Lysobacter capsici AZ78 can be combined with copper to effectively control Plasmopara viticola on grapevine

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ContentslistsavailableatScienceDirect

Microbiological

Research

jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r e s

Lysobacter

capsici

AZ78

can

be

combined

with

copper

to

effectively

control

Plasmopara

viticola

on

grapevine

Gerardo

Puopolo

∗

,

Oscar

Giovannini,

Ilaria

Pertot

DepartmentofSustainableAgro-EcosystemsandBioresources,ResearchandInnovationCentre,FondazioneEdmundMach(FEM),ViaE.Mach1,38010S. Micheleall’Adige,Trento,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received20August2013 Receivedinrevisedform 16September2013 Accepted21September2013 Availableonline27September2013 Keywords: Lysobactercapsici Plasmoparaviticola Bioﬁlm Copper Environmentalstress

a

b

s

t

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ThebacterialgenusLysobacterrepresentsastillunderdevelopedsourceofbiocontrolagentsableto protectplantsagainstpathogenicoomycetes.InthisworktheL.capsicistrainAZ78wasevaluatedwith regardtothebiologicalcontrolofPlasmoparaviticola,thecausalagentofgrapevinedownymildew.L. capsiciAZ78isabletoresistcopperionsanditsresistancetothismetalisprobablyduetothepresence ofgenescodingforcopperoxidase(copA)andcopperexportingPIB-typeATPases(ctpA).Thepresenceof

bothgeneswasalsodetectedinothermembersoftheLysobactergenus.Resistancetocopperallowed L.capsiciAZ78tobecombinedwithalow-doseofacopper-basedfungicide,leadingtomoreeffective controlofgrapevinedownymildew.Notably,prophylacticapplicationofL.capsiciAZ78alonetograpevine leavesreduceddownymildewdiseasetothesamedegreeasacopper-basedfungicide.Furthermore,L. capsiciAZ78persistsinthephyllosphereofgrapevineplantsandtoleratesenvironmentalstressessuch asstarvation,freezing,mildheatshockandUVlightirradiation.ThesetraitssuggestthatL.capsiciAZ78 couldbeasuitablecandidatefordevelopinganewbiofungicidetobeusedincombinationwithcopper tocontrolgrapevinedownymildew.

1. Introduction

Downymildewisoneofthemostseriousdiseasesofgrapevine (Vitisvinifera)worldwide.Itiscausedbythebiotrophicoomycete Plasmoparaviticola,whichcanattackallgreenpartsoftheplant (Gessleretal.2011).Thesedays,controlofdownymildewrelies mainlyon frequent applicationsof chemical fungicides in con-ventional agriculture or copper in organic production (Wong et al. 2001; Gessler et al. 2011). Growing concerns regarding the negative impactof copperin agricultural soils (Wightwick et al. 2008; Komarek et al. 2010) are giving rise to a search for new natural active ingredients against P. viticola. Naturally occurringmicroorganismswithfavourabletoxicologicaland eco-toxicologicalproﬁlesofferapotentialsolution.Notonlyorganic, butalsoconventionalagriculturemaybeneﬁtfromtheuseofsuch low-impactbiofungicides.

Althoughin recent years severalbacterialstrains havebeen selectedforbiological controlofplantdiseases causedby fungi and oomycetes(Lugtenberg andKamilova 2009), noneof them havereachedthemarketfor thecontrolof P.viticola.The liter-aturereports astrainofErwiniaherbicola showinginhibition of

∗ Correspondingauthor.Tel.:+3904615502.

E-mailaddress:gerardo.puopolo@fmach.it(G.Puopolo).

germinationofP.viticolasporangiainvitro(Tilcheretal.1994)and somebacterialstrainsbelongingtoErwinia,Pseudomonasandother generahaveyieldedpromisingresults(Tilcheretal.2002),butno follow-upstudiesonthesebacteriahavebeenreported,inspiteof enormousresearcheffortsdedicatedtoﬁndingalternativestothe useofcopperagainstP.viticola.Someoftheweaknessesof micro-bialfungicidesregardtheirpoorsurvivalinthephyllosphereand theirincompatibilitywithcopper-basedfungicides,sothey can-notbeintegratedwithinastrategyaimingtoreducecopperdosage (Dagostinetal.2011).

The bacterialgenus Lysobacter (Christensenand Cook1978) includesspeciesthat maybepotentiallydeveloped as biofungi-cides(Haywardetal.2010).SomeLysobacterstrainshavealready beenshowntoactivelyprotectplantsfromattackbysoil-borne oomycetes. For example L. enzymogenes strain 3.1T8 inhibits mycelial growthof Phytophthora(Ph.)capsici,Pythium(Py.) ulti-mumandPy.aphanidermatuminvitro.Productionofextracellular proteases,lipasesandunidentiﬁedbiosurfactantsandantifungal moleculesbyL.enzymogenesstrain3.1T8isinvolvedinthecontrol ofinfectionscausedbyPy.aphanidermatumoncucumberplantlets (Folmanetal.2003,2004).TheLysobactersp.strainSB-K88 syn-thesisesXanthobaccinA,BandC,macrocycliclactams,whichare highlyeffectiveinvitroagainstAphanomycescochlioides,Ph. vig-naef.sp.adzukicolaandPy.ultimum(Nakayamaetal.1996)and insuppressingdamping-offofsugarbeetcausedbyPythiumspp.

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insoil(Hommaetal. 1993;Nakayamaet al.1996).SimilarlyL. capsiciYC5194inhibitsthegrowthofPy.ultimumandother phy-topathogenicfungiinvitro(Parketal.2008).Anothermemberof thisspecies,theL.capsicistrainPG4,reducesmycelialgrowthof severalfungiandoomycetesinvitroand,whenappliedtotomato seeds,controlstomatofootandrootrotcausedbyFusarium oxys-porumf.sp.radicis-lycopersici(Puopoloetal.2010).Recentlyanew L.capsicistrain,namedAZ78,isolatedfromthetobaccoplant rhi-zospherewascharacterised(unpublished).Thus,inordertoﬁnda newbiofungicidewhichcanreplacecopper,weassessedthe efﬁ-cacyofL.capsiciAZ78incontrollingP.viticolaongrapevine.

Nostudiesexistregardingthepossibilityofcombiningbacterial strainswithcopper.Fortheﬁrsttimeweshowthatresistanceto copperisatraitsharedbyLysobacterspeciesandassociatedwith thepresenceofgenescodingforcopperoxidase(copA)and cop-perexportingPIB-typeATPase(ctpA).Wehavedemonstratedthat

resistancetocopperinL.capsiciAZ78allowsthisbacterialstrain tobeappliedwithlowdosesofacopper-basedfungicideandthis combinationledtoanincreaseintheefﬁcacyofgrapevinedowny mildewcontrol.Sincesurvivalofmicrobialbiocontrolagentsinthe plantphyllosphereiscrucialforachievingconsistentactivityandas verylittleisknownaboutthepersistenceofLysobactermembers intheplantphyllosphere(GieslerandYuen1998;Jochumetal. 2006)atthemoment,weevaluatedthistraitinL.capsiciAZ78. Fur-thermore,wealsoassessedL.capsiciAZ78tolerancetostarvation, freezing,mildheatshockandUVlightirradiation,whichare impor-tantcharacteristicsthatmaybehelpfulforthedevelopmentofL. capsiciAZ78asabiofungicideforthecontrolofgrapevinedowny mildew.

2. Materialsandmethods 2.1. Bacterialstrains

Thebacterialstrainsusedinthiswork,L.antibioticusDSM2044, L.enzymogenesDSM2043,L.gummosusDSM6980,L.capsiciYC5194 (typestrain),L.capsiciAZ78,L.capsiciM143 andL.capsiciPG4, werestoredatlengthinglycerol40%at−80◦_C_and_routinely_grown

onLuriaBertaniAgar(LBA)inPetridishes(90mmdiameter).The L.capsiciAZ78andM143strainswererespectivelyisolatedfrom tobaccoandtomatoplantrhizosphereandidentifiedonthebasis of theirgene coding for 16S rRNA, accordingto theprocedure describedbyPuopoloetal.(2010).Alltheexperimentswerecarried outat27◦Cexceptwhenotherwiseindicated.Ineachexperiment bacterialcellsuspensionswerepreparedaccordingtothefollowing procedure:after72hgrowth,Petridisheswerefloodedwith5ml ofsterilesalinesolution(0.85%NaCl)andcellswerescrapedfrom themediumsurfaceusingsterilespatulas.Bacterialcellswerethen collectedinsterile15mltubes.Bacterialcellsuspensionswere cen-trifuged(10,000rpm,5min)andpelletsweresuspendedinsterile distilledwatertoafinalopticaldensityat600nm(AOD600nm)of

0.1correspondingto1×108_CFU_ml−1_and_used_in_all_experiments,

exceptwhenotherwiseindicated.

2.2. EvaluationofresistancetocopperinLysobactermembers ThesurvivalofL.capisiciAZ78andtheotherLysobacterstrains reportedaboveonagarmediumamendedwithcopperions(Cu2+₎

wasassessed according to Ritchie and Dittanpongpitch (1991), withsomemodifications.Briefly,volumesofafilter-sterilized cop-per sulphate solution (CuSO4, Sigma) were added to LBA then

poured into Petri dishes in order to obtainthe following ﬁnal concentrations of CuSO4: 100, 200, 300, 400 and 500␮gml−1.

Foreachcopperconcentration,threePetridisheswerespot inoc-ulated withthree drops (30␮l) of a Lysobacter cell suspension

andplateswereincubatedfor72h. Thedevelopmentof macro-coloniesonthemediumreﬂectedtheabilityofLysobacterstrains totolerateincreasingly highcopperconcentrations(Ritchieand Dittanpongpitch1991).

TofurthercharacterisethelevelofresistanceofL.capsiciAZ78 tocopper,100␮lofaserialdilution(10−1to10−7)ofthecell sus-pensionwerespreadontoLBA,amendedwithcoppersulphateat theabovementionedconcentrations.CFUswerecountedafterfour daysof incubation.Threereplicates(Petridishes)of each com-bination(dilutionandcopperconcentration)wereprepared.The survivalratio(SR)wasobtainedbydividingtheCFUsgrownon LBAamendedwithCuSO4 bytheCFUsgrownonLBA(Stockwell etal.2009).

2.3. Detectionofgenesassociatedwithresistancetocopperin Lysobactermembers

ThegenomicDNAofL.capsiciAZ78,L.antibioticusDSM2044, L. enzymogenes DSM 2043, L. gummosus DSM 6980, L. capsici YC5194,L.capsiciM143andL.capsiciPG4wasextractedwitha GenomicDNAisolationKit(Qiagen)andusedasthetemplatein PCRreactionsaimingtodetectthepresenceofgenesinvolvedin resistancetocopperions.ThegenecopAcodingforacopper oxi-dasewasdetectedfollowingthemethoddevelopedbyLejonetal. (2007),while thegenectpA coding forcopperPIB-type ATPases

wasdetectedfollowingthemethodofDelaIglesiaetal.(2010)

andPavissichetal.(2010).TheprimerpairscopAUF(5-GGTGCT GATCATCGCCTG-3)/copAUR(5-GGGCGTCGTTGATACCGT-3), CoprunF2(5-GGSASBTACTGGTRBCAC-3)/CoprunR1(5-TGN GHCATCATSGTRTCRTT-3)andthesamemixturecomposition andtemperaturecyclesasreportedrespectivelybyPavissichetal. (2010)andLejonetal.(2007)wereusedinthePCRreactions.An additionalprimerpair(LctpAF:5-CTGTTGTTCGGTCAGCAC TG-3/LctpAR:5-CGGCGTCCTTGATCAGAATG-3)wasemployed inPCRreactionsforthedetectionofctpAhomologs.InthesePCR reactions,twomicrolitersofgenomicDNAwasusedastemplatein 25␮lreactionincluding1XDreamTaqGreenPCRMastermix (Fer-mentas,Lithuania)and0.2␮MofprimerLctpAF/LctpAR.These PCRreactionsinvolvedaﬁrstcycleat94◦Cfor3minfollowedby 35cyclesof94◦Cfor1min,57◦Cfor1minand72◦Cfor1minwith aﬁnalextensionstepat72◦Cfor5min.

PCR productswere purified using Exo-Sap (Euroclone S.p.a., Italy)accordingtothemanufacturer’sinstructions.Oncepurified, DNAampliconsweresequencedusingBigDyeTerminatorv3.1and theresultingnucleotidesequenceswereanalysedusingBLASTNto findhomologies withDNAsequencesalreadydepositedin Gen-Bank.

2.4. CloningofcopAandctpAgenesfromLysobactercapsiciAZ78 andphylogeneticanalysis

GenecodingforCopAandCtpAwereclonedinL.capsiciAZ78 byusinga setof differentprimerpairs and thePCRconditions reportedinTableS1.PCRproductswerepurified usingExo-Sap (EurocloneS.p.a.,Italy)accordingtothemanufacturer’s instruc-tionsandtheresultingpurifiedDNAfragmentsweresequenced usingBigDye Terminatorv3.1.Once sequenced,thenucleotide sequenceswerethenassembledbyusingthesoftwareGeneious version 6.1.4(Biomatters). Theassembled sequenceswere ana-lysedbyBLASTNsearchtofindhomologieswithDNAsequences inGenBank.

SeveralcopAandctpAgenesbelongingtootherbacterialstrains werecollectedfromGenBank and usedfor phylogenetic analy-ses.ThesesequenceswerealignedwithClustalX(Thompsonetal. 1997)and thealignmentproﬁlewasthen usedtoestablishthe evolutionarydistancesbyapplyingKimura’stwo-parametermodel

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(Kimura1983)implementedintheMEGA3program(Kumaretal. 2004).Thesamesoftwarewasusedtoconstructthebest phylo-genetictreewiththeneighbour-joiningmethod(SaitouandNei 1987).Bootstrapanalysiswith1000replicateswasperformedto assessconﬁdencelevelsforthebranches(Felsenstein1985). 2.5. EvaluationofefﬁcacyofLysobactercapsiciAZ78in combinationwithcopperforthecontrolofPlasmoparaviticola

EvaluationoftheabilityofL.capsiciAZ78toprotectgrapevine plantsagainstP.viticolawascarriedoutontwo-year-oldV.vinifera cv. Pinot Noir grapevine plants, grafted onto Kober 5BB. The plantsweregrowninagreenhouseundercontrolledconditions (20±0.5◦C;70±10%relativehumidity,RH)in2.5Lpots contain-ingamixtureofpeatandpumice(3:1)fortwomonths,untilthe plantshadproducedtwoshootswithatleastnineleaveseach.

P.viticolawasisolatedfromanuntreatedvineyardinS.Michele all’Adige(Italy)in2012andmaintainedongrapevineplantsby sub-sequentweeklyinoculations.Toobtainsporangia,plantsshowing oilspotsymptomswerekeptovernightin thedarkat20–21◦C and100%RH.TheinoculumofP.viticolawaspreparedby wash-ingthefreshlysporulatinglesionsontheabaxialleafsurfacewith cold(4–5◦C)distilledwater.Thesporangiasuspensionwasthen adjustedtoaconcentrationof2.5×105_sporangia_ml−1_by_counting

withahaemocytometerunderalightmicroscope.

L.capsiciAZ78wascombinedwithafungicidebasedon cop-perhydroxidecontaining15%ofcopper(Kocide®3000,DuPontde Nemours,USA)at1.25and0.6125gL−1.Kocide®3000wasapplied aloneasapositivecontrolat2.5,1.25and0.6125gL−1 correspond-ingrespectivelyto375,187.5and93.75␮gofcopperml−1.The untreatedcontrolwastreatedwithwater.Treatmentswereapplied onadaxialandabaxialleafsurfacesusingahandsprayer.L.capsici AZ78cellsuspensionwasapplied24and6hbeforeinoculationwith P.viticola.Kocide®3000andwaterwereappliedsixhoursbefore inoculation.Eachplantwassprayedwith40mlofeachtreatment preparation.

P.viticolainoculumwassprayedontotheabaxialsurfaceofeach fullyexpandedleafusingahandsprayer.Inoculatedplantswere subsequentlyincubatedat20±0.5◦C(80–99%RH)inthedarkfor 24h,thenmaintainedat25◦C(60–80%RH)witha16/8-hday/night lightregime.Sevendaysafterinoculation,theplantswere incu-batedovernightinthedarkat20±0.5◦Cand80–99%RHtoinduce sporulation.

Diseaseseverity(percentageofabaxialleafareacoveredwith sporulatinglesions)and diseaseincidence(percentageof leaves withvisiblesporulation)wereevaluatedsevendaysafterP.viticola inoculation.DiseasewasassessedbasedontheEPPOstandardscale (2004)andexpressedaspercentages.Eachtreatmentwascarried outonﬁveplants(replicates).

2.6. LysobactercapsiciAZ78persistenceongrapevineleaves TheL.capsiciAZ78populationongrapevineleaveswasassessed onehourbeforeandsevendaysafterP.viticolainoculationusingthe dilutionplatingmethod.Atthesetwotimepoints,5gleafsections werecollectedfromtheplantsineachtreatment.Theywere fur-thercutandputindividuallyinto100mlbottlescontaining45mlof sterilesalinesolutionandshaken(200rpm)for2hatroom temper-ature.Aliquotsofthesuspensionswereseriallydilutedandspread ontothesurfaceofLBAamended withkanamycin(25␮gml−1), sinceL.capsiciAZ78,similarlytoL.capsiciPG4,isnaturallyresistant tothisantibiotic(Puopoloetal.2010).Plateswerethenincubated for72handcoloniesresistanttokanamycinwithL.capsiciAZ78 colonymorphologywerecountedtoestimatethenumberof bac-terialcellsg−1onthegrapevineleaves.

Additionalgreenhouseexperimentswerecarriedouttomonitor L.capsiciAZ78persistenceongrapevineleavesattwoRHlevels. TengrapevineplantsweresprayedwithL.capsiciAZ78suspension followingtheproceduredescribedaboveandmaintainedat25◦C. HalfoftheAZ78-treatedplantswerekeptat70±10%RHandthe otherhalfat90±10%RH.At1,3,6,8and10daysafterapplication, L.capsiciAZ78persistenceongrapevineleaveswasassessedusing thedilutionplatingmethoddescribedabove.

2.7. Bioﬁlmproduction

L.capsiciAZ78wasevaluatedforitsabilitytoformbioﬁlmon polystyrenemicrotitreplatesusingamodiﬁedversionofthe pro-ceduredescribedbyMaddulaetal.(2006).Avolumeof1.5␮lof L.capsiciAZ78cellsuspension(1_×107_CFU_ml−1₎_was_inoculated

into150␮lperwellofthreeliquidmedia,LB,King’sB(KB)and NutrientBroth (NB),in 96-well polystyreneplates. Plates were incubatedfor60hwithoutshakingand ﬁnalcelldensitieswere determined(AOD600nm).Unattachedcellswereremovedby

invert-ingtheplateandtappingitontoabsorbentpaper.Theremaining adherentbacterialcellswereﬁxedtotheplatesfor20minat50◦C andthenstainedfor1minwith150␮lperwellofcrystalviolet solu-tion(0.1%insteriledistilledwater).Excessstainwasremovedby invertingtheplate,thenwashingtwicewithdistilledwater(each wash250␮lperwell).Adherentcells weredecolorizedwithan acetone/ethanol(20%/80%)solution(200␮lperwell)for5minto releasethedyeintothesolution.Avolumeof100␮lwas trans-ferredfromeachwelltoanother96-wellplateandtheamount ofdye(proportionaltothedensityofadherentcells)was quan-tiﬁed(AOD540nm).A96-wellpolystyreneplatewasusedforeach

timepointinthetime-courseexperiment.Twentywellswereﬁlled witheachofthetestedgrowthmediaineachmicrotitreplate.L. capsiciAZ78cellsweresimultaneouslyinoculatedintohalfofthe wellscontainingthegrowthmedia,whiletheotherhalfwasnot inoculated(negativecontrol).Celldensityandbioﬁlmformation weredeterminedattimezeroandat12hintervalsuntil60hafter inoculation(sixtimepoints).AOD540nmvalues(adherentcells)were

dividedbyAOD600nmvalues(bacterialgrowth)inordertoobtainthe

speciﬁcbioﬁlmformationvalue(SBF).

2.8. LysobactercapsiciAZ78tolerancetoenvironmentalstresses Stressresponseexperimentswerecarriedoutonsuspensions of L.capsiciAZ78according toStockwelland Loper (2005)and

Stockwelletal.(2009)withsomemodifications.Briefly,tolerance tostarvationstresswasassessedbyinoculatingL.capsiciAZ78into 15mlsteriletubescontaining5mlofsterilepotassiumphosphate buffer(1mM,pH7)and0.8%NaCltoobtainthefinalconcentration of1×108_CFU_ml−1_._Inoculated_tubes_were_maintained_at₂₇◦_C_for

15daysonarotaryshakerat200rpm.L.capsiciAZ78celldensity wasassessedusingthedilutionplatingmethodat0,3,6,9,12and 15dayspost-inoculation.SRwascalculatedbydividingthe popula-tionreachedatdays3,6,9,12and15bythepopulationregistered atthebeginningoftheexperiment(0days).Threetubes(replicates) wereinoculatedateachtimepoint.

Tolerancetomildheatshockwasmeasuredbyincubating ster-ile1.5mlmicrofugetubescontaining100␮lofL.capsiciAZ78cell suspensionfor20minatthefollowingtemperatures:30,33,36, 39and42◦C.Afterthisperiodoftime,avolumeof900␮lof ster-ilepotassiumphosphatebuffer(1mM,pH7)wasaddedandthe suspensionwasmixedinavortexfor30spriortodilution plat-ing.SRwascalculatedbydividingtheL.capsiciAZ78population afterexposuretoeachtemperaturebythepopulationafter expo-sureto27◦C.Threemicrofugetubes(replicates)wereusedforeach temperature.

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Tolerance to freezing was assessed by transferring 100␮l aliquots of L. capsici AZ78 cell suspension into sterile 1.5ml microfugetubesthatweremaintainedat−20◦_C_for₂₄_h._The

via-bilityofL.capsiciAZ78cellswasassessedat6,12,18and24h. Atthesetimepoints,a volumeofsterile900␮lof10mM phos-phatebuffer(pH7)wasaddedtoeachtubeimmediatelyafterbeing removedfromthefreezer.Samplesweregentlymixedby pipet-tingandseriallydilutedinsterilesalinesolution(10−1 to10−7). Volumesof100_␮lofthesedilutionswerespreadontoLBAand enu-merablecolonieswerecountedafterfourdaysofincubation.Three microfugetubes(replicates)wereusedforeachtimepoint.SRwas calculatedbydividingthepopulationafterexposureto−20◦_C_by

thepopulationwhichwasnotexposedtofreezing.

Inordertoevaluatetolerancetoultravioletirradiation,dilutions from10−1 to10−7ofL.capsiciAZ78cellsuspensionwerespread ontoLBAandimmediatelyexposedtoUVirradiation(254nm)at thefollowingdoses:20,40,60,80and100Jm−2.Following expo-sure,plateswereincubatedinthedarkforfourdays,afterwhich colonyformingunitswerecounted.Threereplicates(Petridishes) wereusedforeachdilution.SRwascalculatedbydividingthe popu-lationafterexposuretoUVirradiationbythepopulationofL.capsici AZ78cellswhichwerenotexposedtoUV.

2.9. Statisticalanalysis

Each experiment was carried out twice, except theefﬁcacy trialinthegreenhouse,whichwascarriedoutthreetimes.Asthe experiment factorwasnot signiﬁcant,thedata oftherepeated experimentswerepooledand analysedbyANOVAusing Statis-tica7.1(StatSoft,Tulsa,OK,USA)andmeanswerecomparedwith Tukey’stest(˛=0.01).SRvaluesanddiseaseincidenceandseverity werelog10andarcsintransformedbeforehand.

3. Results

3.1. EvaluationofresistancetocopperofLysobactercapsiciAZ78 anddetectionandcloningofgenesassociatedwithresistanceto copper

PhylogeneticrelationsamongtheLysobacterstrainsusedinthis workaccordingtoanalysisoftheir16SrRNAgenesarereported inFig.S1.During thiswork,thesebacterialstrainswere evalu-atedfortheresistancetocopper.WhenspottedontoLBAamended withthismetalatconcentrationsrangingfrom100to500␮gml−1 bothL.capsiciAZ78andalltheremainingLysobacterstrains devel-opedmacrocolonies.AmendmentoftheLBAwithcoppersulphate upto300␮gml−1 didnotaffectthesurvivalofL.capsiciAZ78.A slightdecreaseinSRwasnoticedat400␮gml−1 ofcopper(log value−0.42±0.16),while a tenfoldreduction waspresent at a concentrationof500␮gml−1 (Fig.1).L.capsiciAZ78released a brownpigmentintothemediumwhengrownonLBAamended withCuSO4at400and500␮gml−1concentrations.Thereleaseof

abrownpigmentwasalsoobservedinthecaseofL.capsiciM143, butnotfortheotherLysobacterstrainsevaluated(Fig.2).

Anucleotideregionof≈1000bpwasampliﬁedbyusingthe primerpairspeciﬁcforcopA(CopRunF2/R1)geneinallthe Lysobac-terstrains,theresultingnucleotidesequencesshowedthehighest sequenceidentitywiththegenecodingforacopperoxidasefrom thecompletegenomeofthebacterialstrainsPseudoxanthomonas suwonensis11-1,StenotrophomonasmalthophiliaD457andJV3.The partialsequencesofcopAgenefromLysobacter memberstested in this workformed two clusters, one containingsequences of L.antibioticusand L.capsicistrainsandtheothercontainingthe sequencesfromL.enzymogenesandL.gummosusstrains(Fig.S2A).

Fig.1.LysobactercapsiciAZ78resistancetocopperionsatdifferentconcentrations. AZ78resistancetocopperionsisexpressedasthelogarithmicvalueofthe sur-vivalratio(SRlog).Survivalratio(SR)wascalculatedastheratiooftheAZ78CFU developedonLBAamendedwithcoppersulphateatdifferentconcentrationstothe AZ78CFUdevelopedonLBA.Pointswiththesamelettersdonotdiffersigniﬁcantly accordingtoTukey’stest(˛=0.01).

PCRreactionsusingprimerpairCopAUF/RtodetecttheDNA region associated withthe copperPIB-type ATPase resulted in

ampliﬁcationofan726bpampliconinL.capsiciAZ78only,which showedthehighest sequenceidentityvaluewiththectpA gene fromthecompletegenomeofP.suwonensisstrain11-1.ThectpA gene from P.suwonensins strain 11-1 wasthus used to design anotherprimerpair(LctpAF/R)inordertodetectthepresenceof thectpAgeneinallLysobacterstrains.Theuseofthisnewprimer pairallowedampliﬁcationofan840bpregionofthectpAgenein alltheLysobacterstrainsusedinthiswork.OnthebasisofBlastN analyses, the amplicons showed the highest sequence identity withthegenecodingforacopperPIB-typeATPasefrombacterial

strainsBurkholderiagladioliBSR3,MethylibiumpetroleiphylumPM1 andP.suwonensisstrain11-1.AllctpAsequencesoriginatedfrom Lysobacterstrainswerelocatedinasingleclusterwiththeexclusive exceptionofthectpAsequenceofL.antibioticusDSM2044,which clusteredwithB.vietnamiensisG4(Fig.S2B).

Onthebasisofsequenceidenty,newprimerpairsweredesigned inordertoobtainthecompletenucleotidesequenceofcopAand ctpAfromL.capsiciAZ78.Oncedeterminedthecompletenucleotide sequences,phylogeneticanalysisofthesetwogenesindicatedthat thecopAsequenceofL.capsiciAZ78clusteredwithgenehomologs from Azotobacter vinelandi strain CA6, Pseudomonas ﬂuorescens strainPfO-1andP.stutzeristrainCCUG29243,bacterialspeciesthat arephylogeneticallydistantfromthegenusLysobacter(Fig.3A). Similarly,thectpAgenesequencefromL.capsiciAZ78was cluster-ingwithgenehomologsfromtheB.cenocepaciastrainJ2315and theB.gladiolistrainABSR3andwasdistantfromctpAhomologs of closely related bacterial genera,such as Pseudoxanthomonas, StenotrophomonasandXanthomonas(Fig.3B).

3.2. EvaluationoftheefﬁcacyofLysobactercapsiciAZ78in combinationwithcopperforthecontrolofPlasmoparaviticolaon grapevine

Inthelight oftheresultsobtainedinvitro,theefﬁcacyofL. capsiciAZ78incontrollingP.viticolawasevaluatedaloneandin combinationwithalow doseof acopper-basedfungicide (cop-perhydroxide),undercontrolledconditions(greenhouse).Allthe concentrationsofcopperhydroxideand L.capsiciAZ78reduced incidenceofdownymildewascomparedtotheuntreatedcontrol. Incidencerangedfrom63±16% to73±11%(average±standard

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Fig.2.ReleaseofabrownpigmentinLBAgrowthmediumamendedwithCuSO4400␮gml−1.(A)LysobactercapsiciYC5194;(B)LysobactercapsiciAZ78;(C)Lysobactercapsici

M143.

deviation)incoppertreatments,andwas63±9%and100±3%inL. capsiciAZ78treatedplantsandtheuntreatedcontrolrespectively. ThecombinationofL.capsiciAZ78withcopperfurtherreducedthe incidenceofthedisease(30±14%and39±13%withtheadditionof 1.25and0.6250gL−1),indicatinganadditiveeffect(Table1).L. cap-siciAZ78stronglyreducedtheseverityofdownymildewonleaves, similarlytocopperatallthetested dosages.Inparticularwhen plantsweretreatedwithL.capsiciAZ78severitywas5%,whereas itwas67%intheuntreatedcontrol(Table1).ThecombinationofL. capsiciAZ78withcopperdidnotfurtherreduceseverity,probably becauseitwasalreadyverylow.

3.3. SurvivalofLysobactercapsiciAZ78cellsinthegrapevine phyllosphere

Atneitherofthetwotimepoints(1hbeforeand7daysafter pathogen inoculation) L. capsici AZ78 cells were isolated from untreatedcontrolplantsortreatedwiththedifferentcopperdoses alone,buttheywererecoveredfromleavesofplantstreatedwith thebacterium.Thebacterialcellpopulationrecoveredfromleaves collectedonehourbeforeP.viticolainoculationwas5.07±0.16 log10CFUg−1ofleaf,whileattheendoftrialtheAZ78cell

pop-ulationreached5.22±0.02log10CFUg−1ofleaf.Inplantstreated

withacombinationofL.capsiciAZ78andcopper,areductionofone orderofmagnitudeinthebacteriumpopulationsizewasrecorded: attheendoftheexperimentsthepopulationwas4.36±0.12and at4.28±0.14log10CFUg−1ofleafonplantstreatedwithcopperat

dosesof1.25andat0.6125gL−1respectively.

In the experiments assessing L. capsici AZ78persistence on grapevineleavesovertendays,L.capsiciAZ78wasrecoveredone dayafteritsapplicationat5.41±0.07and6.57±0.50log10CFUg−1

ofleafforplantskeptatnormal(60–80%)andhigh(80–99%) rel-ativehumidityrespectively(Fig.4).AZ78persistedataconstant

Table1

BiocontrolofPlasmoparaviticolathroughprophylacticapplicationofLysobacter cap-siciAZ78ongrapevineleaves.Diseaseincidenceisexpressedasthepercentageof symptomaticleaveswhilediseaseseverityisexpressedasthepercentageofleaf areacoveredwithsporulatinglesions.

Treatments Disease incidenceb (%) Disease severityb (%) Untreateda ₁₀₀_±₃_a ₆₇_±₂₇_a Kocide®₃₀₀₀_(2.5_g_L−1₎ ₆₃_±₁₆_b ₃_±₂_b Kocide®₃₀₀₀_(1.25_g_L−1₎ ₆₆_±₁₀_b ₈_±₂₂_b Kocide®₃₀₀₀_(0.6125_g_L−1₎ ₇₃_±₁₁_b ₅_±₅_b

L.capsiciAZ78+Kocide®3000(1.25gL−1) 30±14c 2±2b L.capsiciAZ78+Kocide®3000(0.6125gL−1) 39±13c 1±1b

L.capsiciAZ78 63±9b 5±5b

a_Untreated:_plants_treated_with_water_only.

b_Mean_values_±_standard_deviations_are_reported_for_each_treatment._The_same

lettersindicatevalueswhichdonotdiffersigniﬁcantlyaccordingtoTukey’stest (˛=0.01).Dataoriginatingfromthreeindependentexperimentswerepooled.

ratefor10daysonplantsexposedtohighhumidity(6.47±0.16 log10CFUg−1ofleafaftertendays)whileatnormalhumiditythe

AZ78populationwasconstantuntilthe6thdaypost-inoculation andthendecreasedto2.39±0.04log10CFUg−1ofleafafter8days;

AZ78wasstillpresentaftertendays,butatalowconcentration (2.24±0.24log10CFUg−1ofleaf).

3.4. AssessmentoftheabilityofLysobactercapsiciAZ78toform bioﬁlmandresistenvironmentalstress

TheabilityofL.capsiciAZ78toformbioﬁlmoninertsurfaces wasinvestigatedinthiswork.L.capsiciAZ78grewdifferentlyin thethreemediausedinthebioﬁlmproductionassayandKBwas foundtobethemedium sustainingthehighest cellproduction (Fig.5A).Bacterialgrowthintheothertwoliquidmedia(LBandNB), wasalmostidenticalandafter60hL.capsiciAZ78reachedsimilar AOD600nmvalues(Fig.5A).Nonetheless,atthistimepointtheSBF

valueregisteredinNBwashigherthanthequantityreachedinLB (Fig.4B).BiofilmwasnotproducedinLBuntil48handitattainedits highestvalue(SBF=2.62±0.04)after60h;L.capsiciAZ78startedto formbiofilmbetween24and36hinNB(Fig.5B);thehighestSBF valueinKBwasreachedat12handthereafterdecreased(Fig.5B). WealsowishedtoinvestigatewhethertheabilityofL.capsici AZ78topersistongrapevineleaveswasassociatedwithresistance todifferentabioticstresses,soaseriesofexperimentswerecarried outinvitro.Intheexperimentsaimedatassessingresistanceto starvation,whenL.capsiciAZ78wasincubatedinphosphatebuffer andtheconcentrationwasmonitoredoverfifteendays,its viabil-ityslowlydecreasedandbytheendoftheexperiments(15thday) thetotal reduction was0.70_±0.13 log10 (Fig.S3A). Whenmild

heatshockwasinducedbyexposingL.capsiciAZ78cell suspen-siontotemperaturesrangingfrom30to42◦Cfortwentyminutes, exposureto30and33◦Cdidnotresultinanylossofcellviability, whereasexposureto36,39and42◦Creducedviabilitybylog10

−0.19±0.06,−0.12±0.08and−0.35±0.18respectively(Fig.S3B). Incubationat−20◦_C _slightly_decreased_the_viability_of_L._capsici

AZ78cells,althoughbylessthanoneorderofmagnitudeatallthe timepointsinthisexperiment,andthedifferenceswerenot signif-icant(Fig.6A).WithrespecttoL.capsiciAZ78tolerancetoUVlight irradiation,thesurvivalratiodrasticallydecreasedwhenthestrain wasexposedto60,80and100Jm−2,thereductionbeingalmost six-tenfold,whileexposureto20and40Jm−2reducedviabilityby log10−1andlog10−3respectively(Fig.6B).

4. Discussion

Identiﬁcationofnewmicroorganismsthatcaneffectively con-trolP. viticola mayplay an important role in the development of biofungicides helping to reduce the use of copper against downy mildew on organic grapevines. Previous studies have shownthatotherLysobacterstrainscancontrolsoil-borneplant

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Fig.3.PhylogenetictreesderivingfromanalysisofthecopA(A)andctpA(B)genefromLysobactercapsiciAZ78.Thetreeswereobtainedusingtheneighbour-joiningmethod andevolutionarydistanceswerecalculatedusingKimuratwoparametermodel(Kimura1983).Bootstrapvalues(Felsenstein1985)higherthan50areshownatthebranch points.

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Fig.4. LysobactercapsiciAZ78persistenceongrapevineleaves.Thelinewith trian-glesrepresentspersistenceonplantsmaintainedat25◦_C_with₇₀_±_10%_RH;_the_line

withsquaresrepresentspersistenceonplantsmaintainedat25◦_C_with₉₀_±_10%

RH.Bacterialcelldensityisexpressedaslog10CFUg−1ofleaf.Pointswiththesame

lettersdonotdiffersigniﬁcantlyaccordingtoTukey’stest(˛=0.01).a_dpi₌_days_post

inoculation.

pathogenicoomycetessuchasA.choclioidesandPy. aphaniderma-tum(Nakayamaetal.1996;Folmanetal.2001,2003,2004;Islam etal. 2005).However tothebestof ourknowledge,this isthe firstevidenceofthebiologicalcontrolofgrapevinedownymildew achievedthroughtheapplicationofamemberofgenusLysobacter. Moreover,L.capsiciAZ78isoneofthefewbacterialstrains identi-fiedsofarshowingefficacyagainstthisdisease(Tilcheretal.1994, 2002).Interestingly,applicationofL.capsiciAZ78cellstograpevine leavesresultedinareductionofP.viticolaincidenceandseverity comparabletotheapplicationofacopper-basedfungicide.Inorder

Fig.5.AbilityofLysobactercapsiciAZ78toproducebioﬁlmindifferentgrowth media.(A)BacterialcelldensitywasmonitoredbyscoringtheAOD600nmvalueevery

twelvehours.(B)SpeciﬁcBioﬁlmFormation(SBF)wascalculatedastheratioof adherentcells(AOD540nmvalue)tobacterialcelldensity(AOD600nmvalue).The

liq-uidmediawere:KB(triangles),LB(squares)andNB(circles).Pointswiththesame lettersdonotdiffersigniﬁcantlyaccordingtoTukey’stest(˛=0.01).

Fig.6.LysobactercapsiciAZ78tolerancetoabioticstress.(A)Toleranceto24h expo-sureto−20◦_C._(B)_Survival_of_AZ78_cells_exposed_to_increasing_UV_light_irradiation.

TheabilityofAZ78totolerateabioticstressisexpressedasthelogarithmicvalueof thesurvivalratio(SRlog).Survivalratio(SR)wascalculatedastheratioofAZ78 treatedcellstoAZ78untreatedcells.Pointswiththesamelettersdonotdiffer signiﬁcantlyaccordingtoTukey’stest(˛=0.01).

todevelopacommercialbiofungicidefutureresearchshould con-ﬁrmthehighlevelofefﬁcacyattainedundercontrolledconditions andidentifythemechanismofactionofL.capsiciAZ78involvedin controllinggrapevinedownymildew.

However,buildingonthisknowledge,ourstudyprovidesthe ﬁrstevidenceofcopper-resistanceinmembersoftheLysobacter genus.Thisbiologicaltraitisprobablyassociatedwiththe pres-enceofnucleotideregionshavinghighsequencehomologywith genescodingforcopperoxidase(copA)andcopperPIB-typeATPase

(ctpA).Thelatterproteinbelongstotheheavymetaltransporter ATPases,ubiquitousmembraneproteinsdeputedtotheefﬂuxof copperionsinvariousmicroorganisms(Fuetal.1995;Geetal. 1995;PetersenandMøller2000;Arguelloetal.2007),while cop-peroxidaseisdeputed totheoxidation ofcopperionsandthis activityprotectsperiplasmicenzymesfromcopper-induced dam-age(Solomonetal.1996;GrassandRensing2001).Escherichiacoli strainssharingthemulti-copperoxidasegene(pcoA)onthe plas-midpRJ1004produced browncolonies whengrown ongrowth mediacontainingCuSO4(TetazandLuke1983).Similarly,L.capsici

AZ78andM143releasedabrownpigmentonLBAplatesamended withCuSO4400␮gml−1althoughthisbehaviourwasnotobserved

inthestrainL.capsiciYC5194.

ItisworthnotingthatcopAandctpAgenesfromL.capsiciAZ78 clusteredwiththegenesofbacterialstrainsbelongingtospecies thatarephylogeneticallydistantfromthegenusLysobacter(i.e.P.

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ﬂuorescens).BothcopAandctpAcouldbelocatedonthebacterial chromosomeoronplasmids,asinthecaseofcopABCDoperoninP. syringae,containedintheplasmidpPT23D(ChaandCooksey1991). Fromanecologicalpointofview,itwouldbeofgreatinterestto assesswhethercopAandctpAinL.capsiciAZ78arelocatedonthe chromosomeoronplasmids,inordertoassesswhetherhorizontal genetransfereventshaveoccurredbetweenaLysobactermember andotherbacterialspecies.

Ontheotherhandandfromapracticalpointofview,resistance tocopperisa highlydesirabletrait, becausecopperisroutinely applied in the control of P. viticola in organic agriculture, and theexistenceofabiofungicidewhichtoleratescopperionsopens upthepossibilityofcombiningitwithlow dosesofcopperand graduallyreducingitsuse(Dagostinetal.2011).Wefoundthat concurrentapplicationwithL.capsiciAZ78wasmoreeffectiveas comparedtotheapplication ofL.capsici AZ78or copperalone. Moreover,theapplicationofcoppertoL.capsiciAZ78-treatedplants reducedthebacterialpopulationbyonlyoneorderofmagnitude, showingthatthedecreaseincellviabilityobservedinvitrois par-tiallyconservedinplanta.

TheL.capsiciAZ78populationsongrapevineleavesunder con-trolledconditionsweresimilaratthebeginningand attheend oftheexperiments.Inconcurrentexperimentsaimedat monitor-ingthepersistence ofL.capsici AZ78cellsongrapevine leaves, we showed that the L. capsici AZ78 populationremained con-stantlyhighuntilsixdaysafterapplicationonplantsmaintained at25◦Cwitha60–80%RH.However,thereweresome discrepan-ciesbetweenthedegreeofpersistencemeasuredinthegreenhouse efﬁcacytrialandthat measuredin theexperimentsspeciﬁcally designedtoassessL.capsiciAZ78persistence.Inthelatter exper-iments,theL.capsiciAZ78populationdecreasedover8daysfrom 105_to₁₀2_cells_per_gram_of_leaf,_while_after_the_same_period₁₀5

cellspergramofleafwererecoveredfromplantsinthegreenhouse efficacytrials.Thisdiscrepancywasprobablyduetothedifferent conditionsthebacteriumencounteredduringthetwoexperiments. Whilehumidityremainedconstantoverthetendaysofthe per-sistenceexperiments,duringthebiocontrolexperimentsrelative humiditywasincreasedtwice, upon infectionand upon sporu-lationofP.viticola.Thishypothesisisconfirmedbytheconstant persistenceofL.capsiciAZ78onplantsmaintainedathighHRfor theentiredurationof thepersistenceexperiments.Humidityis knowntoinfluencebacterialgrowth(Leben1988;Wilsonetal. 1999;Cooleyetal.2003),sohighhumiditycontributestowards sustainingalargeL.capsiciAZ78populationonleaves.

ItisalsoworthnotingthatL.capsiciAZ78surviveswellinthe grapevinephyllosphere,althoughitwasisolatedfromthe rhizo-sphereoftobaccoplants.Atthemoment,mostoftheLysobacter strainsevaluatedforbiologicalcontrolofplantdiseaseshavebeen isolatedfromthesoilorrhizosphereofcultivatedplants,withthe singleexceptionofL.enzymogenesC3,whichwasobtainedfrom thephylloplaneofKentuckybluegrass (GieslerandYuen 1998). Interestingly,onlythis strain hasbeenevaluatedfor biocontrol ofplantpathogens attackingpartsoftheplantthatgrowabove ground (Kilic-Ekici and Yuen 2003; Kobayashiand Yuen 2005; Jochum et al. 2006), while most of theLysobacter strains have beenevaluatedforbiocontrolofsoil-bornepathogenicfungiand oomycetes(Nakayama et al.1996; Rondon etal. 1999;Folman etal.2003;Postmaetal.2008;Puopoloetal.2010).Theresults ofourstudysuggestthatsoil-borneLysobacterspeciesmightalso beassessed,atleastundercontrolledconditions,forbiological con-trolofpathogenicmicroorganismsattackingtheaerialpartsofthe plants.

LittleisknownaboutbioﬁlmformationbyLysobacterstrains.

Islametal.(2005)showedthatstrainSB-K88formsdense micro-coloniesontherhizoplaneofsugarbeetplantletsgrownfromseeds coatedwiththis bacteriumand theyalso reportedthatSB-K88

adherestotheplantsurfacebyformingfimbriae.Sinceitiswell documentedthat bacteriasurvive onplantsurfacesbyforming largeaggregatesindicatedasbiofilm(Morrisetal.1997;Dullaand Lindow2008)andbecauseofthehighpersistenceofL.capsiciAZ78 ontheleafsurface,weinvestigateditsbiofilmability.Theabilityof L.capsiciAZ78toformbiofilmwastestedoninertsurfacesandour resultsshowedforthefirsttimethataL.capsicistraincandothis, atleastinvitro.However,thisabilitydependedonthecomposition ofthegrowthmedium,asshownbythefactthattheKBmedium sustaineddevelopmentofthegreatestquantityofbacterialcells, withouthoweverleadingtotheformationofbiofilm.

Since KB contains low amounts of available iron ions (King etal.1954),itisreasonabletoassumethatthismetalaffectsthe formationofbiofilmbyL.capsiciAZ78,asisreportedinother Gram-negativebacteriasuchasAcinetobacterbaumanniiandP.aeruginosa, wherebiofilmformationishighlyinfluencedbyironsource, con-centrationand bioavailability (Tomaraset al.2003; Baninetal. 2005).Moreover,ithaspreviouslybeenshownthattheotherL. cap-sicimember,strainPG4,isunabletoproducesiderophoresandthat supplementingtheKBmediumwithFeCl3enhancesits

antibacte-rialproperties(Puopoloetal.2010).Onthebasisofthesedata,we surmisethatironavailabilitymayplayanimportantroleinterms ofpersistenceintheenvironmentandthebiocontrolpotentialofL. capsicimembers.

Whenbacteriaformabioﬁlmtheybecomemoreresistantto var-iousenvironmentalfactorsthataffecttheirpersistence(Ophirand Gutnick1994;Perrotetal.1998;ElasriandMiller1999).Themost frequentlyinvestigatedlimitingenvironmentalfactorsfor bacte-rialpersistenceinthephyllospherearestarvation,temperatureand exposuretoUVlightirradiation(Wilsonetal.1999;Stockwelletal. 2009).Sincelittleinformationisavailableregardingtheabilityof Lysobacterspeciestosurvivefollowingexposuretothesefactors, weinvestigatedhowAZ78respondstothem.

TheviabilityofL.capsiciAZ78cellswasnotnegativelyaffected bystresscausedbylackofnutrients(starvation)nordidtheysuffer followingexposuretoincreasingtemperatures(mildheatshock). Interestingly,L.capsiciAZ78couldtolerateexposuretoUV irradi-ationandfreezingtemperatures(−20◦_C)._The_survival_ratio_of_L.

capsiciAZ78cellsafterexposuretotheseenvironmentalstresses wascomparablewiththatoftheepiphyticstrainP.ﬂuorescens122 andthesoil-bornestrainP.ﬂuorescensPf5,asreportedbyStockwell etal.(2009).

L.capsiciAZ78cellviabilitywasnotnegativelyaffectedbythe lowestUVirradiationdosetestedinthisstudy(20Jm−2).Sundin and Jacobs (1999) reported that the minimum inhibitory dose fortheUV-sensitivestrainP.aeruginosaPAO1was5Jm−2,from which we can conclude that thesensitivity threshold of strain L.capsiciAZ78ishigherthanthatofasensitivebacterialstrain. Recently,Wangetal.(2013)haveshownthatL.enzymogenesstrain OH11synthesisesa yellowpigment that shows similarity with xanthomonadinproducedbyXanthomonadaceaemembers.These xanthomonadin-likearylpolyenemetaboliteshavebeenshownto beinvolvedintheprotectionofOH11cellsagainstUVirradiation andH2O2(Wangetal.2013),thusshowingfortheﬁrsttimethat

membersoftheLysobactergenushaveevolvedmolecular mecha-nismsassociatedwithresistancetothesefactors.

Theresultspresented hereexplain someaspectsof the biol-ogyand ecology ofL. capsiciAZ78.We show for theﬁrst time thatresistancetocopperassociatedwiththepresenceofgenes coding for CopA and CtpAis a commonbiological trait infour Lysobacterspeciesandthatatleastonememberofthisbacterial genus,L.capsiciAZ78,cantolerateabioticstressessuchas starva-tion,coldtemperature,mildheatshockandUVirradiation.Allthese characteristicsmakeL.capsiciAZ78asuitablecandidatefor devel-oping newsustainablestrategies for controllingdowny mildew inthegrapevine,sinceitisresistanttocopperandcanestablish

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itselfinthegrapevinephyllosphere,cantolerateenvironmental stressand,mostimportantly,drasticallyreducestheseverityof downymildew.Thenextstepswillbethedevelopmentofa suit-ableformulationforL.capsiciAZ78inordertoevaluateitinopen ﬁeldconditionsandtoassessitsefﬁcacyagainstdownymildewin vineyards,incombinationoralternationwithlowdosesof copper-basedfungicides.

Acknowledgements

The authors wish to thank Dr. D. Angeli for the fruitful discussion and D. Ress for technical assistance. The present researchwassupportedby“ENVIROCHANGE”projectfundedby theAutonomousProvinceofTrentoandtheEU-projectCO-FREE (themeKBBE.2011.1.2-06,grantagreementnumber289497).

AppendixA. Supplementarydata

Supplementarymaterial relatedto thisarticle canbe found, in the online version, at http://dx.doi.org/10.1016/j.micres. 2013.09.013.

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