTERIAL DISEASES

Broadly, the mushroom is definedas macro-fungus with distinctivefruiting body which can be eitherepigeous or hypogeous. In this articlethe term mushroom has been usedto denote edible cultivatedmushrooms. More than 2,000species of fungi are reported to beedible throughout the world (Changand Miles, 1982). Out of these about16 genera representing more than25 species have been successfullydomesticated.

In India, three mushroomsnamely white button mushroom(Agaricus bisporus), dhingri oroyster mushroom (Pleurotusspecies) and paddy straw mushroom(Volvariella volvacea) are beingexploited for commercial cultivation.In addition to this, recently Calocybeindica which is commonly known asmilky mushroom is also gainingpopularity in some parts of thecountry and is suited for cultivationin warmer areas where A. bisporuscan not be cultivated. Thesemushrooms like any other livingorganism are attacked by several

pathogens. The present chapterdeals mainly with the bacterialpathogens which producerecognizable symptoms and causesignificant crop losses. Theexpression of disease symptoms inmushroom depends upon the stageof development of the fruit body atthe time of infection and cause of thedisease/inoculum potential present.

The bacterial diseases have beenreported from all over the world onfruit bodies of A. bisporus, A.bitorquis, Pleurotus species,Volvarella species, Lentinus edodes,Flammulina velutipes andAuricularia species and are givenalong with their causal organism(s)and distribution in Table 1.

The bacterial pathogens inducedvarieties of symptoms like blotch,mummy, pit, drippy gill, soft rot,yellowing and immature browningbut in India, bacterial diseases hasbeen reported only on fruit bodies ofA. bisporus and species of Pleurotusand Auricularia. The variousbacterial diseases reported fromIndia are discussed as under:

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Table 3: Bacterial diseases of edible cultivated mushrooms

Mushroom Disease Causal organism Distribution Reference

Agaricus bisporus Bacterial blotch Pseudomonas tolaasii Worldwide Fletcher et al.P. fluorescens (1986)

Ginger blotch P. gingeri** UK, Netherlands Fletcher et al.(1986)

Drippy gill** P. agarici UK, Netherlands Fletcher et al.(1986)

Mummy P. aeruginosa UK Wuest andZarkower(1991)

A. bitorquis Bacterial blotch P. tolaasii Worldwide Fletcher et al.(1986)

Soft rot Bukholdria gladioli Worldwide Guleria et al.pv. agaricicola (1987)

Oyster mushroom Bacterial rot P. alcaligens** India Biswas et al.(Pleurotus spp.) (1983)

Brown blotch P. tolaasii Japan, Fermor (1986)Australia Ferri (1985)Netherlands

Yellow blotch P. agarici India, USA Jandaik et al.(1993b)Bessette et al.(1985)

Fist-shaped P. fluorescens Belgium, Italy Poppe et al.Fruit bodies* and Europe (1985)

Other mushrooms

Volvariella spp. Bacterial rot Pseudomonas sp. India KannaiyanIndonesia (1974) Fermor

(1986)

Lentinus edodes Browning* P. fluorescens Japan Komatsu andGoto (1974)

Flammulina Brown soft rot* Erwinia sp. Japan Phawicitvelutipes (1985)

* Not recorded from India

** Invalid names

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Bacterial disease(s) of Agaricusspecies

Bacterial blotch

Bacterial blotch of mushrooms isalso known as brown blotch andbacterial spot.

Occurrence and losses

Blotch is one of the most commonand serious diseases of A. bisporusand is responsible for considerablelosses. The disease also affects. A.bitorquis. The disease was firstdescribed by Tolaas (1915) fromAmerica and later Paine (1919)identified the organism as P. tolaasii.From India, it was first reported in1976 (Guleria, 1976). Bacterialblotch disease reduces crop yieldbecause lesions develop on thesurface of mushroom caps makingthe mushrooms unmarketable. Thedisease has been reported fromalmost all mushroom growingcountries of the world. The diseasecauses 5 to 10 per cent losses in yield(Fermor, 1986; Vantomme et al.,1989). In Australia, bacterial blotchis second in economic importanceonly to the virus disease complex(Nair, 1969) and substantial lossesoccurred particularly after harvestand overnight storage of mushroomsat low temperature.

Symptoms

Bacterial blotch of white buttonmushroom is characterized by brownspots or blotches on the pilei and inmore severe cases, on the stipes.Circular or irregular yellowish spotsdevelop on or near the margins of thecap which enlarges rapidly underfavourable conditions and coalesce toform rich chocolate brown blotchesthat are slightly depressed. The mostcharacteristic symptom of bacterialblotch is the occurrence of darkbrown areas of blotches on thesurface of the cap. These may beinitially light in colour but mayeventually become dark brown.Severely affected mushrooms may bedistorted and the caps may splitwhere the blotch symptoms occur.Brown and slightly concaved spotsappeared on the surface of thediseased fruit bodies. Light infectionof mushroom caps produced a yellowlight brown spotting on the surface,but the common symptom associatedwith infection was appearance ofbrown, slightly sunken lesions ofvariable size and mushroom tissueswere usually affected to a depth of 1to 3 mm. Mushrooms often becomeinfected at a very early stage in theirdevelopment. The enlargement ofthe spots on the cap surface isdependent upon environmentalconditions and is favoured by

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temperatures of at least 20oCtogether with the presence of waterfilm.

Casual organism

Tolaas (1915) described a causalorganism as a pathogenic strain ofPseudomonas fluorescens, but Paine(1919) while working with otherisolates found differences in theiraction on nitrates and starch and assuch proposed the name P. tolaasiiPaine. Lelliot et al. (1966) showedthat P. tolaasii was indistinguishablefrom some isolates of P. fluorescensand suggested that P. tolaasii couldbe considered as one of the naturalconstituents of microflora ofmushroom beds. Fahy (1981)observed that members of P. tolaasiicontained both pathogenic and non-pathogenic strains which werecommon on mushroom.

Olivier et al. (1978) reported theappearance of both smooth andrough forms of P. tolaasii and claimedthat the smooth form was non-pathogenic. Wong and Preece (1979)proposed the white line in agar andmushroom tissue rapid pitting testsfor the identification of P. tolaasii.They observed that a sharply definedwhite line of precipitate was formedin Pseudomonas agar F between theopaque white colonies of P. tolaasii

and translucent colonies of certainunidentified pseudomonads. Thevisible interaction has been utilizeda s specific and reliable method forthe identification of P. tolaasii.

Epidemiology

Casing and airborne dust are theprimary means of introducing theblotch pathogen into a mushroomhouse. Even after pasteurization thebacterial pathogen is present in mostcasing materials. Occurrence of thedisease is associated with the rise inthe bacterial population on themushroom cap rather than in thecasing. Blotch can develop on cap,stipe or both at any stage ofmushroom development. Bacteriapresent on mushroom surfacereproduce in moist conditionsespecially when moisture or freewater film persists for more than 3hours. Once the pathogen has beenintroduced at the farm, it maysurvive between crops on thesurfaces, in debris, on tools andvarious other structures. It is also anatural inhabitant of both peat andchalk. When the disease is presenton the farm, its secondary spreadmay take place through workers,implements, ingredients, mushroomspores, debris etc. Sciarids and mitesare also important carriers of thepathogen beside water splashes.

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Management

Ecological management :Manipulation of relative humidity,temperature, air velocity and airmovements are of great significancein managing the disease.Temperature above 20oC andrelative humidity of more than 85per cent should be avoided.Additional ventilation and aircirculation after watering canensure the quick drying ofmushrooms. Temperaturefluctuations at higher relativehumidity leading of watercondensation must be avoided.

Biological management : Isolatesof P. fluorescnes and otherantagonistic bacteria have resultedin 30 to 60 per cent control ofbacterial blotch. Many selectivebacteriophages have also been foundeffective against P. tolaasii withoutany significant effect of P.fluorescens. Spraying the casing soilwith a mixture of P. fluorescens andbacteriophage has resulted in morethan 80- per cent control of blotchsymptoms.

Chemical management :Application of terramycin 9 mg persquare foot, streptomycin (200 ppm),oxytetracycline (300 ppm),kasugamycin and kanamycin has

been found effective in managing thedisease.

Physical management :Pasteurization of casing soils bysteam/air mixture and short wavelength irradiation have beenreported effective in eliminating thebacterial pathogen but over-heatingshould be avoided otherwisebiological vacuum will be createdand successive invasion of mouldswould be very high. The introductionof water retentive acrylic polymersas a component of casing soilmixture is also claimed to reduce thedisease.

Other bacterial diseases ofAgaricus species

Bacterial pathogen other than P.tolaasii recorded on Agaricus speciesare P. agarici, P.aeruginosa andBukholder gladioli pv. agaricicola.However, P. gingeri is considered tobe a part of the P.tolassii (Miller andSpear, 1995).

Bacterial disease(s) of oystermushroom

Till date, four bacterialpathogens namely, Pseudomonasalcaligens, P. tolaasii, P. agarici andP. fluorescens have been reportedparasitising Pleurotus fruit bodies

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and causing considerable economiclosses to the growers. Among these,P.agarici and P. alcaligens (not validname) have been reported fromIndia and are described as under:

Yellow blotch

Occurrence and losses

In India, heavy incidence ofyellow blotch was reported (Jandaiket al., 1993) which resulted incomplete failure of crop in some ofthe mushroom units.

Symptoms

The disease appears as blotchesof varying sizes on pilei sometimesdepressed, yellow, hazel-brown, fawnor orange in colour. When thedisease appears at primordialformation or pinhead stage, it affectsthe total group of early fruit bodiesor only a part of them. Infected fruitbodies turn yellow and remainstunted. The slimy appearance of theinfected fruit bodies under highrelative humidity (more than 90%)is a common symptom. If the relativehumidity is less than 75 per cent, theblotched fruit bodies give appearanceof burnt ulcers.

Causal organism

Pseudomonas agarici is a gramnegative rod shaped and motile. The

colony is buff, circular, pulvionate,semiopaque and 2 to 6 mm indiameter. Oxzidase and catalase testswere positive and starch hydolysisand nitrate reduction were negative.The bacteria can utilize benzoate,citrate and gluconate efficiently. Incarbohydrate media, acid wasproduced from glucose, maltose andfructose. There was no acidproduction in sucrose, sorbitol,inositol and cellobiose.

Epidemiology

The disease incidence is moreunder warm and humid conditions.The pathogen is easily spread insidethe mushroom farm through watersplashes, workers, tools andmushroom flies. When the humidityis more than 90 per cent the fruitbodies gave slimy appearance andfinally fruit bodies start rotting andsmelling foul within next twentyfour hours. Presence of water film onthe surface of fruit bodies is quitefavourable for earlier appearance ofsymptoms.

Management

Environmental manipulation :High relative humidity andcontinuous persistence of water filmon the surface of pilei enhancebacterial multiplication. Hence,proper ventilation and careful

75


watering coupled with monitoring ftemperature in the mushroom unithelp in limiting the diseaseincidence.

Use of chemicals : The regularapplication of chlorinated watercontaining 100-150 ppm of freelyavailable chlorine (FCA) at 3 to 5days interval help in minimizinglosses due to bacterial pathogen, Useof oxytetracycline and streptocyclinehave also been reported.

Biological management :Biocontrol of yellow blotch of oystermushroom appears to offer a viableproposition, especially with theincreasing awareness amongconsumers about the use ofchemicals in mushroom units. Thepossibility of using bacteriophages ascontrol agent for plant diseasescaused by various bacterialpathogens including Pseudomonadshas been reported and it may haveapplication in mushroom industry aswell.

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VI. SELECTED REFERENCES

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3. Bhavani Devi and Nair, MC.1986. Popularization ofVolvariella spp. in the tropics. In:Beneficial Fungi and TheirUtilization (Nair MC andBalakrihnan eds) Scientific Pub.Jodhpur pp 25-33

4. Bhatt, N and Singh, RP. 2000.Incidence and lossess in yield byfungal pathogens encounteredfrom the beds of A. bisporus.Indian. J. Mush. 18: 46-49

5. Biswas, P, Sasrkar, BB,Chakravarty, DK and Mukherjee,N. 1983. A new report onbacterial rotting of Pleurotussajor-caju. Indian Phytopath. 36:564

6. Doshi, A, Sharma SS and Trivedi,A. 1991. Problems of competitormoulds and insect-pests andtheir control in the beds ofCalocybe indica P&C. Adv. Mush.Sci.p57

7. Earana,N, Mallesha, BC andShetty, KS. 1991. Brown spotdisease of oyster mushroom andits control. Mush. Sci. 11(2): 293-311

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9. Garcha, HS. 1984. A Manual ofMushroom Growing. PAUpublication 71p

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11.Goltapeh, EM, Jandaik, CL,Kapoor, JN and Prakash, V. 1989.Cladobotryum verticillatum – a

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new pathogen of Jew’s earmushroom causing cobwebdisease. Indian Phytopath. 42:305

12.Goltapeh, EM and Kapoor, JN.1990. VLP’s in white buttonmushroom in North India.Indian Phytopath 43: 254

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14.Guleria, DS and Seth, PK. 1977.Laboratory evaluation of somechemicals againstCephalothecium mould infectingmushroom beds. Indian J. Mush.3(1): 24-25

15.Guleria, DS, Thapa, CD andJandaik, CL. 1987. Occurrence ofdiseases and competitors duringcultivation of A. bitorquis andtheir control. Natl. Symp. Adv.Mycol. PU Chandigarh pp55-56

16.Gupta, GK, Bajaj, BS andSuryanarayana, D. 1975. Studieson the cultivation of paddy strawmushroom( Volvariella volvaceaand V. diplasia) IndianPhytopath 23: 615-620

17.Jandaik, CL, Sharma, VP andRaina, R. 1993. Yellow blotch ofPleurotus sajor-caju (fr) Singer-a bacterial disease new to India .Mush. Res. 2: 45-48

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21.Kumar, S and Sharma, SR. 1998.Transmission of parasitic andcompetitor moulds of buttonmushroom through flies.Mush.Res. 7(1):25-28.

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23.Kumar S and Sharma SR 2002.Prospects of IPM in themanagement of mushroom pests.In Current Vistas in MushroomBiology and Production(Upadhyay RC, Singh SK and RaiRD eds), MSI, NRCM, Solan pp213-224

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26.Nair, NG. 1976. Diagnosis ofmusheroom virus diseases. Austr.Mush. Growers Assoc. J. 2(5): 22-24

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31.Seth, PK and Dar, GM. 1989.Studies on Cladobotryumdendroides causing cobwebdisease of A. bisporus and itscontrol. Mush. Sci. 12(2): 711-723

32.Seth, PK and Munjal, RL. 1981.Studies on Lilliputia rufala(Berk. and Br.) Hauges and itscontrol. Mush. Sci. 11(2): 427-441

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34.Sharma , AD and Jandaik, CL.1983. Some preliminaryobservation on the occurrence ofSibirina rot of cultivatedmushrooms in India and itscontrol. Taiwan Mush.6(1,2): 38-42

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35.Sharma, SR. 1991. Viruses inmushrooms-a review. Adv. Mush.Sci. p61

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37.Sharma, SR. 1994. Survey fordiseases in cultivatedmushrooms. Ann. Rep. NRCM,pp23

38.Sharma, SR. 1994. Viruses inmushrooms. In: MushroomBiotechnology (Nair MC,Gokulapalan C, Das L eds). Pp658-685. Indus PublishingCompany, New Delhi.

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40.Sharma, SR and Kumar, S. 2000.Viral diseases of mushrooms. In:Diseases of Horticultural CropsVegetables, Ornamentals andMushrooms (Verma, LR andSharma RC eds). Pp 166-178.Sceintific Publishers Jodhpur.

41.Sharma, SR and Vijay, B. 1993.Competitor moulds- a serious

threat to A. bisporus cultivationin India. Proc. Golden jubileeSymp. Hort. Soc. India. Bangalorep 312-313

42.Sharma SR and Vijay, B. 1996.Prevelance and interaction ofcompetitor and parasitic mouldsin A. bisporus. Mush. Res. 5(1):13-18

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in India and their control. IndianJ. Mycol. Pl. Path. 18: 1-18

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