Can tomato bacterioses be managed with plant resistance inducers?!

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resistance inducers?!

Andana Barrios

To cite this version:

Andana Barrios. Can tomato bacterioses be managed with plant resistance inducers?!. Life Sciences [q-bio]. 2017. �dumas-01653303�

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Can!tomato!bacterioses!be!managed!

with!plant!resistance!inducers?!

!

Par$:$Andana$BARRIOS$

$ $ $ $ $ ! ! ! ! $ Soutenu'à'Angers'le! 12/10/2017! ' Devant'le'jury'composé'de':'' Président$:$Patrice$CANNAVO$ Maître$de$stage$:$Ana$María$ROMERO$ Enseignant$référent$:$Alexandre$DEGRAVE$ $ $ Autres$membres$du$jury$(Nom,$Qualité)$:$ Nicolas$CHEN,$maître$de$conférence$ Caroline$BONNEAU,$experte$Végépolys! Les!analyses!et!les!conclusions!de!ce!travail!d'étudiant!n'engagent!que!la!responsabilité!de!son!auteur!et!non!celle!d’AGROCAMPUS!OUEST$ AGROCAMPUS! OUEST$ $ ! ! Année$universitaire$:$2016S2017$ Spécialité$:$Horticulture$ Spécialisation$(et$option$éventuelle)$:$$ Gestion$Durable$du$Végétal$(GDV)$

Mémoire!de!Fin!d'Études!

$ d’Ingénieur$de$l’Institut$Supérieur$des$Sciences$agronomiques$_{agroalimentaires,$horticoles$et$du$paysage$} $ de$Master$de$l’Institut$Supérieur$des$Sciences$agronomiques$ agroalimentaires,$horticoles$et$du$paysage$ $ d’un$autre$établissement$(étudiant$arrivé$en$M2)$ $ CFR$Angers CFR$Rennes díIngÈnieur+de+líInstitut+SupÈrieur+des+Sciences+agronomiques,+ agroalimentaires,+horticoles+et+du+paysage díIngÈnieur+de+líInstitut+SupÈrieur+des+Sciences+agronomiques,+ agroalimentaires,+horticoles+et+du+paysage díIngÈnieur+de+líInstitut+SupÈrieur+des+Sciences+agronomiques,+ agroalimentaires,+horticoles+et+du+paysage Ce$document$est$soumis$aux$conditions$d’utilisation$$ «PaternitéSPas$d'Utilisation$CommercialeSPas$de$Modification$4.0$France»$$

Agradecimentos!F!Remerciements! $

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Primero,$ quiero$ presentarles$ a$ Ana$ María$ Romero$ y$ a$ Raúl$ Zapata$ mi$ inmensa$ gratitud.$ El$ hospedaje$ que$ me$ ofrecieron$ en$ el$ equipo$ de$ fitopatología$ de$ la$ FAUBA$ es$ inolvidable.$ Les$ agradezco$ por$ la$ atención$ que$ me$ dieron$ y$ el$ tiempo$ que$ me$ dedicaron$ durante$mis$días$en$la$cátedra.$Aprendí$muchísimas$cosas$gracias$a$ellos$y$la$pasión$que$ tienen,$me$la$trasmitieron.$Nunca$olvidaré$estos$tiempos,$fue$un$gusto$trabajar$con$ambos.$ Saludo$particularmente$el$ejemplar$profesionalismo$de$Ana$y$la$amplia$sabiduría$de$Raúl.$ Son$personas$admirables,$¡hasta$hablar$francés$saben!$Gracias$por$todo.$

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Agradezco$ también$ a$ Hernan$ von$ Baczko,$ que$ fue$ un$ colega$ muy$ agradable$ y$ siempre$disponible$para$ayudarme$cuando$podía.$Además$de$trucos$científicos$me$aprendió$ el$esencial:$cebar!el!maté.$A$pesar$de$ser$un$grande$fitopatólogo,$es$un$gran$asador.$Gracias$ y$mucha$suerte$para$lo$que$viene.$

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Quiero$ agradecer$ a$ todos$ los$ que$ trabajan$ en$ la$ cátedra$ de$ Fitopatología,$ Alice,$ Ulises,$Eliana,$Rocío,$Damián$y$todos$los$demás$también,$por$su$buen$humor$y$aprenderme$ más$palabras$de$lunfardo$y$de$guaraní$cada$día.$$

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Pasé$ una$ parte$ de$ mi$ tiempo$ también$ en$ la$ cátedra$ de$ Microbiología,$ donde$ Irma$ Roberts$acepto$de$trabajar$conmigo.$Me$guió$muy$bien$y$aprendí$mucho$en$este$laboratorio.$ Fue$un$placer$trabajar$con$ella$y$la$agradezco$mucho.$Saludo$también$a$todos$los$chicos$y$ chicas$de$ahí,$que$siempre$fueron$dispuestos$a$responder$a$mis$interrogaciones$múltiples$y$ a$compartir$unas$dulzuras$siempre$ricas.$¡Les$deseo$todo$lo$mejor!$ $ No$habré$tenido$la$oportunidad$de$hacer$esta$tesis$sin$la$ayuda$de$Mariela$Andreozzi,$ de$las$direcciones$internacionales$de$la$FAUBA,$y$la$de$Patrice$Cannavo,$responsable$de$los$ convenios$ con$ Argentina$ en$ Agrocampus$ Ouest.$ Les$ expreso$ todo$ mi$ reconocimiento$ por$ permitir$esta$experiencia,$que$para$mí$fue$muy$importante$y$decisiva$para$mi$futuro.$$

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S$ $

$ Je$ voudrais$ également$ remercier$ grandement$ Alexandre$ Degrave,$ pour$ avoir$ fait$ naître$ cette$ collaboration$ scientifique$ entre$ la$ France$ et$ l’Argentine$ sur$ un$ sujet$ aussi$ intéressant$que$les$stimulateurs$de$défense.$Merci$à$Christelle$et$Romain$pour$m’avoir$aidée$ durant$les$trois$semaines$que$j’ai$passées$à$l’INRA,$ainsi$qu’à$toute$l’équipe$ResPom$de$leur$ accueil$professionnel$et$sympathique.$

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$ Enfin$ je$ souhaiterais$ remercier,$ con! todo! mi! corazón,$ ma$ famille$ et$ mes$ amis,$ d’Argentine$et$de$France,$pour$m’avoir$soutenue$moralement$durant$tous$ces$instants.$

LIST!OF!ABREVIATIONS! $ ASM:$AcidbenzolarSSSmethyl$ $ AUPDC:$Area$Under$Disease$Progress$Curve$ $ cDNA:$complementary$DNA$of$messenger$RNA$ $ CFU:$Colony$Forming$Unit$ $ Cmm:$Clavibacter$michiganensis$subsp.$michiganensis$ $ DEPC:$Diethyl$pyrocarbonate$ $ IR:$Induced$Resistance$ $ PCR:$Polymerase$ChainSReaction$ $ PR:$PathogenesisSRelated$(protein)$ $ PRI:$Plant$Reaction$Inducer$ $ ROS:$Reactive$Oxygen$Species$ $ RT:$Retro$Transcription$ $ SA:$Salicylic$Acid$ $ SAR:$Systemic$Acquired$Resistance$ $ UPS:$Ultrapure$Simple$(water)$ $ Xv:$Xanthomonas$vesicatoria$ $ YDC:!Yeast$extractSdextroseScalcium$carbonate$ $ $

SUMMARY!

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INTRODUCTION)...)1

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MATERIAL)AND)METHODS)...)4

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I.

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Biologic!material!and!growing!conditions!...!4

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II.

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Treatments!...!4

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III.

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Preparation!of!bacterial!inoculi!and!inoculation!...!5

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a.! Preparation!of!bacterial!inoculi!...!5! b.! Inoculation!...!5! i.

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Clavibacter!michiganensis!subsp.!michiganensis!...!5

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ii.

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Xanthomonas!vesicatoria!...!5

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IV.

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Experimentation!scheme!...!6

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a.! Symptoms!evaluation!...!6! i.

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Clavibacter!michiganensis!subsp.!michiganensis!...!6

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ii.

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Xanthomonas!vesicatoria!...!7

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b.! Quantification!of!bacterial!population!...!8! i.

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Clavibacter!michiganensis!subsp.!michiganensis!...!8

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ii.

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Xanthomonas!vesicatoria!...!8

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c.! Analysis!of!resistance!genes!expression!...!8! i.

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RNA!extraction!...!9

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ii.

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DNase!treatment!...!9

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iii.

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cDNA!synthesis!...!9

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iv.

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Transport!and!conservation!of!cDNA!...!9

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v.

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cDNA!homogeneity!...!10

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vi.

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Expression!of!resistance!genes!via!qPCR!...!10

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V.

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Statistical!analysis!...!11

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RESULTS)...)11

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I.

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Symptoms!evaluation!...!11

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a.! Symptoms!caused!by!Clavibacter+michiganensis+subsp.+michiganensis!...!11! b.! !Symptoms!caused!by!Xanthomonas!vesicatoria!...!13! II.

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Monitoring!of!bacterial!populations!...!14

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a.! Clavibacter+michiganensis!subsp.!michiganensis!...!14

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b.! Xanthomonas!vesicatoria!...!15! III.

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Resistance!genes!expression!...!16

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DISCUSSION)...)19

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REFERENCES)...)21

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SITOGRAPHY)...)24

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ANNEXE)...)25

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LIST!OF!FIGURES!AND!TABLES! $ $Table$1:$Synthetic$comparison$of$Clavibacter$michiganensis$subsp.$michiganensis$and$ Xanthomonas$vesicatoria$...$2$ Figure$1:$Chemical$formulas$of$acibenzolarSSSméthyl$(A),$phosphite$(B)$and$chitosan$(C)$...$2$ Figure$2:$Conditioning$of$young$tomato$plants$inoculated$with$Xanthomonas$vesicatoria$...$6$ Table$2:$Visual$criteria$of$severity$evaluation$of$symptoms$caused$by$Clavibacter$ michiganensis$subsp.$michiganensis$...$7$ Figure$3:$Stem$totally$affected$by$Cmm$on$the$left$(positive$control)$and$healthy$stem$on$the$ right$(negative$control).$...$7$ Figure$4:$Emerging$canker$on$the$surface$of$an$affected$stem$...$7$ Figure$5:$Leaves$numeration$for$Xv$leaf$symptoms$observation$...$8$ Figure$6:$Localization$of$the$leaflet$studied$for$Xv$necrosis$counting,$the$black$bar$ corresponds$to$2$cm.$...$8$ Table$3:$Description$of$the$20$genes$selected$for$qPCR$analysis$...$10$ Figure$7:$Evolution$of$the$incidence$of$Clavibacter$michiganensis$subsp.$michiganensis$ symptoms$on$leaf.$...$11$ Table$4:$Means$of$the$areas$under$disease$progress$curves.$...$12$ Figure$8:$Severity$of$Clavibacter$michiganensis$subsp.$michiganensis$symptoms$on$leaf.$.$12$ Figure$9:$Severity$of$Clavibacter$michiganensis$subsp.$michiganensis$symptoms$on$ stem.$...$12$ Figure$10:$Stem$lenght$of$plants$inoculated$with$Clavibacter$michiganensis$subsp.$ michiganensis.$...$12$ Figure$11:$Severity$of$Xanthomonas$vesicatoria$symptoms$on$leaf$6$(left$column)$and$7$ (right$column).$13$ Table$5:$Means$of$the$severities$for$each$treatment.$...$13$ Figure$12:$Severity$of$Xanthomonas$vesicatoria$symptoms$on$leaf$3$(left$column)$and$4$ (right$column).$...$13$ Figure$13:$Number$of$necrotic$lesions$caused$by$Xanthomonas$vesicatoria$on$one$leaflet.$14$ Figure$14:$Evolution$of$Clavibacter$michiganensis$subsp.$michiganensis$populations$at$24$ dpi$(left$column)$and$30$dpi$(right$column).$...$14$ Figure$15:$Evolution$of$Clavibacter$michiganensis$subsp.$michiganensis$populations$at$24$ dpi$(left$column)$and$30$dpi$(right$column).$...$15$ Table$6:$HeatSmap$illustrating$intensity$of$the$expression$of$the$genes$selected$regarding$ antimicrobial$action,$cellSwall$modification$and$signalization.$...$16$ Figure$16:$Representation$in$boxplots$of$the$levels$of$expression$of$the$genes$involved$in$ antimicrobial$action..$...$17$ Figure$17:$Representation$in$boxplots$of$the$levels$of$expression$of$the$genes$involved$in$ cell$wall$modification.$...$18$ Figure$18:$Representation$in$boxplots$of$the$levels$of$expression$of$the$genes$involved$in$ signalization.$...$18)

!

INTRODUCTION! $

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Served$ with$ mozzarella$ or$ in$ couscous$ salsa:$ you$ will$ find$ tomato,$ also$ known$ as$ Solanum!lycopersicon,!nearly$everywhere$in$the$world.$This$fruit$is$an$essential$ingredient$in$ most$ cultures$ and$ in$ consequence$ the$ demand$ is$ huge.$ Therefore,$ it$ represents$ the$ first$ horticultural$ production$ without$ considering$ potato$ as$ one,$ with$ in$ total$ up$ 170$ 750$ Mt$ produced$per$year$worldwide,$including$668$Mt$produced$in$Argentina$(FAO,$2014).$Tomato$ production$ is$ generally$ managed$ as$ a$ monoculture,$ whether$ in$ field$ or$ greenhouse,$ with$ usually$two$cycles$per$year$in$Argentina.$The$problem$is$that$monoculture$rimes$with$absence$ of$rotations,$intense$use$of$pesticides$and$lacunary$conscience$of$the$environment$and$these$ conditions$lead$to$a$poor$environment$for$plants$and$creates$a$favourable$one$for$pathogens.$ $ Some$of$them$can$have$a$tremendous$economic$impact,$among$them$some$bacteria.$ Nowadays$and$since$a$couple$of$years,$Clavibacter!michiganensis$subsp.$michiganensis$and! Xanthomonas!spp.$are$on$the$podium$of$bacterial$pathogens$of$tomato.$In$fact,$Clavibacter! michiganensis$subsp.$michiganensis,$the$causal$agent$of$bacterial$tomato$canker,$appears$ to$be$one$of$the$worst$infection$for$tomato$producers$in$Argentina$(Romero$et!al.,$2014)$and$ including$in$the$world$(de$León$et!al.,$2011).$In$Europe,$this$bacterial$disease$makes$part$of$ A2$quarantine$pest$list$(EPPO,$2016).$First$described$in$1909$by$Erwin$F.$Smith$in$the$state$ of$Michigan$in$the$United$States$of$America,$this$GramSpositive$bacterium$infects$the$vascular$ system$of$tomato$plant$(Bryan$M.$K.,$1930),$causing$its$wilting$and$death.$This$pathogenic$ bacterium$is$particularly$problematic$due$to$frequent$latent$infections,$leading$to$a$difficulty$to$ detect$it$in$the$field$or$greenhouse$(Gitaitis,$1991).$Plus,$contaminations$in$freeSCmm$fields$ are$ often$ due$ to$ contaminated$ seeds,$ allowing$ pathogens$ to$ spread$ over$ large$ distances$ (Tsiantos,$ 1987).$ Hence,$ this$ disease$ is$ difficult$ to$ manage.$ Using$ certified! seeds$ and$ transplants$is$the$first$measure$to$adopt$(Jones,$1991),$coupled$with$good$culture$practices,$ meaning$ disinfection$ of$ tools$ during$ pruning$ and$ manipulations$ in$ general$ (Chang$ et! al.,$ 1991),$vigilance$regarding$potential$reservoirs$plants$around$and$elimination$of$residues$after$ every$harvest$(Gleason$et!al.,$1991).$It$is$also$recommended$to$plant$tomatoes$in$spring$and$ rotate$with$a$nonSsusceptible$crop$in$summer$to$reduce$Cmm$inoculum$(Vega,$2016).$

$

Nevertheless,$ because$ these$ measures$ require$ consequent$ investments,$ they$ are$ rarely$applied$and$even$though$it$is$the$case,$the$pathogen$still$find$a$flaw$to$contaminate$and$ cause$ damages$ even$ in$ a$ wellSgroomed$ crop.$ To$ tackle$ bacterial$ infections,$ few$ solutions$ exist:$ no$ cultivar$ has$ so$ far$ proved$ satisfactory$ resistance$ (Gartemann$ et! al.,$ 2003)$ and$ antibiotics$seem$to$be$less$and$less$efficient$due$to$phenomenon$of$bacterial$resistance.$Plus,$ there$use$is$expensive$and$can$be$problematic$for$animal$health,$including$human$health.$ Classical$control$products$as$mancozeb$are$only$preventive$and$are$not$efficient$once$the$ pathogen$ is$ present.$ Interesting$ solutions$ emerged$ these$ last$ years$ as$ plant$ growthS promoting$ rhizobacteria$ (PGPR)$ use,$ allowing$ a$ reduction$ of$ the$ water$ stress$ induced$ by$ Clavibacter! michiganensis$ subsp.$ michiganensis$ (Romero,$ 2014).$ The$ use$ of$ antagonist$ microorganisms$ as$ Bacillus! subtilis$ can$ also$ be$ recommended$ as$ an$ additional$ tool$ to$ classical$treatments$(Roberts$et!al.,$2008).$Different$composts$proved$a$remarkable$efficiency$ in$controlling$Cmm$populations$(Yogev,$2008).$Another$relevant$perspective$is$considered:$ plant$resistance$inducers$(PRI),$also$known$as$plant$defence$stimulators$and$which$modus! operandi$will$be$described$in$an$instant.$Among$these$PRI,$acidbenzolarSSSmethyl$(ASM)$and$ βSaminobutyric$acid$(BABA)$significantly$enhanced$resistance$factors$against$Cmm$in$tomato$ seedlings$(Baysal$et!al.,$2005x$Hassan$et!al.,$2008x$Alexandersson,$2016).$All$these$studies$ give$evidence$of$the$progress$made$over$the$years$and$the$progress$that$still$must$be$done$ to$find$solutions$for$the$control$of$bacterial$canker$of$tomato.$ $ $

!Table!1:!Synthetic!comparison!of!Clavibacter!michiganensis!subsp.!michiganensis!and!Xanthomonas! vesicatoria! $ $ Bacterium$ Clavibacter!michiganensis!subsp.$ michiganensis! Xanthomonas!vesicatoria!

Phylum$ Actinomycete$ Proteobacteria$

Proprieties$ GRAM$+$ GRAM$S$

Cultivate$host$ Solanum!lycopersicum! Solanum!lycopersicum!

Capsicum!annuum!

Infection$ Vascular$ Foliar$

Virulence$ mechanisms$$$

Genetic$sequences$coding$for$enzymes$ causing$degradation$of$vessels$

(Eichenlaub$and$Gartemann,$2011)$and$ virulence$ factors,$ responsible$ of$ aggregates$formation$in$xylem$vessels$ (Chapulowicz,$2012)$

LowSlevel$ type$ III$ secretion$ system$ allowing$secretion$and$translocation$of$ protein$ effectors$ into$ the$ vegetal$ cell$ (Zhang$et!al.,$2009)$

Symptoms$ Unilateral$ yellowing$ and$ wilting$ of$ the$ leaf,$then$of$the$entire$leaf$until$affecting$ the$whole$plant,$causing$its$death$ Possible$ apparition$ of$ necrotic$ canker$ on$the$stem$(De$León,$2011)$

Necrotic$ lesions$ on$ the$ leaves,$ stems,$ petals$and$fruits$(Jones$et!al.,$1991)$

Main$ sources$ of$ inoculum$ Seeds$ Culture$residues$ Survival$in$adventitious$plant$ Seeds$ Survival$in$volunteer$plants$$ Culture$residues$ (Jones$et!al.,$1986)$ Dissemination$ Seeds$

Leaf$ removal$ and$ pruning$ with$ contaminated$tools$ (Chang$et!al.,$1991)$ Seeds$ Rain,$overhead$irrigation$$ $ Optimal$ conditions$T,$H$ 23$to$28°C$ Middle$to$high$relative$humidity$

(Kendrick$ and$ Walker,$ 1948x$ Basu,$ 1966x$Forster$and$Echandi,$1973)$ 20$to$30°C$ High$relative$humidity$ (Basu,$1966x$Araújo$et!al.,$2010)$ Consequences$ on$production$ Reduction$of$the$yield$ Death$of$tomato$plants$

Reduction$ of$ the$ yield$ (due$ to$ photosynthesis$surface$decrease)$ Quality$loss$of$fruits$

Aspect$ of$ the$ colonies$ on$ YDC$ plates$ (Buenos$ Aires,$April$2017,$ personal$ production)$ $ $ Figure!1:!Chemical!formulas!of!acibenzolarSSSméthyl!(A),!phosphite!(B)!and!chitosan!(C)! A$ $ $ $ $ B$ $ $ $ $ C$

Xanthomonas!spp.,!causal$agents$of$bacterial$leaf$spot$of$tomato,$is$another$serious$ economic$problem$for$tomato$producers.$These$bacteria$are$also$classified$as$A2$quarantine$ pests$in$European$Union$(EPPO,$2016).$It$was$first$discovered$on$tomato$in$South$Africa$in$ 1914$as$one$bacterium,!Bacterium!vesicatorium$and$is$now$identified$as$a$worldwide$spread$ complex$of$at$least$four$species$of$same$gender$Xanthomonas!(Jones$et!al.,$2004,$Potnis,$ 2015).$The$species$used$in$this$study$is$Xanthomonas!vesicatoria$which$is$considered$the$ most$common$in$Argentina$(Romero$et!al.,$2003).$As$Cmm,$this$bacterium$can$be$preserved$ in$seeds,$facilitating$a$large$scope$spreading$(Jones$et!al.,$1986).$It$can$also$infect$a$plant$ carried$ by$ rain$ or$ contaminated$ irrigation$ water$ (Ritchie$ et! al.,! 2000).$ Once$ the$ bacterium$ penetrates$in$the$plant,$necrotic$lesions$start$to$appear$in$leaves,$stems$flowers$and$fruits$ (Jones$et!al.,$1991)$reducing$photosynthesis$effectivity$and$quality$of$fruits.$This$has$a$direct$ impact$on$the$yield,$including$the$high$economic$cost$of$the$products$used$its$control.$

$

If$the$producer$wants$to$reduce$this$negative$impact$as$much$as$possible,$he$should$ respect$ basic$ prophylaxis$ measures,$ as$ for$ bacterial$ canker:$ use$ of$ certified$ seeds$ and$ seedlings,$removal$of$volunteer$and$infected$host$plants,$crop$rotation$with$nonShosts$(Goode$ and$ Sasser,$ 1980x$ Ritchie,$ 2000). Today,$ diverse$ copper$ combinations$ including$ with$ bactericides$ are$ used$ to$ limit$ Xanthomonas’s$ development$ but$ the$ latter$ overcame$ it$ and$ developed$resistance$(Stall,$1986x$Ritchie,$1991).$As$a$matter$of$fact,$new$approaches$are$ studied$ especially$ organic$ products.$ Several$ studies$ tested$ bacteriophages$ and$ demonstrated$ higher$ level$ of$ control$ than$ copperSmancozeb$ when$ their$ formulation$ was$ optimized$(Flaherty,$2000x$Balogh,$2003).$Besides,$PRI$also$stood$as$a$valid$alternative$to$ chemical$pesticides:$ASM$and$natural$extract$of$Solanum$lycocarpum$significantly$decreased$ bacterial$ spot$ development$ and$ activated$ antioxidants$ enzymes$ (Cavalcanti$ et! al.,$ 2006).$ Aqueous$ suspension$ of$ Crinipellis! perniciosa$ mycelium$ containing$ chitosan$ also$ induced$ tomato$ resistance$ to$ Xanthomonas! vesicatoria! (Cavalcanti$ et! al.,$ 2007).$ Control$ of$ this$ pathogen$must$be$done$with$an$integrated$pest$management,$preferably$including$solutions$ respectful$of$the$environment.$

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Hence,$stimulators$of$plant$defence$appear$as$an$interesting$alternative$to$classical$ pesticides.$PRI$are$compounds,$natural$as$well$as$chemical,$that$enhance$induced$resistance$ (IR)$ when$ plants$ are$ infected$ by$ a$ pathogen.$ Consequently,$ no$ case$ of$ resistance$ from$ pathogens$has$been$reported$since$their$use.$Depending$on$the$situation,$the$response$of$ the$plant$to$PRI$can$be$local$or$systemic.$Systemic$plant$resistance$is$so$called$induced$(IRS)$ when$stimulated$by$beneficial$rhizobacteria$and$fungix$or$acquired$(SAR)$when$activated$by$ mobile$signals$from$one$site$to$another.$The$latter$was$studied$by$Ryals$who$defined$it$in$ 1996$as$a$“distinct$signal$transduction$pathway$that$plays$an$important$role$in$the$ability$of$ plants$ to$ defend$ themselves$ against$ pathogens”.$ Various$ works$ pointed$ the$ primordial$ importance$ of$ salicylic$ acid$ in$ SAR$ signalling$ and$ pathogen$ resistance$ (Kessmann$ et! al.,$ 1994x$ Delaney$ et! al.,! 1994).$ The$ commercialization$ of$ a$ functional$ analogue$ of$ SA:$ acibenzolarSSSmethyl$ (ASM,$ Figure$ 1A)$ is$ global,$ under$ the$ name$ of$ Bion!$ in$ Europe$ and$ South$ America,$ Agricard!$ in$ the$ USA.$ This$ synthetic$ agent$ indeed$ induces$ similar$ plant$ reaction$to$that$induced$by$salicylic$acid$(Oostendorp$et!al.,$2001).$Studies$made$on$tomato$ seedlings$underlined$its$role$in$defence$activation$against$Clavibacter!michiganensis$subsp.$ michiganensis$ and$ Xanthomonas! vesicatoria,$ apparently$ due$ to$ activation$ of$ enzymes$ responsible$ of$ activation$ of$ oxygen$ species$ (ROS)$ and$ antioxidant$ enzymes$ (Soylu$ et! al.,$ 2003x$Cavalcanti$et!al.,$2006).$

$

ASM$ is$ the$ most$ studied$ of$ PRIs$ but$ because$ induced$ resistance$ is$ a$ cascade$ of$ biochemical$ reactions,$ a$ lot$ of$ other$ compounds$ can$ act$ upon$ it.$ In$ the$ case$ of$ bacterial$ canker$in$tomato,$previous$studies$made$in$Argentina$presented$efficiency$of$PRI$treatments$ to$ potentially$ manage$ it$ and$ potassium$ phosphite$ and$ chitosan$ appeared$ to$ be$ the$ most$ efficient$(Romero,$2014).$Chitosan$application,$even$in$heterogeneous$suspension,$also$gave$ good$results$in$bacterial$leaf$spot$management$as$said$before$(Cavalcanti$et!al.,$2007).$ $

Phosphite$ is$ an$ oxyanion,$ synthetized$ from$ phosphorous$ acid$ corresponding$ to$ reduced$form$of$phosphatex$its$chemical$formula$is$presented$in$Figure$1B$as$H2PO3S$but$it$

also$exists$as$HPO32S.$It$is$used$against$numerous$Oomycetes$and$Ascomycetes$pathogens$

with,$ depending$ on$ the$ concentration$ used,$ both$ direct$ and$ indirect$ action,$ respectively$ degrading$fungi$cells$and$enhancing$induced$resistance$(Reuveni,$1998x$Massoud,$2012).$ Recently,$studies$demonstrated$that$phosphiteSinduced$resistance$involved$the$expression$of$ antioxydation$ enzymes,$ ethylene$ and$ auxin$ biosynthesis$ genes$ and$ MAPK$ cascade$ (Liu,$ 2016).$ In$ the$ agriculture$ market,$ phosphite$ treatment$ is$ sold$ as$ a$ fertilizer$ but$ scientific$ advances$proved$that$it$is$not$the$only$function$of$this$product.$

$

$ With$respect$to$chitosan,$it$is$an$organic$polymer$of$βS(1→4)$NSacetylSDSglucosamine$ (Figure$ 1C),$ a$ derivate$ of$ chitin$ which$ is$ extracted$ from$ Crustacea’s$ exoskeleton.$ In$ Argentina,$it$is$distributed$and$commercialized$as$a$phytoregulator.$Moreover,$chitosan$has$ also$ been$ reported$ as$ an$ IR$ stimulator$ of$ various$ plants.$ In$ fact,$ it$ enhanced$ defences$ of$ tomato$against$Xanthomonas!vesicatoria$allowing$a$protection$rate$of$nearly$43%$and$against$ Ralstonia! solanacearum,$ a$ vascular$ bacterium$ which$ symptoms$ are$ very$ similar$ to$ Clavibacter!michiganensis$subsp.$michiganensis’$ones.$Chitosan$seems$to$upSregulate$genes$ involved$ in$ signal$ transduction$ and$ plant$ defence,$ as$ pathogenesisSrelated$ (PR)$ proteins$ (Cavalcanti,$2006x$Kiirika,$2013).$ $ PRI$thus$appear$as$a$relevant$alternative$to$classical$methods$for$crop$protection,$or$ at$least$as$an$interesting$complement$to$alleviate$the$use$of$chemical$pesticides.$The$aims$of$ this$study$were:$1)$to$evaluate$the$efficiency$of$three$PRIs:$ASM,$phosphite$and$chitosan$to$ reduce$symptoms$expression$in$greenhouse$grown$tomatoes$inoculated$with$two$very$distinct$ bacteria$ (Table$ 1),$ Clavibacter! michiganensis! subsp.! michiganensis! or! Xanthomonas! vesicatoria,$a$vascular$or$a$foliar$bacteria,$respectively$2)$to$determine$the$effect$of$the$three$ compounds$on$multiplication$of$both$bacteria$in$plantax$and$3)$to$establish$the$mechanisms$ of$defence$involved$by$these$treatments$in$plants$inoculated$with$Clavibacter!michiganensis! subsp.!michiganensis.$ $ $ MATERIAL!AND!METHODS! $ $ I.$ Biologic$material$and$growing$conditions$ The$variety$used$was$Elpida$(Enza$Zadem):$tomato$seedlings$were$provided$by$Babyplant$ (Buenos$ Aires,$ Argentina)$ at$ threeSleaves$ stage.$ There$ were$ maintained$ in$ classical$ greenhouse$ conditions$ (15S25°C),$ in$ the$ greenhouse$ of$ phytopathology$ department$ of$ the$ University$of$Buenos$Aires$during$the$period$April$to$June$2017.$There$were$transplanted$in$ 300$cL$white$plastic$pots,$filled$with$substrate$from$Actis$Juan$Carlos$(Escobar,$Argentina)$ composed$of$soil$and$compost$in$2:1$proportions.$ $ $ II.$ Treatments$ The$three$inducers$were$applied$were$plants$had$4S5$leaves.$ASM’s$mode$of$action$is$ well$ described$ in$ scientific$ literature,$ so$ it$ was$ used$ as$ a$ PRI$ reference$ (positive$ control).$ Distilled$water$was$used$as$negative$control.$

ASM$ treatment$ is$ commercialized$ as$ BION"$ 50WG$ by$ Syngenta.$ In$ this$ study,$ the$ solution$was$elaborated$with$200$mg$of$granules$dissolved$in$1L$of$water$and$pulverized$on$ foliar$area$of$tomato$plants$with$a$classic$sprayer$until$runoff.$Phosphite$treatment$(Phi)$is$ produced$by$Serquim$under$the$name$of$Fosfiser$K$Plus,$and$contains$13%$of$assimilable$ phosphor$derived$from$phosphorous$acid$and$19%$of$potassium.$Here,$Phi$treatment$was$ prepared$ with$ a$ concentration$ of$ 80$ µg/100mL.$ Chitosan$ treatment$ (Chi)$ is$ supplied$ by$

Ftalosur$as$Raisan.$It$was$used$in$a$concentration$of$800µg/100mL.$Phi$and$Chi$were$applied$ directly$and$each$plant$received$1$mL$of$the$solution.$

To$avoid$a$potential$effect$of$the$mode$of$application,$treatment$with$distilled$water$has$ been$done$one$part$on$the$substrate$in$the$same$conditions$of$Chi$and$Phi$treatments,$the$ other$part$was$pulverized$in$the$same$conditions$of$ASM$treatment.$Because$ASM$maximal$ efficiency$ was$ proved$ to$ be$ when$ plants$ are$ treated$ 72$ hours$ before$ inoculation$ of$ Cmm! (Soylu$et!al.$2003),$all$treatments$were$applied$three$days$before$inoculating$the$bacteria.$ $ $ III.$ Preparation$of$bacterial$inoculi$and$inoculation$ a.$ Preparation$of$bacterial$inoculi$ Bacterial$strains$used$for$the$study$are$respectively$Clavibacter!michiganensis!subsp.$ michiganensis$Cmm$9$and$Xanthomonas!vesicatoria!Xv$12,$known$to$be$the$most$aggressive$ and$to$induce$stronger$symptoms$on$plant$than$other$strains$(WASSERMANN,$2017x$Romero$ et!al.,$2003).$Strains$were$cultured$on$yeast$extractSdextroseScalcium$carbonate$agar$(YDC)$ medium$at$28°C$during$48$h.$Bacterial$suspensions$were$prepared$in$sterile$distilled$water$ and$ their$ concentration$ estimated$ by$ measuring$ an$ absorbance$ of$ 0,3$ with$ a$spectrophotometer$at$590$nm$(108$CFU/mL)$(Romero$et$al.$2014).$The$concentration$was$ verified$using$the$serial$dilution$methodx$100$µl$of$each$dilution$(1:10)$was$plated$on$YDC,$ incubated$ at$ 28°C$ during$ 48$ h$ and$ the$ number$ of$ colonies$ counted.$ Dilutions$ (1:10)$ were$ made$to$obtain$concentrations$needed$in$the$different$experiments.$ $ b.$ Inoculation$ Inoculation$was$made$3$days$after$treatment$for$both$bacteria.$ i.! Clavibacter!michiganensis!subsp.!michiganensis! Plants$were$inoculated$with$Cmm$by$injection$in$the$axilla$of$the$first$developed$leaf.$Each$ plant$received$20$µL$of$bacterial$suspension$with$107_{$CFU/mL$and$the$syringe$is$sterilized$} between$two$injections.$Thus,$the$infection$is$likened$to$a$real$contamination$which$happened$ when$tools$are$not$disinfected$during$old$leaves$pruning$(Chang$et!al.,$1991).$ ii.! Xanthomonas!vesicatoria! For$Xv$inoculation,$two$different$techniques$were$used:$ $

-$ Plants$ intended$ for$ bacterial$ population$ quantification$ were$ inoculated$ by$ infiltration$ in$ the$third$developed$leaf$with$a$flat$syringe.$By$this$way,$the$solution$sank$in$the$entire$ leaf$so$we$could$cut$the$disk$everywhere$in$the$leaf$during$the$sampling$without$caring$if$ it$ was$ inoculated$ or$ not.$ The$ solution$ for$ this$ inoculation$ had$ a$ concentration$ of$ 105_$

CFU/mL.$ $ -$ Plants$intended$for$symptoms$evaluation$were$dived$in$a$plastic$bottle$containing$2,5$mL$ of$bacterial$suspension$with$108$_{CFU/mL,$20$µL$of$Silwet$LS77$Ag$(Chemtura)$and$0,5L$} of$distilled$water.$The$Silwet$allowed$a$better$adherence$of$the$solution$drops$in$the$leaf$ area.$Thereafter,$plants$were$covered$by$a$transparent$plastic$bag$during$60$hours$to$ conserve$relative$humidity$of$nearly$100%$(Figure$2).$ $ $

IV.$ Experimentation$scheme$

Spatial$ arrangement$ of$ plants$ was$ randomized.$ Four$ treatments$ were$ distributed:$ distilled$ water$(control),$acibenzolarSSSmethyl$(ASM),$phosphite$(Phi)$and$chitosan$(Chi).$ a.$ Symptoms$evaluation$ i.! Clavibacter!michiganensis!subsp.!michiganensis! As$progress$of$wilting$leaves$and$discoloured$stem$ratio$were$successfully$used$as$a$measure$ of$disease$development$following$measures$were$done$in$this$study:$ $

•$ Plant$ incidence$ and$ leaf$ severity$ bacterial$ canker:$ the$ severity$ of$ the$ disease$ was$ estimated$as$the$proportion$of$the$leaves$of$a$plant$that$were$wilted.$This$evaluation$was$ made$ on$ the$ criteria$ presented$ in$ the$ Table$ 2.$ Incidence$ was$ also$ registered$ and$ corresponds$ to$ the$ proportion$ of$ affected$ plants$ in$ each$ repetition$ of$ one$ treatment.$ Observations$started$when$symptoms$first$appeared$and$were$made$every$3$days$for$4$ weeks.$The$area$under$disease$progress$curve$(AUPDC)$was$calculated$for$leaf$severity,$ using$ the$ trapezoidal$ integration$ of$ the$ disease$ progress$ curve$ over$ time$ (Campbell$ &$ Madden$1990)$and$was$used$to$compare$treatments.$There$were$3$repetitions$of$5$plants$ corresponding$to$15$plants$by$treatment.$ $ !"#$% = ' ()+ ()+, /2 /)− /)+, 1+, )2, $ $

where$(₎$and$(_)+,$are$the$mean$disease$severity$at$time$/₎,$corresponding$to$days$postS inoculation,$and$/)$and$/)+,are$consecutive$observation$dates.$

$ $

Table!2:!Visual!criteria!of!severity!evaluation!of!symptoms!caused!by!Clavibacter!michiganensis!subsp.! michiganensis!(Buenos!Aires,!June!2017,!personal!production)! $ •$ Severity$of$symptoms$on$stem:$as$indicated$above,$Cmm$lives$in$xylem$vessels$of$the$ plant$and$obstructs$them$during$the$colonization.$The$vessels$then$appear$yellow$instead$ of$green$$ when$they$are$healthy$(Figure$3).$Once$the$symptoms$evaluation$has$been$done,$all$plants$ are$plucked$and$the$length$of$each$stem$is$measured$with$a$classical$meter,$stretching$the$ stem$as$good$as$possible$to$make$it$straight.$The$length$of$stem$infected$is$reported$and$ is$ divided$ by$ total$ length$ to$ obtain$ severity$ of$ symptoms$ on$ stem$ for$ Cmm.$ Canker$ occurrence$is$also$reported$(Figure$4).$

$

$

ii.! Xanthomonas!vesicatoria!

•$ Evaluation$of$severity$of$bacterial$spot:$leaves$6$and$7$(Figure$5)$were$harvested$on$all$ plants$ once$ first$ symptoms$ had$ appeared,$ 22$ days$ after$ inoculation.$ Percentage$ of$ necrotic$tissue$was$evaluated$on$inferior$side$of$the$leaf.$ $ •$ Necrotic$lesions$on$leaflet:$on$each$leaf$3$and$4$of$a$plant,$number$of$necrotic$lesions$on$ inferior$side$of$leaflet$3$was$registered$(Figure$6).$ $ Here$there$were$6$biological$repetitions$for$each$treatment$(n=24).$ $ $ $ $

0:$Healthy$leaf$ 0,25:$Leaf$with$one$leaflet$_affected$ 0,5:$Leaf$with$middle$of$its$_{leaflets$affected$} 1:$Leaf$entirely$affected$

Figure!3:!Stem!totally!affected!by!Cmm!on!the!left! (positive!control)!and!healthy!stem!on!the!right! (negative!control).!The!black!bar!corresponds!to! 1!cm.!(Buenos!Aires,!June!2017,!personal! production)! Figure!4:!Emerging!canker!on!the!surface!of!an! affected!stem,!the!black!bar!corresponds!to!1!cm! (Buenos!Aires,!June!2017,!personal!production)!

$ $ b.$ Quantification$of$bacterial$population$ For$both$bacteria,$the$medium$use$is$YDC$(EPPO,$2016x$Doidge,$1921). i.! Clavibacter!michiganensis!subsp.!michiganensis! 24$and$30$days$after$inoculation,$1cm$of$the$second$and$third$internodes,$respectively,$of$ each$plant$was$cut$and$weighed.$Stem$portions$were$then$disinfected$with$70°$alcohol$and$ ground$in$a$sterile$mortar$with$1mL$of$distilled$sterile$water.$Serial$dilutions$of$the$resulting$ macerate$were$prepared$and$then$100$µL$were$deposited$homogeneously$on$each$Petri$dish$ using$a$drigalski$spatula.$The$plates$were$incubated$at$27,5°C$during$3$days.$The$number$of$ CFU$per$plate$was$then$divided$by$the$weight$of$each$sample.$There$were$four$plants$per$ treatment. ii.! Xanthomonas!vesicatoria! Xv$is$a$foliar$bacterium$so$instead$of$stem,$two$oneScentimetre$diameter$disks$were$cut$from$ inoculated$leaves$with$a$sterile$cork$borer$and$ground$in$a$similar$manner$as$Cmm$inoculated$ stems.$Plates$preparation$and$conservation$are$identical$to$Cmm$ones.$Number$of$CFU$per$ plate$was$then$calculated$for$1$cm2_{$of$leaf.$There$were$four$plants$per$treatment.$} $ c.$ Analysis$of$resistance$genes$expression$ This$part$concerned$Clavibacter!michiganensis$subsp.$michiganensis.$The$idea$was$ draw$up$in$collaboration$with$ResPom$team$from$the$INRA$of$Angers,$in$France.$

Three$ distinct$ samplings$ were$ done$ for$ RNA$ extraction$ in$ order$ to$ see$ if$ there$ were$ differences$in$genic$expression$in$response$to$one$of$these$three$treatments:$first$sampling$ was$ made$ on$ inoculated$ plants$ just$ after$ inoculation$ and$ 3$ days$ after$ treatmentx$ second$ sampling$was$made$3$days$after$inoculation$and$6$after$treatment$on$inoculated$plantsx$third$ sampling$was$made$at$the$same$moment$but$on$nonSinoculated$plants.$ $ $ Figure!6:!Localization!of!the!leaflet!studied!for!Xv!necrosis!counting,! the!black!bar!corresponds!to!2!cm.! (Buenos!Aires,!July!2017,!personal!realization)! Figure!5:!Leaves!numeration!for!Xv!leaf! symptoms!observation!

$ $ $ $ $ i.! RNA!extraction! Cmm$is$a$bacterium$residing$in$xylem$vessels.$To$be$the$closest$as$possible$of$a$potential$ expression$of$defence$genes,$RNA$was$extracted$from$first$internode,$corresponding$to$the$ first$portion$of$stem$above$inoculation$point.$Samples$were$immediately$put$in$liquid$nitrogen$ at$S80°C$and$then$conserved$at$S60°C.$RNA$extraction$was$made$with$TransZol$provided$by$ TransGene$Biotech®$on$samples$previously$ground$in$a$sterile$mortar$with$liquid$nitrogen$at$ S80°C.$Result$was$a$little$pellet$containing$RNA,$suspended$in$30$µL$of$DEPC$treated,$RNaseS free.$ Tubes$ were$ then$ conserved$ one$ night$ at$ 4°C$ to$ allowed$ RNA$ correct$ suspension$ in$ water.$Before$DNase$treatment,$integrity$of$RNA$was$verified$by$an$electrophoresis$made$on$ 1,5%$ agarose$ gel:$ bands$ should$ appear$ homogeneously$ intense$ and$ at$ the$ same$ level$ (traducing$the$same$molecular$weight).$

ii.! DNase!treatment!

It$is$necessary$to$clean$all$rest$of$DNA$present$in$the$samples,$so$a$DNase$treatment$was$ done$to$limit$contaminations.$Enzyme$used$is$RQ1$DNase$from$Promega,$1$µL$with$1$µL$of$ buffer$ 5x$ is$ mixed$ with$ 8$ µL$ of$ RNA$ extract.$ Tubes$ were$ maintained$ at$ 37°C$ during$ 30$ minutes,$1$µL$of$STOP$solution$was$then$added$and$tubes$were$incubated$at$65°C$during$10$ minutes.$Once$RNA$was$free$of$DNA,$an$integrity$verification$was$made$by$electrophoresis,$ performed$ on$ a$ 1,5%$ agarose$ gel.$ Nanodrop$ measures$ were$ done$ to$ evaluate$ approximatively$ the$ quantity$ of$ RNA$ in$ ng/µL$ and$ the$ potential$ contamination$ by$ proteins,$ given$by$A260/280$ratio.$

iii.! cDNA!synthesis!

Complementary$ DNA$ synthesis$ of$ previous$ RNA$ was$ executed$ with$ Promega’s$ MLV$ RetroTranscriptase.$The$quantity$of$RNA$used$corresponded$to$1$µg,$adjusted$according$to$ Nanodrop$measures$and$intensity$of$electrophoresis$bands.$$

iv.! Transport!and!conservation!of!cDNA!

cDNA$ was$ synthetized$ and$ lyophilized$ in$ the$ laboratory$ of$ Microbiology$ of$ Buenos$ Aires$ University.$Then,$it$was$sent$in$carboSice$to$Phytopathology$laboratory$of$Angers$INRA$where$ DNA$was$newly$suspended$in$14$µL$of$UPS$water,$RNaseSfree.$

TREATMENT$

2nd$sampling:$

3$samples$per$treatment$from$inoculated$plants$

$ 3rd$sampling:$ 3$samples$per$treatment$from$nonSinoculated$plants$ $ 1st$sampling:$ 3$samples$per$ treatment$from$ inoculated$plants$ *$ 1$ sample$ =$ plot$ of$ 3$ different$ plants$ INOCULATION$

v.! cDNA!homogeneity! A$first$homogeneity$check$was$done$in$Argentina$via$a$classic$PCR$of$actin$tomato$gene.$$A$ photography$of$the$first$electrophoresis$is$available$in$Annexe$1.$A$second$verification$was$ done$in$France,$also$by$PCR$but$with$EF$tomato$gene.$$ vi.! !Expression!of!resistance!genes!via!qPCR! ! !Table!3:!Description!of!the!20!genes!selected!for!qPCR!analysis! $ $

PR,$ pathogenesisSrelated$ proteinx$ CHS,$ chalcone$ synthasex$ Far,$ (E,E)SαSfarnesene$ synthasex$ PAL,$ phenylalanine$ ammoniaSlyase$ x$ HMGR,$ hydroxymethyl$ glutarateSCoA$ reductasex$ FPPS,$ farnesyl$ pyrophosphate$ synthasex$ Apox,$ ascorbate$ peroxidasex$ GST,$ glutathione$ SStransferasex$ POX,$ peroxidasex$ PPO,$ polyphenol$ oxidasex$ CalS,$ callose$ synthasex$ Pect,$ pectin$ methyl$ esterasex$ CAD,$ cinnamylSalcohol$dehydrogenasex$EDS1,$enhanced$disease$susceptibility$1x$JAR,$jasmonate$resistant$ 1x$EIN3,$EIN3Sbinding$F$box$protein$1x$WRKY,$WRKY$transcription$factor$30.$

$

Resistance$ genes$ were$ chosen$ among$ genes$ involved$ in$ three$ main$ pathways$ of$ plant$ defences$(Table$3).$To$evaluate$the$level$of$expression$of$these$genes,$qPCR$was$performed$ on$96Swell$plates$which$were$preSquoted$with$3$µL$of$direct$and$reverse$primers$at$1,5$µM$ and$then$dried$in$a$heatSchamber$at$60°C.$Each$well$was$filled$up$with$15$µL$of$reactional$ mix,$made$in$ice$with$977$µL$of$UPS$water,$330$µL$of$Mesa$Blue$qPCR$MasterMix$and$13$µL$ of$RT$product.$ Each$modality$was$represented$by$3$biological$repetitions,$when$one$repetition$was$in$fact$a$ pool$ of$ three$ different$ plants.$ Each$ repetition$ was$ itself$ represented$ in$ triplicate$ for$ each$ primer$couple$of$the$20$genes$analysed,$more$3$reference$genes$(TuA,$GAPDH$and$Actin).$ qPCR$curves$were$visualized$using$BioSRad$CFX$Manager$program$and$interpreted$with$2S ##Ct_{$ method$ (Vandesompele,$ 2002).$ The$ expression$ of$ a$ gene$ was$ characterized$ by$ its$}

threshold$cycle$of$amplification,$so$referenced$as$Ct.$This$method$consists$in$comparing$the$ expression$of$one$gene$to$that$of$a$calibrator$gene,$following$the$formula:$

$

3454'4(674889:5 = 2;< =>?)@A><BA +;<(D>EF?G)_$ $

This$ number$ was$ then$ normalized$ with$ the$ geometric$ mean$ of$ the$ three$ reference$ genes’$ relative$ expression$ and$ passed$ to$ log2,$ in$ order$ to$ accord$ importance$ to$ amplifications$ as$

much$as$to$repressions.$Effect$of$water$was$subtracted$from$ASM,$Phi$and$Chi$measures.$ $

$ $

Defence!pathway! Mechanism!involved! Gene!concerned!

Antimicrobial! action! ! PR$proteins$ PR1,$PR2,$PR4,$PR8,$PR14,$PR15$ Secondary$metabolism$ $ CHS,$Far,$PAL$ HMGR,$FPPS$(upstream$actor)$ Reactive$oxygen$species$ Apox,$GST,$POX$ PPO$(upstream$actor)$ Cell!wall!

modification! Callose$synthesis$_{Pectates$synthesis$ Pect$}CalS$

Lignin$synthesis$ CAD$

Signalization! Salicylic$acid$ EDS1$

Jasmonic$acid$ JAR$

Ethylene$ EIN3$

V.$ Statistical$analysis$ Statistical$analysis$of$data$was$made$using$the$software$RStudio,$version$3.3.3..,$except$for$ part$I,$were$the$software$used$was$Infostat$(University$of$Córdoba,$Argentina).$ $ $ $ RESULTS! $ I.$ Symptoms$evaluation$ a.$ Symptoms$caused$by$Clavibacter!michiganensis!subsp.!michiganensis$ $ Incidence$of$Cmm$symptoms$is$reported$in$the$graphic$above$(Figure$7).$Leaves$of$ water$treated$plants$are$all$symptomatic$from$46$dpi.$ASM$and$Phi$significantly$reduced$the$ proportion$of$plants$affected$by$the$bacteria$until$50$dpi.$ASMStreated$plants$incidence$then$ raised$ nearly$ 100%$ as$ the$ control$ incidence,$ while$ the$ proportion$ of$ affected$ PhiStreated$ plants$ stay$ significantly$ lower.$ At$ 59$ dpi,$ 100%$ of$ ASM,$ Chi$ and$ control$ plants$ present$ symptoms$of$Cmm$infection.$20%$of$PhiStreated$plants$stayed$without$any$symptom$on$their$ leaves.$

$

Figure$8$presents$the$evolution$in$time$of$the$severity$of$symptoms$caused$by$Cmm$on$leaf.$ No$ treatment$ allowed$ zero$ symptoms,$ but$ two$ were$ significantly$ different$ from$ the$ control$ along$the$eight$observations:$Phi$and$Chi$reduce$severity$of$respectively$38$and$30%$two$ months$after$inoculation.$Plants$treated$with$ASM$raised$same$levels$of$waterStreated$plants’$ severity:$even$if$ASM$curve$is$lightly$below$control’s,$it$seemed$to$be$inefficient.$The$efficiency$ can$then$be$evaluated$with$a$comparison$of$AUDPC$obtained$for$each$treatment$(Table$4).$ AUDPC$of$Phi$and$Chi$were$significantly$different$from$the$control.$As$observed$in$Figure$7,$ AUDPC$of$ASM$was$effectively$not$significantly$different$from$the$control.$ 0 10 20 30 40 50 60 70 80 90 100

36!dpi 39!dpi 42!dpi 46!dpi 50!dpi 53!dpi 56!dpi 59!dpi

In ci de nc e! (% ) Control ASM Phi Chi Figure!7:!Evolution!of!the!incidence!of!Clavibacter!michiganensis!subsp.!michiganensis!symptoms!on!leaf.! Data!are!the!mean!of!incidences!per!treatment,!and!bars!represent!standard!deviation!of!the!means!(n=20).!

Table!4:!Means!of!the!areas!under!disease!progress!curves.!AUDPC!followed!by!same!letter!are!not! significantly!different!based!on!LSD!Fisher!test!(p<0,05)!for!comparison!of!meansd! values!are!means!of!independent!trials!±!standard!errors.! Treatment! AUDPC! ! Control$ 1080,4$±182,4$ a$ ASM$ 835,2$±142,4$ ab$ Phi$ 362,9$±114,3$ c$ Chi$ 556,8$±125,7$ c$ $ $ $ Results$on$stems$underline$that$only$PhiStreatment$reduced$significantly$stem$severity$ of$Cmm,$from$a$third$(Figure$9).$Treatment$with$ASM$had$no$effect$on$stem$health$either.$ChiS treated$plants$didn’t$show$significant$difference$with$waterStreated$plants$regarding$severity.$ Nevertheless,$total$length$of$the$stem$was$slightly$higher$in$ChiStreated$plant$comparing$to$ the$other$plants$(Figure$10)$even$if,$according$to$KruskalSWallis$test$with$p>0,05,$no$treatment$ differs$significantly$from$another$concerning$stem$length.$$ $ $ 19,0 66,7 10,3 63,4 4,4 28,8 7,7 37,1 0 10 20 30 40 50 60 70 80

36!dpi 39!dpi 42!dpi 46!dpi 50!dpi 53!dpi 56!dpi 59!dpi

Le af !se ve rit y! (% ) Control ASM Phi Chi Figure!8:!Severity!of!Clavibacter!michiganensis!subsp.!michiganensis!symptoms!on!leaf.!Data!are!the!means!of! severities!per!treatment,!and!bars!represent!standard!deviation!of!the!means!(n=20).! 0 10 20 30 40 50 60 70 80 90

Control ASM Phi Chi

Se ve rit y: on :s te m :(% ) Figure!10:!Severity!of!Clavibacter!michiganensis!subsp.! michiganensis!symptoms!on!stem.! 0 10 20 30 40 50 60 70 80

Control ASM Phi Chi

St em 8le ng ht 8( cm ) Figure!9:!Stem!lenght!of!plants!inoculated!with! Clavibacter!michiganensis!subsp.!michiganensis.! Data!are!the!means!severities!per!treatment,!and!bars!represent!standard!deviation!of!the!means!(n=20).! Stars!indicate!significant!difference!with!corresponded!control!based!on!Wilcoxon!rank!sum!test!(p<0,05).! *$

b.$ $Symptoms$caused$by$Xanthomonas$vesicatoria$

$ Results$ of$ the$ evaluation$ of$ severity$ on$ leaf$ are$ presented$ in$ Figure$ 11.$ It$ clearly$ indicates$ the$ impact$ of$ ASM$ treatment$ on$ tomato$ plants,$ reducing$ by$ 61%$ and$ 27%$ the$ severity$ of$ Xv! foliar$ symptoms$ on$ leaf$ 6$ and$ 7$ respectively.$ Application$ of$ Chi$ and$ Phi$ apparently$did$not$induce$a$significant$reduction$of$these$symptoms$(Table$5),$neither$on$leaf$ 6$ nor$ on$ leaf$ 7.$ Nevertheless,$ a$ complementary$ essay$ proved$ that$ Chi$ could$ improve$ resistance$against$Xv$in$tomato$seedlings$with$a$little$surface$of$necrotic$tissue$(Figure$12).$ $

Table!5:!Means!of!the!severities!for!each!treatment.!Severities!followed!by!same!letter!are!not!significantly! different!based!on!Wilcoxon!rank!sum!test!(p<0,05)!for!comparison!of!meansd!

values!are!means!of!three!independent!trials!±!standard!errors!

Treatment! Severity!on!leaf!6! ! Severity!on!leaf!7! !

Control$ 74,5$±8,9$ a$ 32,6$±13,8$ a$ ASM$ 13,2$±3,3$ b$ 5,9$±2,4$ b$ Phi$ 56,7$±2,7$ ac$ 15,6$±3,8$ a$ Chi$ 71,6$±17,9$ ac$ 26,4$±7,8$ a$ $ $ 0 10 20 30 40 50 60 70 80 90 100

Control ASM Phi Chi

Le af !se ve rit y! (% ) Leaf!6 Leaf!7 Figure!11:!Severity!of!Xanthomonas!vesicatoria!symptoms!on!leaf!6!(left!column)!and!7!(right!column).!Data! are!the!mean!of!four!independent!experiments,!and!bars!represent!standard!deviation!of!the!means!(n=6).! Stars!indicate!significant!difference!with!corresponded!control!based!on!Wilcoxon!rank!sum!test!(p<0,05).! Figure!12:!Severity!of!Xanthomonas!vesicatoria!symptoms!on!leaf!3!(left!column)!and!4!(right!column).! Data!are!the!means!of!leaf!severities!per!treatment.! Bars!represent!standard!deviation!of!the!means!(n=6).! Stars!indicate!significant!difference!with!corresponded!control!based!on!Wilcoxon!rank!sum!test! (p<0,05).!

*$

*$

*$

*$

0 10 20 30 40 50 60 Control Chi Le af !!s ev er ity !(% ) Leaf!3 Leaf!4

*$

_*$

$

Analysis$of$number$of$necrotic$lesions$showed$results$with$high$errors$due$to$the$unfortunate$ and$ accidental$ disappearance$ of$ 4$ Phi$ and$ 2$ ASMStreated$ plants$ before$ the$ day$ of$ observation,$reducing$n$to$18.$However,$ASM$and$Chi$can$still$be$compared$with$the$control$ and$Chi$reduced$number$of$necrotic$lesions$on$the$leaflet$of$the$leaf$3$(Figure$13).$ $ ASM$and$Chi$thus$allowed$a$reduction$of$apparent$symptoms$of$Xanthomonas!vesicatoria.$ $ $ II.$ Monitoring$of$bacterial$populations$ a.$ Clavibacter!michiganensis$subsp.$michiganensis$ Cmm$populations$are$presents$in$similar$number,$in$nonStreated$stems$as$well$as$in$ treatedSstems$ (Figure$ 14).$ In$ ChiStreated$ plants,$ bacteria$ have$ been$ sensibly$ inhibited$ 3$ weeks$and$then$4$weeks$after$inoculation$but$considering$that$measures$are$in$log10$scale,$it$ does$not$appear$as$a$real$decrease.$$ $ $ $ Figure!14:!Evolution!of!Clavibacter!michiganensis!subsp.!michiganensis!populations!at!24!dpi! (left!column)!and!30!dpi!(right!column).!Data!are!the!means!of!CFU!per!treatment,!and!bars! represent!standard!deviation!of!the!means!(n=4).! 1 100 10000 1000000 100000000 1E+10

Control ASM Phi Chi

lo g!10 CF U .g \1of !st em ! 24!dpi 30!dpi 10! ! 8! ! 6! ! 4! ! 2! ! 0! 0 5 10 15 20 25 30 35

Control ASM Phi Chi

Nu m be r!of !n ec rot ic !le si on s! Leaf!3 Leaf!4

*$

Figure!13:!Number!of!necrotic!lesions!caused!by!Xanthomonas!vesicatoria!on!one!leaflet.!Data!are!the! means!of!number!of!necrotic!lesions!per!treatment.! Bars!represent!standard!deviation!of!the!means!(n=12).! Stars!indicate!significant!difference!with!corresponded!control!based!on!Wilcoxon!rank!sum!test!(p<0,05).! 10! ! 8! ! 6! ! 4! ! 2! ! 0!

b.$ Xanthomonas$vesicatoria$ $

Concerning$ Xv$ growth$ in$ leaves$ (Figure$ 15),$ no$ treatment$ is$ distinguishable$ from$ the$ control$8$days$after$inoculation,$the$mean$of$the$tree$measures$getting$to$approximately$109_$

CFU$ per$ cm2_{$ of$ leaf.$ 11$ days$ after$ inoculation,$ ASM$ slightly$ reduced$ number$ of$ colonies$}

presents$in$leaf$while$PhiStreated$leaves$present$a$little$higher$one.$However,$as$mentioned$ in$II.a.,$it$is$a$log10$scale$so$differences$cannot$be$affirmed$regarding$these$data.$ $ $ Figure!15:!Evolution!of!Clavibacter!michiganensis!subsp.!michiganensis!populations! at!24!dpi!(left!column)!and!30!dpi!(right!column).! Data!are!the!means!of!CFU!per!treatment,!and!bars!represent!standard!deviation!of!the! means!(n=4).!

Control ASM Phi Chi

lo g!10 CF U .c m \2of !le af 8!dpi 11!dpi 10! ! 8! ! 6! ! 4! ! 2! ! 0!

III.$ Resistance$genes$expression$

$

Unfortunately,$qPCR$results$for$the$2nd_$and$3rd_{$samplings$were$not$exploitable,$so$what$}

could$ have$ been$ interpreted$ are$ the$ situation$ of$ plants$ treated$ 3$ days$ in$ advance$ of$ inoculation.$ This$ latter$ was$ made$ just$ before$ the$ harvest,$ so$ we$ can$ consider$ that$ the$ bacterium$did$not$have$the$time$to$induce$any$genetic$modification.$Results$presented$below$ announce$ tendencies$ of$ the$ modification$ some$ defence$ gene$ expression$ due$ to$ the$ only$ effect$of$the$different$treatments.$ $ A$heatSmap$is$made$from$data$obtained$with$Excel$treatment$as$described$in$IV.c.vi.$It$ allows$a$better$visualisation$of$which$gene$is$amplified$and$which$one$is$repressed$(Table$6).$ Simple$application$of$water$activated$expression$of$PR2,$PR15$and$HMGR,$and$repressed$ expression$of$PR4,$PR14,$PPO,$FPPS,$and$POX.$Regarding$effect$of$ASM$it$enhanced$the$ expression$of$nearly$all$PR,$excepted$that$of$PR1.$It$also$stimulated$amplification$of$PPO,$ FPPS,$CalS,$Pect$and$EDS1.$A$few$genes$were$repressed$by$ASM,$and$very$slightly:$it$is$the$ case$ of$ HMGR$ and$ Far.$ Treatment$ with$ Phi$ apparently$ caused$ repression$ of$ two$ genes$ involved$in$microbial$action$(HMGR,$Far)$and$two$involved$in$signalization$(EDS1,$EIN3).$On$ the$other$hand,$it$enhanced$amplification$of$Pect$and$CalS,$two$genes$involved$in$cell$wall$ alteration.$Finally,$Chi$seemed$to$be$an$activator$of$PR14$and$CHS$and$a$repressor$of$PR1,$ PR2,$ PPO,$ HMGR$ and$ POX$ for$ what$ is$ about$ antimicrobial$ action.$ It$ also$ gently$ inhibited$ EIN3$ in$ respect$ of$ signalization.$ Genes$ that$ were$ not$ mentioned$ saw$ their$ expression$ too$ lightly$modified$to$be$noted,$what$corresponds$to$nearly$white$colour$in$Table$6.$ $ $ Table!6:!HeatSmap!illustrating!intensity!of!the!expression!of!the!genes!selected!regarding!antimicrobial!action,! cellSwall!modification!and!signalization.!The!deeper!the!red/blue,!the!more!amplified/repressed!the!gene!is.! Antimicrobial!action!

! PR1! PR2! PR4! PR8! PR14! PR15! PAL! CHS! PPO! HMGR! FPPS! Far! Apox! GST! POX!

Control$ -0,9466 3,0649 -1,3865 0,5755 -1,6072 0,9330 -0,7805 -0,5855 -1,4913 2,9622 -1,3474 -0,3888 -0,2388 0,1482 -2,1170 ASM$ -0,7422 0,9899 1,7347 1,7670 0,2331 0,8945 0,2043 0,6649 2,0326 -1,5896 3,1675 -1,3333 0,5252 0,5800 0,5752 Phi$ -3,6257 -2,5233 0,3947 -1,0417 -0,3893 0,0081 -0,2400 0,3594 -1,4362 0,3947 0,2298 -2,2193 -0,2590 -0,1828 -1,3123 Chi$ -3,5530 -3,0285 -0,9441 -0,2979 1,5752 -0,1573 0,0302 0,8907 -1,1844 -3,1377 -0,5588 -0,8610 -0,5691 -0,4351 -1,4381 $ $ $ $ $ $ $ $ $ $ $ $ It$is$interesting$then$to$look$at$the$data$repartition$for$each$gene.$Representation$of$ data$in$boxplots$were$considering$as$a$good$first$approach$to$describe$modifications$caused$ by$the$treatments.$The$boxplots$in$Figures$16,$17$and$18$represent$the$levels$of$expression$ of$ the$ genes$ selected$ and$ the$ situation$ of$ each$ valour$ of$ the$ samples.$ To$ avoid$ result$ distortion,$FPPS$and$Far$could$not$have$been$represented$in$R$due$to$one$absurd$measure$ for$each.$ Antimicrobial$action$is$the$pathway$that$apparently$was$the$most$stimulated$by$the$PRI$(Figure$ 16),$reaching$the$highest$relative$levels$of$expression,$as$resumed$in$the$previous$tables.$ These$boxplots$allow$to$see$the$repartition$within$a$modality.$ASM$measures$has$the$lowest$ disparity,$while$control,$Phi$and$Chi$were$more$irregular.$Control$measures$were$more$regular$ concerning$ cellSwall$ modification$ genes,$ and$ Phi$ and$ Chi$ still$ have$ an$ important$ disparity$

Cell!wall!modification!

! CalS! Pect! CAD!

Control$ -0,7380 -0,2805 0,0349

ASM$ 1,3802 1,5384 0,1659

Phi$ 0,4937 1,0558 -0,2573

Chi$ -0,0262 -0,2688 -0,6155

Signalization!

! EDS1! WRKY! JAR! EIN3!

Control$ -0,9422 0,0466 -1,0048 0,0443

ASM$ 1,3535 0,3432 0,1175 0,2093

Phi$ -1,6889 -0,3982 0,0054 -1,6912

signalization,$even$if$it$is$difficult$to$compare$with$the$other$pathways$because$the$level$of$ expression$was$little$modified:$it$did$not$exceed$an$amplitude$of$2$(Figure$18).$ $$ $ $ Figure!16:!Representation!in!boxplots!of!the!levels!of!expression!of!the!genes!involved!in!antimicrobial!action.! Data!are!the!means!of!the!expression!calculated!in!triplicate!for!each!biological!repetition’s!gene!(n=3).!

$ !

Figure!17:!Representation!in!boxplots!of!the!levels!of!expression!of!the!genes!involved!in!cell!wall!modification.!

Data!are!the!means!of!the!expression!calculated!in!triplicate!for!each!biological!repetition’s!gene!(n=3).!

Figure!18:!Representation!in!boxplots!of!the!levels!of!expression!of!the!genes!involved!in!signalization.!

DISCUSSION! $

Bacterial$ canker$ and$ bacterial$ leaf$ spot$ of$ tomato$ can$ cause$ serious$ problems$ in$ indoor$productions$as$well$as$in$fields.$Considering$the$growing$awareness$of$environment$ and$health$consequences$of$excessive$use$of$pesticides,$various$studies$are$proposing$ alternatives$to$these$latter.$This$study$embraces$this$common$objective$of$finding$greenS friendly$and$effective$phytosanitary$products$to$reduce$the$number$of$treatments$in$all$crop$ productions,$here$particularly$in$tomato$greenhouses.$A$possible$approach$is$to$stimulate$ natural$defences$that$plants$develop$when$a$pathogen$penetrates$in$its$cells,$what$is$one$ of$the$function$fulfilled$by$plant$resistance$inducers.$Here$three$of$these$stimulators$were$ tested:$acibenzolarSSSmethyl,$phosphite$and$chitosan.$$ $

Significant$ differences$ in$ symptoms$ reduction$ were$ observed$ for$ all$ treatments$ depending$on$the$bacterium.$Phi$was$the$only$treatment$to$reduce$foliar$incidence$of$Cmm.$ Both$ Phi$ and$ Chi$ reduced$ severity$ of$ Cmm$ symptoms$ of$ respectively$ 38%$ and$ 30%.$ Results$ concerning$ stem$ severity$ completed$ and$ comforted$ leaf$ observations:$ Phi$ significantly$ alleged$ symptoms$ affecting$ the$ stem.$ Plants$ inoculated$ with$ Cmm$ that$ received$ Phi$ or$ Chi$ are$ thus$ more$ likely$ to$ use$ less$ energy$ to$ struggle$ against$ the$ pathogen.$Negative$effect$of$the$bacterial$infection$on$yield$would$then$probably$be$less$ important,$ but$ fruit$ measures$ were$ not$ realized$ in$ this$ study,$ what$ could$ have$ been$ interesting.$Surprisingly,$ASM$did$not$have$any$effect$in$reducing$symptoms$occurrence$ either$in$reducing$number$of$CFU$per$gram$of$stem.$According$to$previous$experiments$ published$in$scientific$literature$where$same$concentration$of$Bion!$was$used,$ASM$was$ supposed$to$reduce$both$severity$and$growth$of$Cmm.$In$the$experiments$presented$here,$ first$ symptoms$ appeared$ with$ a$ nonSnegligible$ delay:$ 36$ days$ when$ it$ usually$ takes$ 20$ days.$Well$ASM$action$on$symptoms$is$likely$to$decrease$with$time:$in$the$case$of$Cmm,$ severity$can$raise$25%$14$days$after$inoculation$against$5%$4$days$after$inoculation$(Soylu$ et$al.,$2003).$Thus,$a$hypothesis$to$explain$this$fail$could$be$that$ASM$was$degraded$or$ turned$inactive$before$apparition$of$symptoms.$ $ Otherwise,$no$blatant$diminution$in$bacterial$growth$was$observed$in$treatedSplants$ inoculated$with$Cmm$so$diminution$of$severity$and$incidence$could$not$be$explained$in$this$ case$by$a$decrease$of$bacterial$populations$in$the$vegetal$tissues.$According$to$scientific$ literature,$ direct$ inhibition$ of$ the$ pathogen$ development$ occurred$ depending$ on$ the$ concentrations$used$of$Phi$and$Chi,$but$it$was$in$the$case$of$fungi.$As$Cmm$is$a$bacterium$ invading$ xylem$ vessels,$ it$ could$ be$ relevant$ in$ an$ ulterior$ experiment$ to$ compare$ the$ diameter$of$these$structures$for$each$modality.$This$type$of$measure$has$recently$been$ done$and$it$underlined$the$role$of$Azospirillum!brasilense,$a$beneficial$rhizobacteria,$in$the$ augmentation$of$xylem$sections,$countering$this$way$water$stress$due$to$Cmm$(Romero$ et!al.,$2014).$ $ On$the$other$hand,$plants$treated$with$ASM$and$Chi$alleviated$Xv$manifestation$on$ leaves,$ reducing$ AUDPC$ calculated$ with$ severity$ on$ leaf$ and$ number$ of$ necrosis$ per$ leaflet.$We$can$then$imagine$that$photosynthesis,$CO2$assimilation,$stomatal$conductance$

and$transpiration$(Cavalcanti$et!al.,$2006)$of$ASM$and$Chi$treated$plants$are$thus$improved$ in$ comparison$ with$ nonStreated$ plants.$ As$ well$ as$ for$ Cmm,$ these$ consequences$ in$ symptoms$cannot$be$justified$by$the$inhibition$of$bacterial$development,$as$no$significant$ difference$in$CFU$per$cm2_{$of$inoculated$leaf$was$reported.$}

$

Finally,$ the$ samples$ of$ cDNA$ from$ Argentina$ that$ were$ analysed$ in$ France$ gave$ information$about$the$amplification$of$20$genes$involved$in$plant$defence$depending$on$ the$treatment$used.$ASM,$Phi$and$Chi$may$modify$resistance$gene$expression$in$Solanum! lycopersicon,$activating$expression$or$repression$of$genes$involved$in$antimicrobial$action,$ cell$wall$alteration$and$signalization.$Control$measures$of$amplification$data$presented$a$ nonSnegligible$ disparity$ within$ the$ repetitions,$ what$ could$ lead$ to$ some$ errors$ of$ interpretations$as$it$was$designed$as$calibrator.$However,$an$interesting$tendency$must$be$

noticed:$ASM$and$Chi,$and$ASM$and$Phi$do$not$employ$the$same$biochemical$pathways.$ In$fact,$while$ASM$induced$nearly$all$PR$genes$expression,$Phi$and$Chi$repressed$PR1$ and$PR2$expression.$Only$ASM$activated$the$amplification$of$EDS1,$a$salicylic$marker$so$ Phi$and$chi$may$employ$another$via,$or$act$in$downstream$ways.$

$

$ASM$ also$ seemed$ to$ encourage$ amplification$ of$ genes$ involved$ in$ antimicrobial$ action$as$PPO$and$FPPS$and$in$cell$wall$alteration,$as$Pect$and$CalS.$Previous$studies$ allow$a$comparison$with$the$results$obtained,$mainly$concerning$ASM$which$is$now$largely$ used$ in$ agriculture.$ As$ a$ functional$ analogue$ of$ salicylic$ acid,$ ASM$ was$ confirmed$ to$ activate$$,1S3$ chitinases$ (PR2),$ chitinases$ (PR3),$ thaumatinSlike$ proteins$ expression$ in$ tobacco$plants$(Kessmann,$1994).$It$also$enhances$the$activity$of$superoxide$dismutase$ (SOD)$ and$ glutathione$ S$ transferase$ (GST)$ in$ tomato$ plants$ inoculated$ with$ Cmm.$ Nevertheless,$ GST$ at$ 1$ dpi$ was$ not$ significantly$ different$ from$ control$ when$ measures$ concerned$nonSinoculated$ASM$treated$plants$(Soylu,$2003).$The$results$presented$in$III.$ are$thus$coherent:$GST$expression$observed$in$the$heatSmap$and$in$GST$boxplot$of$ASM$ treated$plants$was$quite$close$to$that$of$waterStreated$ones.$Hence,$GST$activity$may$be$ activated$when$both$ASM$and$bacterium$penetrate$the$plant.$

$

Concerning$ Phi,$ in$ Caspicum! annuum$ plants$ inoculated$ with$ Phytophtora! caspici,$ phosphite$induced$the$expression$of$PR1.$Results$obtained$in$this$study$then$appear$a$ little$curious$if$we$remind$that$tomato$and$pepper$are$both$Solanaceae$and$so$may$present$ similarities$in$their$resistance$pathways.$However,$Phi$treatment$here$comports$potassium,$ and$ the$ effects$ of$ this$ salt$ has$ been$ neglected,$ but$ it$ may$ change$ the$ comportment.$ Phosphite$ balance$ with$ phosphate$ may$ also$ play$ a$ role$ in$ resistance$ enhanced$ by$ Phi$ treatment.$ This$ should$ be$ considered$ in$ future$ studies$ with$ the$ inclusion$ of$ nutrition$ variable$parameters,$to$see$if$efficiency$of$Phi$is$then$modified.$Concerning$Chi,$it$did$not$ particularly$ enhance$ any$ resistance$ gene,$ but$ previous$ studies$ underline$ its$ role$ in$ peroxidase$(from$POX)$and$phenylalanine$ammoniaSlyase$(from$PAL)$activity$in$Nicotiana$ tabacum$leaves$sprayed$with$Chitosan$(Falcón$et!al.,$2007).$

$

In$the$future,$it$could$be$relevant$to$evaluate$the$efficiency$of$these$three$products$in$ combination,$ to$ see$ if$ there$ is$ a$ complementarity$ in$ their$ effects,$ as$ silicon$ had$ with$ chitosan$(Kiirika,$2013).$It$is$important$to$note$that$Phi$and$Chi$treatments$were$made$72$ hours$before$inoculation$based$on$ASM$highest$efficiency$(Soylu$et$al.,$2003),$but$it$could$ be$interesting$to$test$different$times$to$optimize$Phi$and$Chi’s$effect.$Phi$and$Chi$seem$to$ have$ antagonist$ action$ on$ a$ couple$ of$ genes,$ as$ EDS1,$ EIN3,$ PR2$ and$ as$ mentioned$ before,$with$ASM$concerning$PR$genes,$it$could$be$interesting$to$see$how$the$expression$ of$these$genes$changes$when$two$products$are$applied.$

$

Finally,$ considering$ that$ PRI$ are$ studied$ in$ the$ perspective$ of$ sustainable$ development$and$health$conditions$improvement,$their$possible$impact$on$nonStargeted$ pathogen$must$be$figured$out.$In$fact,$side$effects$of$the$use$of$PRI$remain$little$studied,$ even$if$crop$protection$yet$largely$use$it.$$$

$

Some$growers$are$reticent$to$the$use$of$greenSfriendly$products,$often$thinking$that$it$ would$ not$ be$ effective.$ Actually,$ PRI$ do$ not$ eradicate$ pathogens,$ but$ it$ do$ enhance$ resistance$ that$ plant$ naturally$ develops$ against$ abiotic$ or$ biotic$ stress.$ Concerning$ the$ latter,$the$main$asset$of$PRI$is$their$large$pathogen$spectrum$of$influence$upon$defence$ resistance.$ It$ particularly$ allows$ to$ limit$ the$ apparition$ of$ resistance$ from$ pathogens,$ in$ opposition$to$classical$chemicals$products$used$in$plant$protection.$Plant$stimulators$may$ not$yet$replace$pesticides,$but$can$be$included$in$pest$management$programs$to$alleviate$ its$ environmental$ and$ agronomic$ impacts.$ All$ alternative$ solutions$ are$ a$ real$ source$ of$ hopeless$and$a$proof$that$in$agronomic$science$advances$are$in$progress.$

REFERENCES! $

ALEXANDERSSON,$ E.,$ MULUGETA,$ T.,$ LANKINEN,$ Å.,$ et! al.$ Plant$ Resistance$ Inducers$ against$ Pathogens$ in$ Solanaceae$ Species—From$ Molecular$ Mechanisms$ to$ Field$ Application.$International!journal!of!molecular!sciences,$2016,$vol.$17,$no$10,$p.$1673.

$

Anonymous,$2016.$EPPO$A1$and$A2$lists$of$pests$recommended$for$regulation$as$quarantine$ pests,$2016.$PM$1/2$(25),!EPPO$Council,$September$2016.$

$

Anonymous,$ 2016.$ PM$ 7/42$ (3)$ Clavibacter! michiganensis! subsp.! michiganensis.$ OEPP/EPPO.$Bulletin$OEPP/EPPO$Bulletin$46,$p.$202–225.$ $ ARAUJO,$E.$R.,$PEREIRA,$R.$C.,$FERREIRA,$M.$A.$S.$V.,$et!al.$Effect$of$temperature$on$ pathogenicity$components$of$tomato$bacterial$spot$and$competition$between$Xanthomonas! perforans!and!X.!gardneri.$In$:$III!International!Symposium!on!Tomato!Diseases!914.$2010.$ p.$39S42. $

BALOGH,$ B.,$ JONES,$ J.$ B.,$ MOMOL,$ M.$ T.,$ et! al.$ Improved$ efficacy$ of$ newly$ formulated$ bacteriophages$for$management$of$bacterial$spot$on$tomato.$Plant!Disease,$2003,$vol.$87,$no$ 8,$p.$949S954.$

$

BAYSAL,$ Ö.,$ GÜRSOY,$ Y.$ Z.,$ ÖRNEK,$ H.,$ et! al.$ Induction$ of$ oxidants$ in$ tomato$ leaves$ treated$ with$ DLSβSAmino$ butyric$ acid$ (BABA)$ and$ infected$ with$ Clavibacter! michiganensis! ssp.!michiganensis.$European!Journal!of!Plant!Pathology,$2005,$vol.$112,$no$4,$p.$361S369. $

BASU,$ P.K.$ Conditions$ for$ symptomatological$ differentiation$ of$ bacterial$ canker,$ spot,$ and$ speck$on$tomato$seedlings.$Canadian!Journal!of!Plant!Science,$1966,$vol.$46,$no$5,$p.$525S 530. $ BRYAN,$M.K.,$et!al.$Studies$on$bacterial$canker$of$Tomato.$Journal!of!Agricultural!Research,$ 1930,$vol.$41,$no$12.$ $ CAVALCANTI,$F.$R.,$RESENDE,$M.$L.$V.,$LIMA,$J.$P.$M.$S.,$et!al.!Activities$of$antioxidant$ enzymes$ and$ photosynthetic$ responses$ in$ tomato$ preStreated$ by$ plant$ activators$ and$ inoculated$by$Xanthomonas!vesicatoria.$Physiological!and!Molecular!Plant!Pathology,$2006,$ vol.$68,$no$4,$p.$198S208.$

$

CAVALCANTI,$ F.$ R.,$ RESENDE,$ M.$ L.$ V.,$ CARVALHO,$ C.$ P.$ S.,$ et! al.$ An$ aqueous$ suspension$of$Crinipellis!perniciosa$mycelium$activates$tomato$defence$responses$against$ Xanthomonas$vesicatoria.$Crop$Protection,$2007,$vol.$26,$no$5,$p.$729S738.$

$

CHANG,$R.$J.,$RIES,$S.$M.,$PATAKY,$J.$K.,$et!al.$Dissemination$of$Clavibacter!michiganensis! subsp.! michiganensis! by$ practices$ used$ to$ produce$ tomato$ transplants.$ Phytopathology,$ 1991,$vol.$81,$no$10,$p.$1276S1281.$

$

CHALUPOWICZ,$ L.,$ ZELLERMANN,$ E.SM.,$ FLÜGEL,$ Monika,$ et! al.$ Colonization$ and$ movement$ of$ GFPSlabeled$ Clavibacter$ michiganensis$ subsp.$ michiganensis$ during$ tomato$ infection.$Phytopathology,$2012,$vol.$102,$no$1,$p.$23S31.$

$

DELANEY,$ T.P.,$ UKNES,$ S.,$ VERNOOIJ,$ B.,$ et! al.$ A$ central$ role$ of$ salicylic$ acid$ in$ plant$ disease$resistance.$Science,$1994,$p.$1247S1250.$

$

DE$ LEÓN,$ L.,$ SIVERIO,$ F.,$ LÓPEZ,$ M.M.,$ et! al.$ Clavibacter! michiganensis! subsp.! michiganensis,$ a$ Seedborne$ Tomato$ Pathogen:$ Healthy$ Seeds$ Are$ Still$ the$ Goal.$ Plant! disease,$2011,$vol.$95,$no$11,$p.$1328S1338.$

DOIDGE,$E.M.$A$tomato$canker.$Annals!of!Applied!Biology,$1921,$vol.$7,$no$4,$p.$407S430.$ $

EICHENLAUB,$ R.$ and$ GARTEMANN,$ K.H.$ The$ Clavibacter! michiganensis$ subspecies:$ molecular$ investigation$ of$ gramSpositive$ bacterial$ plant$ pathogens.$ Annual! review! of! phytopathology,$2011,$vol.$49,$p.$445S464.$

$

FALCONSRODRÍGUEZ,$ A.B.,$ COSTALESSMENÉNDEZ,$ D.,$ ORTEGASDELGADO,$ E.,$ et! al.$Evaluation$of$chitosan$as$an$inhibitor$of$soilSborne$pathogens$and$as$an$elicitor$of$defence$ markers$and$resistance$in$tobacco$plants.$Spanish!Journal!of!Agricultural!Research,$2007,$ vol.$5,$no$4,$p.$533S541.$

$

FORSTER,$ R.L.$ and$ ECHANDI,$ E.$ Relation$ of$ age$ of$ plants,$ temperature,$ and$ inoculum$ concentration$ to$ bacterial$ canker$ development$ in$ resistant$ and$ susceptible$ Lycopersicon$ spp.$Phytopathology,$1973. $ FLAHERTY,$J.E.,$SOMODI,$G.C.,$JONES,$J.B.,$et!al.$Control$of$bacterial$spot$on$tomato$in$ the$greenhouse$and$field$with$HSmutant$bacteriophages.$HortScience,$2000,$vol.$35,$no$5,$p.$ 882S884.$ $

GARTEMANN,$ K.H.,$ KIRCHNER,$ O.,$ ENGEMANN,$ J.,$ et! al.$ Clavibacter! michiganensis! subsp.! michiganensis:$ first$ steps$ in$ the$ understanding$ of$ virulence$ of$ a$ GramSpositive$ phytopathogenic$bacterium.$Journal!of!Biotechnology,$2003,$vol.$106,$no$2,$p.$179S191. $

GITAITIS,$ R.$ D.,$ BEAVER$ R.$ W.,$ and$ VOLOUDAKIS$ A.$ E..$ Detection$ of$ Clavibacter!

michiganensis! subsp.! michiganensis$ in$ symptomless$ tomato$ transplants.$ Plant! Disease,!

1991$75,$no.$8,$p.$834S838.

$

GLEASON,$ M.L.,$ BRAUN,$ E.J.,$ CARLTON,$ W.M.,$ et! al.$ Survival$ and$ dissemination$ of$ Clavibacter!michiganensis!subsp.!michiganensis!in$tomatoes.$Phytopathology,$1991,$vol.$81,$ no$12,$p.$1519S1523.$

$

GOODE,$ M.J.,$ SASSER,$ M.,$ et! al.! PreventionSthe$ key$ to$ controlling$ bacterial$ spot$ and$ bacterial$speck$of$tomato.$Plant!Disease,$1980,$vol.$64,$no$9,$p.$831S834.$

$

HASSAN,$M.$A.$E.,$and$BUCHENAUER,$H..$Enhanced$control$of$bacterial$wilt$of$tomato$by$ DLS3Saminobutyric$acid$and$the$fluorescent$Pseudomonas!isolate$CW2/Effizientere$Kontrolle$ der$ Bakterienwelke$ der$ Tomate$ durch$ DLS3SAminobuttersäure$ und$ das$ fluoreszierende$ PseudomonasSIsolat$CW2.$Journal!of!Plant!Diseases!and!Protection,$2008,$p.$199S207.

$

JONES,$J.B.,$POHRONEZNY,$K.L.,$STALL,$R.E.,$et!al.$Survival$of$Xanthomonas!campestris! pv.! vesicatoria$ in$ Florida$ on$ tomato$ crop$ residue,$ weeds,$ seeds,$ and$ volunteer$ tomato$ plants.$Phytopathology,$1986,$vol.$76,$no$4,$p.$430S434.$

$

JONES,$J.B.,$JONES,$J.P.,$STALL,$R.E.$and$ZITTER,$T.A.$(eds)$Compendium!of!Tomato! Diseases.$St.$Paul,$MN:$APS$Press.$1991.$

$

JONES,$ J.B.,$ LACY,$ G.H.,$ BOUZAR,$ H.,$ et! al.$ Reclassification$ of$ the$ xanthomonads$ associated$ with$ bacterial$ spot$ disease$ of$ tomato$ and$ pepper.$ Systematic! and! applied!

microbiology,$2004,$vol.$27,$no$6,$p.$755S762.$ $ KENDRICK$JR,$J.$B$and$WALKER,$J.$C.$Plant$nutrition$in$relation$to$disease$development.$ IV.$Bacterial$canker$of$tomato.$American!journal!of!botany,$1948,$p.$186S192. $ KEAN,$T.$and$THANOU,$M.$Biodegradation,$biodistribution$and$toxicity$of$chitosan.$Advanced! drug!delivery!reviews,$2010,$vol.$62,$no$1,$p.$3S11.$ $

KESSMANN,$ H.,$ STAUB,$ T.,$ LIGON,$ J.,$ et! al.$ Activation$ of$ systemic$ acquired$ disease$ resistance$in$plants.$European!Journal!of!Plant!Pathology,$1994,$vol.$100,$no$6,$p.$359S369.$ $

KIIRIKA,$ L.M.,$ STAHL,$ F.,$ et$ WYDRA,$ K..$ Phenotypic$ and$ molecular$ characterization$ of$ resistance$ induction$ by$ single$ and$ combined$ application$ of$ chitosan$ and$ silicon$ in$ tomato$ against$Ralstonia!solanacearum.$Physiological!and!molecular!plant!pathology,$2013,$vol.$81,$ p.$1S12.$

$

LIU,$ P.,$ LI,$ B.,$ LIN,$ M.,$ et! al.$ PhosphiteSinduced$ reactive$ oxygen$ species$ production$ and$ ethylene$ and$ ABA$ biosynthesis,$ mediate$ the$ control$ of$ Phytophthora! capsici$ in$ pepper$ (Capsicum!annuum).$Functional!Plant!Biology,$2016,$vol.$43,$no$6,$p.$563S574.$ $ MASSOUD,$K.,$BARCHIETTO,$T.,$LE$RUDULIER,$T.,$et!al.$Dissecting$phosphiteSinduced$ priming$in$Arabidopsis$infected$with$Hyaloperonospora!arabidopsidis.$Plant!Physiology,$2012,$ vol.$159,$no$1,$p.$286S298.$ $ OOSTENDORP,$M.,$KUNZ,$W.,$DIETRICH,$B.,$et!al.$Induced$disease$resistance$in$plants$by$ chemicals.$European!Journal!of!Plant!Pathology,$2001,$vol.$107,$no$1,$p.$19S28.$ $ POTNIS,$N.,$TIMILSINA,$S.,$STRAYER,$A.,$et!al.$Bacterial$spot$of$tomato$and$pepper:$diverse$ Xanthomonas$ species$ with$ a$ wide$ variety$ of$ virulence$ factors$ posing$ a$ worldwide$ challenge.$Molecular!plant!pathology,$2015,$vol.$16,$no$9,$p.$907S920.$

$

REUVENI,$ R.,$ DOR,$ G.,$ et$ REUVENI,$ M.$ Local$ and$ systemic$ control$ of$ powdery$ mildew$ (Leveillula! taurica)$ on$ pepper$ plants$ by$ foliar$ spray$ of$ monoSpotassium$ phosphate.$ Crop!

protection,$1998,$vol.$17,$no$9,$p.$703S709.$ $ RITCHIE,$D.F.,$DITTAPONGPITCH,$V.,$et!al.$CopperSand$streptomycinSresistant$strains$and$ host$differentiated$races$of$Xanthomonas!campestris!pv.!vesicatoria$in$North$Carolina.$Plant! Dis,$1991,$vol.$75,$p.$733S736.$ $

RITCHIE,$ D.$ F.$ Bacterial$ spot$ of$ pepper$ and$ tomato.$ The$ Plant$ Health$ Instructor.$ Online! publication.$2000,$vol.$10.$DOI:$10.1094/PHISIS2000S1027S01$Updated!2007.$ $ ROBERTS,$P.$D.,$MOMOL,$M.$T.,$RITCHIE,$L.,$et!al.!Evaluation$of$spray$programs$containing$ famoxadone$plus$cymoxanil,$acibenzolarSSSmethyl,$and$Bacillus$subtilis$compared$to$copper$ sprays$for$management$of$bacterial$spot$on$tomato.$Crop!Protection,$2008,$vol.$27,$no$12,$p.$ 1519S1526.$ $

ROMERO$ A.M.,$ ZAPATA$ R.L.,$ VON$ BACZKO$ O.H.,$ CANTEROS$ C.$ and$ SARTORI$ P.,$ Nuevos!productos!potencialmente!aplicables!al!manejo!del!cancro!bacteriano!del!tomate,$3°$ Congreso$Argentino$de$Fitopatología.$Tucumán.$4$al$6$junio$2014.$Resúmenes:$314$

$

ROMERO,$ A.M.,$ VEGA,$ D.$ and$ CORREA,$ O.S.$ Azospirillum! brasilense$ mitigates$ water$ stress$imposed$by$a$vascular$disease$by$increasing$xylem$vessel$area$and$stem$hydraulic$ conductivity$in$tomato.$Applied!soil!ecology,$2014,$vol.$82,$p.$38S43.$

$

RYALS,$ J.A.,$ NEUENSCHWANDER,$ U.H.,$ WILLITS,$ M.G.,$ et! al.$ Systemic$ acquired$ resistance.$The!plant!cell,$1996,$vol.$8,$no$10,$p.$1809.

SOYLU,$ S.,$ BAYSAL,$ Ö.,$ and$ SOYLU,$ E.$ M.$ Induction$ of$ disease$ resistance$ by$ the$ plant$ activator,$acibenzolarSSSmethyl$(ASM),$against$bacterial$canker$(Clavibacter!michiganensis! subsp.! michiganensis)$ in$ tomato$ seedlings.$ Plant! Science,$ 2003,$ vol.$ 165,$ no$ 5,$ p.$ 1069S 1075.$

STALL,$ R.$ E.,$ LOSCHKE,$ D.$ C.,$ JONES,$ J.$ B.,$ et! al.! Linkage$ of$ copper$ resistance$ and$ avirulence$ loci$ on$ a$ selfStransmissible$ plasmid$ in$ Xanthomonas! campestris! pv.! vesicatoria.$Phytopathology,$1986,$vol.$76,$no$2,$p.$240S243.$ $ STRIDER,$David$L.$Bacterial$canker$of$tomato$caused$by$Corynebacterium!michiganensed$a$ literature$review$and$bibliography.$NC!Agr!Exp!Sta!Tech!Bull,$1969.$ $ TSIANTOS,$J.$Transmission$of$bacterium$Corynebacterium!michiganense!pv.!michiganense$ by$seeds.$Journal!of!Phytopathology,$1987,$vol.$119,$no$2,$p.$142S146.$ $ VANDESOMPELE,$J.,$DE$PRETER,$K.,$PATTYN,$F.,$et!al.$Accurate$normalization$of$realS time$ quantitative$ RTSPCR$ data$ by$ geometric$ averaging$ of$ multiple$ internal$ control$ genes.$Genome!biology,$2002,$vol.$3,$no$7,$p.$research0034.$1.$ $ VEGA,$D.$and$ROMERO,$A.M.$Survival$of$Clavibacter!michiganensis$subsp.$michiganensis$ in$tomato$debris$under$greenhouse$conditions.$Plant!Pathology,$2016,$vol.$65,$no$4,$p.$545S 550.$ $ WASSERMANN,$E.,$MONTECCHIA,$M.S.,$CORREA,$O.S.,$et!al.$Clavibacter!michiganensis$ subsp.$ michiganensis$ strains$ virulence$ and$ genetic$ diversity.$ a$ first$ study$ in$ Argentina.$European!Journal!of!Plant!Pathology,$2017,$vol.$149,$no$1,$p.$35S42.$ $ YOGEV,$A.,$RAVIV,$M.,$KRITZMAN,$G.,$et!al.$Suppression$of$bacterial$canker$of$tomato$by$ composts.$Crop!Protection,$2009,$vol.$28,$no$1,$p.$97S103.$ $ $ $ SITOGRAPHY! $ FAOSTAT,$2014:$http://www.fao.org/faostat/en/#data/QC$ $ $