Induced resistance during the interaction pathogen x plant and the use of resistance inducers

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Mini

review

Induced

resistance

during

the

interaction

pathogen

x

plant

and

the

use

of

resistance

inducers

M.D.M.

Oliveira

*

,

C.M.R.

Varanda,

M.R.F.

Félix

LaboratóriodeVirologiaVegetal,InstitutodeCiênciasAgráriaseAmbientaisMediterrânicas,UniversidadedeÉvora,Apartado94,7002-554Évora,Portugal

ARTICLE INFO Articlehistory: Received9June2015

Receivedinrevisedform28October2015 Accepted30December2015

Availableonlinexxx Keywords:

Exogenousresistanceinducers Plantdefence

Pathogens

ABSTRACT

Plants reacttoaggressionsthrough differentdefence responses.Mechanicalbarriersconsistin the increaseofproductionanddepositionofsubstancescapableofcontainingpathogeninvasion.Chemical barriersconsistintheincreaseofconcentrationoractivityofdefenceproteinsandsynthesisofphenolic compoundsandphytoalexins.Elicitorsubstanceshavebeenwidelyusedinplantdiseasecontrolshowing impressiveresultsandalowimpacttotheenvironmentandman.Thisreviewcontainsinformationabout plantdefencemechanismsandshowstheuseofinducersofresistanceinthecontrolofpathogensand prospectsofadvancetowardssustainableagriculture.

ã2016PhytochemicalSocietyofEurope.PublishedbyElsevierB.V.Allrightsreserved.

Contents

1. Introduction ... 152

2. Defencemechanisms ... 153

2.1. Chemicalsignallingandoxidativemetabolism ... 154

2.2. Resistanceinducers ... 155

3. Conclusions ... 157

Acknowledgements ... 157

References ... 157

1.Introduction

During the evolutionary process, plants have developed mechanismstorespondtodifferenttypesofstress,eitherabiotic, suchasdrought,salinityandhightemperature(Shahetal.,2014), orbiotic,suchaspathogens(ShahandZeier,2013).Thesedefence mechanismsremaininactiveorlatentuntiltheyareactivatedafter exposureand/orcontactwithinducingagents(Mandal,2010).

Plants haveseveral defence mechanisms against pathogens. Theoctadecanoidpathwayisoneofthebestknownmechanisms inplantdefence,anditsﬁnalproduct,thejasmonicacidisaplant hormonethatinducestheexpressionofseveralgenesrelatedto defence against stress (Shah et al., 2014). Additionally, this pathway induces the production of H2O2, a reactive oxygen

species(ROS)thatcanactasaﬁrstdefencesignallingmoleculein

plants. A number of other reactive species, as well as several antioxidantenzymes,areconstantlymodulatedinthepresenceof acertainstressinordertoovercomeit(Vandenborreetal.,2011; O’Brienetal.,2012;Davaretal.,2013;RaoniandPratyusha,2013). The infection caused by a pathogen may induce dramatic changesintheactivityofplantcellsaroundthesiteofinvasionand mayleadtotheinductionofresistance,knownasthe hypersensi-tiveresponse(HR),characterisedbyrapidcelldeathatthesiteof infection (Durrant and Dong, 2004). Although this reaction is knownforalmostacentury,itisnotyetclearwhethercelldeath has any direct role in resistance or if it is a consequence of signallingmechanismsthatleadtoeventsthatinhibitpathogen action(ThakurandSohal,2013).

Systemic acquired resistance (SAR) and induced systemic resistance (ISR) designate the mechanisms by which plants activatedefencemechanisms not just inthe inductionsite but also at other places,after being exposed to an inducing agent (Conrathetal.,2006).

*Correspondingauthor.

E-mailaddress:monicadmportella@hotmail.com(M.D.M. Oliveira).

http://dx.doi.org/10.1016/j.phytol.2015.12.011

1874-3900/ã2016PhytochemicalSocietyofEurope.PublishedbyElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

Phytochemistry

Letters

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Advances in research involving ISR in plants have been accompaniedbytheemergenceofnewcommercialproductswith higherefﬁciency,stabilityand withless impactonthe environ-mentthanexistingones.Thesenewproductsareabletoincrease agriculturalproductivitynotonlybyreducingthelossescausedby pathogensbutalsobyincreasingvegetativegrowth(Faroukand Osman,2011).

Acibenzolar-S-methyl(ASM)wastheﬁrstSARinductorreleased forcommercialuse(LyonandNewton,2007).Sincethen,several other products have become available on the market or are currentlybeingresearched(GrahamandMyers,2011).Secondary compoundspresentin medicinalplantshavealsobeenusedas resistanceinducersduetotheirimportantrolesinplant-pathogen interactions, either by direct antimicrobial action or by the inductionofdefencemechanismsinotherplants(Garcia-Brugger etal.,2006).Thisreviewprovidesanupdatedsummaryofplant defenceresponsesanddescribesomeresistanceinducersthatare used to induce systemic responses in plants as well as the associatedoxidativemetabolism.

2.Defencemechanisms

Plants have natural resistance mechanisms for defending themselvesagainst pathogenicorganismsthat arecharacterised by the resistanceability of the plant todecrease or avoid the establishment of certainpopulations of pathogens (Farouk and Osman,2011).

Thebetterunderstandingofplantsignallingpathwayshasled to the discovery of natural and synthetic compounds called resistanceinducersthatinducedefenceresponsesinplantssimilar astheonesinducedbypathogeninfection(Gómez-Vásquezetal., 2004).Different typesofresistanceinducers havebeen charac-terised, including carbohydrate polymers, lipids, glycopeptides, andglycoproteins.Inplants,acomplexarrayofdefenceresponses is induced after detection of microorganismvia recognitionof elicitor molecules released during plant-pathogen interaction. Followingelicitorperception,theactivationofsignaltransduction pathways generally lead to the production of reactive oxygen species,phytoalexinbiosynthesis,reinforcementofplantcellwall associated with phenyl propanoid compounds, deposition of callose,synthesis ofdefenceenzymes,and theaccumulationof pathogenesis-related(PR)proteins,someofwhich with antimi-crobialproperties(VanLoonandVanStrien,1999;Madhusudhan etal.,2008;Aryaletal.,2011;ThakurandSohal,2013).ROSleadto hypersensitiveresponse(HR)(Agrios,2005)inplantswhichisa rapiddeathofoneorfewcellsattheinfectionsitetodelimitthe pathogengrowth.FollowingtheactivationofHR,uninfecteddistal partsoftheplantmaydevelopresistance tofurtherinfection,a phenomenonknownassystemicacquiredresistance(SAR).SARis effective against diverse pathogens, including viruses, bacteria, and fungi (Heil and Bostock, 2002; Graham and Myers, 2011; Elsharkawyetal.,2013).

Whenresistantplantsrecognizeresistanceinducers, intracel-lularsignaltransductionpathwaysareactivated.Thesepathways ultimatelyresultinthederepressionofabatteryofgenescalled defence response genes. These latter genes encode various pathogenesis related (PR) toxic proteins such as chitinases, glucanases,lysozyme-activeproteins, orcellwall strengthening proteins such as hydroxyproline rich glycoproteins. Response proteinsmayalsobeenzymesthatactinbiosyntheticpathwaysfor ligniﬁcation of cell walls or production of phytoalexins, low molecularweighttoxicchemicalsthatantagonizetheinvader.In thefollowingsection,thebiochemicalresponseofplantdefence mechanism related to PR-proteins including chitinase and glucanase, as well as plant lignin content will be explained (Ebrahimetal.,2011).

Theproductionand accumulationof PRproteinsinplants in response to invading pathogen and/or stress situation is very important (Liu and Ekramoddoullah, 2006). Phytoalexins are mainlyproducedbyhealthycellsadjacenttolocalizeddamaged andnecroticcells,butPRproteinsaccumulatenotonlylocallyin theinfectedandsurroundingtissuesbutalsoinremoteuninfected tissues.ProductionofPRproteinsintheuninfectedpartsofplants canpreventtheaffectedplantsfromfurtherinfection(Ryalsetal., 1996;Delaney,1997;Ebrahimetal.,2011).PRproteinsinplants were ﬁrst discovered in tobacco plants infected with Tobacco mosaicvirus(VanLoonandVanKammen,1970).Atpresent,these proteinshavebeenfoundinmanyplants.MostplantPRproteins are acid-soluble,havelow molecularweight, andare protease-resistant(Leubner-MetzgerandMeins,1999;Neuhaus,1999).PR proteins dependingontheirisoelectric pointsmaybeacidic or basic proteins but they have similar functions. Most acidic PR proteinsarelocatedintheintercellularspaces,whereas,basicPR proteinsarepredominantlylocatedinthevacuole(Legrandetal., 1987;Nikietal.,1998;VanLoonandVanStrien,1999).PRproteins have been classicallydividedinto 5 families (Sels et al., 2008) based on molecular mass, isoelectric point, localization and biological activity (Van Loon, 1985). Currently PR-proteins are categorized into 17 families according to their properties and functions,including

b

-1,3-glucanases,chitinases,thaumatin-like proteins, peroxidases, ribosome-inactivating proteins defences, thionins,nonspeciﬁclipidtransferproteins,oxalateoxidase,and oxalate-oxidase-like proteins (Van Loon and Van Strien,1999). AmongthesePRproteins,chitinasesand

b

-1,3-glucanasesaretwo importanthydrolytic enzymesthat areabundantinmanyplant speciesafterinfectionbydifferenttypeofpathogens.Theyplaya main role on defence reactions against fungal pathogens by degrading theircell walls,of whichchitin and

b

-1,3-glucan are major structural components.

b

-1,3-glucanases appear to be coordinatelyexpressedalongwithchitinasesafterfungalinfection. This co-induction of the two hydrolytic enzymes has been described in many plant species, including pea, bean, tomato, tobacco,maize,soybean,potatoandwheat(Mauchetal.,1988a; Mauchetal.,1988b;VogelsangandBarz1993;Jachetal.,1995; Bettiniet al.,1998;LambaisandMehdy,1998;Petruzzellietal., 1999;Cheongetal.,2000;Lietal.,2001;Ebrahimetal.,2011).

Constituentchemicaland anatomicalfactorssuchascuticles and preformed cell walls and inhibitors, may be sufﬁcient to prevent colonization of plant tissues. However, if penetration occurs,theinduceddefencesystemisactivated.Thisincludesthe rapidgenerationofreactiveoxygenspecies,changesincellwall polymers,synthesisoflowmolecularweightmetabolitessuchas phytoalexins,productionofnewclassesofproteinsrelatedtothe defenceandahypersensitivityresponsefollowedbyprogrammed cell death(Thakur andSohal, 2013).Collectively,thesesystems ﬁrstpreventthepathogenentranceandtheninhibitthepathogen establishment(ShewryandLucas,1997;ThakurandSohal,2013). Theinducedresistance(IR)canbeactivatedbyanumberof substances, preventing or delaying the entry or subsequent activityofthepathogenintheirtissues(Abdel-Kaderetal.,2013;

Shah etal., 2014).Severalagentscaninducetheproduction of “signs”inplanttissues,triggeringreactionsthatculminateina lasting protection against a wide range of pathogens. The perception occurs whenthe inducingagent moleculesbind to receptor molecules that are probably located in the plasma membraneoftheplantcell.Thesereactionstriggertheactivation ofseveraldefencemechanisms(GrahamandMyers,2011).TheIR canbedividedintotwocategories,systemicacquiredresistance andinducedsystemicresistance(VanLoonetal.,1998).InSAR, resistance develops systemically in response to a pathogen that causes necrotic lesions or through exogenous application of salicylic acid or synthetic compounds such as ASM and

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2,6-dichloroisonicotinicacid(INA).In SAR,theexpressed resis-tanceisgenerallyeffectiveagainstabroadspectrumofpathogens and is associated with the synthesis of proteins related to pathogenesis,manyofthemhaveantimicrobialactivityandare excellent molecular markers for resistance response. SAR is inducedbypathogensorchemicalsactivators(Mandal,2010),itis characterisedbytheexpressionorproductionofasignalreleased fromthesiteofinfectionthatcausesnecrosisandtranslocationof thissignaltootherpartsoftheplant,inducingdefencereactions thatwillprotecttheplantagainstsubsequentattacks.Cellwall modiﬁcations, phytoalexins production andprotein translation relatedtopathogenesisbyincreasinggeneexpression,areamong theinduceddefencemechanismsbySAR(Ebrahimetal.,2011). ISR is usually induced by rhizobacteria and signalling is mediatedbyjasmonicacidand ethylene(VanLoonetal.,1998; ThakurandSohal,2013).InISR,theinducerdoesnotcausenecrotic symptoms at the site of infection, but induces the plant to a systemicprotection.Inthismechanism,thereisnoaccumulation ofpathogenesis-relatedproteins,theplantthatsuffersinduction doesnot showalterations,theinducing agentisusuallya non-pathogenand induction is not salicylate-dependent,suggesting that theremay beanother signalling pathwaymore associated withjasmonatesandethylene(VanLoonetal.,1998;Faroukand Osman,2011).

Accordingtosomeauthors,althoughtheinductionofresistance hasgreatpotentialinplantprotection,itshouldnotbeseenasa redeemingsolutiontoallproblems,mostlybecauseseveralsteps involvedintheactivationofinducedresistancemechanismsare notyetwellstudied.Inaddition,accordingtosomeresearchers, theuseofresistanceforagivenpathogenmaybeassociatedwith the inducing effect of susceptibility to pests and herbivores (Bostock,1999;Cameron,2000;Mandal,2010).

2.1.Chemicalsignallingandoxidativemetabolism

Differentresponsescanbegiventoalesioncausedbyinjury accordingtothepartoftheplantaffected(Shahetal.,2014).In addition,plantsofdifferentagesmaypresentdifferentlevelsof proteinexpressioninsimilarvegetativeorgans(Buchananetal., 2000)andthephysiologicalstateoftheplantmayalsoinﬂuence theresponse(AlarconandMalone,1995).

Intheprocessofinteractionofthepathogenwiththeplant,it mayoccurcompatibility(susceptibility)orincompatibility (resis-tance)(HeilandBostock,2002).Onceanincompatibleinteraction occurs,a series of responsesinvolving therelease of signalling moleculesistriggered,startingwiththerecognitionorperception, signaltransductionandtranslationfortheactivationof defence genes(Bostock,2005;Waltersetal.,2007).

Plantsactivateseveralbarriersofdefenceagainsttheattackof pathogens (Jonesand Dangl, 2006).The firstline of defenceis triggeredbytherecognitionofinvariantmicrobialepitopesknown as pathogen associated molecular patterns (PAMPs). PAMPs recognizepotentialpathogens in theinnate immune system of bothplantsandanimals.ExamplesofPAMPsareconservedcell surfacestructures likeflagellin, lipopolysaccharidesor peptido-glycanesfromgram-negativebacteriaorfungalcellwall compo-nents like glucan or chitosan. These PAMPs are recognized by distinct cell surface pattern recognition receptors that activate basal or innate immune responses, which is termed PAMP-triggered immunity (PTI). Pathogens (virulent) use effectors (virulencefactors)toblockthisPTIleadingtovirulence.However, someplantcultivarshaveevolvedspecificsurveillanceproteins, theResistance(R)proteins,thatrecognizetheseeffectors(called avirulence– avr – factors and the pathogens being avirulent), mounting a second line of defence called effector-triggered immunity(ETI)(Torres,2010).ETIisstrongerthanPTIandusually

displaysanHRwithcelldeathassociatedtothepathogeninfection (JonesandDangl,2006).

Usuallyofaproteinaceousnature,thereceptorislocatedinthe plasmamembraneandinsidethecell.Thereceptormediatesthe perception signals derived from the elicitor or pathogen, and causesalterationsinthecellularmetabolism,suchasactivationof G proteins, increase of ions ﬂux acrosstheplasma membrane, kinases and phosphatasesactivityand productionof secondary messengers(Garcia-Bruggeretal.,2006).

Gproteinsinteractwithreceptorandincreasetheafﬁnityfor GTP (guaninetriphosphate),and whenthereplacementof GDP (guaninediphosphate)byGTPoccurstheproteinbecomesactive, interacting with otherplasma membrane proteins, suchas the ones that function as ions channels, phosphodiesterases and phospholipases,andthiscomplex(Gprotein–elicitorreceptor– plasmamembraneproteins)gainstheabilitytodiffusethesignal, inducing the activity of speciﬁc intracellular proteins (Bowles, 1990;Ebrahimetal.,2011).

Theopeningofanionchannelintheplasmamembraneoccurs due totheinteractionelicitor/receptorstimulating anions ﬂux acrossthemembrane,alteringthetransmembranepotentialand causingtheextracellularalkalinisationduetotheentryofH+_and

Ca2+_and_exit_of_K+_and_{Cl .}_The_entry_of_Ca2+_in_the_cell_activates

oxidativereactionsthatcanactdirectlyindefenceand/ortrigger other defence reactions (Dixon et al., 1994; Thakur and Sohal, 2013).

Thephosphorylationofcytoplasmicproteinsbykinasesisakey mechanism in the signalling system for intracellular signal transduction,itleadstothestimulationofamembraneoxidase involved inthe generationof thesuperoxide ion, initiating the oxidative burst (production of ROS, including H2O2), direct

inductionofgenetranscription,jasmonicacidbiosynthesis(highly variable in concentrationin plant tissues)and/or ethylene and apoptosis(GillandTujela,2010;O’Brienetal.,2012).

Salicylic acid, jasmonates, ethylene, nitrous oxide, protein kinasesandphosphataseshavebeenreportedasbeinginvolvedin thetransductionofsignalresistanceinducers,convertingreceived signals into speciﬁc responses inside the cells (Mandal, 2010; Farouk and Osman, 2011). The hormones involved in defence-related processes may act alone or together and control each other’s effects (Shah et al., 2014). Among the secondary messengersidentiﬁedasputativemediatorsofdefenceresponses, onecanhighlightcAMP,Ca2+_,_Ca2+_-calmodulins_complex,_inositol

triphosphateproteinkinases(IF3)andROS(O’brienetal.,2012). TheproductionofROSisrequiredforhypersensitivityresponse, HR,orinotherwords,theprogrammeddeathofcellsadjacentto theinfection,limitingthepathogenaccesstowaterandnutrients (ThakurandSohal,2013).ThemainpointsofproductionofROSare chloroplastsandmitochondria,mainlybecausetheseorganelles havehighmetabolicoxidationactivityandintenseelectronﬂow.In chloroplasts,the formation of ROSis related to photosynthesis events. The photorespiration phenomenon in peroxisomes is another form of H2O2 production. In mitochondria1-5% of O2

consumed in dark conditions may be responsible for the production of ROS.Other importantsources of ROSproduction inplantsreceivinglittleattention,arethedetoxiﬁcationreactions catalysedbycytochromeP450inthecytoplasmandendoplasmic reticulum.Thesereactivespeciesarealsogeneratedattheplasma membranelevelorextracellularlyintheapoplast(Garcia-Brugger etal.,2006;GillandTuteja,2010).Incaseofvirusinfectionsis, sometimes,locatedinthecytoplasm.(Fig.1).

Earlystudiesdidnotdetectanyreactiveoxygenspecies(ROS) productioninsystemictissuesduringtheonsetofSAR( Neuensch-wanderetal.,1995;Ryalsetal.,1995).However,In 1998,itwas discovered that H2O2 accumulates in small groups of cells in

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strain of Pseudomonas syringae (Alvarez et al., 1998). These microbursts occur within two hours after an initial oxidative burstintheinoculatedtissueandarefollowedbytheformationof microscopicHRlesions.UsingcatalasetoscavengeH2O2,orDPI

(diphenylene iodonium) to inhibit the NADPH oxidase, it was demonstrated that both the primary and secondary oxidative burstsarerequiredfortheonsetofSAR.Theauthorsproposedthat microburstsofROSmightactivatedefenceresponsesatalowlevel throughouttheplantandthiscontributedtotheSAR-inducedstate (DurrantandDong,2004).

ThemostbiologicallyimportantROSarethefreeradicalsH2O2

(hydrogen peroxide), O2. (superoxide) and OH (hydroxyl

radicals),theseareconsidered“active”or“reactive”becausethey do notrequireenergy inputtoreactwithothermolecules.The superoxide(O2. )canbeproducedinplantsbymanymechanisms

includingtheactivationofNADPHoxidase/synthasesboundtothe membrane,cellwallperoxidases,lipoxygenases,andasaresultof electron transfer from mitochondria or chloroplasts. The O2.

usuallyoxidises organicmolecules suchas ascorbateor metals such as Fe3+ ₍_Saathoff _et _al., _2013; _Thakur _and _Sohal, ₂₀₁₃_).

Hydroxylradicals(OH )areextremelytoxic,theyaregeneratedby H2O2conversioninthepresenceofFe2+andtheyactinproteins

reducingenzymeactivitywithlipidsincreasingthepermeabilityof membranesandwithDNAcausingmutations.

H2O2ispredominantlygeneratedinchloroplaststhroughthe

transfer of energy from a photo-excited chlorophyll to the molecularoxygenelectron.H2O2reactseasilywithdoublebonds

andhasafﬁnity withaminoacidssuchashistidine,methionine, tryptophan,andcysteine.H2O2accumulationcandependontwo

consecutivesteps:reductionofmolecularO2toO2. followedby

spontaneousorcatalyseddismutationofO2. toH2O2(Thakurand

Sohal,2013).Thesuperoxidedismutase(SOD)enzymecatalyses O2. toO2andH2O2.Hydrogenperoxidecanbedirectlytoxicto

pathogensand isinvolvedincellwall strengtheningandin the ligninbiosynthesis process. It alsoactsas a second messenger, beingresponsiblefortheactivationofthebenzoicacidhydrolase, theenzymeresponsiblefor theconversion ofbenzoic acidinto salicylicacid.

ROSarehighlyreactiveandtoxictothecellandtotheattacker, beingquicklyremovedfromthemediumbyantioxidantenzymes such as superoxide dismutases, ascorbate/glutathione cycle enzymes, catalase and

b

-carotene (Raoni and Jyothsna, 2010;

Davaretal.,2013;Shahetal.,2014).SODscanbelinkedtoametal, Cu/Zn, Mn and Fe, present in the cytosol, chloroplasts and mitochondria,respectively.Theascorbateorglutathionecycleis the mainROS removalsystem in thechloroplast. Theenzymes involvedin thiscycleinclude:ascorbateperoxidase, dehydroas-corbate reductase,monodehydroascorbate reductase and gluta-thionereductase(ThakurandSohal,2013;RaoniandPratyusha, 2013). H2O2 can be reduced and removed by the ascorbate

peroxidaseasareducingagenttoformthemonodehydroascorbate radical. This in turn will dismutate to dehydroascorbate and ascorbate.CatalasesareenzymesthatconvertH2O2toH2OandO2

thatarepresentinglycosomesandperoxisomes.Catalasesarethe mainenzymesinplantH2O2detoxiﬁcationandtheycandismutate

H2O2 directly or oxidise substrates such as methanol, ethanol,

formaldehyde and formic acid. The

b

-carotene is an effective detoxiﬁcationagentof1O2.Itparticipatesintheantennaesystem

ofchloroplasts,inlightabsorptionandinenergytransfertothe reactionscentres(Garcia-Bruggeretal.,2006).

In1998,whenAlvarez etal.(1998) workedwithArabidopsis, theyobservedthatH2O2accumulatedinleavesinsmallgroupsof

cellsafterbeinginoculatedwithanavirulentagent,Pseudomonas syringae.Thisaccumulationoccurredwithintwohoursafterthe initial oxidative burst in inoculated tissue, followed by the formation of microscopic lesions of hypersensitivity response. WhenusingcatalasetoremoveH2O2,orDPI(difenilenoiodonium)

toinhibitNADPHoxidase,itwasdemonstratedthattheprimary andsecondaryoxidativeburstswererequiredforthebeginningof SAR. The authors proposed that the oxidative microbursts in reactive oxygen species can activate defence responses and contributetoSAR.

2.2.Resistanceinducers

Exogenousapplicationofplantresistanceinducers(PRIs)able to activate plant defences is an interesting approach for new integratedpestmanagementpractices.ThefullintegrationofPRIs into agricultural practices requires methods for the fast and objectiveupstreamscreeningofefﬁcientPRIsandoptimizationof their application (Bernonville et al., 2014). The induction of resistancehasbeenextensivelystudiedinordertodevelopnew alternativesin diseasecontrol,particularlytodevelopmeasures thatwillminimisetheimpacttotheenvironmentandtoman.The

Fig.1.Speculativemodelshowingpossiblecomponentsinvolvedinreactiveoxygenspeciesgenerationandtheireffectsonthepathogenortheactivationofplantdefence mechanisms(adaptedfromMehdy,1994).

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inductionofresistancecanbeobtainedthroughtheuseofnatural products,withfungalorplantorigin,andithasalsotheadvantage toallowresponses totheattack of a wide range of pathogens mostlybecauseithasnospeciﬁcmechanismsofaction( Madhu-sudhanetal.,2008;GrahamandMyers,2011).

Elicitor or inductor is deﬁned as a molecule or molecules presentinanorganismorproducedbytheplantitselfandwhose functionsinclude thegenerationof defenceresponses(Mandal, 2010). Molecules of fungi,bacteria and viruscan be resistance inducers.Thesearemostlymadeupofproteinsandcarbohydrates presentinthecellsofplantpathogens.

Severalstudieshaveshownthattheuseofexternalresistance inducers activate defence mechanisms in most of the species studied.Madhusudhan etal. (2008) when using Acibenzolar-S-methylpre-treatmenttotomatoandtobaccoplantsreducedthe concentrationofTomatomosaicvirus(ToMV)andTobaccomosaic virus (TMV) through a mechanism that involves RdRp (RNA-dependentRNAPolymerase)andAOX(AlternativeOxidase)gene induction.

Mandal(2010)investigatedaninducedresistanceineggplant (SolanummelongenaL.)inrespecttocellwallstrengtheningand defenceenzymeactivationaffectedbyfourresistanceinducers, chitosan(CHT),salicylicacid(SA),methyl salicylate(MeSA)and methyljasmonate(MeJA).Theincreaseintotalphenoliccontent and lignin eposition in the cell wall of eggplant roots by the resistanceinducers was signiﬁcantly higher. Phenylalanine am-monia-lyase(PAL)activityshowedanincrease.Highestactivityof peroxidase(POD)wasobservedat24hafterelicitationunderthe precise inﬂuence of CHT and SA. The activities of polyphenol oxidase (PPO), cinnamyl alcohol dehydrogenase (CAD) and catalase(CAT)werealsoincreasedseveralfoldsbytheresistance inducers.Accumulationofphenolicsandlignininhighamounts, together with higher activity of major defence enzymes in response to the resistance inducers, may bolster eggplants in mounting practical and effective resistance against Ralstonia solanacearum,adevastatingpathogenthatcausesplantwilt.

DeMeyeretal.(1998)providedargumentsfortheparticipation ofinducedplantdefenceinTrichodermaharzianumT39controlof Botritiscinereaintomato,lettuce,pepper,beanandtobacco,which resultedina25-100%reductionofgreymouldsymptoms,caused byadelayorsuppressionofspreadinglesionformation.

Elsharkawy et al. (2013) evaluated T. asperellum SKT-1 for inductionofresistanceagainstyellowstrainofCucumbermosaic virus(CMV-Y)inArabidopsisplants.Diseaseseveritywasratedat 2weekspostinoculation(WPI).CMVtitreinArabidopsisleaveswas determined by indirect enzyme-linked immunosorbent assay (ELISA) at 2 WPI. Their results demonstrated that among all Arabidopsisplantstreatedwithbarleygraininoculum(BGI)of SKT-1NahGandnpr1plantsshowednosignificantreductionindisease severity and CMV titre as compared with control plants. In contrast,diseaseseverityandCMVtitreweresigni_ficantlyreduced inallArabidopsisplantstreatedwithculturefiltrate(CF)ofSKT– 1 as compared with control plants. RT-PCR results showed increasedexpressionlevelsofSA-induciblegenes,butnot JA/ET-inducible genes, in leaves of BGI treated plants. Moreover, expressionlevelsofSA-andJA/ETinduciblegeneswereincreased inleavesofCFtreatedplants.Inconclusion,BGItreatmentinduced systemicresistanceagainstCMVthroughSAsignallingcascadein Arabidopsisplants.While,treatmentwithCFofSKT-1mediatedthe expressionof a majorityof thevariouspathogenrelatedgenes, which led to the increased defence mechanism against CMV infection.

Harpins are glycine-rich and heat-stable proteins that are secretedthroughtypeIIIsecretionsystemingram-negative plant-pathogenicbacteria.Manystudiesshowthat theseproteinsare mostlytargetedtotheextracellularspaceofplanttissues,unlike

bacterialeffectorproteinsthatactinsidetheplantcells.Thefirst harpin of pathogen origin, HrpN of Erwinia amylovora, was reportedin1992asacell-freeelicitorofhypersensitiveresponse (HR). Some harpins were shown to have virulence activity, probably because of their involvement in the translocation of effector proteins into plant cytoplasm. Based on this function, harpinsarenowconsideredtobetranslocators.Theirabilitiesof pore formation in the artificial membrane, binding to lipid components,and oligomerization areconsistentwith this idea. Whenharpinsareappliedtoplantsdirectlyorexpressedinplant cells,theseproteinstriggerdiversebeneficialresponsessuchas induction of defence responses against diverse pathogens and enhancementofplantgrowth(Choietal.,2006).

Obradovicet al.(2005) studiedtwostrainsofplant growth-promoting rhizobacteria, two systemic acquired resistance inducers(harpin andacibenzolar-S-methyl),host-specific unfor-mulatedbacteriophages,andtwoantagonisticbacteriaforcontrol of tomato bacterialspot incitedbyXanthomonas campestrispv. vesicatoriain greenhouseexperiments.The resultsshowed that whenplantgrowth-promotingrhizobacteriastrainswereapplied alone or in combination with other treatments, they had no significanteffectonbacterialspotintensity.Antagonisticbacterial strains, when applied alone, had negligible effects on disease intensity.Acibenzolar-S-methylcompletelypreventedoccurrence oftypicalsymptomsofthedisease,howevernecroticspotstypical ofahypersensitivereaction(HR)wereobservedonplantstreated withacibenzolar-S-methylalone.Electrolyteleakageand popula-tiondynamicsexperimentsconfirmedthat acibenzolar-S-methyl-treated plants responded to inoculation by eliciting an HR. Applicationof bacteriophagesin combinationwith acibenzolar-S-methylsuppressedavisibleHRandprovidedexcellentdisease control. These authors concluded that thentegrated use of acibenzolar-S-methyl and phages may complement each other asanalternativemanagementstrategyagainstbacterialspoton tomato.

Theefﬁcacyofhexanoicacid(Hx)asaninducerofresistancein tomatoplants againstPseudomonassyringae pv.tomato DC3000 wasdemonstratedbyScalschietal.(2014)andtheplantresponse wascharacterized.Theirworkprovidedtheﬁrstdemonstrationof theresponseofthepathogentothechangesobservedinplants afterHxapplicationnotonlyintermsofthepopulationsizebut also of the transcriptional levels of genes involved in quorum sensing establishment and pathogenesis. It is possible that Hx treatment attenuates the virulence and survival of bacteria by preventing or diminishing the appearance of symptoms and controllingthegrowthofthebacteriainthemesophyll.Hxisable toalterbacteriapathogenesisandsurvivalonlywhenitisapplied asaresistanceinducerbecausethechangesitpromotesinplants affectthebacteria.

ThebestknowncommercialresistanceinducerisASM(Dietrich et al., 2005), which is a structural and functional analogue of salicylicacid.Itinducesresistanceinvariousplantspeciesagainsta broad spectrum of pathogenic viruses, bacteria, fungi and nematodes.Studiesusingthiselicitordemonstraterapid expres-sionofresistance-relatedgenessuchasglucanasesandchitinases (Dietrichetal.,2005).Comparativestudiesarebeingdoneinan attempttoobtainanewcommercialresistanceinducerduetothe wide use of ASM and to understand the defence mechanisms activatedwiththeuseoftheseproducts.

ASMand othercommercialinducersofresistance havebeen extensivelyevaluatedascomponentsforplantdiseasecontrolin theﬁeld(ValladandGoodman,2004);however,theireffectiveness for practical application in disease management has been questioneddue tovariabilityofcontrol(Waltersand Fountaine, 2009). Field studies showing promising results for control of bacterialdiseaseshavebeenconductedwithfoliarspraysofASM

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eitheraloneorincombinationwithcopperontomatoandpepper (Louws et al., 2001; Romero et al., 2001). For citrus, foliar application of ASM was effective against citrus canker under greenhouse conditions but foliar sprays of ASM combined or alternated with copper oxychloride did not contribute to the controlofcankeronsweetorangetreesinﬁeldtrials(Grahamand Leite,2004).ExpressionofthePRprotein(

b

-1,3glucanase)gene, PR-2,incitrusincreasedinresponsetoASMandisonicotinicacid (INA).However,PR-2responseandreductionoflesionsafterfoliar sprayswassustainedforonlyafewweeks(Dekkersetal.,2004). Likewise,ASMinducedacidicPR-1expressionintomatofor7–10 days (Herman and Restrepol, 2007), con_ﬁrming that foliar applicationsat weeklyintervalsarerequiredforﬁeldcontrolof XanthomonasleafspotandPseudomonasbacterialspeckontomato (Louwsetal.,2001).

3.Conclusions

Theprotection of plants against pathogens throughinduced resistancecanbemadethroughtheuseofexogenoussubstances.It islikely thatinducedresistanceagainstdiseaseseitherthrough chemical activators or other means will become an important componentofpest managementprograms,particularlyincases wherecurrentcontrolmeasuresarelittleeffective.Obviously,one oftheoutcomesoftheuseofthis newtechnologyshouldbea reductionoftheuseoftraditionalpesticides,whichisofamajor concernregardingthepreservationoftheenvironment.

However, more studies are needed regarding the defence mechanismsthatareactivatedineachcase,andtheresponsesthat aretriggeredsothattheseresistanceinducersmaybereleasedto themarket.Andmostimportantly,itisessentialtounderstandthat resistanceinducersdonotperformmiraclesanditisstillneeded an integrated management to reduce to the lowest the losses causedbydiseases.

Acknowledgements

MônicaD.M.Oliveirareceivedapost-doctoralfellowshipfrom Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES): CsF/2581/31-2. Carla M. R. Varanda received a post-doctoralfellowshipfromFoundationforScienceandTechnology (FCT):SFRH/BPD/76194/2011,fundedbyQREN–POPH–Typology 4.1–co-fundedbyMESNationalFundingandTheEuropeanSocial Fund.ThisworkhasbeensupportedbyNationalfunds,throughthe ProgramaOperacionalRegionaldoAlentejo(InAlentejo)Operation ALETN-07-0262-FEDER-001871/LaboratóriodeBiotecnologia Apli-cadaeTecnologiasAgro-Ambientais.

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