Potential of antimicrobial volatile organic compounds to control Sclerotinia sclerotiorum in bean seeds

Maurcio Batista Fialho(1), Maria Heloisa Duarte de Moraes(1), Annelise Roberta Tremocoldi(1) and Srgio Florentino Pascholati(1)
(1) Universidade de So Paulo, Escola Superior de Agricultura Luiz de Queiroz, Departamento de Fitopatologia e Nematologia, Caixa Postal09,CEP13418900Piracicaba,SP,Brazil.Email:fialhomb@hotmail.com,mhdmorae@esalq.usp.br,annetremocoldi@hotmail.com, sfpascho@esalq.usp.br

abstract Theobjectiveofthisworkwastoevaluatethepotentialofanartificialmixtureofvolatileorganic compounds(VOCs),producedbySaccharomyces cerevisiae, to control Sclerotinia sclerotioruminvitroand in bean seeds. The phytopathogenic fungus was exposed, in polystyrene plates, to an artificial atmosphere containingamixtureofsixVOCsformedbyalcohols(ethanol,3methyl1butanol,2methyl1butanoland phenylethylalcohol)andesters(ethylacetateandethyloctanoate),intheproportionsfoundintheatmosphere naturally produced by yeast. Bean seeds artificially contamined with the pathogen were fumigated with the mixture ofVOCs in sealed glass flasks for four and seven days. In the in vitro assays, the compounds 2methyl1butanoland3methyl1butanolwerethemostactiveagainstS. sclerotiorum,completelyinhibiting itsmycelialgrowthat0.8LmL-1,followedbytheethylacetate,at1.2LmL-1.Beanseedsfumigatedwiththe VOCsat3.5LmL-1showeda75%reductioninS. sclerotiorumincidenceafterfourdaysoffumigation.The VOCsproducedbyS. cerevisiaehavepotentialtocontrolthepathogeninstoredseeds. Indexterms:Phaseolus vulgaris,alternativecontrol,fumigation,whitemold,yeast.

Potencial de compostos orgnicos volteis antimicrobianos no controle de Sclerotinia sclerotiorum em sementes de feijo
ResumoOobjetivodestetrabalhofoiavaliaropotencialdamisturadecompostosorgnicosvolteis(COVs), produzidosporSaccharomyces cerevisiae,nocontroledeSclerotinia sclerotioruminvitroeemsementesde feijo.Ofungofitopatognicofoiexposto,emplacasdepoliestireno,aumaatmosferaartificialquecontinha umamisturadeseisCOVs,formadaporlcoois(etanol,3metil1butanol,2metil1butanolefeniletilalcool) esteres(acetatodeetilaeoctanoatodeetila)nasproporesencontradasnaatmosferanaturalmenteproduzida pela levedura. Sementes de feijo artificialmente contaminadas com o patgeno foram fumigadas com a misturadeCOVsemfrascosdevidroseladosporquatroesetedias.Nosexperimentosinvitro,oscompostos 2metil1butanol e 3metil1butanol foram os mais ativos sobre S. sclerotiorum, com inibio completa docrescimentomicelialnaconcentraode0,8LmL-1,seguidospeloacetatodeetilanaconcentraode 1,2 LmL-1.AssementesdefeijofumigadascomosCOVsnaconcentraode3,5LmL-1apresentaram 75%dereduodaincidnciadeS. sclerotiorumapsquatrodiasdefumigao.OsCOVsproduzidosporS. cerevisiae possuempotencialdecontroledopatgenoemsementesarmazenadas. Termosdeindexao:Phaseolus vulgaris,controlealternativo,fumigao,mofobranco,levedura.

Introduction
Sclerotinia sclerotiorum (Lib.) de Bary is a broad host range soilborne fungus and the causal agent of white mold in many crops of economic importance. Boland&Hall(1994)reported75families,278genera and408speciesashostsofthispathogen,whichcauses one of the most serious and destructive diseases in irrigatedcrops.

S. sclerotiorum dissemination by infected seeds playsakeyroleintheinfestationofareaspreviously freeofthepathogen.Theproposedtoleranceindexfor thisfungusinseedcertificationprograms,inBrazil,is zero(Mentenetal.,2006),sincetheoccurrenceofthe pathogeninseeds,evenatlowrates,cancauselarge productionlosses,andonceintroducedandestablished inanarea,itseradicationispracticallyimpossibleina shortperiodoftime.

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Seed treatment with fungicides is an important measure to control pathogens carried by them. However, this kind of control is not completely effective, especially if the pathogen is transported in the inner part of the seed. Problems associated with fungicideresistanceandtheconsumersperceptionon thepotentialimpactoftraditionalpracticesofcontrol on human health and environment have led farmers and researchers to consider alternative methods to plantdiseasecontrol(Punja&Utkhede,2003). During plantpathogen interaction, microbial antagonists can interrupt some stages of the disease or the pathogens life cycle. This can occur through parasitism, competition for space and nutrients, productionofhydrolyticenzymes(Punja&Utkhede, 2003) and through antimicrobial compounds (Vinale etal.,2006),includingvolatiles(Strobel,2006). Volatile organic compounds (VOCs) usually have lowmolecularweightandhighvaporpressure.They are active at very low concentrations and belong to severalchemicalgroups,suchasalcohols,aldehydes, ketones,esters,lactones,terpenesandsulfurcompounds (Wheatley, 2002). Because of their volatility, these compoundscantravellargedistancesinastructurally heterogeneous environment composed of solids, liquidsandgases(Wheatley,2002),whichisamajor advantageforthiskindofantimicrobialagent. The VOCs have received limited attention in comparison to the antagonism caused by mediumdiffusiblecompounds;however,newfindings have recently brought these volatile metabolism productsintofocus.Mostofthestudiesontheproduction ofantimicrobialVOCsarerelatedtoMuscodor albus, Trichodermaspp.andBacillusspp.(Humphrisetal., 2002;Grimmeetal.,2007;Leelasuphakuletal.,2008) controllingphytopathogenicandwooddecayfungi. An alternative method for S. sclerotiorum control is seed fumigation by antimicrobial VOCs in closed chambers, during storage. Muscodor albus, an endophytic fungus isolated from the cinnamon tree, isawellknownproducerofantimicrobialVOCsand hasbeentestedtocontrolseveralpathogensininfested soils,fruitsandseedsinstorage(Strobel,2006).Natural and artificial mixtures of VOCs have inhibited by 100%thegrowthofseveralplantpathogens,including S. sclerotiorum, and effectively served as fumigants offungalcontaminatedseeds(wheat,chickpea,corn, barley and canola) during storage. Propanoic acid,

2methyl and several related esters were associated withVOCsbioactivity(Ezra&Strobel,2003). Previous work has shown that the yeast Saccharomyces cerevisiae isolated from fermentation processesforfuelethanolproductionproducesVOCs that are able to inhibit the in vitro development of Guignardia citricarpa, the causal agent of citrus black spot (Fialho et al., 2010). That was the first known report on the production of VOCs by yeasts with antimicrobialactivityagainstplantpathogens.TheVOC composition was identified by gas chromatography with mass spectrometric detection (GCMS) and the artificial mixture mimicked the effects of the natural VOCsproducedbyyeast(Fialhoetal.,2010). The objective of this work was to evaluate the potential of an artificial mixture of volatile organic compounds,madeofsixcompoundsfromS. cerevisiae, to control S. sclerotiorum in vitro and in inoculated beanseeds.

Materials and Methods

TheeffectoftheVOCmixturewasevaluatedinvitro in separated experiments.The tested plant pathogens evaluatedwere:S. sclerotiorum, Fusarium oxysporum f. sp. phaseoli and Colletotrichum lindemuthianum, which were isolated from infected bean seeds, and maintained at the Departamento de Fitopatologia e Nematologia da Escola Superior deAgricultura Luiz de Queiroz, Universidade de So Paulo, Piracicaba, SP, Brazil. Based on the results obtained by GCMS analysis of the gaseous atmosphere produced by S. cerevisiae(Fialhoetal.,2010),anartificialmixture of VOCs was made using authentic commercial standards(SigmaAldrich,St.Louis,Missouri,USA). Thesixcompoundsusedwerepositivelyidentifiedin themixture,andtheconcentrationofeachoneofthem wasbasedonitsrelativeproportion(%)inrelationto allothercomponents(Table1). Twosectiondividedpolystyreneplates(90x15mm, 60mLvolume) (J.Prolab,SoJosdosPinhais,Paran, Brazil)wereusedforbioassays.A5mmagarplugwith themyceliumofeachphytopathogenwastransferred to one side of the plate, containing 10 mL of potato dextroseagar(PDA)medium.Apieceofsterilecotton wool,containing100L(2LmL-1airspace)ofthe artificial mixture, was added to the opposite side of the plate. The control consisted of plates containing

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the pathogen in the absence of the artificial mixture. Theplateswereimmediatelysealedwithparafilmand kept at 26C under a 12 h photoperiod (fluorescent light). The mycelial growth was evaluated based on theaveragebetweenthetwoopposingmeasurements whenthecolonyincontrolplatesreachedtheborders. Attheendoftheassays,theagarplugsweretransferred totheplatescontainingPDAmediumintheabsenceof theartificialmixtureinordertoverifyfungalviability. Theassayswerecarriedoutinfivereplicates. Due to the higher in vitro susceptibility of S. sclerotiorum to the VOCs and to its economical impact on several crops, in another experiment, bean seeds with S. sclerotiorum were fumigated with the artificial mixture ofVOCs. For the artificial inoculation, bean seeds (Phaseolus vulgaris L.) from the cultivar Carioca, free of S. sclerotiorum, were used. Seeds were submitted to surface sterilization withsodiumhypochlorite(1%)for2min,washedin sterile distilled water and dried at room temperature for 24 h. For inoculation, seeds were placed in Petri disheswithPDAmedium,colonizedwithafivedays old S. sclerotiorum culture (Peres et al., 2002). The incubation was performed at 21C, in the dark, for 48h.Seedsweredriedatroomtemperaturefor72h, mixedwithhealthyseedsintheproportion1:2(w/w), andhomogenized. Forfumigation,200gofseedswereplacedin310mL glass flasks, and different volumes of the artificial mixturewereaddedtoapieceofcottonwoolfixedto the cap in order to obtain the desired concentrations inside the flasks (0.875, 1.75 and 3.5 L mL-1 air space). The flasks were closed, sealed with parafilm andstoredat25Cinthedark,forfourandsevendays. The control treatment consisted of flasks without the

artificial mixture. After the fumigation period, the seeds were removed from the flasks, placed in paper bagsandstoredat4Cuntilmicrobialanalysis. The seeds were submitted to seed health test for detectionofS. sclerotiorumusingtheblottermethod. Two hundred seeds per treatment (four replicates of 50seedsforeachtreatment)wereanalyzed.Tenseeds weredistributedequidistantlyinsidepolystyreneplates (90x15mm)onthreesheetsoffilterpaperpreviously moistenedwithsteriledistilledwater.Theplateswere wrapped in paper and placed inside a dark plastic bagtomaintainhumidityandpreventfromlight,and keptat10C(Koch&Menten,2000).Theevaluation wasperformedafter7and14daysofincubation,by observing the development of typical mycelium and theformationofsclerotiabythepathogen. Toevaluatetheindividualeffectofeachcompound intheVOCmixture,anotherinvitroassaywascarried out, exposing the pathogen to the different VOCs at 1LmL-1airspace.Inanotherassay,themostbioactive compounds were tested at different concentrations (0.0 to 2.0 L mL-1 air space). The control consisted of plates with the pathogen in the absence of volatiles. Inbothassays,theplatesweresealed,incubatedand evaluated as described above. Four replicates were usedforeachtreatment. The experimental design used in this study was completelyrandomized,andthemeanswerecompared bytheTukeytest,at1%probability.

Results and Discussion

S. sclerotiorum, C. lindemuthianumandF. oxysporum f. sp. phaseoli used in the previous in vitro control experiment, showed different susceptibility to the artificial mixture of VOCs used (Table 2). The highest inhibition level was observed on S. sclerotiorum (77% of inhibition), while the mycelial growth of C. lindemuthianum andF. oxysporum f. sp. phaseoli was
Table 2. Influence of the volatile organic compounds artificial mixtureontheinvitrodevelopmentofbeanpathogens(1).
Fungus Sclerotinia sclerotiorum Colletotrichum lindemuthianum Fusarium oxysporum Mycelialgrowth(cm) Control Volatiles 5.51a 1.29b 4.43a 1.55b 4.48a 3.00b

Table 1. Composition of the volatile organic compounds producedbySaccharomyces cerevisiae.

Compound(1) Ethanol Nonidentified Ethylacetate 3methyl1butanol 2methyl1butanol Phenylethylalcohol Ethyloctanoate Relative% 85.3 1.5 1.8 6.9 2.4 0.7 1.4

(1) Identificationbygaschromatographywithmassspectrometricdetection (GCMS)(Fialhoetal.,2010).

(1) Meansfollowedbyequalletters,inthelines,donotdifferbytheTukey test,at1%probability.

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inhibited65and33%,respectively.Fialhoetal.(2010), usingthesameartificialmixtureofVOCs,verifiedthat themycelialgrowthofG. citricarpa wasreducedmore than87%.Fusariumspp.seemstobemoreresistantto antimicrobial volatiles. Stinson et al. (2003) reported thattheVOCsproducedbyGliocladium sp. fungus or their artificial mixtures inhibited F. oxysporum only partially. Similarly, F. solani was the less susceptible amongseveraltestedfungitotheVOCsproducedby M. albus (Strobel et al., 2001). Atmosukarto et al. (2005) studied the susceptibility of plant fungal pathogens, from several taxonomic groups, to the artificial mixture of VOCs produced by M. albus. The artificial mixture was able to inhibit 100% of the mycelial growth of all tested microorganisms, exceptF. culmorum.Inthatstudy,Colletotrichum spp. also showed less susceptibility to the VOCs, since C. coccodes and C. gloeosporioides remained alive despite the total inhibition of mycelial growth. The same behavior was observed by Mercier & Jimnez (2004)forC. coccodesandC. acutatumwhenexposed totheVOCsproducedbyM. albus. A possible determinant factor in the sensibility differences among microorganisms to antimicrobial compounds,includingalcoholsandesters,istheplasma membranecompositionandtheresistancemechanisms usedtocounteractdeleteriouseffects,suchastheability tochangethecompositionandstructureoftheplasma membrane (Weber & Bont, 1996), and processes of detoxification, efflux transport and storage (Duffy et al., 2003). However, the particular mechanisms involvedintheprotectionagainstvolatilesarenotyet knownindetail. In the control treatment, the white mold incidence in bean seeds was 29.5% (Table 3). The fumigation with the VOCs at 0.875 L mL-1 and 1.75 L mL-1

caused significant incidence reduction. Nevertheless, the highest fungal reduction (75%) was observed at 3.5 L mL-1. There was no statistical difference in relationtotheexposuretime(fourorsevendays). Althoughtheseedtreatmentundertheseconditions did not eradicatethe fungus, the fumigationwith the artificial mixture of VOCs was considered effective. The fungus incidence was about 30%, but in natural conditionsitsincidenceismuchlower,around0.2%on average(Mentenetal.,2006).Sincethepathogenwas artificiallyinoculatedintheseeds,thefungusmayhave penetrated more deeply into the seed and the VOCs mightnothavereachedallpathogen.Poorqualityor heavily contaminated seeds will most likely not be keptforuse.However,seedswithslightcontamination wouldnormallybetreatedinclosedchambersduring storage to eliminate unwanted fungal contamination. According to Ezra & Strobel (2003), the fumigation ofwheat,corn,barley,canolaandchickpeaseedswith the VOCs produced by M. albus contributed to the incidence reduction of the natural infection with the fungi Aspergillus sp., Penicillium sp., Fusarium sp. Leptosphaeria maculans,andA. ochraceus,artificially inoculatedinwheatseeds.Theauthorsfoundthatthe treatment was ineffective in seeds with high fungal contamination,probablyduetodeeperinfection. In relation to the individual effect of each compoundoftheVOCmixture(Table4),themycelial growth of the phytopathogen was inhibited nearly 100% by the compounds 2methyl1butanol and 3methyl1butanol. The ethyl acetate showed high inhibitoryactivity(87%),whilethecompoundsethyl octanoate and phenylethyl alcohol showed lower activity on the fungus (34 and 8% of inhibition,
Table 4. Influenceofthedifferentvolatileorganiccompounds and of their mixture (1 L mL-1 air space) on the in vitro developmentofSclerotinia sclerotiorum(1).
Compound Control Ethanol Phenylethylalcohol Artificialmixture Ethyloctanoate Ethylacetate 2methyl1butanol 3methyl1butanol
(1)

Table 3. Influence of the concentration of volatile organic compoundsartificialmixtureontheincidenceofSclerotinia sclerotiorumartificiallyinoculatedinbeanseeds(1).

Dose (LmL-1) Control 0.875 1.75 3.5 Incidence(%) Fourdays Sevendays 29.5Aa 29.5Aa 19.5Ba 20.0Ba 20.0Ba 17.0Ba 17.0Ca 16.5Ca

(1) Meansfollowedbyequalletters,uppercaseinthecolumnsandlowercase inthelines,donotdifferbytheTukeytest,at1%probability.

Mycelialgrowth(cm) 5.25a 5.03a 4.82ab 4.35b 3.45c 0.70d 0.07e 0.05e

MeansfollowedbyequallettersdonotdifferbytheTukeytest,at1% probability.

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respectively).Theethanoldidnotaffectsignificantlythe fungalgrowth,despitebeingthemaincomponent(85%) ofthemixtureofVOCs.TheVOCs2methyl1butanol, 3methyl1butanol and ethyl acetate, identified in S. cerevisiae, were the main ones responsible for the antimicrobial activity on S. sclerotiorum. This information is important to develop an improved syntheticmixtureforthepathogencontrolinseeds. The pathogen S. sclerotiorum was completely inhibited by the alcohols 2methyl1butanol and 3methyl1butanol at 0.8 L mL-1, and the ethyl acetatehadthesameeffectat1.2LmL-1 (Figure1). Independently of the concentration tested, the compounds exhibited no fungicide activity, and the fungusresumedgrowthafterthetransfertonewPDA medium in the absence of volatiles. The compounds tested alone, but in higher concentrations than those naturallyproducedbyyeast,wereabletocompletely suppressfungaldevelopment.However,thesynergism between the different components of the mixture cannotbediscarded.ThefungusM. albusproducesa complex mixture of about 30 VOCs (Strobel, 2006). Theuseofartificialmixturesshowedthatthepresence of3methyl1butanol,naphthaleneandpropanoicacid was necessary to keep the inhibitory activity against the pathogens S. sclerotiorum, Pythium ultimum and Rhizoctonia solani, indicating the synergy between the different components of the mixture (Ezra et al., 2004). Very little is known about the microbial volatile modeofactiononthecontroloffungi.Itislikelythat volatilesactbychangingproteinexpression(Humphris et al., 2002) and the activity of specific enzymes

(Wheatley, 2002), which may reflect on growth. Anotherprobableprimarysiteofactionistheplasma membrane,inwhichtheaccumulationofsolventscould affecttheorganizationandstabilityofthelipidbilayer, and increase the proton permeability leading to the dissipationoftheprotonmotiveforce.Damagetothe plasmamembranecauseslossofessentialmetabolites, changesinthecontroloftheinternalpH,reducesthe activity of membraneassociated enzymes (Weber & Bont,1996;McDonnell&Russell,1999),andimpairs nutrient uptake (Jacobsen, 1995). The compounds 2methyl1butanoland3methyl1butanolhavehigh affinityfortheplasmamembraneand,therefore,higher toxicitywhencomparedtolesslipophiliccompounds, such as ethanol, which has toxic effects only in high concentrations(Weber&Bont,1996).

Conclusions
1.The artificial mixture of volatile organic compounds produced by Saccaromices cerevisiae is abletoinhibittheinvitrodevelopmentofSclerotinia sclerotiorum. 2.Fumigationwiththevolatilecompoundsreduces significantly the incidenceof S. sclerotiorum in bean seeds. 3.The compounds 2methyl1butanol, ethyl acetate and 3methyl1butanol are the main ones responsible for the antimicrobial activity against the phytopathogen.

Acknowledgments
To Coordenao de Aperfeioamento de Pessoal de Nvel Superior and Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico, for the support.

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ReceivedonSeptember22,2010andacceptedonDecember20,2010

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