Fungal Endophytes of Vitis vinifera—Plant Growth Promotion Factors

Abstract

Endophytes are microorganisms that live asymptomatically inside plant tissues. They are beneficial to their host in many aspects, especially as a defense against foreign phytopathogens through the production of a variety of metabolites. These substances can serve as sources of new natural products for medicinal, agricultural, and industrial purposes. This article is focused on endophytic fungi from Vitis vinifera. The purpose of the research was their isolation and identification during the Vitis vinifera growing season. Subsequently, the isolates were tested for the production of biotechnologically interesting metabolites (siderophores, antioxidants, and antifungal compounds). In total, 24 endophytic fungi were isolated, the most represented genus was Cladosporium sp. The results of the test for antioxidant and antifungal properties, as well as siderophore production, have shown that the population of Vitis vinifera endophytic microscopic fungi could serve as a promising source of metabolites with a wide range of applications.

Full text

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Agriculture2021,11,1250.https://doi.org/10.3390/agriculture11121250www.mdpi.com/journal/agriculture
Article
FungalEndophytesofVitisvinifera—PlantGrowth
PromotionFactors
MarkétaKulišová
1,
*,MariaVrublevskaya
1
,PetraLovecká
2
,BlankaVrchotová
2
,MilenaStránská
3
,
MiroslavKolařík
4
andIrenaKolouchová
1

1
DepartmentofBiotechnology,UniversityofChemistryandTechnology,Technická5,16628Prague,
CzechRepublic;maria.vrublevskaya@vscht.cz(M.V.);irena.kolouchova@vscht.cz(I.K.)
2
DepartmentofBiochemistryandMicrobiology,UniversityofChemistryandTechnology,Technická5,
16628Prague,CzechRepublic;petra.lovecka@vscht.cz(P.L.);blanka.vrchotova@vscht.cz(B.V.)
3
DepartmentofFoodAnalysisandNutrition,UniversityofChemistryandTechnology,Technická5,
16628Prague,CzechRepublic;milena.stranska@vscht.cz
4
InstituteofMicrobiology,AcademyofSciencesoftheCzechRepublic,Vídeňská1083,14220Prague,
CzechRepublic;mkolarik@biomed.cas.cz
*Correspondence:marketa.kulisova@vscht.cz
Abstract:Endophytesaremicroorganismsthatliveasymptomaticallyinsideplanttissues.Theyare
beneficialtotheirhostinmanyaspects,especiallyasadefenseagainstforeignphytopathogensthrough
theproductionofavarietyofmetabolites.Thesesubstancescanserveassourcesofnewnaturalproducts
formedicinal,agricultural,andindustrialpurposes.Thisarticleisfocusedonendophyticfungifrom
Vitisvinifera.ThepurposeoftheresearchwastheirisolationandidentificationduringtheVitisvinifera
growingseason.Subsequently,theisolatesweretestedfortheproductionofbiotechnologically
interestingmetabolites(siderophores,antioxidants,andantifungalcompounds).Intotal,24endophytic
fungiwereisolated,themostrepresentedgenuswasCladosporiumsp.Theresultsofthetestfor
antioxidantandantifungalproperties,aswellassiderophoreproduction,haveshownthatthe
populationofVitisviniferaendophyticmicroscopicfungicouldserveasapromisingsourceof
metaboliteswithawiderangeofapplications.
Keywords:microscopicfungi;endophytes;Vitisvinifera;antifungalactivity;antioxidants;siderophores

1.Introduction
Thegrapevine(Vitisvinifera)isoneofthemosteconomicallyimportantcrops,beingused
mainlyforwineproduction(approximately80%ofharvestedwinegrapesisusedforthis
purpose).GrapesandotherpartsofVitisviniferacontainanumberofhealthpromoting
metabolites[1].Aswithotherplants,thetissuesofthegrapevineareinhabitedbyvarious
typesofmicroorganisms.Theseorganismscanbeepiphytic,i.e.,superficial,orcolonizing
internaltissues,i.e.,endophytic[2].Theinteractionsbetweenendophytesandtheirplant
hostsarediverse.Plantsprovideprotectionandendophyticmicroorganismsarecapableof
producingusefulmetabolitesthatincreasenutrientuptake,induceresistancetopathogens,
increasetolerancetoosmoticstress,heavymetals,xenobioticcontaminants,andotherforms
ofabioticstress[3].Mostendophytesarerepresentedbybacteria,butmicroscopicfungiand
yeastsalsoformasignificantpartoftheendophyticpopulation.Endophytesareisolatedfrom
avarietyofplantspecies,andalmostallstudiedplantspecieshavebeenfoundtohostatleast
oneendophyticmicroorganism[4].Colonizationofthehostplantwithuptoahundred
Citation:Kulišová,M.;
Vrublevskaya,M.;Lovecká,P.;
Vrchotová,B.;Stránská,M.;
Kolařík,M.;Kolouchová,I.Fungal
EndophytesofVitisvinifera—Plant
GrowthPromotionFactors.
A
griculture2021,11,1250.
https://doi.org/10.3390/
agriculture11121250
AcademicEditor:OfirDegani
Received:11November2021
Accepted:9December2021
Published:10December2021
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.

Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
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/by/4.0/).

Agriculture2021,11,12502of14
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differentspeciesisnoexception.Geographiclocation,season,climate,andtypeofplanttissue
areamongthefactorsthataffectspeciescompositionandfrequencyofendophyte
colonization[5,6].
Endophytesactasimportantsourcesofstructurallyuniquebioactivenaturalmetabolites
withawidebiotechnologicalpotential.Theyrepresentanattractivesourceofnatural
productsthatcanbeusedinagriculture,industry,andmedicine[7–13].Theformationof
antibacterial,antifungal,antiviral,cytotoxic,andimmunosuppressivemetabolites,aswellas
antioxidantsandsiderophores,hasbeenpreviouslyfound[14,15].Nowadays,whennew
diseasescausedbymicroorganismsareemergingandresistancetoknowndrugsisspreading,
itisthesiderophoresofarelativelypoorlystudiedendophyticpopulationthatcanbeusedto
developactivesubstancesinthepharmaceuticalindustry[16].
Severalresearchershaverecentlyinvestigatedgrapevinefungalendophytestoclarify
theirdiversityandecologicalroleinthisplant.Theuseofchemicalsasfertilizersin
agriculture,endophytesproducingantibacterialandantifungalcompoundscouldbean
interestingalternativetotheseactivesubstances.Phomaglomerata,Chaetomiumglobosum,
Aureobasidiumpullulans,Epicoccumnigrum,andAcremoniumspp.haverepeatedlyexhibited
antibacterialandantifungalpropertieseffectiveagainstanumberofplantdiseases[6,17–22].
AlternariaalternataandFusariumproliferatumhavealsobeenidentifiedaspromisingbiocontrol
agentsagainstspecificpathologicalconditionsofVitisvinifera,suchasgrapevinedowny
mildewcausedbyPlasmoparaviticola[6,22,23].
Resveratrol,asanantioxidantcompoundknowntoincreaseresistancetostressand
prolongthelifeofavarietyoforganisms,fromyeaststovertebrates,isabundantinVitis
viniferagrapes.Manyendophytesshowtheabilitytoproducethesamefunctionalcompounds
astheirhostswhilelivingasymptomaticallyinplanttissues.Fungalendophytescapableof
resveratrolproductionincludePenicillium,Aspergillus,Mucor,Alternaria,Cephalosporium,and
Geotrichum.Alternariaspeciesappeartobethebestproducersduetothestableproductionof
resveratrol[24].TheresearchworkofYangetal.[25]investigatedtheroleofendophytesin
theformationofsecondarymetabolites(totalflavonoidsandresveratrol)andtheinfluenceof
physio‐chemicaltraitsingrapesandleaves.FungalendophytesoriginallyisolatedfromVitis
viniferawerere‐inoculatedongrowinggrapevineplantsandtheireffectongrapesandleaves
wasevaluated.Thisinoculationincreasedthecontentofreducingsugar,totalflavonoids,
polyphenols,andtrans‐resveratrolinparticularpartsofVitisvinifera.Nigrosporasp.and
Fusariumsp.appearedtobethemostpromisingofthefungalgenerastudied.
Theaimofourstudywastoisolateandcharacterizetheendophyticfungithatoccurin
Vitisviniferaleaves,canes,andberriesgrowninvineyardswithintheCzechRepublic.Another
goalwastoinvestigatetheirpotentialtoactasplantgrowthpromotersanddiseaseprotective
agents.Thiswasdonebytestingtheirabilitytoproduceantioxidants,siderophores,and
antifungalcompounds.
2.MaterialsandMethods
2.1.Samples
SamplesofMullerThurgau,PinotGris,PinotNoirandRieslingRheinhessengrapevine
varietieswerecollectedfromtwodifferentvineyardswithintheCzechRepublic,KutnaHora
(49.9336N,15.2889E;grapevinegrownaccordingtotheprinciplesoforganicfarming)and
Prague(50.0690N,14.4454E;conventionallygrowngrapevine).Threedifferentexperimental
plantslocatedatdifferentsiteswithinthevineyardswereselectedforcontinuoussampling
duringtheentirevegetationyear.Thesamplingofleavesandcanesaslignifiedstemsofthe
plantswascarriedoutinJanuary,May,AugustandOctober2019inapproximateamounts
between3and10gofleaves,dependingonthesamplingseason(leaveswerenotsampledin
Januaryduetotheirfallduringtheautumn),andcaneswerecollectedinapproximate

Agriculture2021,11,12503of14
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amountsof50g.BerriesweresampledinSeptember2019inanamountof500g,onlyfrom
thePraguevineyard(strongstormsinKutnaHoraregionruinedthecropsandsamplingwas
notpossible).Thesampleswerestoredat−80°Cbeforeprocessinginthelaboratory.
CharacterizationofthesamplesfromwhichwerefungalendophytesisprovidedinTable1.
Table1.CharacterizationofVitisviniferaleaves/canes/berriesfromwhichfungalendophyteswereisolated.
SampleCodeSamplingPeriodGrapevineVarietyPlantPartGrowingLocality(FarmingSystem)
Z‐MT‐G‐S6January2019MullerThurgaucanesPrague(conventional)
Z‐RR‐G‐SJanuary2019RieslingRheinhessencanesPrague(conventional)
Z‐MT‐KH‐S1January2019MullerThurgaucanesKutnaHora(organic)
Z‐MT‐KH‐S2January2019MullerThurgaucanesKutnaHora(organic)
Z‐RM‐KH‐S1January2019PinotNoircanesKutnaHora(organic)
Z‐RM‐KH‐S2January2019PinotNoircanesKutnaHora(organic)
J‐MT‐G‐L2May2019MullerThurgauleavesPrague(conventional)
J‐RR‐G‐S2May2019RieslingRheinhessencanesPrague(conventional)
J‐RS‐KH‐L1May2019PinotGrisleavesKutnaHora(organic)
J‐RS‐KH‐L2May2019PinotGrisleavesKutnaHora(organic)
J‐MT‐KH‐S4May2019MullerThurgaucanesKutnaHora(organic)
J‐RM‐KH‐S3May2019PinotNoircanesKutnaHora(organic)
J‐RM‐KH‐S4May2019PinotNoircanesKutnaHora(organic)
J‐RM‐KH‐S5May2019PinotNoircanesKutnaHora(organic)
J‐RR‐KH‐S2May2019RieslingRheinhessencanesKutnaHora(organic)
J‐RR‐KH‐S3May2019RieslingRheinhessencanesKutnaHora(organic)
L‐MT‐KH‐L5August2019MullerThurgauleavesKutnaHora(organic)
L‐RS‐KH‐L4August2019PinotGrisleavesKutnaHora(organic)
L‐RR‐KH‐L4August2019RieslingRheinhessenleavesKutnaHora(organic)
L‐RM‐KH‐S6August2019PinotNoircanesKutnaHora(organic)
P‐RM‐G‐L1October2019PinotNoirleavesPrague(conventional)
P‐RS‐G‐S2October2019PinotGriscanesPrague(conventional)
P‐MT‐KH‐L7October2019MullerThurgauleavesKutnaHora(organic)
P‐RM‐KH‐L7October2019PinotNoirleavesKutnaHora(organic)
MT‐M1September2019MullerThurgauberriesPrague(conventional)
MT‐M4September2019MullerThurgauberriesPrague(conventional)
RR‐M1September2019RieslingRheinhessenberriesPrague(conventional)
RR‐M2September2019RieslingRheinhessenberriesPrague(conventional)
RS‐M2September2019PinotGrisberriesPrague(conventional)
2.2.FungalEndophytesIsolationandCultivation
Theplantmaterialwassurfacesterilizedbysequentialimmersionin0.625%aqueous
sodiumhypochloritewithadropofTween80(7min),followedby70%aqueousethanol(3
min).Aftertheseprocedures,thesampleswererinsedfourtimeswithsterilizedwater(15
min).Thesurface‐sterilizedtissueswerehomogenizedandusedtoinoculateYGCmedium
(yeastextractglucosechloramphenicolagar)andincubatedat20°Cfor72hormore.
2.3.MolecularGeneticIdentificationofEndophytes
GenomicDNAwasisolatedfrompurefungusculturebyusingtheArchivePureDNA
YeastandGram‐+Kit(5PRIME,Hamburg,Germany).Subsequently,thenuclearribosomal
ITS1‐5,8S‐ITS2regionwasdeterminedforallstrainsaccordingtoKolaříketal.[26].Dueto

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thelowresolutionoftheITSregioninsomefungalspecies,thesequencingofothersections
wasmadetoclarifytheidentification.Elongationfactor1alpha(EF1α)wasamplifiedand
sequencedusingprimersEF‐728F/EF‐986RandEF1‐983F/EF1‐2218RaccordingtoKolaříket
al.[26].Thepartialβ‐tubulin(TUB2)genewasamplifiedusingT1/T2accordingtoPíchováet
al.[27].Thesequencesobtainedweremanuallycutfromunreadablesectionsandthehighest
probabilityoftheacquiredsequencewassearchedintheGenBankdatabase.
2.4.DeterminationofSiderophoresProductionoftheIsolates
ThemethodofMarquesetal.[28]wasfollowedtodeterminesiderophoreproduction.
FungalcultureswereinoculatedonChromeazurolS(CAS)agar,andcultivatedat28°Cfor
7days.Aftercultivation,thecolorchange(bluetoyellow)wasevaluatedandscaled(0=blue
mediumsurface,nosiderophoreproduction;1=30%yellowmediumsurface—low
siderophoreproduction;2=60%yellowmediumsurface—mediumsiderophoreproduction,
3=yellowmediumsurface,highsiderophoreproduction(Figure1.)).

Figure1.FungalendophyteculturedonCASagarwithhighsiderophoreproductionactivity(color
changefrombluetoyellow).
2.5.DeterminationofAntioxidantActivityoftheIsolates
ThefungalendophyteisolatesweregrowninPDBmediumat30°Cfor7dayswith
constantshaking.Theantioxidantactivityofthesupernatantofthefilteredculturewas
determinedaccordingtoFidlerandKolářová[29].Theanalyseswereperformedonthe
microtiterplatesinthreeparallelsforeachsample.Analiquotof100μLofthesamplewas
pipettedtogetherwith200μLofDPPHataconcentrationof52mgL−1(inmethanol)inthe
wells.Distilledwaterwasusedasablank.Theplatewasincubatedinthedarkfor15min.
Theabsorbancewasmeasuredat517nm.Theresultsoftheanalysiswereexpressedasthe
percentdecreaseinthediscolorationofthesolutionagainsttheblank.Theresultswere
expressedasanascorbicacid(AA)equivalent,whichwaschosenasananalyticalstandardin
theconcentrationrangeof2.5–25mgL−1.
2.6.DeterminationofAntifungalActivityoftheIsolates
AntifungalactivitywastestedonPDAagaraccordingtoBelletal.[30].Twowells(7.5
mmdiameter)wereexcavatedintheagaratthesamedistancefromthecenter.Oneofthe
wellswasfilledwithagarwithagrownfungalendophyteandtheotherwithagarcontaining
afungalphytopathogen.Theseplateswerecultivatedat28°Cfor7days.Testingwascarried
outwiththreefungalphytopathogens—BotrytiscinereaDBM1246,FusariumsolaniCCF2967,
andMucorplumbeusCCF2626.Thephytopathogeniccultureitselfservedasacontrolsample.
Antifungalactivitywasdisplayedbyslowingorstoppingthegrowthofafungal
phytopathogeninthevicinityofthegrowthofanendophyticfungus.Thedegreeoffungal
antagonismwasevaluatedonascaleof5–1(5—theendophytecompletelyoutgrowsthe
phytopathogen;4—theendophytecolonizes2/3ofthemediumsurface;3—theendophyteand
thephytopathogenbothcolonizehalfofthemediumsurface(Figure2);2—phytopathogen

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colonizes2/3ofthemediumsurface;1—thephytopathogencompletelyoutgrowsthe
endophyte).

Figure2.Fungalendophytewiththedegreeofantagonismoflevel‘3’tophytopatogen(colonizationof
halfofthemediumsurface).
2.7.StatisticalAnalysis
Dixon’sQtestwasperformedtodetectoutliersindatasetsobtainedbythedetermination
ofantioxidantactivity(thedeterminationwasperformedinfiveparallels.Thedeviationof
thefivedeterminationswaslessthan5%).Thedeterminationofabilitytoproduce
siderophoresandantifungalactivitywasperformedinthreeparallels.
3.Results
3.1.FungalEndophytesCharacterizationandMolecularGeneticIdentification
Forcanesandleaves,atotalof24endophyticmicroscopicfungibelongingto14fungal
generawereisolatedfrombothvineyards.Sixisolateswereobtainedduringthewinterfrom
bothconventionalandorganicfarminglocalities,tenisolateswereobtainedfromthespring
collection,withthemajorityofendophytesoriginatingfromorganicallygrownplants,four
fungalendophyticspecieswereisolatedfromsummersamplesfromtheorganicfarming
region,andfourisolatescamefromtheautumnsampling,frombothfarmingsystem
localities.ThegenusCladosporiumwasrepresentedbytwospecies,Cladosporiumcladosporioides
andCladosporiumherbarum.ThefurthermorerepresentedgenerawereDidymellasp.,
Aspergillussp.,Aureobasidiumsp.andAlternariasp.Allfiveisolatesobtainedfromtheberries
belongtoPenicilliumsp.,specificallytothespeciesPenicilliumcructosum(fordetails,seeTable
2).


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Table2.Thespeciesoffungalendophytesisolatedfromcanes,leavesandberriestaxonomyidentification,togetherwith
biologicalactivitiesofparticularisolates.
SampleCode1Sample
Matrix
EndophyteSpecies
Taxonomy
AbilitytoProduce
Siderophores2
Antioxidant
Activity
(mgAAL−1)
AntifungalActivity3to:
     Botrytis
cinerea
Fusarium
solani
Mucor
plumbeus
Z‐MT‐G‐S6 canesCladosporiumcladosporioides112.4222
Z‐RR‐G‐Scanes
A
lternariaarborescens013.8323
Z‐MT‐KH‐S1canesDiatrypestigma317.5222
Z‐MT‐KH‐S2canesDidymellanegriana04.8322
Z‐RM‐KH‐S1canesAspergilluspseudodeflectus221.8332
Z‐RM‐KH‐S2canesAspergillusniger313.4244
J‐MT‐G‐L2leavesEpicoccumnigrum217.6332
J‐RR‐G‐S2canesPleurophomaossicola02.7233
J‐RS‐KH‐L1leavesSporocadusrosigena16.6332
J‐RS‐KH‐L2leavesDendrophomajuglandina20222
J‐MT‐KH‐S4canesPseudogymnoascus
pannorum 00232
J‐RM‐KH‐S3canesAureobasidiumpullulans16.4222
J‐RM‐KH‐S4canesDidymellasancta18.3222
J‐RM‐KH‐S5canesCladosporiumherbarum07.5332
J‐RR‐KH‐S2canesPhaeosphaeriaceaesp.07.8222
J‐RR‐KH‐S3canesNeosetophomashoemakeri 26.7233
L‐MT‐KH‐L5leavesAspergillusfumigatus 19.3243
L‐RS‐KH‐L4leavesLophiostomacorticola18.3411
L‐RR‐KH‐L4leavesCladosporiumherbarum08.1232
L‐RM‐KH‐S6canesAureobasidiumpullulans10332
P‐RM‐G‐L1leaves
A
lternariaastroemeriae07.5332
P‐RS‐G‐S2canesAureobasidiumpullulans17.1332
P‐MT‐KH‐L7leavesCladosporiumherbarum 00111
P‐RM‐KH‐L7leavesDidymellasancta13.4333
MT‐M1berriesPenicilliumcrustosum110.5332
MT‐M4berriesPenicilliumcrustosum39.3452
RR‐M1berriesPenicilliumcrustosum113.9232
RR‐M2berriesPenicilliumcrustosum323.9453
RS‐M2berriesPenicilliumcrustosum319.1452
Boldformattingvalues—highabilitytoproducesiderophores,orhighantioxidantactivityorhighantifungalactivity;1See
Table1.forsamplecharacterization;2‘0’—nosiderophoreproduction;‘1’—lowsiderophoreproduction;‘2’—medium
siderophoreproduction,‘3’—highsiderophoreproduction;3Degreeofantagonism:5—theendophytecompletelyoutgrows
thephytopathogen;4—theendophytecolonizes2/3ofthemediumsurface;3—endophyteandphytopathogencolonizeeach
½ofthemediumsurface;2—phytopathogencolonizes2/3ofthemediumsurface;1—thephytopathogencompletelyoutgrows
theendophyte.
3.2.ProductionofSiderophores
Siderophoreproductionwasestablishedfor83%ofisolatesfromthewinterbiomass
collection,in60%ofisolatesfromthespringsampling,in75%ofisolatesfromsummer,in
50%offungalendophytesspeciesbeingisolatedfromautumnleavesandcanesamples,and

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inallendophytesisolatedfromberries.Thehighestabilitytoproducesiderophores(degree
‘3’)wasdetectedforendophytesfromwintersamplingandorganicallygrowncanes,in
particularforDiatrypestigma(Z‐MT‐KH‐S1isolate)andAspergillusniger(Z‐RM‐KH‐S2
isolate),andforendophytesoriginatingfromberries,i.e.,Penicilliumcrustosum(MT‐M4
isolate,RR‐M2isolate,RS‐M2isolate).
IsolatesJ‐MT‐G‐L2(Epicoccumnigrum),J‐RS‐KH‐L2(Dendrophomajuglandina)andJ‐RR‐
KH‐S3(Neosetophomashoemakeri)fromspringsamplingshowedthemediumabilityto
producesiderophores(degree‘2’),thehighestoneforthegivenperiod.Regardingthe
summerandautumnsampling,allisolateshadloworzerosiderophoreproductionability
(degree‘1’or‘0’),withtheexceptionofthethreeabove‐mentionedisolatesfromberries.The
detailsaresummarizedinTable2.
3.3.AntioxidantActivity
Theabilitytoproduceantioxidantsintothemediumwasidentifiedinallendophytes
isolatedfromwintercanes,in80%ofendophytesisolatedfromthespringbiomasscollection,
in75%ofisolatesoriginatingfromthesummerandautumnV.viniferabiomass,andinallof
theisolatesfromberries.Thecontentofantioxidantsexpressedasascorbicacid(AA)
equivalentwasdeterminedintherangeof4.8–21.8mgAAL−1forthewinterisolates,0–17.6
mgAAL−1forthespringisolates,0–9.3mgAAL−1forthesummerisolates,0–7.5mgAAL−1forthe
autumnendophytesfromleavesandcanesand9.3–23.9mgAAL−1fortheautumnisolatesfrom
berries.Theendophyteswiththehighestantioxidantproductionwereisolatesfromberries,
i.e.,Penicilliumcrustosum(RR‐M2isolateandRS‐M2isolate),with23.9and19.1mgAAL−1,
respectively,andAspergilluspseudodeflectus(Z‐RM‐KH‐S1isolate)fromwinterorganically
farmedcanes(21.8mgAAL−1).Fordetails,seeTable2.
3.4.AntifungalActivity
Thehighestdegreeofantagonism,level‘5’explainingthehighestantagonismwherethe
endophytecompletelyoutgrowsthephytopathogen,wasagainstF.solaniandwasobserved
forPenicilliumcrustosum(MT‐4,RR‐M2,RS‐M2)isolatedfromberries.Allthesethree
endophyticisolatesalsoshowedsignificantantifungalactivityagainstB.cinerea(level‘4’).
TheotherrelativelystrongantagonistofB.cinereaDBM4111wastheendophyte
Lophiostomacorticola(L‐RS‐KH‐L4)isolatedfromorganicallygrownleavescollectedin
summer.ForthephytopathogenF.solaniCCF2967,theotherhighlyeffectiveendophytes
wereAspergillusniger(Z‐RM‐KH‐S2)isolatedfromorganicallyfarmedwintercanesand
Aspergillusfumigatus(L‐MT‐KH‐L5)isolatedfromorganicallyfarmedsummerleaves.For
MucorplumbeusCCF2626,thehighestantagonistwasAspergillusniger(Z‐RM‐KH‐S2)isolated
fromorganicallygrownwintercanes.Fordetails,seeTable2.
4.Discussion
Recently,endophyticmicroorganismsandtheirproductshavebeenattractingthe
attentionofthescientificcommunityasarelativelypoorlyunderstoodsourceofawiderange
ofchemicallydiversenaturalsubstancespotentiallyusableinbiotechnology,pharmaceutical
andfoodindustry.Theincreasedattentionforstudyingendophyticpopulationsisbasedon
thedesiretoproducenon‐chemicalbasedsolutions.Comparingendophyticpopulationsin‐
situintermsofthepresenceofindividualmicroorganismsortheformationoftheir
metabolitesischallengingduetothemanyfactors(altitude,temperature,totalprecipitation,
rhizospherecomposition,orpesticideuse)thataffectthesepopulations.Despitethis,the
methodsfortestingtheendophyticisolatesinlaboratoryconditionsarewellestablished.


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4.1.FungalEndophytesCharacterizationandMolecularGeneticIdentification
Anotherdegreeofvariabilityintheendophytecompositionisthephysiologicalstateof
thehostplantitself,itsgrowthphase,andthetissuefromwhichthesampleistaken.Thus,
evenisolatesfromthesamegeographicallocationmaybediametricallydifferent[31–34].This
variabilitywasconfirmedinthiswork,whereisolatesoffungalendophytesfromtwo
vineyardsofdifferentfarmingsystemswereexamined.TheKutnaHoravineyardsgrow
grapevinesaccordingtotheprinciplesoforganicfarming,andthePraguevineyardsgrow
theirgrapevinesinaconventionalway.Thetotalnumberoffungalendophytesisolatedfrom
canesandleavesfromorganicallygrownplantswasapproximatelythreetimeshigherthan
thenumberofendophytesisolatedfromconventionalvineyards,whichisconsistentwith
previousstudies[35,36].Thisdifferencecouldberelatedtotheuseofchemicalororganic
fertilizersandherbicidesthatdirectlyaffectmicroorganismsoralterthephysiologyofthe
hostplant[37,38].Theresponseofendophyticmicrobialcommunitiestotheseexternal
productsisverybeneficialforcomparingorganicandconventionalagriculture,andfurther
researchcouldgointhisdirection.
Precipitationisoneofthemainabioticfactorsthataffectthedensityofendophytesinthe
hostplant[39–41].Theproportionofendophytesintheleavesoftreesthathavebeen
protectedfromrainislowerthanintheleavesofidenticaltrees,butunprotectedfromrainfall.
Suryanarayananetal.dealtwiththisissueintherainforestenvironmentduringtherainy
seasonanddrought.InalltestedleafsamplesofBauhiniaracemosa,Ixoranigricans,Erythroxylon
monogynumandElaeodendronglaucum,anincreasedrepresentationoftheendophytic
communitywasdetectedduringtherainyseason[40].Themoreabundantcolonizationof
plantsbyfungalmicroorganismsathigherprecipitationratesmayberelatedtothe
consequentincreasednumberofendophytesinthehostplant.Precipitationisalsooneofthe
majortypesofendophyticsporetransmission.Sofar,thereisalittleinformationonwhere
endophytesporesareproduced,wheretheycanhibernateandwhatthemodeoftransmission
is.R.parkerisporulatesprolificallyonContariniamidgegallsonDouglasfirneedles,andthere
weremeasured1200spores/mLinwaterdrippingfromaheavilygalledbranchlet.R.parkeri
anditsanamorphalsosporulateinthefallinabscisedneedles.Thesporemassesofthis
endophyteareproducedinmucilage,whichisindicativeofwatertransmission.Further,
newlyflushedneedlesinthespringdonotbecomeinfecteduntiltheyarerainedoninthe
fall.Fromthesefindingswecouldsuggestthattheassociationbetweenhigherendophyte
countsandmoistureisnotaccidental[41].Ifwefollowthenumberofcaneandleafisolates
obtainedfromindividualperiodsoftheVitisviniferagrowingyear,thepredominanceof
springsamplingisevident(Table1.).May2019(springsampling)waswellabovethelong‐
termprecipitationaverage.August2019(summersampling)wasonlyslightlybelowthis
averageandOctober2019(autumnsampling)wasquiteaverage,whichcorrespondstoa
lowerendophyticproportion(Figure3).However,heavyrainandstormscouldcompletely
destroythegrapevinecrop,whichunfortunatelyoccurredinautumn2019intheKutnaHora
vineyards.Thesenaturalphenomenamadethesamplingofberriesfromthisbiodynamic
vineyardinSeptember2019impossible.

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Figure3.AverageprecipitationfromFebruary2019toOctober2019comparedtothelong‐termaverage(1981–2010)(data
fromtheCzechHydrometeorologicalInstitute).
TheamplificationofITSrDNAandsubsequentcomparisonoftheobtainedsequence
withthedatabaseiscurrentlythemostwidelyusedmethodfortheidentificationoffungal
endophytes[42,43].All29isolatesbelongingto15generaweresuccessfullyidentified,andall
ofthembelongedtotheAscomycotaphylum.Thisphylumsignificantlypredominatesinthe
proportionoffungalendophytesinVitisvinifera,regardlessofthegeographicallocationofthe
hostplant[34,44].ThemostabundantgenerawerePenicilliumsp.,Cladosporiumsp.,Didymella
sp.,Aspergillussp.,Aureobasidiumsp.,andAlternariasp.Alternariasp.andCladosporiumsp.are
oneofthemostabundantendophytesofVitisvinifera[34,43],whichisinaccordancewithour
results.
4.2.ProductionofSiderophores
Siderophoreshavereceivedgreatattentioninmedicine,biotechnology,and
environmentalresearchduetotheirhighaffinityandspecificityforFe3+.Theonlyfungal
endophyteisolatesfromcanesandleaveswithahighabilitytoproducesiderophorescame
fromthewinterperiodof2019.Theabilitytoformthesecompoundshasbeendecliningsince
winter,withonlyloworzeroproductionactivityinsummerandautumn.Thehighestresult
ofsiderophoreproductioninwintercanbeexplainedbythereducedmovementofnutrients
inthesoilduetolowtemperaturesandthereforeirondeficiencyinboththeendophyteand
thehostplantandtheincreasedneedforuptakeofthesenutrientsbyothermechanisms[45].
DiatrypestigmaandAspergillusnigerwereisolateswiththehighestdetectedsiderophore
productionactivity.Inthispaper,theproductionofsiderophoresbythegenusDiatrypewas
provedforthefirsttime.Withregardtothehighactivityofproductionofthesecompounds
identified,itwouldbeinterestingtopayfurtherattentiontothespeciesofthismicrobial
genusintheresearch.Aspergillusspeciesarewell‐researchedproducersofsiderophores,
servingasamodelorganismtoelucidatethebiosynthesis,absorption,anddegradationof
thesesecondarymetabolites[46].Threeberryisolatesalsoshowedahighabilitytoproduce
siderophores.AlloftheseisolatesbelongtoPenicilliumcrustosum,whichisinagreementwith
thefindingsintheliteraturethatthisgenusiscapableofsiderophoreproduction[47].Ina
studyonthecharacterizationofsiderophoresproducedbyendophytesfromCymbidium
aloifolium,thegenusPenicilliumwasfoundtobethebestproducerofthesecompounds[48].

0
20
40
60
80
100
precipitation[mm]
averagemonthlyprecipitation(2019)
long‐termaveragemonthlyprecipitation(1981‐2010)

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4.3.AntioxidantActivity
Thereisgrowingevidenceofoxidativedamagetobiomoleculesbyfreeradicals.These
injuriescouldcausemuchtissueharm.Antioxidantsareconsideredhighlyeffectivein
defendingtissueagainstdamagecausedbyreactiveoxygenspecies[49].Fungalendophytes
canbeapotentiallyverygoodsourceofantioxidants[50]whichhasbeenconfirmedinisolates
inthisstudy.Theisolatewiththehighestantioxidantactivity(21.7mgAAL−1)ofcanesand
leaveswasAspergilluspseudodeflectus.AroraandChandra[51]investigatedtheantioxidant
activityofthegenusAspergillus,specificallyAspergillusfumigatus.Thedatahaveshownthat
thismicroorganismcanserveasapromisingsourceofantioxidantcompounds.Ourresults
showanevenhigherantioxidantactivityforthefungalendophyteAspergilluspseudodeflectus
thanwasmentionedinthearticle.Otherstudiesalsomentionthehighantioxidantactivityof
endophytesofAspergillussp.andthepossibilitiesoffurtheruseoftheseproperties[52,53].
Therefore,thisfungalgenuscouldservetomoreeasilyadjusttheproductionandpurification
ofnaturalantioxidants.
Penicilliumisanotherfungalendophyticgenusstudiedinmoredetailwithhigh
antioxidantactivity[7,54].Inberries,twoofthePenicilliumcrustosumisolatesshowedhigh
antioxidantactivity(23.9and19.1mgAAL−1)whichisinconnectionwithpreviousstudies
[7,55,56].OtherfungalendophyteswithhighantioxidantactivityareFusariumsp.[57,58]and
Burkholderiaphytofirmans[59].Inotherstudy,fungalendophytesDiaporthesp.,Colletotrichum
sp.,andArthiniumsptendtogenerateawidearrayofbioactivecompounds(β‐dihydro
agarofuran,α‐agarofuran,δ‐eudesmol,β‐agarofuran,andoxo‐agarospirol)withstrong
antioxidantactivity[60].AccordingtoHamiltonandBauerle,theantioxidantactivityin
plantswithendophytesunderabioticstressishigherthaninplantswithoutthese
microorganisms[61].
Ingeneral,theproportionofgenerawithantioxidantactivityinVitisviniferaisrelatively
high,whichcorrelateswiththeassumptionoftheformationofsimilarsecondarymetabolites
betweenthehostplantanditsendophytes.Grapevineitselfisanimportantsourceof
antioxidants,especiallyphenolicsubstances.
4.4.AntifungalActivity
ThefungalendophytesofVitisviniferacouldhaveanantagonisticeffectonsome
importantphytopathogens.Studiesmappingthisantifungalabilityofendophytic
communitiesareessentialtoshapepestcontrolstrategiesbutalsotopotentialproductionof
high‐qualityagriculturalproducts.Inthecaseofgrapevine,oneofitsmostcommon
pathogensisthefungusBotrytiscinerea,whichcausesBotrytisbunchrot.Themosteffective
antifungalagentsagainstthisphytopathogenaretheendophytesAlternariasp.andEpicoccum
sp.BotharealsopromisingbiocontrolagentsagainstPlasmoparaviticola,anotherimportant
sourceofVitisviniferadiseases[34].TheantifungalabilityfoundagainstBotrytiscinereain
AlternariaandEpicoccumisolatesinthisresearchworkwasexpressedbythedegreeof
antagonismatlevel‘3’,whichcouldbeexpressedas50%.Thementionedgeneradidnotshow
aboveaverageactivityevenagainsttheothertwotestedphytopathogensFusariumsolaniand
Mucorplumbeus.Fusariumsolaniisanimportantplantpathogenthatmostoftencausesrotin
therootsystem.Ithastheabilitytopenetratecellwallsandthereforecauseplanttissuetorot
[62].Mucorplumbeusisassociatedwiththegrowthoffungiincereals,rice,soybeans,nuts,
fruits,herbs,andothers[63].
Thehighestdegreeofantagonism(level‘4’)againstBotrytiscinereawasdetectedin
isolatesfromcanesandleavesinonlyoneendophyte,Lophiostomacorticola.Thisisthefirst
papertoshowtheabilityofthisfungustoproduceantifungalcompounds.Theabilityofthe
Lophiostomagenushasbeenshowntoproducemetabolitesthatareeffectiveonlyagainst
pathogenicbacteria[64,65].However,Lophiostomacorticolahadadegreeofantagonismof

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level‘1’againstFusariumsolaniandMucorplumbeusandthereforezeroantifungalactivity.For
suchaquestionableresult,itwouldbeidealtoperformantifungaltestswithother
phytopathogenstodetecttheantifungalactivityofthisgenus.Whenwecontinuewiththe
antifungalresultsfromisolatesconnectedwithcanesandleaves,thehighestactivityagainst
FusariumsolaniwasdetectedintwoisolatesofthegenusAspergillus,Aspergillusnigerand
Aspergillusfumigatus.Aspergillusnigerwastheonlyspeciestoshowthehighestactivityalso
againstMucorplumbeus.AntifungalactivityagainstthephytopathogensGiberellazeae,
Thanatephoruscucumerisandsixothernonpathogenicmicroscopicfungiwasdetectedinthe
endophyteAspergillusfumigatusisolatedfromHyoscyamusmuticus[66].Aspergillusflavus,an
endophyteofLanneacoromandelica,showedhighantifungalactivityagainstCandidaalbicans
andMalasseziapachydermis.Aspergillusnigerisolatedfromthesamehostplantshoweda
moderateabilitytoformantifungalmetabolitesagainstthementionedphytopathogens[67].
Fromthementionedstudies,itcanbeconcludedthattheantifungalactivityofthegenus
Aspergillusishigh,whichisinaccordancewithourresults.ThegenusPenicilliumisknown
foritsantifungaleffectonBotrytiscinerea[7,68,69]andalsoshowsthiseffectonFusariumsp.
[70,71].Penicilliumcrustosumisolatesfromberriesconfirmedthesefindings,astheyexhibited
highantifungalactivityagainstbothBotrytiscinereaandFusariumsolani.
5.Conclusions
ThepopulationofendophyticfungiofVitisviniferahasproventobeapromisingsource
ofgrowth‐promotingandprotectivepropertiesusefulfortheplant.Furtherstudiesare
neededtoinvestigateendophyticfungiindetailasapotentialsourceofsecondary
metabolites.Asitisaverypoorlyresearchedsourceofmetabolites,itwouldbeinterestingto
usethefindingsofthisresearchwork,choosethemostproductiveendophyticspecies,and
conductdetailedresearch.Acloserfocusontheformationofsiderophorescouldbevery
usefulinconjunctionwithenhancementofplantgrowthandbiocontrolagainst
phytopathogens.Thestudyofantifungalmetabolitescouldbeusedtodevelopeffective
biopesticidesthatcouldbeamoreenvironmentallyfriendlyoptionforboththeplantandthe
environment.
AuthorContributions:Conceptualization,I.K.,P.L.andM.K.(MarkétaKulišová);methodology,P.L.,
I.K.,M.K.(MiroslavKolařík)andB.V.;formalanalysis,M.K.(MarkétaKulišová)andI.K.;investigation,
M.K.(MarkétaKulišová),B.V.,M.V.andM.K.(MiroslavKolařík);resources,P.L.,M.S.andI.K.;data
curation,M.K.(MarkétaKulišová),M.V.,P.L.andI.K.;writing—originaldraftpreparation,M.K.
(MarkétaKulišová)andM.S.;writing—reviewandediting,M.K.(MarkétaKulišová);visualization,
M.K.(MarkétaKulišová)andM.V.;supervision,P.L.andI.K.;projectadministration,I.K.andM.S.All
authorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:ThisresearchwasfundedbyTheCzechScienceFoundation(GACR),grantnumber18‐26463S.
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
Acknowledgments:WeacknowledgePavelBulánekforthepossibilityofcollectingVitisvinifera
samplesonvineyardsinPragueandStanislavRudolfskýonvineyardsinKutnaHora.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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