Protective effect of organic substrates against soil-borne pathogens in soilless cucumber crops

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Protective effect of organic substrates against soil-borne pathogens in soilless cucumber crops

Virginie Montagne, Hervé Capiaux, Patrice Cannavo, Sylvain Charpentier, Sophie Renaud, Emilie Liatard, Claire Grosbellet, Thierry Lebeau

To cite this version:

Virginie Montagne, Hervé Capiaux, Patrice Cannavo, Sylvain Charpentier, Sophie Renaud, et al..

Protective effect of organic substrates against soil-borne pathogens in soilless cucumber crops. Scientia

Horticulturae, Elsevier, 2016, 206, pp.62-70. �10.1016/j.scienta.2016.04.035�. �hal-01523673�

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ContentslistsavailableatScienceDirect

Scientia Horticulturae

j o u r n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / s c i h o r t i

Protective effect of organic substrates against soil-borne pathogens in soilless cucumber crops

Virginie Montagne

^a,b,c,d,∗

, Hervé Capiaux

^b

, Patrice Cannavo

^b

, Sylvain Charpentier

^b

, Sophie Renaud

^c

, Emilie Liatard

^c

, Claire Grosbellet

^c

, Thierry Lebeau

^d,∗∗

aPlateformed’AnalyseMoléculaireBiodiversité-Environnement,IUT,85035LaRochesurYon,France

bIRSTV-AGROCAMPUSOUEST,UPEPHor,49042Angers,France

cFLORENTAISEcompany,44850Saint-MarsduDésert,France

dUMR6112CNRS,LPG,44322Nantes,France

a r t i c l e i n f o

Articlehistory:

Received21December2015 Receivedinrevisedform8April2016 Accepted29April2016

Availableonline11May2016

Keywords:

Fusarium Suppressivity Greenhouse Trichoderma TTGE

a b s t r a c t

Thedevelopmentofsustainablecropprotectionisexpectedbyvegetableproducersandhighlyencour- agedbyauthorities.Forcropsgrowninsoillesssystems,vegetableﬁbersarerelevantforbothagronomical andplantprotectionpurposes.Thisworkexaminestheirpotentialagainstthesoil-bornepathogenFusar- iumoxysporumf.sp.radicis-cucumerinum.

Woodﬁber,coirﬁberandpeatweretestedovertwocucumbercroppingperiods.Fusariumblight symptomsweremonitoredoncucumber,andfungalcommunitystructure(PCR-TTGE)insubstrates.

Substratesterilizationandbio-augmentationwithantagonisticstrainswerealsostudied;theydidnot modifyprotection.Comparedtotheothersubstrates,woodfiberincreasedprotectionattheendofthe firstassay,butdidnotduringthesecondassay.Differencesincropseasonandplantdensitymayhave impactedoncucumberphysiologyandmayhaveindirectlymodifiedrhizospherefungalcommunity structure.

Thesoledeterminationofmicrobialactivityinsubstratesisnotsufﬁcienttopredictprotection.Growth conditions,substratetypeandthemicrobiomealtogetherimpactedontheprotectionofcucumber.

1. Introduction

Consumersaremore andmoremindfulofthequalityofthe vegetablestheyconsume.In parallel, theimpactofagricultural practicesontheenvironmentismoreandmoreunderfocus.New agriculturalsystems, suchas soilless culture,can increase pro- ductivitywhatevertheclimateconditions,butalsooptimizethe managementofinputs(fertilizers,pesticides),withinagiveneco- nomicandenvironmentalframework.Theyalsoaimatcontrolling diseasesmoreefﬁciently(Gullinoetal.,2015).Mineralsoillesscul- turesubstrates,which hardlycontainanymicroorganisms, have been widely used. Organic substrates are also in use because theyappearmorenatural,theyhostmicroorganismslikelytobe usefulforplanthealth andthustheycansupply partof plants’

∗Correspondingauthorat:AgrocampusOuest−Centred’Angers,2rueLeNôtre, 49045Angerscedex1,France.

∗∗Correspondingauthor.

E-mailaddresses:montagne.v@live.fr,virginie.montagne@dijon.inra.fr (V.Montagne),thierry.lebeau@univ-nantes.fr(T.Lebeau).

nutritionalneeds. Peatis the mostcommonly usedmaterial in organic substrates because it exhibits unequalled stability and physico-chemicalproperties(AlNaddafetal.,2011).Compostsare addedtothegrowingmediatoimprovemicrobialactivity.They cansubstantiallyreducediseaseseverity(Khalil,2013).However, peatbogsrepresentaslowlyrenewablepoolandsanitaryissues remainlingeringdespitetheuseofpesticides.Othermaterials(coir orwoodﬁbers)arenowbeingusedinsteadofpeat(Olleetal.,2012;

Robin,1997).Theirphysicalresilienceisconsideredassufﬁcient undercroppingconditions,butlittleiscurrentlyknownabouttheir physico-chemicalandmicrobiologicalproperties.

Soillesscultureofcucumberrepresentsasubstantialeconomic stake roundthe world(2,114,000 tons produced everyyear in Europe).Knowingi)howseveresomepathogenscanbe(especially theformaespecialesformsofFusarium oxysporumoncucumber;

Abeysinghe,2012),ii)therestricteduseofseveralactivemolecules, andiii)theproducers’wish toadoptagro-ecologicalproduction practices,othermeansofprotectionneedtobedeveloped.

Phytopathogenicity is related to the microbial populations present in the culture substrates. Yet, organic matter type appears as a determining factor for the development of both http://dx.doi.org/10.1016/j.scienta.2016.04.035

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Table1

Physico-chemicalandmicrobialcharacteristicsofthesubstrates(±:standarddeviation,n=3).

Substrate Substrate origin

Process C:Nratio pH^a (water)

Organic matter^b (gdwkg⁻¹)

Drybulk density^c (kgdwm⁻³)

Waterstorage capacity^c (mll⁻¹)

Microbial organicC^d (mgdwkg⁻¹)

BacterialCFUs^e (substrateg⁻¹)

FungalCFUs (substrateg⁻¹)

PiF1s Pine1 Screw

grinding

612 4.7 996 72.4 101 457.5

(±216.8)

2.1×10⁶ (±5.2×10⁵)

5.7×10⁷ (±2.6×10⁷)

CoF Coconut Unknown 130 6.4 962 59.4 116.8 388.6

(±103.3)

9×10⁶ (±9×10⁵)

2.19×10⁵ (±1.04×10⁴)

SpPn Sphagnum Extracted 48 6.9 867 110.2 284.9 875.9

(±30.8)

9.3×10⁶ (±3.16×10⁶)

1.23×10⁶ (±4.06×10⁵)

aNFEN13037(2000)standardmethod.

bNFEN13039(2011)standardmethod.

c NFEN13041(2000)standardmethod.

d NFISO14240-2(2011)standardmethod.

eEstimationofthenumberofculturablebacteria(TSA+cycloheximide)andfungi(PDA+streptomycinandtetracycline).

phytopathogenicmicroorganismsandtheirantagonists(Dome ˜no etal.,2011;Kleiberetal.,2012;Pérezetal.,2002)

Environment-friendlycontrolstrategies,amongwhichmicrobi- ologicalcontrol,areofgrowinginterestforthesector.Manystudies have addressed therole of microorganisms in plantprotection againstpathogens,includingsoil-borneones(MercierandManker, 2005; Shanmugam and Kanoujia, 2011). The specific physico- chemical properties of organicsubstrates are believed to drive microbialdevelopment,andsometimesleadtoaprotectiveeffect againstopportunisticpathogens(Clematisetal.,2009;Martínez etal.,2013).Competitionfornutrientsand/orspaceandantibiosis phenomenaoccuramongmicroorganisms(Benítezetal.,2004),but ourunderstandingofthesemechanismsstillremainstobefurther examined.Inthiscontext,wepreviouslycomparedthebiochemical composition of different organicsubstrates, the related micro- bialactivities,andthemicrobialcommunitystructures(Montagne etal.,2015).Ourresultsconfirmedthatspecificmicrobial activ- itiesandmicrobialpopulationstructuresarerelated tomaterial type(woodfiber,coirfiber,peat)andthereforetospecificorganic compositions. Hence it appeared interesting to investigate the involvementofspecificmicrobialcommunitiesinthecontrolof soil-bornepathogensofsoillesscrops.

Thepresentstudyaims attesting theresponsivenessofpine woodﬁber,coirﬁberandpeattothehost/pathogenpairCucumis sativus L.-Fusarium oxysporum,f.sp.radicis-cucumerinum (FORC), apathogenofcucumber.Twobioassayswereperformed:onein summer2014,andtheotherinspring2015,undergreenhousepro- ductionconditions.Plantgrowth,pathogenattacksymptoms,and theevolutionoftherhizosphericfungalcommunitystructurewere monitoredthroughoutthetwobioassays.

2. Materialsandmethods

2.1. Organicsubstrates

Thefollowingsubstrateswerestudied:pinewoodﬁber(PiF1s), coirﬁber(CoF),and peat (SpPn)(Florentaisecompany,France).

Their physico-chemicaland microbiological properties are pre- sentedinTable1.

2.2. Plant:croppingandmanagement

Cucumber (Cucumis sativus L. Galaxy F1, ENZA Zaden, Enkhuizen, The Netherlands)non-treated seedswere placed in vermiculiteshelves(25^◦C, 12hphotoperiod).Aftergermination, plantletsweretransferredtopotsﬁlledwith1.1lofsubstrate.A drop-irrigation-fertilizationsystemwasused.Thenutrientsolution (pH5.6,electricalconductivity1.5dSm⁻¹)waspreparedfrommin- eralfertilizers(PlantProd229and216,Fertil,Boulogne-Billancourt,

France)toreachtheequivalentofa10-2-8nitrogen-phosphorus- potassiumratio.

Crops were grown under the greenhouse, minimum temperature was 18^◦C, and openings allowed for aeration when temperaturesreached24^◦C.Duringthecultivationperiod,plants werepropped,fruitwerecollectedattheendoftheassays,andbio- logicalprotectionwasappliedtocontrolthedevelopmentofpest insects byusingtheauxiliaryinsects Euseiusgallicus,Phytoseiu- luspersimilis,andSteinernemafeltiae(Biobest,Belgium).Climatic parametersaresummarizedinTable3.

2.3. Microorganisms:growthconditionsandinoculum preparation

Twoantagonisticstrainsandonepathogenicstrainwerestudied inthebioassays:

− the antagonistic strain Fusarium oxysporum(MIAE 00047, UMR1347Agroécologie,INRADIJON;Alabouvetteetal.,1987)iso- latedfromsoilfromChâteaurenard(France)in1976(Fo47);

−theTrichodermaatroviridaestrain(MUCL45632,NIXELabora- toire,SophiaAntipolis,France)(Tricho);

− the pathogenic strain Fusarium oxysporum f. sp. radicis- cucumerinum (FORC), first isolated from diseased plants at a producer’sfarm,andthen identifiedbyPCRusingspecificFORC primers(Lievensetal.,2008).

Microbialcultureandsubstrateinoculationwereperformedas follows:

−theFo47strainwasgrownonPotatoDextroseBroth(PDB,Lab- oratoriosCONDA,Spain)undershakingat100rpmat26^◦C.Itwas inoculatedwhenseedsweresown(mixedwithvermiculite)and whenplantletswerepotted,ataconcentrationof10³conidiaperml ofnon-tyndallizedsubstrate.Conidiawerecollectedafterﬁltration ona48-␮mnylonﬁlter(Buisine,France),andwereenumeratedon aMalassezcountingchamber;

−theTrichodermastrainMUCL45632wasfirstgrownonwood fiber for 3 weeks toreach a highconcentration, and then the inoculum wasmixed withthe PiF1s substrate to reacha final concentration of approximately 10⁵cellsg⁻¹ of non-tyndallized substrate;

−theFORCstrainwasgrowninthesameconditionsastheFo47 strain.Conidiawerecollectedandenumeratedasdescribedabove.

FORCwasinoculatedatthesurfaceofthepotsataconcentration of5×10³conidiapermlofsubstrate(tyndallizedornot),when plantletswereatthe3–4leafstage.

Topreventmicroorganismsleachingfromthesubstrates,inthe weekfollowinginoculationplants wereirrigatedwiththesame amountofwaterasinthefollowingweeks,butitwasprovidedin smallerquantitiesandmorefrequently.

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Table2

Conditionsofthetwobioassays.

Treatment Bioassay1 Bioassay2

FORC-free FORCadded FORC-free FORCadded

Withpriortyndallization PiF1s/FORC-free/Tynd PiF1s/FORC/Tynd PiF1s/FORC-free/Tynd PiF1s/FORC/Tynd

CoF/FORC-free/Tynd CoF/FORC/Tynd – –

SpPn/FORC-free/Tynd SpPn/FORC/Tynd – –

Withoutpriortyndallization PiF1s/FORC-free/NoTynd PiF1s/FORC/NoTynd PiF1s/FORC-free/NoTynd PiF1s/FORC/NoTynd CoF/FORC-free/NoTynd CoF/FORC/NoTynd CoF/FORC-free/NoTynd CoF/FORC/NoTynd SpPn/FORC-free/NoTynd SpPn/FORC/NoTynd SpPn/FORC-free/NoTynd SpPn/FORC/NoTynd Withoutpriortyndallization,

withbio-augmentation

PiF1s/FORC-free/Fo47 PiF1s/FORC/Fo47 PiF1s/FORC-free/NoTynd/Tricho PiF1s/FORC/NoTynd/Tricho

CoF/FORC-free/Fo47 CoF/FORC/Fo47 – –

SpPn/FORC-free/Fo47 SpPn/FORC/Fo47 – –

Table3

Temperatureandlightintensityinthegreenhouseduringthebioassays.

Temperature(^◦C) Lightintensity(Jcm²day⁻¹) Bioassay Bioassay1 Bioassay2 Bioassay1 Bioassay2

Min 9.5 10.7 482 530

Max 36.7 42.7 2644 3064

Mean 21.4 22.1 1775 2017

2.4. Microbialanalysis

Differentmicrobiologicalanalyseswereperformedateachsam- plingtimeandattheendofeachassay:

–F.oxysporumwasenumeratedintherhizosphereandtheroots of5plantspercondition,inbioassay1only:therhizospheresfrom threeplantsfromoneblockwereused,andfromoneplantfromthe othertwoblocks.Suspensions/dilutionswereprepared,using1g ofvortexedsubstratein45mlofsterilewater.Thentheappropri- atedilutionswereenumeratedinPetridishesbyspreading1ml ofmicrobialsuspensionmixed withKomadamedium (Komada, 1975).TheF.oxysporum(Fo47orFORC)densitywasexpressedas colony-formingunits(CFU)/gofsubstrate.

F.oxysporumwasalsoenumeratedintheplants’roots.Roots werefirst disinfectedby dipping30sin70% ethanol, and then rinsedbybathing15sin twosuccessivesterilewater baths.At leastfiverootfragmentsfromasameplantwerelaidonKomada agarmedium.Petridishescontainingmicrobialsuspensionsorroot fragmentswereallreadafterfivedaysofincubationat25^◦Cinthe dark,followedby3daysunderlighttorevealthepurplepinkF.

oxysporumcolonies(DavetandRouxel,1997);

−thestructureofrhizosphericfungalcommunitieswasdeter- mined: Rhizospheric substrate was kept at −20^◦C and then analyzed by PCR-TTGE (Polymerase Chain Reaction−Temporal TemperatureGelElectrophoresis; Jaffrèsetal.,2009; Montagne etal.,2015).Briefly,microbialDNAextractedfromthesubstrate wasamplifiedaccordingtoJoly’sprotocol(Jolyetal.,2012).This step amplifiespartof fungal ITS 1, based onthefact that cer- tainITS1zonesvaryaccordingtomicroorganisms.Theamplicons migratedthroughanacrylamidegelunderatemporal tempera- turegradienttorevealsequence differencesamong thevariable regionsoffungalDNA.ThegelswerestainedwithGelRed(Biotium, UnitedStates),andthenfungalprofilesweredevelopedunderUV light.Geneticfingerprints(number,position,andintensityofthe bands)werecompared usingFPQuestSoftware(Bio-Rad,United States).

2.5. Experimentaldesign

Twoassayswereconductedattwodifferentseasons.Theﬁrst cropwas grown in late summer 2014, and the second one in spring2015.Theconditionsstudiedinthetwoassaysaredetailed

in Table 2. Some substrateswere ﬁrst sterilized by tyndallization(Marchal,1976)whileotherswerebio-augmentedbyadding pathogenicorantagonisticmicroorganisms(FORC,F.oxysporum Fo47,andT.atroviridaeMUCL45632).

Alight/temperaturegradientwasappliedinthegreenhouse.

Thatiswhya3-blocksystemwasused,eachblockcontainingone replicateofeachoftheconditions.Ineachblock,3plantswere grownperconditionandpersamplingtime-point,i.e.9plantsin total.Twosamplings(S1andS2)wereperformedduringthebioas- says,andathirdone(S3)attheend.Firstsamples(S1)weretaken sevendaysafterinoculationwithFORC,i.e.after32daysofculti- vationinbioassay1,and34inbioassay2.SamplesS2weretaken 21daysafterinoculationwithFORC,i.e.after46daysofcultivation inbioassay1,and44inbioassay2.SamplesS3weretakenattheend ofthebioassays,i.e.after69daysofcultivationinbioassay1,and78 inbioassay2.SubstratepH,electricalconductivity(followingthe standardNFEN13038,2000)andmicrobialanalysiswererecorded atthebeginningofthecroppingperiods,duringsamplings,andat theendofthecroppingperiods.

The different conditions were randomly distributed in each block.Cropdensitywas45pots(1.1-l)perm²inbioassay1,and 12.5potsperm²inbioassay2.Cropdensitywasreducedinthe secondassaybecauseplantsweretoodifﬁculttohandleattheend ofﬁrstassay.

Plantsize,leafandfruitnumberswereevaluatedoverthetwo croppingperiods.Inparallel,theoutbreakofsymptomsrelatedto FORCattackwasmonitoredbyevaluatingi)thenumberofhealthy plants,ii)thenumberofdiseasedplantsexhibiting ayellowish, narrowerstem baseand pronouncedwiltingof theaerialparts, andiii)thenumberofdeadplantsinwhichmorethanhalfofthe aerialpartwasdryandyellow.

2.6. Dataanalysis

Analysesofvariance(ANOVA)wereperformedattheendofthe 2bioassays,usingTukey’stestwitha5%signiﬁcancethreshold.

OnlyFORC-freeconditionswereanalyzedtotesttheeffectsofthe

“substratetype”,“tyndallization”,“Fo47”and“Trichoderma”fac- torsonplantgrowth.Forplanthealthdata,ANOVAanalyseswere supplementedby a Newman-Keulsmultiplecomparison testto comparethenumbersofdeadplantsamongconditionsattheend ofthetwobioassays.Varianceanalysesandmultiplecomparison testsweregeneratedusingR3.1.1(FreeSoftwareFoundation)and theSNK.testfunctionoftheagricolaepackage.

Fungalcommunitystructureanalysiswasperformedbasedon geneticﬁngerprints.Pearson’stestwasusedtocomparethem,and UnweightedPairGroupMethodwithArithmeticMean(UPGMA) wasusedtocreateadendrogrambasedonsimilaritycoefﬁcients (Ibekweetal.,2010).

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3. Results

3.1. Initialsubstratecharacteristicsandplantgrowth

Whateverthesubstrate,organicmattercontentswerehigh,but C/Nsigniﬁcatlydifferedbetweensubstrates(Table1).Becausepeat hadahigherwaterstoragecapacitythantheothertwosubstrates, waterregimeswereadaptedaccordingly(PaulandLee,1976).

Initialmicrobialcarbonwastwicehigherinpeatthaninwood ﬁberandcoirﬁber.

Themeanconcentrationofculturablebacteriainthesubstrates was10⁶–10⁷CFUsg⁻¹,andthemeanconcentrationofculturable fungivariedfrom10⁵to10⁷CFUsg⁻¹.PiF1scontained100times morefungithanCoFand10timesmorethanSpPn,buthalfasmany bacteriaastheothersubstrates.

Climaticconditionsweremonitoredthroughoutthetwocrop- pingperiods(Table3).Onaveragetemperaturewassimilarforthe twocrops,butmeanlightintensitywashigherduringbioassay2.

AsforsubstratepHandelectricalconductivity,theyremainedclose to7.5and200␮Scm⁻¹throughoutthe2bioassays,respectively.

Theheightofcontrol(FORC-free)plantswasmeasuredduring thebioassays.Meanﬁnalsizewas225cminbioassay1,and170cm inbioassay2.Controlplantsdevelopedinahomogeneousmanner overtime inthetwo bioassays:there wasnosigniﬁcantdiffer- enceamongconditionsoramongblocks;p-valueswere0.1185in bioassay1,and0.4616inbioassay2.Moreover,attheendofthe bioassays,leafandfruitnumbersweresimilaramongconditions, butthenumberoffruitwashigherinbioassay2,with4fruitsper plantinbioassay2vs.2inbioassay1.Potdensitydifferedtoo:itwas lowerinbioassay2,with12.5potsm⁻²vs.45potsm⁻²inbioassay 1.

3.2. Diseasesuppressioninthedifferenttreatments

Thepathogendosewasinoculatedatthe3–4trueleafstageto allowforgradualandsufﬁcientexpressionofthedisease.Whatever thebioassay,FORC-relatedsymptomscameoutonemonthafter inoculation,i.e.after55daysofcultivation.Statisticaldataanal- ysisdidnotrevealanyblockeffect.Meanpercentagesofhealthy, diseasedanddeadplantsweredeterminedattheendofeachbioas- say.Intheﬁrstbioassay(Fig.1),noplantwasfounddiseasedinthe FORC-freeconditions.

Signiﬁcantdifferences in the number of dead plants among conditions were determined by one-way ANOVA (P-value:

7.3×10⁻¹⁰).Thenamultiplecomparisonwasperformedtoform homogeneousgroupsbasedonthenumbersof deadplants.No deadplantwasobservedintheFORC-freecontrols,whichclustered ingroupc.Inthe9FORC-treatedconditions,woodﬁber-grown plantsdisplayedaveragemortalityratesrangingbetween22and 33%dependingonthecondition,coirﬁber-grownplantsbetween 55 and 77%, and peat-grown plants between 55 and 77% too.

Themortalityratesofnon-tyndallized woodfiber-grownplants (PiF1s/FORC/NoTynd.)andbioaugmentedwoodfiber-grownplants (PiF1s/FORC/Fo47)didnotsignificantlydiffer fromthecontrols, with 33 and 22%, respectively. However, bioaugmented wood fiber-grownplants also displayed a significantly lower number ofdeadplantsthantheplantsof2outofthe3peattreatments (SpPn/FORC/Tyndand SpPn/FORC/Fo47,with77% mortalityrate each),and2outofthe3coirfibertreatments(CoF/FORC/NoTynd andCoF/FORC/Tynd,with77%mortalityrateeach).

A two-way ANOVA solely applied to the FORC conditions showedaneffectofthesubstrate(P-value:0.005204).Thiscon- firmedtheexistenceofasignificantdifferencebetweenthemean highestmortalityratesandthemeanlowestmortalityrates.Conse- quently,theplantsgrownonwoodfiberwerelessseverelyattacked bythepathogenthanthosegrownoncoirfiberandpeat.Bycon-

trast, treatments(tyndallizationor bioaugmentationwithFo47) altogetherhadnoeffectonmortalityrates.

Inthesecondbioassay(Fig.2),amongcontrolconditions(i.e.

FORC-freesubstrates), only oneplantexhibited Fusarium blight symptomsalthoughithadnotbeeninoculatedwithFORC.Ascom- paredtotheﬁrstbioassay,thepercentageofhealthyplantswas lower,andsowasthepercentageofdeadplants,butthepercentage ofdiseasedplantswashigher.

Aone-wayANOVAwasappliedwhosevariablewasthenumber ofdeadplantsandwhosefactorwasthe10conditions,andthen amultiplecomparisontestwasperformed(Fig.2).Thehomoge- neousgroupscorrespondingtodeadplantsareindicatedbyletters.

Onthisbasis,theFORC-inoculatedrawsubstrates(withorwithout prior tyndallization)sharedthesamegroupasthecontrolsub- strates.

Furtherin detail, consideringthenon-tyndallized conditions and FORC-inoculated substrates, no dead plant was observed amongCoF-treatedplants,andPiF1sandSpPntreatmentsdidnot showsigniﬁcantdifferencesascomparedtoprevioustreatments.

Thepercentageofdiseasedplantswassimilaramongthethree substrates,with66%.Asregardsthetyndallizationfactor,wenoted the same tendency as in bioassay 1 for substrate PiF1s only, namelythatpriortyndallizationofthesubstrate(PiF1s/FORC/Tynd) appearedtoincreasethepercentageofdeadplants,with33%vs.

11%forthesamenon-tyndallizedsubstrate(PiF1s/FORC/NoTynd).

Nevertheless, these differences were not signiﬁcant, as shown by ANOVAresults (groupsab and b, respectively).By contrast, bioaugmentationwithMUCL45632(PiF1s/NoTynd/Tricho)hada signiﬁcantnegativeeffectonplantmortality(66%)ascomparedto FORC/PiF1s/NoTynd(11%).

Thepercentagesofdiseasedand/ordeadplantsdifferedfrom thoseobtainedinbioassay1.Inotherwords,thetime-courseof thediseaseappearedfasterinbioassay1,withahigherpercentage ofdeadplants,whileinbioassay2plantswereonlydiseased,with asimilarpercentageofhealthyplants.

3.3. F.oxysporumplatecount

F.oxysporumpopulationswereenumeratedintheﬁrstbioassay (Table4).Fo47andFORCwerenotphenotypicallydiscriminatedon theKomadamediumusedtoassesstheCFUcount.

AtS1,theFo47-free,FORC-inoculatedconditions,andtheFo47- bioaugmentedconditions(bothFORC-freeandFORC-inoculated) displayed a highF.oxysporumdensity (ca.3 log(CFU)g⁻¹).By contrast,theFORC-andFo47-free conditionsdidnot revealthe presenceofF.oxysporum.ThereforethepresenceofF.oxysporum correspondedtotheFORC-inoculatedorFo47-bioaugmentedconditions.

AtS2,populationswereatabalanceataround3log(CFU)g⁻¹ fortheFORCandFo47conditions.

Ascomparedtopeatand coirﬁber,in thepresence ofFORC wood ﬁber appeared to be more responsive to the F. oxysporumstrains,whetherpathogenicornot(PiF1sFORC/NoTyndand PiF1s/FORC/Tynd).

AtS3(the end oftheassay), F.oxysporumwasfoundin the

“FORC-free/NoTynd.”and“FORC-free/Tynd”conditions,suggesting contaminationfrom theair or fromspores present in thesub- strates.However,thesamewasnotedwithtyndallizedsubstrates, andtyndallizationprovedefﬁcientsincefungalampliﬁcationatthe beginningoftheassayswasnotpossible(datanotshown).Contam- inationfromtheairthereforeappearsasarelevanthypothesis.

TomonitorthedevelopmentofF.oxysporumstrains,detection testswerecarriedoutinplantroots(datanotshown).Atleast20%

oftherootsystems,allconditionsincluded,containedF.oxysporum.

F.oxysporumdistributionaccordingtothedifferentconditionswas similarintherootsandintherhizosphere.Amaximumof100%

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Fig.1.Percentagesofhealthy,wiltedanddeadplantsattheendofbioassay1(n=3,9plants).

Differentlettersofthehomogenousgroups(a,c,ab,bcandabc)indicatesigniﬁcantdifferencesamongtreatmentsforthenumberofdeadplants(blackbarsonly)according toNewman-Keulstest(P=0.05)

Fig.2.Percentagesofhealthy,wiltedanddeadplantsattheendofbioassay2(n=3,9plants).

Differentlettersofthehomogenousgroups(a,bandab)indicatesigniﬁcantdifferencesamongtreatmentsforthenumberofdeadplants(blackbarsonly)accordingto Newman-Keulstest(P=0.05)

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Fig.3.FungalcommunitystructuresofPiF1s,CoFandSpPnthroughoutthetwobioassaysB1(in2014)andB2(in2015).Greyrectanglessymbolizethethreeclustersnamed 1,2and3onFig.S1FigS.S1,S2andS3aresamples.(F)indicatesthesamplesthatreceivedthephytopathogenFORC.−1and−2arereplicates.Technicaldifﬁcultiesdidnot allowustoobtaintheSpPnB1(2014)proﬁle.

infectedplantswasfoundintheFORC/Tynd,FORC/Fo47andFORC- free/Fo47substrates.

3.4. Characterizationofthefungalpopulationstructure

Thefungalstructuresof the3FORC-freeorFORC-inoculated substrates(NoTynd./FORCorFORC-free)weresampledatS1,S2and S3duringthetwobioassays(Fig.3).Foragivencondition,replicates noted−1and−2displayedratherlowsimilaritypercentages.Nev- ertheless,comparisonsbetweenthefungalstructuresofthetwo bioassayscanbemade,allconditionsincluded.Aboveall,fungal structuredependedonsubstratenature(CoFrepresentedbyrect- angle1,PiF1srepresentedbyrectangle2,andSpPnrepresentedby

rectangle3),whatevertheyearorthepresence/absenceofFORC.

Moreprecisely,inrectangle1whichmainlyrepresentsCoFsub- strates,aPiF1sclusterwaspresentanddifferedagainfromtheCoF cluster.

AsregardsCoF(rectangle1),similaritybetweenbioassay1(B1) and bioassay2 (B2)was low.Therefore the fungalstructure of CoFwasrelatedtotheyear.Themodiﬁcationofthefungalcom- munitystructureofCoFinbioassay1ascomparedtobioassay2 couldexplainthedifferentprotectionlevelssuppliedbythiskind ofsubstrate.

IfwespeciﬁcallyanalyzePiF1s(Fig.4)attheﬁrst(S1)andlast (S3)samplingtime-pointsofbioassays1and2,samplestructures rankaccordingtosamplingdate,notaccordingtotheyear.

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Table4

CFUsofFusariumoxysporumpergofrhizosphericsubstrateafter32days(S1),46days(S2),andattheendofthecroppingseason(after69days,S3)inbioassay1 (means±standarddeviations;n=5).Dataareexpressedinlog-transformedvalues.

PiF1s CoF SpPn

32d 46d 69d 32d 46d 69d 32d 46d 69d

FORC-free/NoTynd. 0.00 0.00 2.83 0.00 0.00 2.37 0.00 0.00 0.00

±0.00 ±0.00 ±2.58 ±0.00 ±0.00 ±2.18 ±0.00 ±0.00 ±0.00

FORC/NoTynd. 3.88 1.98 4.63 2.59 0.53 4.51 0.79 2.21 4.02

±0.72 ±2.87 ±0.93 ±2.41 ±1.19 ±0.48 ±1.77 ±2.04 ±0.49

FORC-free/Tynd. 0.00 0.73 0.53 0.00 0.00 1.71 0.00 0.00 0.59

±0.00 ±1.63 ±1.19 ±0.00 ±0.00 ±1.58 ±0.00 ±0.00 ±1.32

FORC/Tynd. 3.72 2.65 4.46 3.58 1.40 4.89 2.71 2.80 4.45

±0.85 ±2.66 ±2.53 ±0.56 ±1.92 ±0.75 ±1.61 ±1.72 ±0.32

FORC-free/Fo47 2.96 1.12 3.22 1.24 1.85 3.68 1.85 0.73 3.33

±1.80 ±1.54 ±2.04 ±1.70 ±1.73 ±0.50 ±1.73 ±1.63 ±0.41

FORC/Fo47 2.98 2.11 2.17 3.50 0.53 2.21 2.63 2.88 4.12

±0.32 ±1.96 ±2.04 ±2.04 ±1.19 ±2.05 ±1.52 ±1.71 ±0.89

Fig.4.FungalcommunitystructureofPiF1sthroughoutthetwobioassaysB1(in2014)andB2(in2015).Greyrectanglessymbolizethetwoclustersnamed1and2onFig.

S1andS3aresamples.(F)indicatesthesamplesthatreceivedthephytopathogenFORC.−1and−2arereplicates.

4. Discussion

Thisstudyunderlinestheimplicationoforganicsubstratesin cropbioprotectionagainsta pathogenofcucumber(FORC),and onthereproducibilityofthatprotectionunderconditionsnearing large-scaleproductionconditions.

Plantsgrewwell,whateverthesubstratetype.Oursubstrates aresuited for soilless culture,although somephysico-chemical andmicrobiologicalparameters(Table1:waterretentioncapacity, biochemicalcomposition,granulometry,specialmicrobialpopula- tions,etc.)differedamongthethreesubstrates.

Apartfromoneplant affectedbyFORC inthesecond bioassay,control(FORC-free)plantsdidnotdisplayanyFusariumblight symptoms,whereasFORC-inoculatedplantsdid(thediseasecame outafter 55days).The onlydifferencebetweenthesetwo con- ditionswastheadditionofFORC,sothesymptomswereindeed relatedtothepresence ofFORC,andwerenotrelated topossi- bleindigenouspathogenicstrains.F.oxysporumdensityonKomada mediumduringtheﬁrstbioassayactuallyshowedthatFORCand/or Fo47hadsettledinthesubstrates(Table4).

Intheﬁrstbioassay,tyndallizationhadastrongerimpactwhen appliedtoPiF1s,withalowernumberofhealthyplantsasaresult ofwoodﬁbertyndallization.Thatiswhythatfactoronlywastested againonPiF1sinthesecondbioassay.TheFo47factordidnotyield

homogeneousresultsacrossallsubstrates,soitwasreplacedby Trichoderma(onlytestedonPiF1s),astrainknownforitsgrowth- stimulatingeffects,whichseemstosettlewellinwoodfiber.Peat initiallycontainedthegreatestamountofmicroorganisms(ithad thehighestrateofmicrobialcarbon).Theculturablefungi/bacteria ratio wasapproximately 1, and wasthe highest (>1) in wood fiber.Theseinitialdifferencesimplieddifferentplantprotection potentials.Fusariumblightdifferedinitsexpressiondependingon thesubstratethroughoutthetwo bioassays.Plantfiberprocess- ing(screwgrindingforwoodfiber,unknownforcoirfiber)could explainthislessermicrobialcolonization,whilepeatwassimply extractedmechanically.

Lookingatthenumberofdeadplantsattheendofthebioas- says,it appearedthat substratetypehad asignificantinfluence onmortality.Mortalitywaslowerinplantsgrownonwoodfiber (PiF1s),particularlyinthefirstassay,despitethehighnumberof F.oxysporumCFUs.Thismayhaveresultedfromthepresenceof differentF. oxysporumpopulations, withpredominanceof non- pathogenicones.Competitionamongthemmayexplainthislow mortalityrate(Dhingraetal.,2006).Moreover,thesepopulations possibly triggeredplantresistance beforeFORC attack.Besides, distinguishingbetweendiseasedand deadplantsallowedusto assessthetime-courseofthediseaseinthetwobioassays.Mor- talityoccurredlaterinbioassay2,butaddingTrichodermacaused

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ittooccurearlier.FORCattackoccursunderplantweaknesscon- ditions;itisallthemorevirulentasplantsareweakenedortheir innerresourcesaredepleted.Inourbioassays,symptomsprobably ﬁrstappearedconcomitantlywithcombinedstresses,suchasplant fruitload—forexample,attheendofbioassay2,plantbiomass (vegetativeparts+fruit)reached2250gonaverage—orincreases intemperatureandlightintensity(Table3).Inbioassay1,thedis- easesymptomsburstoutallofasudden,andplantsrapidlydied.

Conversely,symptomscameoutmore progressivelyinbioassay 2.Temperatureincreasedmoregradually,soplantsprobablygot acclimatedmoreeasily.Asaresult,fewerplantsdied,moreplants becamediseased(22–66%dependingontheconditions,vs.0–44%

inbioassay1).Moreover,thehigherplantdensityinbioassay1 probablyintensiﬁedthesestresses.

Tyndallizationdrasticallyreducesinitialmicrobialpopulation densities in the substrates, while maintaining their physico- chemicalandstructuralproperties.Itdidnotsigniﬁcantlymodify plantprotectionascomparedtonon-tyndallizedsubstrates.This meansthatthemicrobialpopulationsnaturallypresentinthesub- strateshavelittleinﬂuenceonplantprotection,whichissurprising andcontradictspreviouslypublishedresults(Clematisetal.,2009).

Theanalysisofthefungalcommunitystructureovertimedidnot evidenceanynoticeabledifferencebetweentyndallizedandnon- tyndallizedsamples.Thisconﬁrmsthatinthecaseoftyndallized substrates,re-colonizationisunderthecontrolofthesubstrate,and thereforeofitsphysico-chemicalcharacteristics(Table1).Ascrops grew,CoFfungalcommunitystructuredivergedfromPiF1sfungal communitystructure(Fig.3).Competitionforspaceand/oraccess tosubstratesprobablyexplainwhyprotectionwasmodulated.

IfwespecificallyanalyzePiF1s(Fig.4)atthefirst(S1)andlast (S3)samplingpointsofthe2B1andB2bioassays,wecannotethat thesamplingdateinfluencedfungalcommunitystructureindepen- dentlyoftheyearortheaddition(ornot)ofthephytopathogen FORC.Theevolutionofthefungalcommunitystructureaccordingto cultivationstagecouldbeexplainedbyquantitativeand/orqualita- tivevariationsofrhizodepositsinthecourseofthecroppingperiod (Zhangetal.,2014).Plantsgrewunderdifferentgrowthconditions inthe2bioassays,sowecanassumethatrootexudatecomposition wasdifferenttoo.Whenassociatedtootherfactorssuchassub- stratetemperatureandhumidity,theseparametersmayexplain thattherhizospheric microbialcommunitystructurewasmodi- fiedinthecourseofthe2bioassays,andinturntheresponsetothe pathogen.Wecanindeednotethat,especiallyforCoF,thefungal populationsofthetwoassaysevolved(Fig.3).Differentpopulations inCoFB1andCoFB2couldexplainwhytheyprovideddifferent levelsofprotectioninthe2bioassays.Gaoetal.(2015)workedon cucumbercrops;theyshowedthatmicrobialcommunitiesdiffered dependingontheseasonandtheculturalpractices(e.g.grafting).

Fo47wasonlystudiedintheﬁrstbioassay,onthe3substrates.F.

oxysporumFo47antagonizesthecausalagentsofFusariumblighton tomatoandmelon(Fuchsetal.,1997;Mighelietal.,2000).Besides, othernon-pathogenicF.oxysporumstrainscompetefornutrients withFORC(Alabouvetteetal.,2009;Mandeeland Baker,1991).

Inbioassay1, Fo47didnot inﬂuenceonmortality.Inthesame way,Fo47hadnoeffectinCoForSpPn,probablyduetoatoolow Fo47/FORCratio(Olivainetal.,2004).

MUCL45632 wasalsostudied asan antagonistic agent,only in PiF1sin bioassay 2.Numerous Trichoderma species canpro- tectcropsbyproducingchitinases(antibioticeffect)orcellulases (competitionfornutrients)andallowforplantstodevelopprop- erlyby improvingmineralbio-availability(Benítezet al.,2004;

Boehmetal.,1993;Johnetal.,2010;Lemanceauetal.,2012).Yet, underourexperimentalconditionstheTrichodermastrainweused (MUCL45632)hadnopositiveeffectoncucumbergrowth.Onaver- age,thenumberofdeadplantsinPiF1swasevenhigherinthe presenceofMUCL45632(Fig.2).TheMUCL45632patentpresents

itasastimulantofplantgrowth(Nixe,2008).Wedidnotspeciﬁ- callymonitorthisstraininthiswork,sowecannotconcludeabout itsabilitytosurviveandtobeactiveinPiF1s.

5. Conclusions

Plant sensitivitytopathogens remains greatlydependenton cropping conditions that are in turn dependentonthe season, even though the organic composition of substrates canreduce pathogenvirulence.Microbiologicalcontrolbasedontheuseof organicsubstratesthereforeappearstobeefﬁcientuptoa cer- tainphytopathogenthreshold.Our2bioassaysalsoshowedhow importantitistostudybio-controlmethodscasebycase,andhow difﬁcultitistoreproducesuppressivenesseffects.

Conﬂictofinterest

Noconﬂictofinterestdeclared.

Acknowledgements

We would like to thank the Florentaise Company and the ANRT (Association Nationale de la Recherche et de la Technologie—convention CIFRE N^◦ 2012/1062) for funding this work. We thank La Roche sur Yon Agglomération for funding thebead grinder.We are more particularlygrateful tothefol- lowingpeopleandorganizationsfortheiroccasionalhelpinthe experiments and their advice: IUT la Roche sur Yon for pro- vidingmaterials;LaurentTosten fromFLORENTAISECo.,Claude AlabouvettefromAGRENECo. inDijon; ChristianSteinbergand CharlineLecomtefromtheINRACenterofDijon;MarcLollierfrom theUniversitédeHaute-Alsace;andChristianDouillardfrom“Les 3Moulins”company,atSaint-Philbert-de-Grand-Lieu.

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