ArticlePDF AvailableLiterature Review

Molecular Mechanisms Underlying Fungicide Resistance in Citrus Postharvest Green Mold

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
J.Fungi2021,7,783.https://doi.org/10.3390/jof7090783 www.mdpi.com/journal/jof
Review
MolecularMechanismsUnderlyingFungicideResistance
inCitrusPostharvestGreenMold
PalomaSánchezTorres
DepartmentofFoodBiotechnology,InstituteofAgrochemistryandFoodTechnology,SpanishNational
ResearchCouncil(IATACSIC),CalleCatedráticoAgustínEscardino7,Paterna,46980 Valencia,Spain;
psanchez@iata.csic.es;Tel.:+34963900022
Abstract: The necrotrophic fungus Penicillium digitatum (Pd) is responsible for the green mold
diseasethatoccursduringpostharvestofcitrusandcausesenormouseconomiclossesaroundthe
world.Fungicidesremainthemainmethodusedtocontrolpostharvestgreenmoldincitrusfruit
storagedespitenumerousoccurrencesofresistancetothem.Hence,itisnecessarytofindnewand
moreeffectivestrategiestocontrolthistypeofdisease.Thisinvolvesdelvingintothemolecular
mechanisms underlying the appearance of resistance to fungicides during the plant–pathogen
interaction.Althoughmechanismsinvolvedinresistancetofungicideshavebeenstudiedformany
years,therehavenowbeengreatadvancesinthemolecularaspectsthatdrivefungicideresistance,
which facilitates the design of new means to control green mold. A wide review allows the
mechanismsunderlyingfungicideresistanceinPdtobeunveiled,takingintoaccountnotonlythe
chemicalnatureofthecompoundsandtheirtargetofactionbutalsothegeneralmechanismthat
couldcontributetoresistancetootherscompoundstogeneratewhatwecallmultidrugresistance
(MDR)phenotypes.Inthiscontext,fungaltransportersseemtoplayarelevantrole,andtheirmode
of action may be controlled along with other processes of interest, such as oxidative stress and
fungalpathogenicity.Thus,themechanismsforacquisitionofresistancetofungicidesseemtobe
part of a complex framework involving aspects of response to stress and processes of fungal
virulence.
Keywords:citrus;fungicideresistance;postharvest;Penicilliumdigitatum;infection
1. Introduction
Citrusfruitsareimportantfruitcropsaroundtheworldbecausetheyprovide
numerousnutrientsthatpromotehumanhealth[1,2].Citrusfruitsaresubjecttodifferent
bioticorabioticstressesduringthepostharvestperiod,whichincludeshandling,
shipping,storage,andmarketing.Inthiscontext,fruitspoilageandfoodsafetyrisksdue
topostharvestfungaldiseasesconstitutesomeofthemostsignificantthreats[3].
Penicilliumdigitatum(Pers.:Fr)Sacc.(Pd),whichcausesgreenmold,isthemajor
postharvestrotofcitrusfruits[1].Postharvestgreenmoldcauseshugeeconomiclosses
worldwideeveryyearandcanaccountforupto90%ofthetotalpostharvestdamageto
citrusfruits,especiallyindryareasandsubtropicalclimates[4,5].
Pdiscapableofinvadingandinfectingthefruitthroughwoundsthatareproduced
byenvironmentalfactorsorduringtheharvestdevelopment,transport,andfurther
treatments[6].Thisfungusextendsinoilglandsthroughrindwounds[7],wheretheycan
accessnutrientsthatpromotegerminationoftheconidia.Thestartingpointofinfection
isthesofterandmorewateryareaonthesurfaceoftheskinwhere,withsuitable
temperatureandoptimalconditions,itprogressestogiverisetoawhitemyceliumthat
laterturnstoolivecolorduetotheappearanceofconidia[8,9](Figure1).
Citation:SánchezTorres,P.
MolecularMechanismsUnderlying
FungicideResistanceinCitrus
PostharvestGreenMold.J.Fungi
2021,7,783.
https://doi.org/10.3390/jof7090783
AcademicEditor:IvanM.
Dubovskiy
Received:2September2021
Accepted:20September2021
Published:21September2021
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(http://creativecommons.org/licenses
/by/4.0/).
J.Fungi2021,7,7832of18
Figure1.GreenmolddecayinorangefruitinfectedwithPenicilliumdigitatum.(a)Orangefruitwith
greenmoldsymptomscausedbyP.digitatum;(b)typicalaspectP.digitatumgrowingonPDAplates;
(c)P.digitatumconidiphorewithconidiaborneterminallyinchainsobservedbybrightfieldoptical
microscopy;(d)P.digitatumwithfluorescenceafterstainingwithCFW.
Pdwholegenomesequencinghasrecentlyopenedupnewpossibilitiestoinvestigate
fungalfactorsrelatedtohost–pathogeninteractionsrangingfromvirulencefactorstogenes
involvedinfungicideresistancemechanisms.Understandingtheinfectionprocessandthe
fungalstrategytoovercomefungicidesisonewaytodevelopnewformsofcontrol[10,11].
Thecontrolofgreenmoldiscurrentlyachievedthroughtheapplicationofsynthetic
compounds,suchasimazalil,thiabendazole,pyrimethanil,andfludioxonil[12].However,
thecontinueduseofchemicalstopreventfungaldiseaseshasrestrictedtheireffectiveness
andshelflife.Theemergenceofpathogenicfungithatisresistanttosyntheticfungicides
mainlyusedforthecontroloffungalinfectionsposesarisktotheenvironmentand
consumerhealth[3],thuspromptingresearchtodevelopnew,moreeffectivecontroltools.
2.FungicideResistanceHasBecomeaMajorProblem
Fungicidesarecrucialtopreservehealthy,consistent,highqualityagricultural
goods.Until1970,almostallchemicalsusedtomanageplantpathogensweremultisite
inhibitorsthatworkedasprotectorsofdiseases.Inspiteoftheirextensiveuseinsome
cases,resistancehasnotevolvedtolargelynonsystemicprotectantfungicidesduetotheir
multisitemodesofaction[13].However,sincetheintroductionofsitespecificfungicides
inthelate1960s,fungicideresistanceinplantpathogenicfungihasemergedasamajor
problemincropcontrol[14].Sincethe1970s,therehasbeenanimprovementincrop
protectionowingtosystemicsinglesitefungicidesthatpossessbothprotectiveand
eradicatingcharacteristics,suchasmethylbenzimidazolecarbamates(MBC),sterol
biosynthesisinhibitors(DMIs),externalquinoneinhibitors(QoI),andsuccinate
dehydrogenaseinhibitors(SDHI)(Table1).
Resistancetofungicidesresultsinreductioninsensitivitytocertaincompoundsand
iscausedbyaninheritedadjustmentofthefungustothatcompound.Itisnormallydue
toeithersingleormultiplegeneticmutations.Theidentificationofresistantisolates
appearswithanaturalrateofgeneticmutation,sothenumberofresistantstrainsis
generallynotaffectedbytheapplicationofafungicide[15].

a
b
c
d
J.Fungi2021,7,7833of18
Table1.Typesoffungicidesusedincitruscontrolprogramsandtheirtargets.
FRACCodeFungicideClassCelularFunctionAffectedTargetProteinRiskResistance
Development
1Methylbenzimidazoles
(MBCs)CytoskeletontubulinHigh
3Demethylationinhibitors
(DMIs)
Membrane
biosynthesis
Sterol14
demethylase
(CYP51)
Medium
11
Quinoneoutside
inhibitors
(QoIs)
RespirationMitochondrial
cytochromebHigh
7Succinatedehydrogenase
inhibitors(SDHIs)RespirationSuccinate
dehydrogenaseMediumtoHigh
12Phenylpyrroles(PPs)
Alteredtargetsite(protein
kinaseinvolvedin
osmoregulation)
ProteinkinaseLowtomedium
9Anilinopyrimidines
(APs)
Alteredtargetsite(protein
kinaseinvolvedin
osmoregulation)
ProteinkinaseMedium
Whilefungicideseffectivelykillsensitivestrains,resistantstrainsbecomedominant
overtimebecausepathogenpopulationsareunderselectionpressurefromcontinued
fungicideuse,leadingtofailuretocontroldisease[16].Thefitnessofthefungicide
resistantfungalisolatesonceselectedwilldeterminethepermanenceoftheresistant
genotypes.Insomecases,ithasbeenobservedthatresistantstrainsmayhaveless
aptitudethansusceptiblestrains,thusrequiringselectionpressureofthefungicideto
survive.Therefore,whentheapplicationoffungicidesceases,thenumberofresistant
isolatesinpathogenpopulationswilldecrease.Ontheotherhand,thestrongisolates
presentafitnesssimilartothesensitiveisolates,andtheycouldremainforalongtime
evenwithouttheapplicationoffungicides[17].
Likeinallorganisms,thereisgeneticvariationinpathogenicfungalpopulations.
Thisvariationprovidesthestartingpointfromwhichfungicideresistanceprogresses.A
completepopulationofresistantpathogenicfungidevelopsowingtonaturalselection,in
whichtheenvironmentfavorsthereproductionandproliferationofresistantforms.
Individualfungicideapplicationscanbeconsideredthe“selectionevents”thatpromote
thisprocess,selectivelykillingsusceptiblefungi.However,anyresistantmutantwill
survivetheseeventsandsubsequentlyhavetheopportunitytogrowandreproduce
withoutcompetitionfromsusceptiblefungi.Afteroneapplication,thisincreasingly
resistantpopulationcanproliferateandreproduce[18](Figure2).
Figure2.Adiagramoftheevolutionofresistancetofungicides.Thisgraphicshowsanexampleofhowselectionpressure
maytakeplace.Initialpopulationwithlittleresistanceevolvesuntilresistancebecomeswidespreadduetorepeated
fungicideapplications.AdaptedfromDeisingetal.[18].
Resistancetofungicidesmightbeduetovariousprocedures[19–22],including(a)
reducedfungicidebindingduetoalterationofthetargetsite,(b)overexpressionofthetarget
protein,(c)reducedfungicideabsorptionduetoeffluxpumpremovingtoxiccompounds,
and(d)metabolicdegradationofthefungicidethroughdetoxification(Figure3).
J.Fungi2021,7,7834of18
Figure3.MainmechanismsofacquiringresistancetofungicidesinP.digitatum.Mechanismsof
resistancetosinglesitefungicides:(a)detoxificationoffungicidethroughmetabolicenzymes;(b)
reducedfungicidebindingduetoalterationofthetargetprotein;(c)overexpressionofthetarget
protein;(d)effluxpumpsremovingfungicideoutofthecell.AdaptedfromLucasetal.[17].
Themechanismsinvolvedintheappearanceofresistancetofungicidesin
populationsoffieldpathogensentailthestudyoftheprocessesthatinterveneinthe
reductionofsensitivitytothecompoundandthegeneticbasisoftheresistancetrait.As
thereareseveralclassesofsinglesiteinhibitors,itislikelythatthereareseveral
mechanismsthatleadtofungicideresistanceinplantpathogens,includingthemajor
citruspathogenPd.
Thenewestbiotechnologyforgenomeeditingisapromisingtoolforthe
developmentofdiseaseresistantcropsinthefuture[23].Thus,investigatingthe
molecularmechanismsunderlyingfungicideresistanceinplant–pathogeninteractionsis
essentialfordevelopingnewandbetterapproachesforefficientlycontrollingplant
diseases.
Thefruit–pathogeninteractionisfundamentalfortheprogressionoffungal
pathogen.Ithasthereforepromptedgreatinterestintheresearchcommunity,and
numerousstudieshavebeenundertakeninrelationtothevirulenceofpathogensandthe
responseofthefruittoinfection[3].Inthecitrus–Pdinteraction,ithasbeenadvantageous
tohavethecompletesequenceofthePdgenomeaswellasthegenetictransformation
systemsforPd[5,8,24].Thishasmassivelyfacilitatedknowledgeofthemolecular
processesunderlyingthepathogenicityofPd[25].
Thisreviewpresentsanoverallviewofrecentadvancesinthefungicideresistance
mechanismsofpostharvestcitrusgreenmold,providingvaluableinformationonthe
molecularproceduresinvolvedintheachievementofresistancetodifferentchemicals,
eithertoasinglecompoundortoseveralcompoundsatthesametimeinthecontextof
thefruit–pathogeninteraction.Thisinformationisbeneficialfordevelopingnoveland
saferstrategiestopreventpostharvestgreenmoldincitrusfruitsandcontributes
substantiallytoknowledgeonfungaldiseasemanagement.
3.MolecularMechanismsofFungicideResistance
Fungicideresistancecanevolvedifferentlybasedonthecharacteristicsofthe
fungicide(fungicideclass)(Table1).
J.Fungi2021,7,7835of18
3.1.MethylBenzimidazoles(MBCs)
Themechanismofbenzimidazoletypefungicides,whichincludesthiabendazole
(TBZ),involvesbindingtoβ‐tubulins.Thispreventstheassemblyofmicrotubulesandcell
divisionduringmitosisandthereforeresultsintoxicitytofungalcells[26,27].
Resistancetobenzimidazolefungicideshasbeendescribedinawidevarietyoffungi.
Frequently,theresponsiblemechanismcorrespondstopointmutationsinthetubulin
gene,whichleadstothemodificationofsomeaminoacids[28–30].Amongthenumerous
changesobservedintheβ‐tubulingeneassociatedwithresistancetoMBCfungicidesin
phytopathogenicfungi,themostfrequenthavebeeninresidues198and200[14].Itshould
benotedthatthereplacementofglutamicacidbyalanine,valine,orglycineatposition
198andphenylalaninebytyrosineatposition200canleadtovaryinglevelsofresistance
toMBCfungicides[31,32].IntheparticularcaseofTBZ,modificationsintheTBZbinding
siteprovidescellularresistancetoit.Suchvariationsusuallyoccuratpositions198or200
ofβ‐tubulin,althoughotherchangesarealsopossible[14,33].InPd,twodifferentpoint
mutationshavebeendescribedasbeingresponsibleforTBZresistance.Mutationat
position198GlutoLyswasdescribedbyMaandMichailides[14]basedonstudies
performedinPenicilliumexpansum[27].InPdisolatesfoundincitrusfruitsfromCalifornia
packinghouses,resistancetoTBZwasduetomodificationatposition200ofβtubulin
[34].ThesamemutationwaslaterdescribedinPdSpanishisolatescollectedfromorchards
andpackinghouses[35],revealingthatresistancemechanismisindependentoffungicide
pressure.AmongPdisolatescollectedfromcitrusinTaiwan,resistancetoTBZwas
associatedwiththemostfrequentβ‐tubulinmutationsatcodon198or200[36].
However,untilnow,nostudyhasdescribedthemolecularprocessbywhichgenetic
variationsinβ‐tubulinpreventthebindingoffungicide.Recently,researchcarriedouton
PodosphaeraxanthiiusingacombinationofdifferentapproachesproposedthattheMBC
fungicidebindingsiteinβtubulindoesnotparticipateintheresiduesresponsiblefor
fungalresistance[37].Asamechanism,itissuggestedthatwhenMBCfungicides
spontaneouslybindtoβtubulininsensitivefungi,theirconformationisalteredand
adequatepolymerizationinmicrotubulesoccurs;however,thisdoesnottakeplacein
resistantstrains,wherethereisaconformationalchangepromotedbyspecific
modifications.
3.2.DemethylationInhbithors(DMIs)
DMIfungicideshampertheactivityofthecytochromeP450dependentsterol14α‐
demethylase(Cyp51)andthusblockC14demethylationoflanosterol,aprecursorof
ergosterolinfungalpathogens[38].DMIsencompassoneofthemostrelevantgroupsof
fungicidesthatpreventdifferentplantdiseasesbyinhibitingtheactivityofcytochrome
P450dependentsterol14α‐demethylase(P45014DM)andwerefirstusedinagriculturein
the1970s[39].Imazalilisademethylationinhibitor(DMI)thatblocksergosterol
biosynthesis[40,41]andisfrequentlyusedtopreventpostharvestdiseasesofcitrusfruits
worldwideduetoitscurativeandantisporulantactionagainstPd[42].CYP51encodes
sterol14demethylase,anenzymeresponsibleforergosterolbiosynthesis[43],andisthe
targetofDMIfungicides.
ThemainmechanismsthatprovideDMIresistanceare(i)modificationsinCYP51or
(ii)highexpressionofCYP51.DifferentprocedurescausingDMIresistancehavebeen
reported.Theyaremediatedeitherbyspecificchangesinthecodingregion[44–46]orby
augmentinggenetranscriptionduetoaninsertioninthepromoter[47].Therearethree
homologuesofthesterol14α‐demethylaseencodedCYP51geneinPd,namely
PdCYP51A[48],PdCYP51B,andPdCYP51C[49].Thefirstmechanisminvolving
modificationsinCYP51hasbeendescribedinseveralpathogens.Asinglechange,suchas
thesubstitutionofaphenylalanineforatyrosineatresidue136(Y136F)ofCYP51,ledto
resistancetoDMIinUncinulanecator[50],Erysiphegraminisf.sp.hordei[51],Erysiphenecator
[52],andP.expansum[44],whiletwosinglenucleotidechangeswerefoundtoresultin
J.Fungi2021,7,7836of18
aminoacidsubstitutionsY136FandK147QinCYP51inBlumeriagraminis[53].Other
changeshavebeendescribedinTapesiasp.[54],Penicilliumitalicum[55],Ustilagomaydis
[56],Blumeriellajaapii[57],andMycosphaerellagraminicola[58].InPd,noPdCYP51Apoint
mutationswerefoundtoberesponsibleforPdresistancetoIMZorotherDMI[35]orto
prochloraz[46].Ontheotherhand,inPdCYP51B,novariationsinthegenewereinitially
detectedinisolatesresistanttoIMZ[59].However,recently,differentsubstitutionsof
PdCYP51Bhavebeenfoundcorrespondingtodifferentlevelsofsensitivitytoprochloraz,
namelyY136HandQ309Hinhighresistantstrains,G459SandF506Iinmediumresistant
strains,andQ309Hinlowresistancestrains[46].
TheotherprocessresponsibleforresistancetoDMIischangeinthelevelofCYP51
transcription[60].Themostfrequentmechanismisthepresenceofinsertionsinthe
promoterregioninthephytopathogenicfungus,aswasthecaseinB.jaapii[57],Venturia
inaequalis[61],Moniliniafructicola[62],andM.graminicola[58].Thisprocesshasalsobeen
linkedtotheimazalilresistanceinPd.Thefirstmechanismdescribedwasthepresenceof
fivetandemrepeatsofa126bptranscriptionalenhancerinthepromoterregionof
PdCYP51A,resultingintheoverexpressionofPdCYP51A[40].Thesespecificrepeats
allowedthedesignofamoleculartooltoidentifyIMZresistantPd.Themethodisbased
onthedetectionofthetandemrepeatofa126bpsequenceinthepromoterregionof
PdCYP51AbyPCR[48].Furthermore,anew199bpsequencewasidentifiedthatdisrupts
the126bptranscriptionalenhancer,resultinginincreasedexpressionofPdCYP51A[63].
Ontheotherhand,inastudycarriedoutin75SpanishstrainsofPd,resistancetoDMIs
inPddidnotcorrelatewiththe126bptandemrepeatsofPdCYP51A[35].Therefore,in
thenewCYP51gene(PdCYP51B)identifiedinPd,auniqueinsertionof199bpwas
observedinthepromoterregionthatwasassociatedwithitsoverexpressionand
resistancetoDMIfungicides[49].Thesameinsertion,butreducedto195bp,was
identifiedinSpanishPdisolates,demonstratingthatoverexpressionofthisgeneisthe
predominantmechanismforresistancetoDMIandinparticulartoIMZ[59].Thisinsert
wasidenticaltothatdescribedbyGhosophetal.[63]inPdCY51A,whichalsoconferred
resistancetoIMZ.Thus,thePdCYP51Benhanceractuallybehaveslikeatransposonthat
actsastheMITEelementPdMLE[64]andismorestableandpredominantthanthe
PdCYP51Aenhancer.Infact,whenpresentinPdCYP51B,itisnotcompatiblewiththe
presenceofthefivetandemrepeatsof126bpenhancerofPdCYP51A[59].
3.3.QuinoneOutsideInhibitors(QoI)
QoIfungicidesimpederespirationbybindingtotheQositeofthecytochromebc1
enzymecomplex,resultinginenergydeficiencyandleadingtothedeathoffungal
pathogens[65].ThismodeofactioninQoIfungicidesresultsinfrequentappearanceof
QoIresistanceinspecificphytopathogenicfungi.
Aswithotherexternalquinoneinhibitor(QoI)fungicides,azoxystrobinishighly
effectiveinpreventingawidevarietyofplantdiseases[20,66],includingcitrusgreenmold
[1].Azoxystrobin(strobilurin)wasregisteredasanewfungicideintheUSAforthecontrol
ofpostharvestdiseasesofcitrus[67,68].However,duetoitssitespecificmodeofaction,
asmentionedabove,ithasahighriskofdevelopingresistanceintargetphytopathogenic
fungalpopulations.PdisolatescollectedfromvariouspackaginginChinawereshownto
behighlysensitivetoazoxystrobineventhoughithadneverpreviouslybeenusedforthe
controlofcitrusdiseases,indicatingthelackofresistantbiotypesinthenaturalpopulation
[69].AlthoughPdhasahighpotentialtodevelopresistancetoazoxystrobin,noresistance
hasbeendescribednaturallysofar.Onlyamoderatelevelofresistancetostrobilurins
werefoundinsomeofthePdisolatesevaluated,whichshowsthatstrobilurinsare
effective[35].
ThemainmechanismofresistancetoQoIisbasedonthetargetsiteandinvolves
changesinthemitochondrialcytochromeb(CYTB)gene,resultinginvariationsinthe
peptidesequencethatpreventfungicidebinding.MutationsaffectingsensitivitytoQoI
fungicideshavebeenidentifiedintwoareasofCYTB,whicharerelatedtoaminoacid
J.Fungi2021,7,7837of18
positions120–155and255–280oftheencodedprotein.Thismechanismthatunderlies
resistancetoazoxystrobinhasbeenreportedinseveralimportantphytopathogenicfungi
[70–75].Inmostcaseswhereresistancetostrobilurinshasbeendescribed,resistancewas
conferredbysubstitutionofasingleaminoacid(alanineforglycine)atcode143(G143A)
inthecytbgene.Furthermore,substitutionincode129forleucinebyphenylalanine
(F129L)wasalsofoundtoconferresistancetoQoIinsomespeciesoffungi,althoughthe
levelofresistancewaslowerthanthatconferredbytheG143Asubstitution[14,76].
Recently,anadditionalaminoacidsubstitutionfromglycinetoarginineatposition137
(G137R)wasalsoassociatedwithresistancetoQoI[77].InPd,onlyUVinduced
azoxystrobinresistantmutantswerefound.ThesePdmutantsweregeneticallystable,
andtheirhighlevelsofazoxystrobinresistancewereconferredbyasinglepointmutation
(G143A)inthePdcytbgene[69].
ThesecondmechanismofresistancetoQoIfungicidesismediatedbytheinductionof
alternativecyanideresistantrespirationsustainedbyalternativeoxidase(AOX)[78].Inthis
rescuemechanism,mitochondrialelectrontransferisdeviated,bypassingtheQoIinhibitory
siteinthecytochromebc1complex.Underfieldconditions,alternativerespirationappears
tohavelimitedimpactontheprotectiveactivitiesofQoIfungicides[79].
3.4.SuccinateDehydrogenaseInhibitors(SDHIs)
Thetargetofboscalidissuccinatedehydrogenase(SDH)inthemitochondrial
electrontransportchain.TheSDHenzymecatalyzestheoxidationofsuccinateto
fumarateinthemitochondrialmatrix,couplingwiththedecreaseinubiquinoneto
ubiquinolinthemembraneduringaerobicrespiration[80,81].SDHIfungicides
specificallyinhibitfungalrespirationbypreventingubiquinonebindingsitesinthe
mitochondrialcomplexII[81].
SDHIs,suchasboscalid,fluxapyroxad,penthiopyrad,isopyrazam,andfluopyram,
haveaspectrumofactivityagainstawidevarietyoffungalpathogensindifferentcrops.
Boscalidisasuccinatedehydrogenaseinhibitor(SDHI)fungicidethatisvery
effectiveinpreventingalargenumberofplantpathogens,includingSclerotinia
sclerotiorum,Botrytiscinerea,Alternariaalternata,andCorynesporacassiicola[82–85].An
aminoacidmodificationinthehighlyconservedsubunitSDHBisdirectlyrelatedtothe
bindingofSDHItothetargetandhasbeendescribedindifferentplantpathogens.In
SDHIresistantisolates,histidineatorthologouspositions277,272,and267were
substitutedinA.alternata(BH277Y/R)[86],B.cinerea(BH272Y/R/L)[87],andlaboratory
mutantsofZ.tritici(BH267Y/L/F/N/Q)[88,89].Recently,consecutivetreatmentsover
severalgenerationswithboscalidinthelaboratorywereshowntoresultinresistancein
Pd.StudiesshowedthatboscalidinhibitedSODactivitywhilePODactivityincreased,
whichmaybethereasonfortheincreasedO2−anddecreasedH2O2concentrationsinPd
[90].HighlevelsofROSareharmfulandcauseoxidativedamagetoorganisms,butthey
alsoplayanimportantroleintheregulationofavarietyofbiologicalfunctions[91].
Boscalidisasinglesitefungicideandisthereforeconsideredtohaveahighpotential
forresistancedevelopmentregardlessofitshighactivityagainstPd.TheFungicide
ResistanceActionCommittee[92]classifiedSDHIfungicidesasmediumtohighriskwith
respecttothedevelopmentofresistance(Table1)basedprimarilyonsinglesitemutations
ofthegeneencodingtheenzymesuccinatetargetdehydrogenase.Thereportedresistance
hasbeenlimitedtogenerationIcarboxinfungicidesaswellasgenerationIISDHIboscalid.
3.5.Phenylpyrroles(PPs)andAnilinopyrimidines(Aps)
Fludioxonilandpyrimethanilareincludedintheclassesofphenylpyrrole(PPs)and
anilinopyrimidine(APs),respectively.Bothfungicidesareveryeffectiveinpreventingthe
germinationofconidiaandtheelongationofthegermtubeofP.expansumandB.cinerea
[93],andbothareregisteredinalargenumberofcropsaspostharvestfungicidesand
incorporatedforpostharvestuseincitrus[94].Whilefludioxonil‐ andpyrimethanil
resistantPdisolateshavebeennaturallyidentifiedinpackinghouses,theyarenot
J.Fungi2021,7,7838of18
associatedwithcropdiseases[1].Fludioxonilisusedaloneorincombinationwith
azoxystrobininthecontrolofgreenmoldandotherpostharvestdiseasesofcitrus.In
Californiacitruspackinghouses,areferencesensitivitytofludioxonilhasbeenestablished
inPdpopulations[1].Nevertheless,itwasnotuntil2015thatthefirstincidenceof
resistancetofludioxonilinPdcollectedfromcommercialcitruspackinghouseswas
reportedaftertheintroductionofthefungicideinthemarket[95].Pyrimethanilresistant
isolateswerealsoobtainedfromdifferentorchards[96].
Themodeofactionandresistancemechanismsforbothclassesoffungicideshave
beencarriedoutinmutantsinducedinthelaboratoryorinfieldisolatesofvariousfungi.
AlthoughtwonucleotidesubstitutionswerefoundinasequenceanalysisoftheN
terminalaminoacidrepeatregionoftheos1relatedhistidinekinasegeneamongPd
isolates,thesewerenotcorrelatedwithfludioxonilresistance.Studiesindicatethatthe
modeofactionoffludioxonilonPdisprobablythemitogenactivatedproteinkinase
pathway,whichstimulatesglycerolsynthesisinsensitiveandresistantstrains[1].In
addition,whilepyrimethanilisbelievedtoinhibitthebiosynthesisofmethionineand
otheraminoacidsandthesecretionofhydrolyticenzymesinvolvedintheinfection
processindifferentfungalpathogens[97],methioninebiosynthesisisnotthemaintarget
ofAPsinPd[1].Therefore,themechanismsofresistancetofludioxonilandpyrimethanil
inPdremaintobeelucidated.
4.ResistanceMediatedDrugEffluxTransporters
Effluxtransporterscanallowfungitosurviveexposuretotoxiccompounds,
eliminatingtheaccumulationofcompoundsintoxicconcentrationswithinfungalcells.
Thesemembraneboundproteinsareknowntoprovideprotectionagainstawiderange
ofnaturallyoccurringandxenobiotictoxiccompounds[98].Manystudieshavereported
linksbetweenenhancedeffluxtransporteractivityandtheappearanceofresistancein
differentfungalpathogens[41,99–102]includingPd,indicatingthateffluxtransporters
mayhaveacommonandcriticalroleinfungicidesensitivity.Inaddition,coincident
resistancetomanychemicaltypesoffungicideswasfoundtobeattributableto
overexpressionofeffluxpumpsinsomeimportantfungalpathogens.
Drugeffluxtransportersareintegralmembraneboundproteinsthattransportan
extensivevarietyofcompounds,suchasproteinmacromolecules,ions,orsmallmolecules
inabiologicalmembrane[103].Twomajorgroupsofdrugtransportershavebeen
characterizedinfungi,includingABC(ATPbindingcassette)transportersandMFS
(majorfacilitatorsuperfamily)transporters.Multidrugandtoxiccompoundextrusion
(MATE),anothertypeoftransporterthathasbeenmainlyreportedinbacteria[104],is
relatedtoresistancetoantimicrobialagentsandwasrecentlyreportedtobeinvolvedin
prochorazresistanceinPdintrancriptomicanalysis[105].Inthissection,thegeneral
functionofdrugeffluxtransportersrelatedtoresistancetofungicidesinthePd–citrus
pathosystemarereviewed(Figure4).
Figure4.ABCandMFStransporters.ABC:ATPbindingcassettetransportersuperfamily,MFS:
majorfacilitatorsuperfamily.
J.Fungi2021,7,7839of18
4.1.ATPBindingCassetteTransporters(ABC)
ATPbindingcassettetransporters(ABC)makeuponeofthelargestproteinfamilies
describedtodate.ThefamilyofABCtransportersisoneofthemostrelevanteffluxpumps
thatexertprotectionoffungiagainstchemicalcompounds[106,107].Thesetransporters
constituteprimaryactivetransportsystemsastheyobtaintheenergyrequiredfortransport
owingtothehydrolysisofATP(Figure4).Infilamentousfungi,ABCtransporterscanact
againstsyntheticfungicidesorcompoundsproducedbycompetingmicroorganisms[108].
Thephenomenon,describedasthesimultaneousresistancetoseveralchemicallyunrelated
compounds(MDR),isrelatedtotheoverexpressionofABCtransportersduetotheresulting
pleiotropiceffects.FourABCtransportershavebeenidentifiedinPd:PMR1,PMR3,PMR4,
andPMR5.Ofthem,onlyPMR1[48,109]andPMR5[110]appeartoberelatedtomultidrug
resistanceinPd.Amoreexhaustivecharacterizationofthefourtransportersshowedthat
whilenogeneticchangesweredetectedbetweenisolatesinPMR1,PMR3,andPMR4,some
specificmodificationswereobservedinthepromoterandcodingregionsofPMR5instrains
resistanttobothTBZanddifferentDMIfungicides[35].Furthermore,thepresenceoftoxic
substancesselectivelyactivatestheexpressionofPMR1andPMR5.Specifically,triflumizole
andimazalilactivatePMR1transcription,whilebenzimidazoles,dithianone,and
resveratrolpromotePMR5transcription.Thus,Pdresistancecanbedeterminedbyselective
transcriptionalactivationofABCtransportergenestoatoxiccompound.[110].Moreover,
anexhaustivesearchofputativeABCgenesinPdidentifiedatotalof46chromosome
encodedABCfamilytransporters.AnalysisofthesegenesrevealedthatfivemoreABC
transportersmaybeinvolvedindrugresistanceastheywereupregulatedinimazalil
inducingexpressionanalysis[64].Furthermore,transcriptomeanalysisofprochloraz
treatedPdstrainsrevealedthreenewABCtransportersthatweremoreinvolvedin
prochlorazresistance[111].
4.2.MajorFacilitatorSuperfamilyTransporters(MFS)
MFStransportersarepartofthefamilyofactivesecondarytransportersthatcan
transportsubstancesinresponsetoionicgradients.MFStransporterscanmediate
antiport,uniport,andsymportofdifferentproducts[112](Figure4).MFStransporterscan
serveasdrugtransportersowingtoaprotongradient,whichallowsittoconfermultidrug
resistance(MDR).ManyoftheseMFStransporterstransportsmallmoleculesinresponse
toionicgradientsinsuchawaythattheyfunctionasanH+antiporterinmicroorganisms,
regulatingtheirgrowthunderstressconditionsastheyaffectthemembranepotentialand
internalpH[113].
Thesetransporterscouldplayaroleinsensitivitytodifferentcompoundsasthey
usuallyhaveanarrowsubstrateaffinitythatguaranteesanimportantcontributioninthe
transactionofawiderangeofsubstrates.Theeffectontoxineffluxandfungicide
sensitivityhasbeenobservedinmanyfungalMFStransporters.Forinstance,suppression
oftheCercosporanicotianaeMFStransporterreducedthecercosporintoxin[114].InB.
cinerea,BcMfs1affectedsensitivitytocamptothecinandcercosporinandresistancetoDMI
[115]andmfsM2showedgreatereffluxfungicidalactivity[99].TheeliminationofMgMfs1
fromM.graminicolacontributedtostrobilurinfungicideresistancebutnototherevaluated
fungicides[116,117].InZymoseptoriatritici,theMgMFS1transporterparticipatedinthe
MDRphenotype[110].Furthermore,theAaMFS19MFStransporterwasshowntoplaya
roleinresistancetooxidativestressandchemicalsinthephytopathogenicfungus
Alternariaalternata[118].TranscriptomeanalysisofMDRstrainsofP.expansumreported
overexpressionofMFStransportergenesbeforeorafterexposuretofludioxonil[119].
InPd,morethan100MFShavebeenidentifiedduetotheavailabilityofthePd
genome[5].Sofar,ofalltheidentifiedPd–MFStransporters,sevenhavebeen
characterizedmorethoroughly,namelyPdMfs1[120],Pdmfs2[121],PdMFS1[101],
PdMFS2,PdMFS3,PdMFS4,andPdMFS5[102].Allareinvolvedinsomewayinresistance
tochemicalfungicidesandinsomecasesmaycontributetoanincreaseinfungal
J.Fungi2021,7,78310of18
virulence.AnanalysisofeachoftheproteinsencodedbytheseMFSgenesshowsthatthey
sharelittlehomologybetweenthem,whichalsoaffectstheirfunctionality.Thus,while
PdMfs1hasacleareffectagainstimazalil,Pdmfs2andPdMFS1playanimportantrolein
prochlorazresistance.Botharealsoinvolvedinprocessesdevelopedduringthefruit–
pathogeninteraction,suchasconidiaandtheprogressoffungaldisease.Furthermore,
PdMFS1isabletoconferMDRphenotypeasitcontributestotheoutputofawiderange
offungicidalcompounds[101].AmongthelatestidentifiedPd–MFStransporters,only
PdMFS2andPdMFS3appeartoparticipateinfungicideresistance.Bothgenescontribute
tosimultaneousresistancetoseveralunrelatedtoxiccompounds(MDRphenotype),as
previouslyreportedforotherfungalMFStransporters[101,113,122].Thephylogenetic
analysisofalargenumberoftheseMFStransportersinPdrevealedallthegeneshad
differentgeneticstructuresandencodedproteinsofdifferentsizesandthatonly
PdMFS2pappearedtogetherwiththegroupthatcomprisedtheMFStransporters
assignedasdrugeffluxtransporters[102].Ontheotherhand,thetranscriptomicanalysis
carriedoutinPdaftertreatmentwithprochlorazhighlightedoverexpressionof14
differentMFStransporters[111].
MFStransportershavebeenlinkedtoQoIresistance.InMgMfs1,whichencodesan
MFStransportergenefromM.graminicola,thedeletionofMgMfs1showedinsensitivity
toQoI[116].However,untilnow,nodecreaseinsensitivityorresistancetoQoIfungicides
hasbeenidentifiedinPdmediatedbyanMFStransporter.However,thecontributionof
theseenergydependentmechanismsinadaptationtofungicidesbyphytopathogenic
fungishouldbeseriouslyconsidereddespitethescarcityofdataonresistancetoefflux
transporterbasedQoIfungicides.
Untilnow,thecontributionofMFStransportersasadecisivefactorintheplant–
pathogeninteractionisunknown[37],andfurtherfunctionalcharacterizationofmore
differentMFStransporterswillbenecessarytoestablishtheirroleinthePd–citrus
interaction.
5.RegulationofFungicideResistance
5.1.TranscriptionFactorsinPdFungicideResistance
Transcriptionfactors(TF)areinvolvedintranscriptionalregulationandplaya
relevantroleinfungalinteractions.TFscancontributetoprimaryorsecondary
metabolism[123],alongwithstressresponsesandsensitivitytopleiotropicdrugs[124].
SREBPtranscriptionfactors,whichcontainabHLHdomain,functionascritical
controllersofsterolhomeostasisandareuniversallyfoundinfungi.Inmostfungi,SREBPs
playacrucialroleincontrollingergosterolbiosynthesis[125].InPd,theSREBPprotein
SreAwasinitiallyidentifiedandcharacterized,whichplaysanimportantrolein
prochlorazresistanceandinthetranscriptionofergosterolsynthesisgenes[111].Evidence
onthetranscriptionalregulationofthesetargetgeneshasemergedtoexplainthedrug
resistantmechanismsofPd.InthecitruspostharvestpathogenPd,thereisanotherSREBP
homolog,PdsreB,whichappearstobeinvolvedinfungicideresistanceandinthecontrol
ofCYP51geneexpression[126].Functionalcharacterizationshowedthetwogenes
(PdsreAandPdsreB)actasglobalcontrollersinagreatvarietyofbiologicalfunctions,
especiallyinaspectsthatmediateergosterolbiosynthesisandresistancetofungicides.
Thus,theexpressionoftheERG1gene(intheergosterolpathway)isregulatedbyboth
thePdsreAandPdsreBgenes,whileonlyPdsreAisinvolvedintheexpressionofERG2.As
bothgenesregulatedifferentaspects,ashasbeenshownwithsingleanddoublemutations
ofthegenes,itispossiblethatthereareothertranscriptionfactorsinvolvedinergosterol
biosynthesisthatcouldbeactivatedwhenbothSREBPsareinhibited[126].Furthermore,
itispossiblethattheSREBPgenesplayarelevantroleinthecontrolofcertainMFS
transportersinPdassomeofthemwerefoundtobeoverrepresentedingenetranscription
studies[126].
J.Fungi2021,7,78311of18
Fungiareknowntousethetranscriptionalregulationofgenesencodingefflux
transporterstodetoxifycertaincompounds.Theexpressionofeffluxtransportersis
controlledmainlybyfungalzincgrouptranscriptionfactors(TF[Zn2Cys6])[127].Fungi
apparentlyregulateandmanagedifferentstagesofthedetoxificationsystemby
modificationsinparticulartranscriptionfactors,andthisregulatorysystemseemstobe
conservedinfilamentousfungi[128].Modificationsintheactivityoftranscriptional
regulatorselicitoverproductionofMFStransporters[129].
TranscriptionalregulatorSte12mightfunctionasaregulatorofpathwayspecificgenes
[130].InPd,PdSte12mightbeinvolvedinthecontrolofexpressionofseveralgenesthrough
repressionoractivation,triggeringmultipleresponses,suchasdetoxification.PdSte12acts
asanegativeregulatorinseveralgenesinvolvedintransport,includingtheprimaryABC
transporters(PMR1andPMR5)andthesecondaryMFStransporters(PdMFS16).PdSte12
alsopositivelycontrolssteroldemethylases(CYP51andPdCYP51B)[131].
Skn7isahighlyconservedstressresponsivetranscriptionfactorand,apartfrom
Ssk1/SskA,thesecondresponseregulatorthatcanbeactivatedviathephosphotransfer
proteinYpd1.Skn7playsawellestablishedroleintheoxidativestressresponse.Skn7is
involvedinmaintenanceofthecellwallintegrityofS.cerevisiaeandotherfungi.Although
thesegeneshavenotbeenidentifiedtodateinPd,intheMFStransportersofA.alternata
(AaMFS19andAaMFS54),geneexpressionissimultaneouslyregulatedbythestress
sensitivetranscriptionfactorYap1.TheexpressionofAaMFS19isalsocontrolledbythe
stressrelatedregulatorSkn7[118,132],butthisregulatordoesnotaffectAaMFS54.ROS
resistanceinA.alternatais,atleastinpart,mediatedbymembraneboundtransportersas
regulatorsYap1andSkn7havebeenshowntoplayacriticalroleinresistancetooxidative
stress[133](Figure5).
Figure5.Schematicofregulatorymechanismsinvolvedinfungalresistancethatarealsorelatedto
fungicidevirulenceandoxidativestress.
5.2.ProteinKinasesinPdFungicideResistance
Fungiprocessingiscontrolledbyproteinkinase(PK)cascades[134].MAPKsare
involvedinsignalingpathwaysthatarehighlyconservedinalleukaryoticorganisms.There
arethreeorthologousMAPKsinfilamentousfungi,namelyHog1,Slt2,andFus3/Kss1[135].
TheMAPKHog1,Slt2,andFus3/Kss1orthologousinPd,calledPdos2,PdSlt2,andPdMpkB,
havebeenidentifiedandcharacterized[46,136–138].Hog1likeMAPKs,whicharehighly
conservedamongvariousfungi,possessdifferentphysiologicalfunctions,includingahigh
osmolarityadaptation[139].Pdos2isinvolvedinosmoticadaptationandisassociatedwith
positivecontrolofglycerolsynthesisandnegativeregulationofergosterolsynthesis[138].
J.Fungi2021,7,78312of18
ThemodeofactionoffludioxonilonPdisprobablyviathemitogenactivatedproteinkinase
pathway,whichpromotesglycerolsynthesis[1].
PdSlt2functionsasanegativeregulatorofdifferentgenesinvolvedintransport,
comprisingprimarytransporters(ABCtransportersPMR1andPMR5)andsecondary
transporters(MFStransportersPdMFS16).Incontrast,PdSlt2positivelycontrolssterol
demethylasesPdCYP51AandPdCYP51B[137].Inthissense,thecontrolofimportant
genesinvolvedinfungicideresistancehighlightstheroleofthisMAPKinmediatingthe
processinvolvedinresistancetofungicides.
Figure5illustrateshowtranscriptionalregulationplaysanimportantroleinfungal
interactionsandhowsignaltransductionpathwayscanbeinterconnected.Furthermore,
oxidativestressandtheROSresponsemayalsobepartoffungal–plantinteractionasthey
aresimultaneouslyinvolvedinfungalpathogenicityandresistancetofungicides.
6.Conclusions
ThemechanismsresponsibleforacquiringresistancetofungicidesinPdaredueto
numerousgenesthatinsomecasesdependonthetypeoffungicide.Resistancetoacertain
fungicidecanbedefinedbymodificationsinasinglegene.However,ingeneral,different
mechanisms,suchasvariationsthatmodifythebindingtargetofthefungicideorchanges
ingeneexpression,determinetheappearanceofresistancetotoxiccompounds.
ThecomplexityofgenomicpathwaysinPdpopulationshasallowedthemtorespond
andadapttofungicidesinmultipleways.Atleastsevenmechanismsthatcancause
resistancetovariousclassesofchemicalfungicidesinPdhavebeendescribed(Figure6).
Remarkably,resistancecausedbyincreasedactivityofdrugeffluxpumpshasbeenthe
mostnotoriousforallmajorchemicalclasses,indicatingitscommonroleinresistance
evolution.Othermechanismsmentionedabove,suchasdetoxificationandtranscription
factors,havealsobeenfoundtoberelevant.
Figure6.Mechanismsofresistancetomaintypesoffungicidesusedforgreenmolddisease
management.Eachorangerepresentsachemicalclass,whereasthecryptogramswithintheoranges
correspondtoaresistanceprocessasshowninthefigure.MBCs:methylbenzimidazolecarbamates,
DMIs:demethylationinhibitors;QoIs:quinoneoutsideinhibitors;SDHIs:succinatedehydrogenase
inhibitors;APs:anilinopyrimidines;PPs:phenylpyrroles.AdaptedfromHuandChen[105].
Fungicidesplayadeterminingroleinthecontrolofcroppathogens,anditislikely
thattheywillcontinuetobeoneofthemostrelevantmeansinthefuturetopreventthe
developmentofdiseases.Anapproachthatintegratesplantbreedingandbiotechnology,
thedevelopmentofchemicalcompounds,andpoliciesthatensuretheuseoffungicides
J.Fungi2021,7,78313of18
inasustainablewaythroughinnovationinalternativetechnologiesisessentialtoachieve
thechallengeoffoodsafetyinachangingenvironmentandcounteractrisksinplanthealth
andpostharvestcitrusfruitsinparticular.
Expandingknowledgeoffungalresistancemechanismsnotonlyallowsthedesign
offastermoleculartoolstorapidlydetectfungalresistancebutcanalsoallowthe
identificationofnaturalsecondarymetabolitesandthedesignofnewantifungal
compoundsthataremoreefficientandspecific.
Funding:Thisresearchreceivednoexternalfunding.
InstitutionalReviewBoardStatement:Notapplicable.
InformedConsentStatement:Notapplicable.
ConflictsofInterest:Theauthordeclarenoconflictofinterest..
References
1. Kanetis,L.;Forster,H.;Adaskaveg,J.E.BaselinesensitivitiesforthenewpostharvestfungicidesagainstPenicilliumspecieson
citrusandmultipleresistanceevaluationsinP.digitatum.PlantDis.2008,92,261–269.
2. Palou,L.PenicilliumdigitatumandPencilliumitalicum(GreenMold,BlueMold).InPostharvestDecay:ControlStrategies;Bautista
Baños,S.,Eds.;AcademicPress:Cambridge,MA,USA,2014;pp.45–102.
3. Tian,S.;Torres,R.;Ballester,A.R.;Li,B.;Vilanova,L.;GonzalezCandelas,L.Molecularaspectsinpathogenfruitinteractions:
Virulenceandresistance.PostharvestBiol.Technol.2016,122,11–21.
4. Macarisin,D.;Cohen,L.;Eick,A.;Rafael,G.;Belausov,E.;Wisniewski,M.;Droby,S.Penicilliumdigitatumsuppressesproduction
ofhydrogenperoxideinhosttissueduringinfectionofcitrusfruit.Phytopathology2007,97,1491–1500.
5. MarcetHouben,M.;Ballester,A.R.;delaFuente,B.;Harries,E.;Marcos,J.F.;GonzálezCandelas,L.;Gabaldón,T.Genome
sequenceofthenecrotrophicfungusPenicilliumdigitatum,themainpostharvestpathogenofcitrus.BMCGenom.2012,13,646.
6. Perez,M.F.;Ibarreche,J.P.;Isas,A.S.;Sepulveda,M.;Ramallo,J.;Dib,J.R.Antagonisticyeastsforthebiologicalcontrolof
Penicilliumdigitatumonlemonsstoredunderexportconditions.Biol.Control2017,115,135–140.
7. Ghooshkhaneh,N.G.;Golzarian,M.R.;Mamarabadi,M.DetectionandclassificationofcitrusgreenmoldcausedbyPenicillium
digitatumusingmultispectralimaging.J.Sci.FoodAgric.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.Ahighlyefficient
AgrobacteriumtumefaciensmediatedtransformationsystemforthepostharvestpathogenPenicilliumdigitatumusingDsRedand
GFPtovisualizecitrushostcolonization.J.Microbiol.Meth.2018,144,134–144.
9. Ismail,M.A.;Zhang,J.Postharvestcitrusdiseasesandtheircontrol.OutlooksPestManag.2004,15,2935.
10. DeRamónCarbonell,M.;SánchezTorres,P.ThetranscriptionfactorPdSte12contributestoPenicilliumdigitatumvirulence
duringcitrusfruitinfection.PostharvestBiol.Technol.2017,125,129–139.
11. Zhang,T.;Qian,X.;Sun,X.;Li,H.ThecalcineurinresponsivetranscriptionfactorCrz1isrequiredforconidation,fullvirulence
andDMIresistanceinPenicilliumdigitatum.Microbiol.Res.2013,168,211–222.
12. Smilanick,J.L.;Mansour,M.;Gabler,F.M.;Sorenson,D.Controlofcitruspostharvestgreenmoldandsourrotbypotassium
sorbatecombinedwithheatandfungicides.PostharvestBiol.Technol.2008,47,226–238.
13. Hollomon,D.W.Fungicideresistance:Facingthechallenge.PlantProtect.Sci.2015,51,170–176.
14. Ma,Z.;Michailides,T.J.Advancesinunderstandingmolecularmechanismsoffungicideresistanceandmoleculardetectionof
resistantgenotypesinphytopathogenicfungi.CropProt.2005,24,853–863.
15. Brent,K.J.;Hollomon,D.FungicideResistanceinCropPathogens.HowCanItBeManaged,2nded.;FRAC:Brussels,Belgium,2007.
16. Hahn,M.;Leroch,M.Multidrugeffluxtransporters.InFungicideResistanceinPlantPathogens;Springer:Berlin/Heidelberg,
Germany,2015;pp.233–248.
17. Lucas,J.A.;Hawkins,N.J.;Fraaije,B.A.Theevolutionoffungicideresistance.Adv.Appl.Microbiol.2015,90,29–92.
18. Deising,H.B.;Reimann,S.;Pascholati,S.F.Mechanismsandsignificanceoffungicideresistance.BrazilianJ.Microbiol.2008,39,
286–295.
19. Gisi,U.;Chin,K.M.;Knapova,G.;KüngFärber,R.;Mohr,U.;Parisi,S.;Sierotzki,H.;Steinfeld,U.Recentdevelopmentsin
elucidatingmodeofresistancetophenylamide,DMI,andstrobilurinfungicides.CropProt.2000,19,863–872.
20. Gullino,M.L.;Leroux,P.;Smith,C.M.Usesandchallengesofnovelcompoundsforplantdiseasecontrol.CropProt.2000,19,1–
11.
21. Fluit,A.C.;Visser,M.R.;Schmitz,F.J.Moleculardetectionofantimicrobialresistance.Clin.Microbiol.Rev.2001,14,836–871.
22. Capote,N.;Pastrana,A.M.;Aguado,A.;SánchezTorres,P.MolecularToolsforDetectionofPlantPathogenicFungiand
FungicideResistance.InPlantPathology;Cumagun,C.J.R.,Ed.;InTech:EastProvidence,RI,USA,2012;pp.151–202.
23. Langner,T.;Kamoun,S.;Belhaj,K.CRISPRcrops:Plantgenomeeditingtowarddiseaseresistance.Annu.Rev.Phytopathol.2018,
56,479–512.
J.Fungi2021,7,78314of18
24. BurónMoles,G.;LópezPérez,M.;GonzálezCandelas,L.;Vinas,I.;Teixido,N.;Usall,J.;Torres,R.UseofGFPtaggedstrains
ofPenicilliumdigitatumandPenicilliumexpansumtostudyhostpathogeninteractionsinorangesandapples.Int.J.FoodMicrobiol.
2012,160,162–170.
25. Costa,J.H.;Bazioli,J.M.;deMoraesPontes,J.G.;Fill,T.P.Penicilliumdigitatuminfectionmechanismsincitrus:Whatdoweknow
sofar?FungalBiol.2019,123,584–593.
26. Holloman,D.W.;Butters,J.A.;Barker,H.;Hall,L.Fungalβ‐tubulin,expressedasafusionprotein,bindsbenzimidazoleand
phenylcarbamatefungicides.Antimicrob.AgentsChemother.1998,42,2171–2173.
27. Baraldi,E.;Mari,M.;Chierici,E.;Pondrelli,M.;Bertollini,P.;Pratella,G.C.StudiesofThiabendazoleresistanceofPenicillium
expansumofpears:Pathogenicfitnessanggeneticcharacterization.PlantPathol.2003,52,362–370.
28. Leroux,P.;Fritz,R.;Debieu,D.;Albertini,C.;Lanen,C.;Bach,J.;Gredt,M.;Chapeland,F.Mechanismsofresistancetofungicides
infieldstrainsofBotrytiscinerea.PestManag.Sci.2002,58,876–888.
29. Malandrakis,A.;Markoglou,A.;Ziogas,B.MolecularcharacterizationofbenzimidazoleresistantB.cinereafieldisolateswith
reducedorenhancedsensitivitytozoxamideanddiethofencarb.Pestic.Biochem.Physiol.2011,99,118–124.
30. Fan,J.;Luo,Y.;Michailides,T.J.;Guo,L.SimultaneousquantificationofallelesE198AandH6Yintheβ‐tubulingeneconferring
benzimidazoleresistanceinMoniliniafructicolausingaduplexrealtime(TaqMan)PCR.PestManag.Sci.2014,70,245–251.
31. Albertini,C.;Gredt,M.;Leroux,P.Mutationsoftheβ‐tubulingeneassociatedwithdifferentphenotypesofbenzimidazole
resistanceinthecerealeyespotfungiTapesiayallundaeandTapesiaacuformis.Pestic.Biochem.Physiol.1999,64,17–31.
32. Yarden,O.;Katan,T.Mutationsleadingtosubstitutionsataminoacids198and200ofbetatubulinthatcorrelateswithbenomyl
resistancephenotypesoffieldstrainsofBotrytiscinerea.Phytopathology1993,83,1478–1483.
33. Koenraadt,H.;Somerville,S.C.;Jones,A.L.Characterizationofmutationsinthebetatubulingeneofbenomylresistantfield
strainsofVenturiainaequalisandotherplantpathogenicfungi.Phytopathology1992,82,1348–1354.
34. Schmidt,L.S.;Ghosoph,J.M.;Margosan,D.A.;Smilanick,J.L.Mutationatβtubulincodon200indicatedThiabendazole
resistanceinPenicilliumdigitatumcollectedfromCaliforniacitruspackinghouses.PlantDis.2006,90,765–770.
35. SánchezTorres,P.;Tuset,J.J.MolecularinsightsintofungicideresistanceinsensitiveandresistantPenicilliumdigitatumstrains
infectingcitrus.PostharvestBiol.Technol.2011,59,159–165.
36. Lee,M.H.;Pan,S.M.;Ng,T.W.;Chen,P.S.;Wang,L.Y.;Chung,K.R.Mutationsofβ‐tubulincodon198or200indicate
thiabendazoleresistanceamongisolatesofPenicilliumdigitatumcollectedfromcitrusinTaiwan.Int.J.FoodMicrobiol.2011,150,
157–163.
37. VelaCorcía,D.;Romero,D.;deVicente,A.;PérezGarcía,A.Analysisofβ‐tubulincarbendaziminteractionrevealsthatbinding
siteforMBCfungicidesdoesnotincluderesiduesinvolvedinfungicideresistance.Sci.Rep.2018,8,1–12.
38. Brent,K.J.;Hollomon,D.W.FungicideResistance:TheAssessmentofRisk,2nded.;GlobalCropProtectionFederation:Brussels,
Belgium,1998;pp.1–48.
39. Aoyama,Y.;Yoshida,Y.Differentsubstratespecificitiesoflanosterol14ademethylase(P45014DM)ofSaccharomycescerevisiae
andratliverfor24methylene24,25dihydrolanosteroland24,25dihydrolanosterol.Biochem.Biophys.Res.Commun.1991,178,
1064–1071.
40. Hamamoto,H.;Hasegawa,K.;Nakaune,R.;Lee,Y.J.;Makizumi,Y.;Akutsu,K.;Hibi,T.Tandemrepeatofatranscription
enhancerupstreamofthesterol14_demethylasegene(CYP51)Penicilliumdigitatum.Appl.Environ.Microbiol.2000,66,3421–
3426.
41. Nakaune,R.;Hamamoto,H.;Imada,J.;Akutsu,K.;Hibi,T.AnovelABCtransportergene,PMR5,isinvolvedinmultidrug
resistanceinthephytopathogenicfungusPenicilliumdigitatum.Mol.Genet.Genom.2002,267,179–185.
42. Holmes,G.J.;Eckert,J.W.RelativefitnessofImazalilresistantand‐sensitivebiotypesofPenicilliumdigitatum.PlantDis.1995,
79,1068–1073.
43. Parks,L.W.;Casey,W.M.Physiologicalimplicationsofsterolbiosynthesisinyeast.Annu.Rev.Microbiol.1995,49,95–116.
44. Ali,E.M.;Amiri,A.Selectionpressurepathwaysandmechanismsofresistancetothedemethylationinhibitordifenoconazole
inPenicilliumexpansum.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
complexityoftheTR46/Y121F/T289Acyp51AazoleresistancemechanisminAspergillusfumigatus.FungalGenet.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.NovelmutationsinCYP51BfromPenicillium
digitatuminvolvedinprochlorazresistance.J.Microbiol.2014,52,762–770.
47. Fan,J.;Urban,M.;Parker,J.E.;Brewer,H.C.;Kelly,S.L.;HammondKosack,K.E.;Fraaije,B.A.;Liu,X.;Cools,H.J.
Characterizationofthesterol14α‐demethylasesofFusariumgraminearumidentifiesanovelgenusspecificcyp51function.New
Phytol.2013,198,821–835.
48. Hamamoto,H.;Nawata,O.;Hasegawa,K.;Nakaune,R.;Lee,Y.J.;Makizumi,Y.;Akutsu,K.;Hibi,T.TheroleoftheABC
transportergenePMR1indemethylationinhibitorresistanceinPenicilliumdigitatum.Pestic.Biochem.Phys.2001,70,19–26.
49. Sun,X.;Wang,J.;Feng,D.;Ma,Z.;Li,H.PdCYP51B,anewputativesterol14α‐demethylasegeneofPenicilliumdigitatum
involvedinresistancetoimazalil