Molecular Mechanisms Underlying Fungicide Resistance in Citrus Postharvest Green Mold

Abstract

The necrotrophic fungus Penicillium digitatum (Pd) is responsible for the green mold
disease that occurs during postharvest of citrus and causes enormous economic losses around the
world. Fungicides remain the main method used to control postharvest green mold in citrus fruit
storage despite numerous occurrences of resistance to them. Hence, it is necessary to find new and
more effective strategies to control this type of disease. This involves delving into the molecular
mechanisms underlying the appearance of resistance to fungicides during the plant–pathogen
interaction. Although mechanisms involved in resistance to fungicides have been studied for many
years, there have now been great advances in the molecular aspects that drive fungicide resistance,
which facilitates the design of new means to control green mold. A wide review allows the
mechanisms underlying fungicide resistance in Pd to be unveiled, taking into account not only the
chemical nature of the compounds and their target of action but also the general mechanism that
could contribute to resistance to others compounds to generate what we call multidrug resistance
(MDR) phenotypes. In this context, fungal transporters seem to play a relevant role, and their mode
of action may be controlled along with other processes of interest, such as oxidative stress and
fungal pathogenicity. Thus, the mechanisms for acquisition of resistance to fungicides seem to be
part of a complex framework involving aspects of response to stress and processes of fungal
virulence.

Full text

J.Fungi2021,7,783.https://doi.org/10.3390/jof7090783 www.mdpi.com/journal/jof
Review
MolecularMechanismsUnderlyingFungicideResistance
inCitrusPostharvestGreenMold
PalomaSánchez‐Torres
DepartmentofFoodBiotechnology,InstituteofAgrochemistryandFoodTechnology,SpanishNational
ResearchCouncil(IATA‐CSIC),CalleCatedráticoAgustínEscardino7,Paterna,46980 Valencia,Spain;
psanchez@iata.csic.es;Tel.:+34‐963‐900‐022
Abstract: The necrotrophic fungus Penicillium digitatum (Pd) is responsible for the green mold
diseasethatoccursduringpostharvestofcitrusandcausesenormouseconomiclossesaroundthe
world.Fungicidesremainthemainmethodusedtocontrolpostharvestgreenmoldincitrusfruit
storagedespitenumerousoccurrencesofresistancetothem.Hence,itisnecessarytofindnewand
moreeffectivestrategiestocontrolthistypeofdisease.Thisinvolvesdelvingintothemolecular
mechanisms underlying the appearance of resistance to fungicides during the plant–pathogen
interaction.Althoughmechanismsinvolvedinresistancetofungicideshavebeenstudiedformany
years,therehavenowbeengreatadvancesinthemolecularaspectsthatdrivefungicideresistance,
which facilitates the design of new means to control green mold. A wide review allows the
mechanismsunderlyingfungicideresistanceinPdtobeunveiled,takingintoaccountnotonlythe
chemicalnatureofthecompoundsandtheirtargetofactionbutalsothegeneralmechanismthat
couldcontributetoresistancetootherscompoundstogeneratewhatwecallmultidrugresistance
(MDR)phenotypes.Inthiscontext,fungaltransportersseemtoplayarelevantrole,andtheirmode
of action may be controlled along with other processes of interest, such as oxidative stress and
fungalpathogenicity.Thus,themechanismsforacquisitionofresistancetofungicidesseemtobe
part of a complex framework involving aspects of response to stress and processes of fungal
virulence.
Keywords:citrus;fungicideresistance;postharvest;Penicilliumdigitatum;infection
1. Introduction
Citrusfruitsareimportantfruitcropsaroundtheworldbecausetheyprovide
numerousnutrientsthatpromotehumanhealth[1,2].Citrusfruitsaresubjecttodifferent
bioticorabioticstressesduringthepostharvestperiod,whichincludeshandling,
shipping,storage,andmarketing.Inthiscontext,fruitspoilageandfoodsafetyrisksdue
topostharvestfungaldiseasesconstitutesomeofthemostsignificantthreats[3].
Penicilliumdigitatum(Pers.:Fr)Sacc.(Pd),whichcausesgreenmold,isthemajor
postharvestrotofcitrusfruits[1].Postharvestgreenmoldcauseshugeeconomiclosses
worldwideeveryyearandcanaccountforupto90%ofthetotalpostharvestdamageto
citrusfruits,especiallyindryareasandsubtropicalclimates[4,5].
Pdiscapableofinvadingandinfectingthefruitthroughwoundsthatareproduced
byenvironmentalfactorsorduringtheharvestdevelopment,transport,andfurther
treatments[6].Thisfungusextendsinoilglandsthroughrindwounds[7],wheretheycan
accessnutrientsthatpromotegerminationoftheconidia.Thestartingpointofinfection
isthesofterandmorewateryareaonthesurfaceoftheskinwhere,withsuitable
temperatureandoptimalconditions,itprogressestogiverisetoawhitemyceliumthat
laterturnstoolivecolorduetotheappearanceofconidia[8,9](Figure1).
Citation:Sánchez‐Torres,P.
MolecularMechanismsUnderlying
FungicideResistanceinCitrus
PostharvestGreenMold.J.Fungi
2021,7,783.
https://doi.org/10.3390/jof7090783
AcademicEditor:IvanM.
Dubovskiy
Received:2September2021
Accepted:20September2021
Published:21September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
(http://creativecommons.org/licenses
/by/4.0/).

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Figure1.GreenmolddecayinorangefruitinfectedwithPenicilliumdigitatum.(a)Orangefruitwith
greenmoldsymptomscausedbyP.digitatum;(b)typicalaspectP.digitatumgrowingonPDAplates;
(c)P.digitatumconidiphorewithconidiaborneterminallyinchainsobservedbybright‐fieldoptical
microscopy;(d)P.digitatumwithfluorescenceafterstainingwithCFW.
Pdwholegenomesequencinghasrecentlyopenedupnewpossibilitiestoinvestigate
fungalfactorsrelatedtohost–pathogeninteractionsrangingfromvirulencefactorstogenes
involvedinfungicideresistancemechanisms.Understandingtheinfectionprocessandthe
fungalstrategytoovercomefungicidesisonewaytodevelopnewformsofcontrol[10,11].
Thecontrolofgreenmoldiscurrentlyachievedthroughtheapplicationofsynthetic
compounds,suchasimazalil,thiabendazole,pyrimethanil,andfludioxonil[12].However,
thecontinueduseofchemicalstopreventfungaldiseaseshasrestrictedtheireffectiveness
andshelflife.Theemergenceofpathogenicfungithatisresistanttosyntheticfungicides
mainlyusedforthecontroloffungalinfectionsposesarisktotheenvironmentand
consumerhealth[3],thuspromptingresearchtodevelopnew,moreeffectivecontroltools.
2.FungicideResistanceHasBecomeaMajorProblem
Fungicidesarecrucialtopreservehealthy,consistent,high‐qualityagricultural
goods.Until1970,almostallchemicalsusedtomanageplantpathogensweremultisite
inhibitorsthatworkedasprotectorsofdiseases.Inspiteoftheirextensiveuseinsome
cases,resistancehasnotevolvedtolargelynonsystemicprotectantfungicidesduetotheir
multisitemodesofaction[13].However,sincetheintroductionofsite‐specificfungicides
inthelate1960s,fungicideresistanceinplantpathogenicfungihasemergedasamajor
problemincropcontrol[14].Sincethe1970s,therehasbeenanimprovementincrop
protectionowingtosystemicsingle‐sitefungicidesthatpossessbothprotectiveand
eradicatingcharacteristics,suchasmethylbenzimidazolecarbamates(MBC),sterol
biosynthesisinhibitors(DMIs),externalquinoneinhibitors(QoI),andsuccinate
dehydrogenaseinhibitors(SDHI)(Table1).
Resistancetofungicidesresultsinreductioninsensitivitytocertaincompoundsand
iscausedbyaninheritedadjustmentofthefungustothatcompound.Itisnormallydue
toeithersingleormultiplegeneticmutations.Theidentificationofresistantisolates
appearswithanaturalrateofgeneticmutation,sothenumberofresistantstrainsis
generallynotaffectedbytheapplicationofafungicide[15].

a
b
c
d

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Table1.Typesoffungicidesusedincitruscontrolprogramsandtheirtargets.
FRACCodeFungicideClassCelularFunctionAffectedTargetProteinRiskResistance
Development
1Methylbenzimidazoles
(MBCs)Cytoskeleton‐tubulinHigh
3Demethylationinhibitors
(DMIs)
Membrane
biosynthesis
Sterol14‐
demethylase
(CYP51)
Medium
11
Quinoneoutside
inhibitors
(QoIs)
RespirationMitochondrial
cytochromebHigh
7Succinatedehydrogenase
inhibitors(SDHIs)RespirationSuccinate
dehydrogenaseMediumtoHigh
12Phenylpyrroles(PPs)
Alteredtargetsite(protein
kinaseinvolvedin
osmoregulation)
ProteinkinaseLowtomedium
9Anilino‐pyrimidines
(APs)
Alteredtargetsite(protein
kinaseinvolvedin
osmoregulation)
ProteinkinaseMedium
Whilefungicideseffectivelykillsensitivestrains,resistantstrainsbecomedominant
overtimebecausepathogenpopulationsareunderselectionpressurefromcontinued
fungicideuse,leadingtofailuretocontroldisease[16].Thefitnessofthefungicide‐
resistantfungalisolatesonceselectedwilldeterminethepermanenceoftheresistant
genotypes.Insomecases,ithasbeenobservedthatresistantstrainsmayhaveless
aptitudethansusceptiblestrains,thusrequiringselectionpressureofthefungicideto
survive.Therefore,whentheapplicationoffungicidesceases,thenumberofresistant
isolatesinpathogenpopulationswilldecrease.Ontheotherhand,thestrongisolates
presentafitnesssimilartothesensitiveisolates,andtheycouldremainforalongtime
evenwithouttheapplicationoffungicides[17].
Likeinallorganisms,thereisgeneticvariationinpathogenicfungalpopulations.
Thisvariationprovidesthestartingpointfromwhichfungicideresistanceprogresses.A
completepopulationofresistantpathogenicfungidevelopsowingtonaturalselection,in
whichtheenvironmentfavorsthereproductionandproliferationofresistantforms.
Individualfungicideapplicationscanbeconsideredthe“selectionevents”thatpromote
thisprocess,selectivelykillingsusceptiblefungi.However,anyresistantmutantwill
survivetheseeventsandsubsequentlyhavetheopportunitytogrowandreproduce
withoutcompetitionfromsusceptiblefungi.Afteroneapplication,thisincreasingly
resistantpopulationcanproliferateandreproduce[18](Figure2).

Figure2.Adiagramoftheevolutionofresistancetofungicides.Thisgraphicshowsanexampleofhowselectionpressure
maytakeplace.Initialpopulationwithlittleresistanceevolvesuntilresistancebecomeswidespreadduetorepeated
fungicideapplications.AdaptedfromDeisingetal.[18].
Resistancetofungicidesmightbeduetovariousprocedures[19–22],including(a)
reducedfungicidebindingduetoalterationofthetargetsite,(b)overexpressionofthetarget
protein,(c)reducedfungicideabsorptionduetoeffluxpumpremovingtoxiccompounds,
and(d)metabolicdegradationofthefungicidethroughdetoxification(Figure3).

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Figure3.MainmechanismsofacquiringresistancetofungicidesinP.digitatum.Mechanismsof
resistancetosingle‐sitefungicides:(a)detoxificationoffungicidethroughmetabolicenzymes;(b)
reducedfungicidebindingduetoalterationofthetargetprotein;(c)overexpressionofthetarget
protein;(d)effluxpumpsremovingfungicideoutofthecell.AdaptedfromLucasetal.[17].
Themechanismsinvolvedintheappearanceofresistancetofungicidesin
populationsoffieldpathogensentailthestudyoftheprocessesthatinterveneinthe
reductionofsensitivitytothecompoundandthegeneticbasisoftheresistancetrait.As
thereareseveralclassesofsingle‐siteinhibitors,itislikelythatthereareseveral
mechanismsthatleadtofungicideresistanceinplantpathogens,includingthemajor
citruspathogenPd.
Thenewestbiotechnologyforgenomeeditingisapromisingtoolforthe
developmentofdisease‐resistantcropsinthefuture[23].Thus,investigatingthe
molecularmechanismsunderlyingfungicideresistanceinplant–pathogeninteractionsis
essentialfordevelopingnewandbetterapproachesforefficientlycontrollingplant
diseases.
Thefruit–pathogeninteractionisfundamentalfortheprogressionoffungal
pathogen.Ithasthereforepromptedgreatinterestintheresearchcommunity,and
numerousstudieshavebeenundertakeninrelationtothevirulenceofpathogensandthe
responseofthefruittoinfection[3].Inthecitrus–Pdinteraction,ithasbeenadvantageous
tohavethecompletesequenceofthePdgenomeaswellasthegenetictransformation
systemsforPd[5,8,24].Thishasmassivelyfacilitatedknowledgeofthemolecular
processesunderlyingthepathogenicityofPd[25].
Thisreviewpresentsanoverallviewofrecentadvancesinthefungicideresistance
mechanismsofpostharvestcitrusgreenmold,providingvaluableinformationonthe
molecularproceduresinvolvedintheachievementofresistancetodifferentchemicals,
eithertoasinglecompoundortoseveralcompoundsatthesametimeinthecontextof
thefruit–pathogeninteraction.Thisinformationisbeneficialfordevelopingnoveland
saferstrategiestopreventpostharvestgreenmoldincitrusfruitsandcontributes
substantiallytoknowledgeonfungaldiseasemanagement.
3.MolecularMechanismsofFungicideResistance
Fungicideresistancecanevolvedifferentlybasedonthecharacteristicsofthe
fungicide(fungicideclass)(Table1).

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3.1.MethylBenzimidazoles(MBCs)
Themechanismofbenzimidazole‐typefungicides,whichincludesthiabendazole
(TBZ),involvesbindingtoβ‐tubulins.Thispreventstheassemblyofmicrotubulesandcell
divisionduringmitosisandthereforeresultsintoxicitytofungalcells[26,27].
Resistancetobenzimidazolefungicideshasbeendescribedinawidevarietyoffungi.
Frequently,theresponsiblemechanismcorrespondstopointmutationsinthe‐tubulin
gene,whichleadstothemodificationofsomeaminoacids[28–30].Amongthenumerous
changesobservedintheβ‐tubulingeneassociatedwithresistancetoMBCfungicidesin
phytopathogenicfungi,themostfrequenthavebeeninresidues198and200[14].Itshould
benotedthatthereplacementofglutamicacidbyalanine,valine,orglycineatposition
198andphenylalaninebytyrosineatposition200canleadtovaryinglevelsofresistance
toMBCfungicides[31,32].IntheparticularcaseofTBZ,modificationsintheTBZbinding
siteprovidescellularresistancetoit.Suchvariationsusuallyoccuratpositions198or200
ofβ‐tubulin,althoughotherchangesarealsopossible[14,33].InPd,twodifferentpoint
mutationshavebeendescribedasbeingresponsibleforTBZresistance.Mutationat
position198GlutoLyswasdescribedbyMaandMichailides[14]basedonstudies
performedinPenicilliumexpansum[27].InPdisolatesfoundincitrusfruitsfromCalifornia
packinghouses,resistancetoTBZwasduetomodificationatposition200ofβ‐tubulin
[34].ThesamemutationwaslaterdescribedinPdSpanishisolatescollectedfromorchards
andpackinghouses[35],revealingthatresistancemechanismisindependentoffungicide
pressure.AmongPdisolatescollectedfromcitrusinTaiwan,resistancetoTBZwas
associatedwiththemostfrequentβ‐tubulinmutationsatcodon198or200[36].
However,untilnow,nostudyhasdescribedthemolecularprocessbywhichgenetic
variationsinβ‐tubulinpreventthebindingoffungicide.Recently,researchcarriedouton
PodosphaeraxanthiiusingacombinationofdifferentapproachesproposedthattheMBC
fungicidebindingsiteinβ‐tubulindoesnotparticipateintheresiduesresponsiblefor
fungalresistance[37].Asamechanism,itissuggestedthatwhenMBCfungicides
spontaneouslybindtoβ‐tubulininsensitivefungi,theirconformationisalteredand
adequatepolymerizationinmicrotubulesoccurs;however,thisdoesnottakeplacein
resistantstrains,wherethereisaconformationalchangepromotedbyspecific
modifications.
3.2.DemethylationInhbithors(DMIs)
DMIfungicideshampertheactivityofthecytochromeP450‐dependentsterol14α‐
demethylase(Cyp51)andthusblockC14‐demethylationoflanosterol,aprecursorof
ergosterolinfungalpathogens[38].DMIsencompassoneofthemostrelevantgroupsof
fungicidesthatpreventdifferentplantdiseasesbyinhibitingtheactivityofcytochrome
P450‐dependentsterol14α‐demethylase(P45014DM)andwerefirstusedinagriculturein
the1970s[39].Imazalilisademethylationinhibitor(DMI)thatblocksergosterol
biosynthesis[40,41]andisfrequentlyusedtopreventpostharvestdiseasesofcitrusfruits
worldwideduetoitscurativeandantisporulantactionagainstPd[42].CYP51encodes
sterol14demethylase,anenzymeresponsibleforergosterolbiosynthesis[43],andisthe
targetofDMIfungicides.
ThemainmechanismsthatprovideDMIresistanceare(i)modificationsinCYP51or
(ii)highexpressionofCYP51.DifferentprocedurescausingDMIresistancehavebeen
reported.Theyaremediatedeitherbyspecificchangesinthecodingregion[44–46]orby
augmentinggenetranscriptionduetoaninsertioninthepromoter[47].Therearethree
homologuesofthesterol14α‐demethylase‐encodedCYP51geneinPd,namely
PdCYP51A[48],PdCYP51B,andPdCYP51C[49].Thefirstmechanisminvolving
modificationsinCYP51hasbeendescribedinseveralpathogens.Asinglechange,suchas
thesubstitutionofaphenylalanineforatyrosineatresidue136(Y136F)ofCYP51,ledto
resistancetoDMIinUncinulanecator[50],Erysiphegraminisf.sp.hordei[51],Erysiphenecator
[52],andP.expansum[44],whiletwosinglenucleotidechangeswerefoundtoresultin

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aminoacidsubstitutionsY136FandK147QinCYP51inBlumeriagraminis[53].Other
changeshavebeendescribedinTapesiasp.[54],Penicilliumitalicum[55],Ustilagomaydis
[56],Blumeriellajaapii[57],andMycosphaerellagraminicola[58].InPd,noPdCYP51Apoint
mutationswerefoundtoberesponsibleforPdresistancetoIMZorotherDMI[35]orto
prochloraz[46].Ontheotherhand,inPdCYP51B,novariationsinthegenewereinitially
detectedinisolatesresistanttoIMZ[59].However,recently,differentsubstitutionsof
PdCYP51Bhavebeenfoundcorrespondingtodifferentlevelsofsensitivitytoprochloraz,
namelyY136HandQ309Hinhighresistantstrains,G459SandF506Iinmediumresistant
strains,andQ309Hinlowresistancestrains[46].
TheotherprocessresponsibleforresistancetoDMIischangeinthelevelofCYP51
transcription[60].Themostfrequentmechanismisthepresenceofinsertionsinthe
promoterregioninthephytopathogenicfungus,aswasthecaseinB.jaapii[57],Venturia
inaequalis[61],Moniliniafructicola[62],andM.graminicola[58].Thisprocesshasalsobeen
linkedtotheimazalilresistanceinPd.Thefirstmechanismdescribedwasthepresenceof
fivetandemrepeatsofa126bptranscriptionalenhancerinthepromoterregionof
PdCYP51A,resultingintheoverexpressionofPdCYP51A[40].Thesespecificrepeats
allowedthedesignofamoleculartooltoidentifyIMZ‐resistantPd.Themethodisbased
onthedetectionofthetandemrepeatofa126bpsequenceinthepromoterregionof
PdCYP51AbyPCR[48].Furthermore,anew199bpsequencewasidentifiedthatdisrupts
the126bptranscriptionalenhancer,resultinginincreasedexpressionofPdCYP51A[63].
Ontheotherhand,inastudycarriedoutin75SpanishstrainsofPd,resistancetoDMIs
inPddidnotcorrelatewiththe126bptandemrepeatsofPdCYP51A[35].Therefore,in
thenewCYP51gene(PdCYP51B)identifiedinPd,auniqueinsertionof199bpwas
observedinthepromoterregionthatwasassociatedwithitsoverexpressionand
resistancetoDMIfungicides[49].Thesameinsertion,butreducedto195bp,was
identifiedinSpanishPdisolates,demonstratingthatoverexpressionofthisgeneisthe
predominantmechanismforresistancetoDMIandinparticulartoIMZ[59].Thisinsert
wasidenticaltothatdescribedbyGhosophetal.[63]inPdCY51A,whichalsoconferred
resistancetoIMZ.Thus,thePdCYP51Benhanceractuallybehaveslikeatransposonthat
actsastheMITEelementPdMLE[64]andismorestableandpredominantthanthe
PdCYP51Aenhancer.Infact,whenpresentinPdCYP51B,itisnotcompatiblewiththe
presenceofthefivetandemrepeatsof126bpenhancerofPdCYP51A[59].
3.3.QuinoneOutsideInhibitors(QoI)
QoIfungicidesimpederespirationbybindingtotheQositeofthecytochromebc1
enzymecomplex,resultinginenergydeficiencyandleadingtothedeathoffungal
pathogens[65].ThismodeofactioninQoIfungicidesresultsinfrequentappearanceof
QoIresistanceinspecificphytopathogenicfungi.
Aswithotherexternalquinoneinhibitor(QoI)fungicides,azoxystrobinishighly
effectiveinpreventingawidevarietyofplantdiseases[20,66],includingcitrusgreenmold
[1].Azoxystrobin(strobilurin)wasregisteredasanewfungicideintheUSAforthecontrol
ofpostharvestdiseasesofcitrus[67,68].However,duetoitssite‐specificmodeofaction,
asmentionedabove,ithasahighriskofdevelopingresistanceintargetphytopathogenic
fungalpopulations.PdisolatescollectedfromvariouspackaginginChinawereshownto
behighlysensitivetoazoxystrobineventhoughithadneverpreviouslybeenusedforthe
controlofcitrusdiseases,indicatingthelackofresistantbiotypesinthenaturalpopulation
[69].AlthoughPdhasahighpotentialtodevelopresistancetoazoxystrobin,noresistance
hasbeendescribednaturallysofar.Onlyamoderatelevelofresistancetostrobilurins
werefoundinsomeofthePdisolatesevaluated,whichshowsthatstrobilurinsare
effective[35].
ThemainmechanismofresistancetoQoIisbasedonthetargetsiteandinvolves
changesinthemitochondrialcytochromeb(CYTB)gene,resultinginvariationsinthe
peptidesequencethatpreventfungicidebinding.MutationsaffectingsensitivitytoQoI
fungicideshavebeenidentifiedintwoareasofCYTB,whicharerelatedtoaminoacid

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positions120–155and255–280oftheencodedprotein.Thismechanismthatunderlies
resistancetoazoxystrobinhasbeenreportedinseveralimportantphytopathogenicfungi
[70–75].Inmostcaseswhereresistancetostrobilurinshasbeendescribed,resistancewas
conferredbysubstitutionofasingleaminoacid(alanineforglycine)atcode143(G143A)
inthecytbgene.Furthermore,substitutionincode129forleucinebyphenylalanine
(F129L)wasalsofoundtoconferresistancetoQoIinsomespeciesoffungi,althoughthe
levelofresistancewaslowerthanthatconferredbytheG143Asubstitution[14,76].
Recently,anadditionalaminoacidsubstitutionfromglycinetoarginineatposition137
(G137R)wasalsoassociatedwithresistancetoQoI[77].InPd,onlyUV‐induced
azoxystrobin‐resistantmutantswerefound.ThesePdmutantsweregeneticallystable,
andtheirhighlevelsofazoxystrobinresistancewereconferredbyasinglepointmutation
(G143A)inthePdcytbgene[69].
ThesecondmechanismofresistancetoQoIfungicidesismediatedbytheinductionof
alternativecyanide‐resistantrespirationsustainedbyalternativeoxidase(AOX)[78].Inthis
rescuemechanism,mitochondrialelectrontransferisdeviated,bypassingtheQoIinhibitory
siteinthecytochromebc1complex.Underfieldconditions,alternativerespirationappears
tohavelimitedimpactontheprotectiveactivitiesofQoIfungicides[79].
3.4.SuccinateDehydrogenaseInhibitors(SDHIs)
Thetargetofboscalidissuccinatedehydrogenase(SDH)inthemitochondrial
electrontransportchain.TheSDHenzymecatalyzestheoxidationofsuccinateto
fumarateinthemitochondrialmatrix,couplingwiththedecreaseinubiquinoneto
ubiquinolinthemembraneduringaerobicrespiration[80,81].SDHIfungicides
specificallyinhibitfungalrespirationbypreventingubiquinonebindingsitesinthe
mitochondrialcomplexII[81].
SDHIs,suchasboscalid,fluxapyroxad,penthiopyrad,isopyrazam,andfluopyram,
haveaspectrumofactivityagainstawidevarietyoffungalpathogensindifferentcrops.
Boscalidisasuccinatedehydrogenaseinhibitor(SDHI)fungicidethatisvery
effectiveinpreventingalargenumberofplantpathogens,includingSclerotinia
sclerotiorum,Botrytiscinerea,Alternariaalternata,andCorynesporacassiicola[82–85].An
aminoacidmodificationinthehighlyconservedsubunitSDH‐Bisdirectlyrelatedtothe
bindingofSDHItothetargetandhasbeendescribedindifferentplantpathogens.In
SDHI‐resistantisolates,histidineatorthologouspositions277,272,and267were
substitutedinA.alternata(B‐H277Y/R)[86],B.cinerea(B‐H272Y/R/L)[87],andlaboratory
mutantsofZ.tritici(B‐H267Y/L/F/N/Q)[88,89].Recently,consecutivetreatmentsover
severalgenerationswithboscalidinthelaboratorywereshowntoresultinresistancein
Pd.StudiesshowedthatboscalidinhibitedSODactivitywhilePODactivityincreased,
whichmaybethereasonfortheincreasedO2−anddecreasedH2O2concentrationsinPd
[90].HighlevelsofROSareharmfulandcauseoxidativedamagetoorganisms,butthey
alsoplayanimportantroleintheregulationofavarietyofbiologicalfunctions[91].
Boscalidisasingle‐sitefungicideandisthereforeconsideredtohaveahighpotential
forresistancedevelopmentregardlessofitshighactivityagainstPd.TheFungicide
ResistanceActionCommittee[92]classifiedSDHIfungicidesasmediumtohighriskwith
respecttothedevelopmentofresistance(Table1)basedprimarilyonsingle‐sitemutations
ofthegeneencodingtheenzymesuccinatetargetdehydrogenase.Thereportedresistance
hasbeenlimitedtogenerationIcarboxinfungicidesaswellasgenerationIISDHIboscalid.
3.5.Phenylpyrroles(PPs)andAnilinopyrimidines(Aps)
Fludioxonilandpyrimethanilareincludedintheclassesofphenylpyrrole(PPs)and
anilinopyrimidine(APs),respectively.Bothfungicidesareveryeffectiveinpreventingthe
germinationofconidiaandtheelongationofthegermtubeofP.expansumandB.cinerea
[93],andbothareregisteredinalargenumberofcropsaspostharvestfungicidesand
incorporatedforpostharvestuseincitrus[94].Whilefludioxonil‐ andpyrimethanil‐
resistantPdisolateshavebeennaturallyidentifiedinpackinghouses,theyarenot

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associatedwithcropdiseases[1].Fludioxonilisusedaloneorincombinationwith
azoxystrobininthecontrolofgreenmoldandotherpostharvestdiseasesofcitrus.In
Californiacitruspackinghouses,areferencesensitivitytofludioxonilhasbeenestablished
inPdpopulations[1].Nevertheless,itwasnotuntil2015thatthefirstincidenceof
resistancetofludioxonilinPdcollectedfromcommercialcitruspackinghouseswas
reportedaftertheintroductionofthefungicideinthemarket[95].Pyrimethanil‐resistant
isolateswerealsoobtainedfromdifferentorchards[96].
Themodeofactionandresistancemechanismsforbothclassesoffungicideshave
beencarriedoutinmutantsinducedinthelaboratoryorinfieldisolatesofvariousfungi.
AlthoughtwonucleotidesubstitutionswerefoundinasequenceanalysisoftheN‐
terminalaminoacidrepeatregionoftheos‐1‐relatedhistidinekinasegeneamongPd
isolates,thesewerenotcorrelatedwithfludioxonilresistance.Studiesindicatethatthe
modeofactionoffludioxonilonPdisprobablythemitogen‐activatedproteinkinase
pathway,whichstimulatesglycerolsynthesisinsensitiveandresistantstrains[1].In
addition,whilepyrimethanilisbelievedtoinhibitthebiosynthesisofmethionineand
otheraminoacidsandthesecretionofhydrolyticenzymesinvolvedintheinfection
processindifferentfungalpathogens[97],methioninebiosynthesisisnotthemaintarget
ofAPsinPd[1].Therefore,themechanismsofresistancetofludioxonilandpyrimethanil
inPdremaintobeelucidated.
4.Resistance‐MediatedDrugEffluxTransporters
Effluxtransporterscanallowfungitosurviveexposuretotoxiccompounds,
eliminatingtheaccumulationofcompoundsintoxicconcentrationswithinfungalcells.
Thesemembrane‐boundproteinsareknowntoprovideprotectionagainstawiderange
ofnaturallyoccurringandxenobiotictoxiccompounds[98].Manystudieshavereported
linksbetweenenhancedeffluxtransporteractivityandtheappearanceofresistancein
differentfungalpathogens[41,99–102]includingPd,indicatingthateffluxtransporters
mayhaveacommonandcriticalroleinfungicidesensitivity.Inaddition,coincident
resistancetomanychemicaltypesoffungicideswasfoundtobeattributableto
overexpressionofeffluxpumpsinsomeimportantfungalpathogens.
Drugeffluxtransportersareintegralmembrane‐boundproteinsthattransportan
extensivevarietyofcompounds,suchasproteinmacromolecules,ions,orsmallmolecules
inabiologicalmembrane[103].Twomajorgroupsofdrugtransportershavebeen
characterizedinfungi,includingABC(ATP‐bindingcassette)transportersandMFS
(majorfacilitatorsuperfamily)transporters.Multidrugandtoxiccompoundextrusion
(MATE),anothertypeoftransporterthathasbeenmainlyreportedinbacteria[104],is
relatedtoresistancetoantimicrobialagentsandwasrecentlyreportedtobeinvolvedin
prochorazresistanceinPdintrancriptomicanalysis[105].Inthissection,thegeneral
functionofdrugeffluxtransportersrelatedtoresistancetofungicidesinthePd–citrus
pathosystemarereviewed(Figure4).

Figure4.ABCandMFStransporters.ABC:ATP‐bindingcassettetransportersuperfamily,MFS:
majorfacilitatorsuperfamily.

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4.1.ATP‐BindingCassetteTransporters(ABC)
ATP‐bindingcassettetransporters(ABC)makeuponeofthelargestproteinfamilies
describedtodate.ThefamilyofABCtransportersisoneofthemostrelevanteffluxpumps
thatexertprotectionoffungiagainstchemicalcompounds[106,107].Thesetransporters
constituteprimaryactivetransportsystemsastheyobtaintheenergyrequiredfortransport
owingtothehydrolysisofATP(Figure4).Infilamentousfungi,ABCtransporterscanact
againstsyntheticfungicidesorcompoundsproducedbycompetingmicroorganisms[108].
Thephenomenon,describedasthesimultaneousresistancetoseveralchemicallyunrelated
compounds(MDR),isrelatedtotheoverexpressionofABCtransportersduetotheresulting
pleiotropiceffects.FourABCtransportershavebeenidentifiedinPd:PMR1,PMR3,PMR4,
andPMR5.Ofthem,onlyPMR1[48,109]andPMR5[110]appeartoberelatedtomultidrug
resistanceinPd.Amoreexhaustivecharacterizationofthefourtransportersshowedthat
whilenogeneticchangesweredetectedbetweenisolatesinPMR1,PMR3,andPMR4,some
specificmodificationswereobservedinthepromoterandcodingregionsofPMR5instrains
resistanttobothTBZanddifferentDMIfungicides[35].Furthermore,thepresenceoftoxic
substancesselectivelyactivatestheexpressionofPMR1andPMR5.Specifically,triflumizole
andimazalilactivatePMR1transcription,whilebenzimidazoles,dithianone,and
resveratrolpromotePMR5transcription.Thus,Pdresistancecanbedeterminedbyselective
transcriptionalactivationofABCtransportergenestoatoxiccompound.[110].Moreover,
anexhaustivesearchofputativeABCgenesinPdidentifiedatotalof46chromosome‐
encodedABCfamilytransporters.AnalysisofthesegenesrevealedthatfivemoreABC
transportersmaybeinvolvedindrugresistanceastheywereupregulatedinimazalil‐
inducingexpressionanalysis[64].Furthermore,transcriptomeanalysisofprochloraz‐
treatedPdstrainsrevealedthreenewABCtransportersthatweremoreinvolvedin
prochlorazresistance[111].
4.2.MajorFacilitatorSuperfamilyTransporters(MFS)
MFStransportersarepartofthefamilyofactivesecondarytransportersthatcan
transportsubstancesinresponsetoionicgradients.MFStransporterscanmediate
antiport,uniport,andsymportofdifferentproducts[112](Figure4).MFStransporterscan
serveasdrugtransportersowingtoaprotongradient,whichallowsittoconfermultidrug
resistance(MDR).ManyoftheseMFStransporterstransportsmallmoleculesinresponse
toionicgradientsinsuchawaythattheyfunctionasanH+antiporterinmicroorganisms,
regulatingtheirgrowthunderstressconditionsastheyaffectthemembranepotentialand
internalpH[113].
Thesetransporterscouldplayaroleinsensitivitytodifferentcompoundsasthey
usuallyhaveanarrowsubstrateaffinitythatguaranteesanimportantcontributioninthe
transactionofawiderangeofsubstrates.Theeffectontoxineffluxandfungicide
sensitivityhasbeenobservedinmanyfungalMFStransporters.Forinstance,suppression
oftheCercosporanicotianaeMFStransporterreducedthecercosporintoxin[114].InB.
cinerea,BcMfs1affectedsensitivitytocamptothecinandcercosporinandresistancetoDMI
[115]andmfsM2showedgreatereffluxfungicidalactivity[99].TheeliminationofMgMfs1
fromM.graminicolacontributedtostrobilurinfungicideresistancebutnototherevaluated
fungicides[116,117].InZymoseptoriatritici,theMgMFS1transporterparticipatedinthe
MDRphenotype[110].Furthermore,theAaMFS19MFStransporterwasshowntoplaya
roleinresistancetooxidativestressandchemicalsinthephytopathogenicfungus
Alternariaalternata[118].TranscriptomeanalysisofMDRstrainsofP.expansumreported
overexpressionofMFStransportergenesbeforeorafterexposuretofludioxonil[119].
InPd,morethan100MFShavebeenidentifiedduetotheavailabilityofthePd
genome[5].Sofar,ofalltheidentifiedPd–MFStransporters,sevenhavebeen
characterizedmorethoroughly,namelyPdMfs1[120],Pdmfs2[121],PdMFS1[101],
PdMFS2,PdMFS3,PdMFS4,andPdMFS5[102].Allareinvolvedinsomewayinresistance
tochemicalfungicidesandinsomecasesmaycontributetoanincreaseinfungal

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
virulence.AnanalysisofeachoftheproteinsencodedbytheseMFSgenesshowsthatthey
sharelittlehomologybetweenthem,whichalsoaffectstheirfunctionality.Thus,while
PdMfs1hasacleareffectagainstimazalil,Pdmfs2andPdMFS1playanimportantrolein
prochlorazresistance.Botharealsoinvolvedinprocessesdevelopedduringthefruit–
pathogeninteraction,suchasconidiaandtheprogressoffungaldisease.Furthermore,
PdMFS1isabletoconferMDRphenotypeasitcontributestotheoutputofawiderange
offungicidalcompounds[101].AmongthelatestidentifiedPd–MFStransporters,only
PdMFS2andPdMFS3appeartoparticipateinfungicideresistance.Bothgenescontribute
tosimultaneousresistancetoseveralunrelatedtoxiccompounds(MDRphenotype),as
previouslyreportedforotherfungalMFStransporters[101,113,122].Thephylogenetic
analysisofalargenumberoftheseMFStransportersinPdrevealedallthegeneshad
differentgeneticstructuresandencodedproteinsofdifferentsizesandthatonly
PdMFS2pappearedtogetherwiththegroupthatcomprisedtheMFStransporters
assignedasdrugeffluxtransporters[102].Ontheotherhand,thetranscriptomicanalysis
carriedoutinPdaftertreatmentwithprochlorazhighlightedoverexpressionof14
differentMFStransporters[111].
MFStransportershavebeenlinkedtoQoIresistance.InMgMfs1,whichencodesan
MFStransportergenefromM.graminicola,thedeletionofMgMfs1showedinsensitivity
toQoI[116].However,untilnow,nodecreaseinsensitivityorresistancetoQoIfungicides
hasbeenidentifiedinPdmediatedbyanMFStransporter.However,thecontributionof
theseenergy‐dependentmechanismsinadaptationtofungicidesbyphytopathogenic
fungishouldbeseriouslyconsidereddespitethescarcityofdataonresistancetoefflux
transporter‐basedQoIfungicides.
Untilnow,thecontributionofMFStransportersasadecisivefactorintheplant–
pathogeninteractionisunknown[37],andfurtherfunctionalcharacterizationofmore
differentMFStransporterswillbenecessarytoestablishtheirroleinthePd–citrus
interaction.
5.RegulationofFungicideResistance
5.1.TranscriptionFactorsinPdFungicideResistance
Transcriptionfactors(TF)areinvolvedintranscriptionalregulationandplaya
relevantroleinfungalinteractions.TFscancontributetoprimaryorsecondary
metabolism[123],alongwithstressresponsesandsensitivitytopleiotropicdrugs[124].
SREBPtranscriptionfactors,whichcontainabHLHdomain,functionascritical
controllersofsterolhomeostasisandareuniversallyfoundinfungi.Inmostfungi,SREBPs
playacrucialroleincontrollingergosterolbiosynthesis[125].InPd,theSREBPprotein
SreAwasinitiallyidentifiedandcharacterized,whichplaysanimportantrolein
prochlorazresistanceandinthetranscriptionofergosterolsynthesisgenes[111].Evidence
onthetranscriptionalregulationofthesetargetgeneshasemergedtoexplainthedrug‐
resistantmechanismsofPd.InthecitruspostharvestpathogenPd,thereisanotherSREBP
homolog,PdsreB,whichappearstobeinvolvedinfungicideresistanceandinthecontrol
ofCYP51geneexpression[126].Functionalcharacterizationshowedthetwogenes
(PdsreAandPdsreB)actasglobalcontrollersinagreatvarietyofbiologicalfunctions,
especiallyinaspectsthatmediateergosterolbiosynthesisandresistancetofungicides.
Thus,theexpressionoftheERG1gene(intheergosterolpathway)isregulatedbyboth
thePdsreAandPdsreBgenes,whileonlyPdsreAisinvolvedintheexpressionofERG2.As
bothgenesregulatedifferentaspects,ashasbeenshownwithsingleanddoublemutations
ofthegenes,itispossiblethatthereareothertranscriptionfactorsinvolvedinergosterol
biosynthesisthatcouldbeactivatedwhenbothSREBPsareinhibited[126].Furthermore,
itispossiblethattheSREBPgenesplayarelevantroleinthecontrolofcertainMFS
transportersinPdassomeofthemwerefoundtobeoverrepresentedingenetranscription
studies[126].

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Fungiareknowntousethetranscriptionalregulationofgenesencodingefflux
transporterstodetoxifycertaincompounds.Theexpressionofeffluxtransportersis
controlledmainlybyfungalzincgrouptranscriptionfactors(TF[Zn2Cys6])[127].Fungi
apparentlyregulateandmanagedifferentstagesofthedetoxificationsystemby
modificationsinparticulartranscriptionfactors,andthisregulatorysystemseemstobe
conservedinfilamentousfungi[128].Modificationsintheactivityoftranscriptional
regulatorselicitoverproductionofMFStransporters[129].
TranscriptionalregulatorSte12mightfunctionasaregulatorofpathway‐specificgenes
[130].InPd,PdSte12mightbeinvolvedinthecontrolofexpressionofseveralgenesthrough
repressionoractivation,triggeringmultipleresponses,suchasdetoxification.PdSte12acts
asanegativeregulatorinseveralgenesinvolvedintransport,includingtheprimaryABC
transporters(PMR1andPMR5)andthesecondaryMFStransporters(PdMFS1‐6).PdSte12
alsopositivelycontrolssteroldemethylases(CYP51andPdCYP51B)[131].
Skn7isahighlyconservedstress‐responsivetranscriptionfactorand,apartfrom
Ssk1/SskA,thesecondresponseregulatorthatcanbeactivatedviathephosphotransfer
proteinYpd1.Skn7playsawell‐establishedroleintheoxidativestressresponse.Skn7is
involvedinmaintenanceofthecellwallintegrityofS.cerevisiaeandotherfungi.Although
thesegeneshavenotbeenidentifiedtodateinPd,intheMFStransportersofA.alternata
(AaMFS19andAaMFS54),geneexpressionissimultaneouslyregulatedbythestress‐
sensitivetranscriptionfactorYap1.TheexpressionofAaMFS19isalsocontrolledbythe
stress‐relatedregulatorSkn7[118,132],butthisregulatordoesnotaffectAaMFS54.ROS
resistanceinA.alternatais,atleastinpart,mediatedbymembrane‐boundtransportersas
regulatorsYap1andSkn7havebeenshowntoplayacriticalroleinresistancetooxidative
stress[133](Figure5).

Figure5.Schematicofregulatorymechanismsinvolvedinfungalresistancethatarealsorelatedto
fungicidevirulenceandoxidativestress.
5.2.ProteinKinasesinPdFungicideResistance
Fungiprocessingiscontrolledbyproteinkinase(PK)cascades[134].MAPKsare
involvedinsignalingpathwaysthatarehighlyconservedinalleukaryoticorganisms.There
arethreeorthologousMAPKsinfilamentousfungi,namelyHog1,Slt2,andFus3/Kss1[135].
TheMAPKHog1,Slt2,andFus3/Kss1orthologousinPd,calledPdos2,PdSlt2,andPdMpkB,
havebeenidentifiedandcharacterized[46,136–138].Hog1‐likeMAPKs,whicharehighly
conservedamongvariousfungi,possessdifferentphysiologicalfunctions,includingahigh
osmolarityadaptation[139].Pdos2isinvolvedinosmoticadaptationandisassociatedwith
positivecontrolofglycerolsynthesisandnegativeregulationofergosterolsynthesis[138].

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ThemodeofactionoffludioxonilonPdisprobablyviathemitogen‐activatedproteinkinase
pathway,whichpromotesglycerolsynthesis[1].
PdSlt2functionsasanegativeregulatorofdifferentgenesinvolvedintransport,
comprisingprimarytransporters(ABCtransportersPMR1andPMR5)andsecondary
transporters(MFStransportersPdMFS1‐6).Incontrast,PdSlt2positivelycontrolssterol
demethylasesPdCYP51AandPdCYP51B[137].Inthissense,thecontrolofimportant
genesinvolvedinfungicideresistancehighlightstheroleofthisMAPKinmediatingthe
processinvolvedinresistancetofungicides.
Figure5illustrateshowtranscriptionalregulationplaysanimportantroleinfungal
interactionsandhowsignaltransductionpathwayscanbeinterconnected.Furthermore,
oxidativestressandtheROSresponsemayalsobepartoffungal–plantinteractionasthey
aresimultaneouslyinvolvedinfungalpathogenicityandresistancetofungicides.
6.Conclusions
ThemechanismsresponsibleforacquiringresistancetofungicidesinPdaredueto
numerousgenesthatinsomecasesdependonthetypeoffungicide.Resistancetoacertain
fungicidecanbedefinedbymodificationsinasinglegene.However,ingeneral,different
mechanisms,suchasvariationsthatmodifythebindingtargetofthefungicideorchanges
ingeneexpression,determinetheappearanceofresistancetotoxiccompounds.
ThecomplexityofgenomicpathwaysinPdpopulationshasallowedthemtorespond
andadapttofungicidesinmultipleways.Atleastsevenmechanismsthatcancause
resistancetovariousclassesofchemicalfungicidesinPdhavebeendescribed(Figure6).
Remarkably,resistancecausedbyincreasedactivityofdrugeffluxpumpshasbeenthe
mostnotoriousforallmajorchemicalclasses,indicatingitscommonroleinresistance
evolution.Othermechanismsmentionedabove,suchasdetoxificationandtranscription
factors,havealsobeenfoundtoberelevant.

Figure6.Mechanismsofresistancetomaintypesoffungicidesusedforgreenmolddisease
management.Eachorangerepresentsachemicalclass,whereasthecryptogramswithintheoranges
correspondtoaresistanceprocessasshowninthefigure.MBCs:methylbenzimidazolecarbamates,
DMIs:demethylationinhibitors;QoIs:quinoneoutsideinhibitors;SDHIs:succinatedehydrogenase
inhibitors;APs:anilinopyrimidines;PPs:phenylpyrroles.AdaptedfromHuandChen[105].
Fungicidesplayadeterminingroleinthecontrolofcroppathogens,anditislikely
thattheywillcontinuetobeoneofthemostrelevantmeansinthefuturetopreventthe
developmentofdiseases.Anapproachthatintegratesplantbreedingandbiotechnology,
thedevelopmentofchemicalcompounds,andpoliciesthatensuretheuseoffungicides

J.Fungi2021,7,78313of18
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
inasustainablewaythroughinnovationinalternativetechnologiesisessentialtoachieve
thechallengeoffoodsafetyinachangingenvironmentandcounteractrisksinplanthealth
andpostharvestcitrusfruitsinparticular.
Expandingknowledgeoffungalresistancemechanismsnotonlyallowsthedesign
offastermoleculartoolstorapidlydetectfungalresistancebutcanalsoallowthe
identificationofnaturalsecondarymetabolitesandthedesignofnewantifungal
compoundsthataremoreefficientandspecific.
Funding:Thisresearchreceivednoexternalfunding.
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
ConflictsofInterest:Theauthordeclarenoconflictofinterest..
References
1. Kanetis,L.;Forster,H.;Adaskaveg,J.E.BaselinesensitivitiesforthenewpostharvestfungicidesagainstPenicilliumspecieson
citrusandmultipleresistanceevaluationsinP.digitatum.PlantDis.2008,92,261–269.
2. Palou,L.PenicilliumdigitatumandPencilliumitalicum(GreenMold,BlueMold).InPostharvestDecay:ControlStrategies;Bautista‐
Baños,S.,Eds.;AcademicPress:Cambridge,MA,USA,2014;pp.45–102.
3. Tian,S.;Torres,R.;Ballester,A.R.;Li,B.;Vilanova,L.;Gonzalez‐Candelas,L.Molecularaspectsinpathogen‐fruitinteractions:
Virulenceandresistance.PostharvestBiol.Technol.2016,122,11–21.
4. Macarisin,D.;Cohen,L.;Eick,A.;Rafael,G.;Belausov,E.;Wisniewski,M.;Droby,S.Penicilliumdigitatumsuppressesproduction
ofhydrogenperoxideinhosttissueduringinfectionofcitrusfruit.Phytopathology2007,97,1491–1500.
5. Marcet‐Houben,M.;Ballester,A.R.;delaFuente,B.;Harries,E.;Marcos,J.F.;González‐Candelas,L.;Gabaldón,T.Genome
sequenceofthenecrotrophicfungusPenicilliumdigitatum,themainpostharvestpathogenofcitrus.BMCGenom.2012,13,646.
6. Perez,M.F.;Ibarreche,J.P.;Isas,A.S.;Sepulveda,M.;Ramallo,J.;Dib,J.R.Antagonisticyeastsforthebiologicalcontrolof
Penicilliumdigitatumonlemonsstoredunderexportconditions.Biol.Control2017,115,135–140.
7. Ghooshkhaneh,N.G.;Golzarian,M.R.;Mamarabadi,M.DetectionandclassificationofcitrusgreenmoldcausedbyPenicillium
digitatumusingmultispectralimaging.J.Sci.FoodAgric.2018,98,3542–3550.
8. Vu,T.X.;Ngo,T.T.;Mai,L.T.D.;Bui,T.T.;Le,D.H.;Bui,H.T.V.;Nguyen,H.Q.;Ngo,B.X.;Tran,V.T.Ahighlyefficient
Agrobacteriumtumefaciens‐mediatedtransformationsystemforthepostharvestpathogenPenicilliumdigitatumusingDsRedand
GFPtovisualizecitrushostcolonization.J.Microbiol.Meth.2018,144,134–144.
9. Ismail,M.A.;Zhang,J.Post‐harvestcitrusdiseasesandtheircontrol.OutlooksPestManag.2004,15,29‐35.
10. DeRamón‐Carbonell,M.;Sánchez‐Torres,P.ThetranscriptionfactorPdSte12contributestoPenicilliumdigitatumvirulence
duringcitrusfruitinfection.PostharvestBiol.Technol.2017,125,129–139.
11. Zhang,T.;Qian,X.;Sun,X.;Li,H.Thecalcineurin‐responsivetranscriptionfactorCrz1isrequiredforconidation,fullvirulence
andDMIresistanceinPenicilliumdigitatum.Microbiol.Res.2013,168,211–222.
12. Smilanick,J.L.;Mansour,M.;Gabler,F.M.;Sorenson,D.Controlofcitruspostharvestgreenmoldandsourrotbypotassium
sorbatecombinedwithheatandfungicides.PostharvestBiol.Technol.2008,47,226–238.
13. Hollomon,D.W.Fungicideresistance:Facingthechallenge.PlantProtect.Sci.2015,51,170–176.
14. Ma,Z.;Michailides,T.J.Advancesinunderstandingmolecularmechanismsoffungicideresistanceandmoleculardetectionof
resistantgenotypesinphytopathogenicfungi.CropProt.2005,24,853–863.
15. Brent,K.J.;Hollomon,D.FungicideResistanceinCropPathogens.HowCanItBeManaged,2nded.;FRAC:Brussels,Belgium,2007.
16. Hahn,M.;Leroch,M.Multidrugeffluxtransporters.InFungicideResistanceinPlantPathogens;Springer:Berlin/Heidelberg,
Germany,2015;pp.233–248.
17. Lucas,J.A.;Hawkins,N.J.;Fraaije,B.A.Theevolutionoffungicideresistance.Adv.Appl.Microbiol.2015,90,29–92.
18. Deising,H.B.;Reimann,S.;Pascholati,S.F.Mechanismsandsignificanceoffungicideresistance.BrazilianJ.Microbiol.2008,39,
286–295.
19. Gisi,U.;Chin,K.M.;Knapova,G.;KüngFärber,R.;Mohr,U.;Parisi,S.;Sierotzki,H.;Steinfeld,U.Recentdevelopmentsin
elucidatingmodeofresistancetophenylamide,DMI,andstrobilurinfungicides.CropProt.2000,19,863–872.
20. Gullino,M.L.;Leroux,P.;Smith,C.M.Usesandchallengesofnovelcompoundsforplantdiseasecontrol.CropProt.2000,19,1–
11.
21. Fluit,A.C.;Visser,M.R.;Schmitz,F.J.Moleculardetectionofantimicrobialresistance.Clin.Microbiol.Rev.2001,14,836–871.
22. Capote,N.;Pastrana,A.M.;Aguado,A.;Sánchez‐Torres,P.MolecularToolsforDetectionofPlantPathogenicFungiand
FungicideResistance.InPlantPathology;Cumagun,C.J.R.,Ed.;InTech:EastProvidence,RI,USA,2012;pp.151–202.
23. Langner,T.;Kamoun,S.;Belhaj,K.CRISPRcrops:Plantgenomeeditingtowarddiseaseresistance.Annu.Rev.Phytopathol.2018,
56,479–512.

J.Fungi2021,7,78314of18


24. Burón‐Moles,G.;López‐Pérez,M.;González‐Candelas,L.;Vinas,I.;Teixido,N.;Usall,J.;Torres,R.UseofGFP‐taggedstrains
ofPenicilliumdigitatumandPenicilliumexpansumtostudyhost‐pathogeninteractionsinorangesandapples.Int.J.FoodMicrobiol.
2012,160,162–170.
25. Costa,J.H.;Bazioli,J.M.;deMoraesPontes,J.G.;Fill,T.P.Penicilliumdigitatuminfectionmechanismsincitrus:Whatdoweknow
sofar?FungalBiol.2019,123,584–593.
26. Holloman,D.W.;Butters,J.A.;Barker,H.;Hall,L.Fungalβ‐tubulin,expressedasafusionprotein,bindsbenzimidazoleand
phenylcarbamatefungicides.Antimicrob.AgentsChemother.1998,42,2171–2173.
27. Baraldi,E.;Mari,M.;Chierici,E.;Pondrelli,M.;Bertollini,P.;Pratella,G.C.StudiesofThiabendazoleresistanceofPenicillium
expansumofpears:Pathogenicfitnessanggeneticcharacterization.PlantPathol.2003,52,362–370.
28. Leroux,P.;Fritz,R.;Debieu,D.;Albertini,C.;Lanen,C.;Bach,J.;Gredt,M.;Chapeland,F.Mechanismsofresistancetofungicides
infieldstrainsofBotrytiscinerea.PestManag.Sci.2002,58,876–888.
29. Malandrakis,A.;Markoglou,A.;Ziogas,B.Molecularcharacterizationofbenzimidazole‐resistantB.cinereafieldisolateswith
reducedorenhancedsensitivitytozoxamideanddiethofencarb.Pestic.Biochem.Physiol.2011,99,118–124.
30. Fan,J.;Luo,Y.;Michailides,T.J.;Guo,L.SimultaneousquantificationofallelesE198AandH6Yintheβ‐tubulingeneconferring
benzimidazoleresistanceinMoniliniafructicolausingaduplexreal‐time(TaqMan)PCR.PestManag.Sci.2014,70,245–251.
31. Albertini,C.;Gredt,M.;Leroux,P.Mutationsoftheβ‐tubulingeneassociatedwithdifferentphenotypesofbenzimidazole
resistanceinthecerealeyespotfungiTapesiayallundaeandTapesiaacuformis.Pestic.Biochem.Physiol.1999,64,17–31.
32. Yarden,O.;Katan,T.Mutationsleadingtosubstitutionsataminoacids198and200ofbeta‐tubulinthatcorrelateswithbenomyl‐
resistancephenotypesoffieldstrainsofBotrytiscinerea.Phytopathology1993,83,1478–1483.
33. Koenraadt,H.;Somerville,S.C.;Jones,A.L.Characterizationofmutationsinthebeta‐tubulingeneofbenomyl‐resistantfield
strainsofVenturiainaequalisandotherplantpathogenicfungi.Phytopathology1992,82,1348–1354.
34. Schmidt,L.S.;Ghosoph,J.M.;Margosan,D.A.;Smilanick,J.L.Mutationatβ‐tubulincodon200indicatedThiabendazole
resistanceinPenicilliumdigitatumcollectedfromCaliforniacitruspackinghouses.PlantDis.2006,90,765–770.
35. Sánchez‐Torres,P.;Tuset,J.J.MolecularinsightsintofungicideresistanceinsensitiveandresistantPenicilliumdigitatumstrains
infectingcitrus.PostharvestBiol.Technol.2011,59,159–165.
36. Lee,M.‐H.;Pan,S.‐M.;Ng,T.‐W.;Chen,P.‐S.;Wang,L.‐Y.;Chung,K.‐R.Mutationsofβ‐tubulincodon198or200indicate
thiabendazoleresistanceamongisolatesofPenicilliumdigitatumcollectedfromcitrusinTaiwan.Int.J.FoodMicrobiol.2011,150,
157–163.
37. Vela‐Corcía,D.;Romero,D.;deVicente,A.;Pérez‐García,A.Analysisofβ‐tubulin‐carbendaziminteractionrevealsthatbinding
siteforMBCfungicidesdoesnotincluderesiduesinvolvedinfungicideresistance.Sci.Rep.2018,8,1–12.
38. Brent,K.J.;Hollomon,D.W.FungicideResistance:TheAssessmentofRisk,2nded.;GlobalCropProtectionFederation:Brussels,
Belgium,1998;pp.1–48.
39. Aoyama,Y.;Yoshida,Y.Differentsubstratespecificitiesoflanosterol14a‐demethylase(P‐45014DM)ofSaccharomycescerevisiae
andratliverfor24‐methylene‐24,25‐dihydrolanosteroland24,25‐dihydrolanosterol.Biochem.Biophys.Res.Commun.1991,178,
1064–1071.
40. Hamamoto,H.;Hasegawa,K.;Nakaune,R.;Lee,Y.J.;Makizumi,Y.;Akutsu,K.;Hibi,T.Tandemrepeatofatranscription
enhancerupstreamofthesterol14_demethylasegene(CYP51)Penicilliumdigitatum.Appl.Environ.Microbiol.2000,66,3421–
3426.
41. Nakaune,R.;Hamamoto,H.;Imada,J.;Akutsu,K.;Hibi,T.AnovelABCtransportergene,PMR5,isinvolvedinmultidrug
resistanceinthephytopathogenicfungusPenicilliumdigitatum.Mol.Genet.Genom.2002,267,179–185.
42. Holmes,G.J.;Eckert,J.W.RelativefitnessofImazalil‐resistantand‐sensitivebiotypesofPenicilliumdigitatum.PlantDis.1995,
79,1068–1073.
43. Parks,L.W.;Casey,W.M.Physiologicalimplicationsofsterolbiosynthesisinyeast.Annu.Rev.Microbiol.1995,49,95–116.
44. Ali,E.M.;Amiri,A.Selectionpressurepathwaysandmechanismsofresistancetothedemethylationinhibitordifenoconazole
inPenicilliumexpansum.Front.Microbiol.2018,9,2472.
45. Snelders,E.;Camps,S.M.T.;Karawajczyk,A.;Rijs,A.J.M.M.;Zoll,J.;Verweij,P.E.;Melchers,W.J.G.Genotype–phenotype
complexityoftheTR46/Y121F/T289Acyp51AazoleresistancemechanisminAspergillusfumigatus.FungalGenet.Biol.2015,82,
129–135.
46. Wang,J.;Yu,J.;Liu,J.;Yuan,Y.;Li,N.;He,M.;Qi,T.;Hui,G.;Xiong,L.;Liu,D.NovelmutationsinCYP51BfromPenicillium
digitatuminvolvedinprochlorazresistance.J.Microbiol.2014,52,762–770.
47. Fan,J.;Urban,M.;Parker,J.E.;Brewer,H.C.;Kelly,S.L.;Hammond‐Kosack,K.E.;Fraaije,B.A.;Liu,X.;Cools,H.J.
Characterizationofthesterol14α‐demethylasesofFusariumgraminearumidentifiesanovelgenus‐specificcyp51function.New
Phytol.2013,198,821–835.
48. Hamamoto,H.;Nawata,O.;Hasegawa,K.;Nakaune,R.;Lee,Y.J.;Makizumi,Y.;Akutsu,K.;Hibi,T.TheroleoftheABC
transportergenePMR1indemethylationinhibitorresistanceinPenicilliumdigitatum.Pestic.Biochem.Phys.2001,70,19–26.
49. Sun,X.;Wang,J.;Feng,D.;Ma,Z.;Li,H.PdCYP51B,anewputativesterol14α‐demethylasegeneofPenicilliumdigitatum
involvedinresistancetoimazalilandotherfungicidesinhibitingergosterolsynthesis.Appl.Microbiol.Biot.20