What are the traits of Phelipanche ramosa (L.) Pomel that contribute to the success of its biological cycle on its host Brassica napus L.?

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What are the traits of Phelipanche ramosa (L.) Pomel that contribute to the success of its biological cycle on

its host Brassica napus L.?

Stéphanie Gibot-Leclerc, Georges Salle, Xavier Reboud, Delphine Moreau

To cite this version:

Stéphanie Gibot-Leclerc, Georges Salle, Xavier Reboud, Delphine Moreau. What are the traits of

Phelipanche ramosa (L.) Pomel that contribute to the success of its biological cycle on its host Brassica

napus L.?. Flora, Elsevier, 2012, 207 (7), pp.512–521. �10.1016/j.flora.2012.06.011�. �hal-01702104�

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ContentslistsavailableatSciVerseScienceDirect

Flora

j ou rna l h o m e p a g e :w w w . e l s e v i e r . d e / f lo r a

What are the traits of Phelipanche ramosa (L.) Pomel that contribute to the success of its biological cycle on its host Brassica napus L.?

Stéphanie Gibot-Leclerc

^a,∗

, Georges Sallé

^b

, Xavier Reboud

^c

, Delphine Moreau

^c

aAgroSupDijon,UMR1347Agroécologie,BP86510,F-21000Dijon,France

bLaboratoiredeParasitologieVégétale,UPMCUnivParis06,F-75005Paris,France

cINRA,UMR1347Agroécologie,BP86510,F-21000Dijon,France

a r t i c l e i n f o

Articlehistory:

Received5December2011 Accepted23March2012

Keywords:

Phelipancheramosa(L.)Pomel=syn.

OrobancheramosaL.

BrassicanapusL.

Parasitism Biologicalcycle Trophicrelationships Competition

a b s t r a c t

InFrance,thefactthattheholoparasiticplantPhelipancheramosahasadaptedtooilseedrapeoverthe pasttwodecadesisdeeplyworrying,asitcancauseover80%yieldlosses.Itsdistributionareaand therangeofitshostplants,whethertheyarecultivatedplantsorweeds,areexpandingdramatically.

Asnonaturalregressionoftheinfestationhasbeenrecordedsofar,weundertookadetailedstudy ofthedifferentstepsofthebiologicalcycleoftheP.ramosa/oilseedrapepathosystemtodetermine theadaptivetraitsthatfavourtheparasite’ssuccess.Wecombinedexperimentalapproachesinvolving controlledconditionswithinvitroandinpotco-cultivationontheonehandandafieldtrialontheother hand.Theseexperimentsallowedustodeterminea4-mmzonearoundhostrootswithinwhichoilseed raperootexudatesstimulatethegerminationofP.ramosa,theearlyactionofhostrootexudatesand therapidfixationoftheparasiteonhostroots.WealsounveiledthatP.ramosawasabletotuneits biologicalcycletothatofoilseedrape.Theimportanttrophicrelationshipsbetweentheparasiteandits hostinducedphenotypic(dwarfism,leafchlorosis,siliqueabortion)aswellasagronomical(90%yield losses)consequencesonoilseedrape.Ourresultscanconstitutearelevantbasisforfurtherexperimental studies.Theresearchperspectivestheyopenwillfocusonkey-processesofthehost–parasiterelationship, andmoreparticularlyonthetrophicrelationshipsthataresetupasfarascarbonassimilatesandminerals suchasnitrogenareconcerned.

Introduction

Amongcropbioagressors,thePhelipancheandOrobanchegenera areobligateparasitesofdicotyledonousrootsandareconsideredas oneofthemainbioticconstraintsoneconomicallyimportantcrops.

Theyarealsoknowntobethecauseofcroplossesrangingfrom5to 100%(Joeletal.,2007;Musselman,1980;ParkerandRiches,1993;

PressandGraves, 1995;Pressand Phoenix,2005; Schneeweiss, 2007).Phelipancheramosa(L.)Pomel(Joel,2009)ismostdevas- tatingand hasby farthe widestrange of hosts, among which Solanaceae,Brassicaceaeandlegumesareprominent(Buschmann etal.,2005;Haidaretal.,2003;Joeletal.,2007;ParkerandRiches, 1993).InFrance,P.ramosagotremarkablyadaptedtooilseedrape, causingdeeplyworryinglossesofabout80%,butalsotohempand tobacco.SofartheadaptationofP.ramosatooilseedrapeseemsto haveremainedlimitedtotheFrenchterritory.Thedistributionarea

∗Correspondingauthorat:AgroSupDijon,UMR1347Agroécologie,Département AgronomieAgroéquipementElevageEnvironnement,26BdDrPetitjean,BP87999, 21079DijonCedex,France.Tel.:+330380772803;fax:+330380772551.

E-mailaddress:stephanie.gibot-leclerc@dijon.inra.fr(S.Gibot-Leclerc).

oftheparasiteandtherangeofitshostplantsappeartohavedra- maticallyexpandedlately,withmorethan70weedspeciesasnew hosts(Bouletetal.,2007;Braultetal.,2007;Gibot-Leclercetal., 2003,2006,2009).

Thebiologicalcycleofepirhizalparasitesisusuallycomposed of two distinct phases (Bouwmeester et al., 2003; Joel et al., 2007;Keyesetal.,2001;ParkerandRiches,1993;Rubiales,2003;

Sauerborn,1991;Yoder,2001).Underground,thefirstphaseisini- tiatedbyseedgerminationandgoesonwiththefixationonhost rootsandthepenetrationintohosttissuesalongwiththedevelop- mentofanabsorptionsystemcalledahaustorium.Aboveground, thesecondphasestartswiththeemergenceandthenthegrowthof floralscapesandendswithfloweringandfructification.Inrecent yearsseveralauthors(Buschmannetal.,2005;Echevarría-Zome ño etal.,2006;Goldwasseretal.,2001;Haidaretal.,2003;Kogan, 1994;Luetal.,2000;PérezdeLuqueetal.,2004;Salléetal.,2000) haveinsistedontheneedtoincreaseourknowledgeaboutparasitic plantbiologyinordertomakeiteasiertodevelopsuitableprotocols foranefficientcontrol.Theknowledgeacquiredaboutbroomrape sofardealswiththebiologyofthemainbroomrape/hostpathosys- tems, i.e. Orobanche cernua/sunflower, O. crenata/faba bean, O.

http://dx.doi.org/10.1016/j.ﬂora.2012.06.011

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P. ramosa/tomato. However, a detailed study of the biological cycleoftheP.ramosa/oilseedrapepathosystemhasneverbeen performed. Acquiring knowledge about the biology of the P.

ramosa/oilseedrapepathosystemisallthemorecrucialasrecent molecularbiologyworkshaveprovedthatthereexistatleasttwo differentP.ramosapathovarswithclearcuthostspeciﬁcities,which makesculturemanagementinseverelyinfestedplotsevenmore complicated.

Inordertodeterminetheadaptivetraitsthatfavourthepar- asite’s success, we characterized the biological cycle of the P.

ramosa/oilseedrapepathosystem.Moreprecisely,wedetermined (1)theextentofthezonewhereoilseedraperootexudatesstimu- lateP.ramosaseedgermination;(2)thesuccessrateofthefirst fixationstepsof P.ramosaonhostroots followingitsgermina- tion;(3)howthedifferentphasesoftheP.ramosadevelopment cycle tune in with the oilseed rape cycle; (4) the phenotypic andagronomicalconsequencesoftheparasitismofP.ramosaon oilseedrape.Inordertomakeiteasiertostudytheearlydevel- opment steps of P. ramosa, we decided to work with in vitro andsandwichco-cultivationmethods.Inparallel,afieldtrialwas

Fig.1.DevelopmentcycleofthePhelipancheramosa/oilseedrapepathosysteminCharente-Maritime(France).

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performedtocharacterize the consequences ofP.ramosa para- sitismonoilseedrapeatthewholeplantlevelandatthecanopy level.

Materialsandmethods

Laboratory and ﬁeld experiments were conducted between 2000and2004.

Seedorigin

The seeds were collected in 1999 from natural populations of P. ramosa that had severely infested ﬁelds of oilseed rape (BrassicanapusL.Brassicaceae)inSaint-Pierre-de-Juillers(Latitude 45,9333; Longitude −0.3667; Charente-Maritime, France). Once harvested,theseedsweresiftedtocleanthem.Oilseedrapeseeds (var.Zénith)wereprovidedbytheCETIOM(Centretechniquedes

Fig.2. (A)MorphologicalaspectofaPhelipancheramosaseedinSEM.Wecannotethewayalveoliarearranged(*)aswellaspunctuationdetails(arrows)ofthesurfacecell innerwalls.(B)LongitudinalsectionofaP.ramosagerminated8daysafterstartinginvitroco-cultivationwithoilseedrape.(C)Phelipancheramosagerminated11daysafter startingsandwichco-cultivationwithoilseedrape.(D)Phelipancheramosafixationonanoilseedraperoot,inthefield28DAE.(E)VeryyoungP.ramosatubercles2months afterstartinginvitroco-cultivationwithoilseedrape.(F)VeryyoungtubercleinSEM,inthefield28DAE.(G)Youngtuberclewithprotuberances(arrows)correspondingto theparasite’sfutureadventitiousroots,inthefield28DAE.(H)YoungtubercleinSEM,inthefield28DAE.al,albumen;ch,chalazazone;hlr,hostlateralroot;mi,micropyle;

ra,radicle;s,seed;tu,tubercle.

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Table1

NumbersofPhelipancheramosaplantsforeachofthe5undergroundontogenicstagesobservedonall30infestedoilseedraperootsystemsinasoilthatisnaturallyinfested bybroomrapeinCharente-Maritime(France).

Samplingdate(daysafteroilseedrapeemergence(DAE)) Phelipancheramosaontogenicdevelopmentalstages

Fixation Youngtubercle Oldtubercle Bud Undergroundstem Total

7DAE 0 0 0 0 0 0

12DAE 0 0 0 0 0 0

19DAE 0 0 0 0 0 0

28DAE 8 46 0 0 0 54

49DAE 1 60 36 12 0 109

70DAE 0 218 368 21 128 735

97DAE 0 0 100 20 90 210

145DAE 0 0 417 267 257 941

194DAE 0 0 0 ++ +++ >1500

244DAE 0 0 0 0 +++ >1500

Whenthehostrootsystemwasinfestedbymorethan50broomrapes,theexactnumberofeachofthebroomrapedevelopmentalstagescouldnotbedetermined.Their relativefrequencyisindicatedbyplussigns:+,lownumbersoftheparasite’sdevelopmentalstage;++,averagenumbers;+++,highnumbers.

oléagineuxmétropolitainsetduchanvreindustriel,France).Phe- lipancheramosaaswellasoilseedrapeseedswerekeptinglass containersatroomtemperature.

Invitroexperiment

Afterdisinfection(70%ethanol,5min,and3%calciumhypochlo- rite solution, 10min), 15–25 seeds of P. ramosa were laid on WhatmanGF/Apaperdiscs(Ø12mm).Eightdiscspreparedthat way wereplaced on a Whatman GF/A paper sheet(Ø 90mm) at the bottom of a Petri dish (Ø 90mm), and then hydrated.

TwentyPetridishes,containing120–200seedseach,wereplaced indarknessat20^◦Cfor14daystopre-conditiontheseeds(obli- gate hydrationperiod). Thereafter, one disinfectedoilseedrape seedwasplacedeachinarectangularplasticboxcontainingMS (1/2)(MurashigeandSkoog,1962)nutrientsolutionwith1%agar.

Twentyboxespreparedthatwaywereplacedinagrowthchamber, at23±1^◦C(day)and18±1^◦C(night),witha16-hphotoperiod, and70␮molm⁻²s⁻¹ photosyntheticphotonﬂuxdensity(PPFD).

Oncetheoilseedraperoot systemhad developed (i.e. 2 weeks afterthebeginningoftheexperiment),the8discsbearingthepre- conditionedP.ramosaseedsweretransferredunderthehostroot system.TheP.ramosa/oilseedrapeco-cultivationwascarriedout inthesametemperatureandlightconditions.Thedevelopmentof theparasiteonoilseedraperootswasmonitoredfor12weeks.The observationswerecarriedoutunderastereomicroscopedailyfor 4weeks,andthenonceaweekforthefollowing8weeks.Foreach observation,thepercentageofP.ramosagerminationinducedby oilseedraperootexudateswascounted,andtheearlydevelopmen- talstagesoftheparasiteonitshostwererecorded.Ourobservations werealsocarriedoutinlightandscanningelectronmicroscopy (SEM).Thewholeexperimentwasperformedtwice.

Sandwichexperiment

Losner-Goshenandcollaborators’method(1998)wasapplied, withslightmodiﬁcations.Afterdisinfection(70%ethanol,5min, and3%calciumhypochloritesolution,10min),441mg-P.ramosa seedsamples(approximately350seedseach)weredriedunder a laminar ﬂow at room temperature for 24h. Then each sam- plewassprinkledontoaGF/Asheet(Ø90mm)atthebottomof aPetri dish(Ø90mm),and thenhydrated.The 44Petri dishes wereplacedindarknessat20^◦Cfor14daystoachieveseedpre- conditioning.Forty-fouroilseedrapeseedswerelaidindividually ontoGF/Asheets(Ø90mm)atthebottomofPetridishes(Ø90mm), andthenhydrated.Afteroilseedrapeseedgerminationindark- ness at 30^◦C, each oilseedrape seedlingwas laid onto a GF/A sheet(Ø90mm)incontact withthepre-conditionedP.ramosa

seeds.Asecondsheetwasaddedtocoveralltheseeds.Thesand- wichedindividualoilseedrapeseedlingandbroomrapeseedswere placedonpiercedplasticPetridishes(Ø90mm)whosesideshad beennotched.Eachof the44 modiﬁed Petridishes wasplaced slantwiseinsideaterra-cottapot(Ø15cm)ﬁlledtwo-thirdsfull witha1vol:1volpottingsubstrateandplantingsubstratesoilmix.

TwentymlofMS(1/2)nutrientsolutionwasaddedtoeachpot.

Theco-cultivationoftheP.ramosa/oilseedrapepathosystemwas carriedoutinthegreenhouse[23±1^◦C(day)and18±1^◦C(night), and55%relativehumidity].Thenaturallightwascomplementedby artiﬁciallight(100␮molm⁻²s⁻¹PPFD)tohavea16-hphotoperiod.

Tenmlofnutrientsolutionwasaddeddaily.Phelipancheramosa developmentonoilseedrapewasassayedfor12weeks.Samples weretakendailyduringtheﬁrstweek,every2daysforthenext 2weeks and thenoncea weekfor theremaining9weeks. For eachsampling,therootsystemof2oilseedrapeplantswastaken outandobservedundera stereomicroscope.Thepercentageof P.ramosagerminationinducedbyoilseedraperootexudateswas countedandtheearlydevelopmentalstagesoftheparasiteonits hostwererecorded.Ourobservationswerealsocarriedoutinlight microscopyandSEM.Thewholeexperimentwasperformedtwice.

Fieldtrial

On28thAugust2000,oilseedrapewassowedonasoil,con- sistingofsilt,clayandsand,severelyinfestedbyP.ramosaand locatedinVillemorin(Latitude:46.003887;Longitude:−0.296944, Charente-Maritime,France).The4-replicatetrialwascarriedoutin wholeFischerblocks.Theexperimentwasledthroughtilloilseed rapeharvest.Samplesweretakenat10differentdates,i.e.7,12, 19,28,49,70,97,145,194and244DAE(daysafteremergence).

Ateachsamplingdate,30oilseedrapeplantsweredugoutand observedundera stereo microscope.Oilseedrapedevelopmen- talstagesweredeterminedandinfestedhostrootswerecounted.

Infestationintensity wasestimated bycounting thenumber of P.ramosaontogeneticstagesfixedonhostroots asfollows: (a) fixation; (b) youngtubercle without adventitious roots;(c) old tuberclewithadventitiousroots;(d)budwithadventitiousroots butwithoutastem;(e)undergroundstem;(f)stememergence;(g) flowering;(h)fructification.Ourobservationswerealsocarriedout inlightmicroscopyandSEM.

Histologicalandcytologicalstudiesunderalightmicroscope andascanningelectronmicroscope(SEM)

Theparasiteonthehostplantwastaken atdifferentdevel- opmentstages,ﬁxedina4%glutaraldehydesolutioninapH7.4 cacodylatebuffer for 24hand then post-ﬁxed in a 1%osmium

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tetroxide solution in the same buffer for 1h (Ledbetter and Porter, 1963).Afterthat, thesampleswere dehydratedbydip- pingthemintoethanolbathsofincreasingethanolconcentrations andaraldite-includedaccordingtoGlauertandGlauert’smethod (1958).Thentheincludedsampleswereslicedintosemi-thinsec- tionsforphotonmicroscopyandstainedwithtoluidineblue.

For SEM observations, samples ﬁrst ﬁxed in a glutaralde- hydesolutionforseveraldaysweredehydratedbydippingthem intobathsofincreasingethanol concentrations.Thentheywere CO₂-treateduntilthecriticalpointandsputter-coatedwithgold (Guillaumin,1980).

Results

Fig.1givesacomprehensiveoverviewofallresultsdetailedin thefollowingandillustratesthedevelopmentalcycleoftheparasite anditshost.

UndergroundlifestageofPhelipancheramosa

Phelipancheramosaseedsareoval-shaped,350␮mlong,250␮m wide,andweigh3␮g.Theyarecoveredbyahardreticulatecoat.

Thenarrowestendcorrespondstothemicropylewhiletheround- estoppositeend correspondstothechalaza(Fig.2A).Thetesta containsalveolarcavitiesinit(Fig.2A,*)whicharecomposedof dehydratedcellsalignedalongthelongaxisoftheseed.Asthecyto- plasmiccontentofcellsisgone,theouterwallhascollapsedand followsthecircularpunctuationsoftheinnerwall(Fig.2A,arrows).

Thealbumen (Fig.2B)iscomposedof3or4layersofcells con- tainingnumerouslipidglobulesandstarchgranules.Itsurrounds theprimitiveembryodevoidofradicle,gemmuleorcotyledons.

Afterpre-conditioning,onlytheP.ramosaseedssituated nearby theoilseedraperoots(lessthan4mminourconditions)wereable togerminate.TheradicleofP.ramosajutsout ofthemicropyle region(Fig.2B)andgrowstowardsthehostroot(Fig.2C).Inthe absenceofﬁxationonoilseedraperoots,P.ramosaseedsdegen- eratedanddiedinlessthan3or4days.Intheinvitromethod,P.

ramosaseedgerminationoccurred3days(61growingday-degrees, base0)afterstartingtheco-cultivation.Onthe8thday,P.ramosa germinationpercentagewas50%anditreached95%3dayslater.

Withthesandwichmethod,thelag-timewaslonger(7days),the germinationpercentagewas50%onthe11thdayanditreached 90%onthe15thday.Takingintoaccountthedifferentlag-times probablyduetotheageoftheoilseedrapeseedlingswhenparasite andhostwereputtogether,wecanconsiderthatP.ramosager- minationoccurredwhenoilseedrapeseedlingswere11–14days old,whilethemaximalgerminationpercentagewasreachedwhen theywere21daysold(Fig.1).

Inthefield,oilseedrapeemergenceoccurred9daysaftersow- ing(Fig.1).Whenthefirstsamplesweretaken7,12and19DAE, hostrootswerenotyetinfectedbyP.ramosa(Table1).Yet,the resultsobtainedwiththeinvitroandsandwichmethodsindicate thatthemaximalgerminationpercentagefor P.ramosaseedsis reachedwhenoilseedrapeis21daysold.Wecanthereforehypoth- esizethatthe21-day-old-oilseedraperootexudates(B6,i.e.6-leaf stage)didinducethegerminationofnumerousP.ramosaseeds, butthesegerminationswereverylikelylostduetotheuproot- ingandwashingstepsoftherootsystems.Parasitefixationsand youngtubercleswereobservedfromthe4thsamplingdateonward (Fig.1,B8,i.e.8-leafstage,28 DAE).Itisimportanttonotethat P.ramosa only fixed itself onthe lateral roots of oilseed rape.

Atthat time, youngtubercles werealready 5 times as numer- ousasﬁxations(Table1).Fieldresultswereinaccordance with thoseobtainedwiththeinvitroandsandwichmethods.Phelipanche ramosaﬁxationsweredetectedwhenoilseedrapewas30daysold

andyoungtuberclesonlydeveloped1monthlater.Inthesecon- trolledconditions,P.ramosadevelopmentdidnotgofurtherthan theyoung-tuberclestagethroughoutthe3-monthlongexperiment (Fig.2D–H).

WhenP.ramosaﬁrstpenetratedintooilseedraperoots,meris- tematiccellproliferationallowedthehaustorium,whichatthat stagewasnotyetfunctional,tomake itswaythroughthehost tissues(Fig.3A).Lateron,onlyapicalcellsremainedabletopro- liferatewhereasdistalcellsgotorganizedintoaparenchymaand xylem elements differentiated. Once haustorialapical cells had madecontactwiththehost’svascularsystemcells,theystopped dividingandmigratingintothehost.Theystucktothehostvessel cells withoutperforatingthem.Thecontinuity betweenbroom- rape haustorium vessels and the host xylem is clearly visible (Fig.3B).

Oldtuberclesandbudswereobservedwhenoilseedrapewas 49daysoldorolder(Fig.1,B12,i.e.12-leafstage).Theprolifera- tionofcellslocatedaroundthecaulinarymeristem,attheorigin ofthefloralscape,ledtotheformationofawreathofadventi- tious roots (Fig.4A, arrows) whichin somecases wereable to makecontactwithotherhostrootsandproducedsecondaryhaus- toria.Onceadventitiousroots wereformed,thetuberclerapidly turnedintoa bud thankstoits apicalcells.Insidethebud, the caulinarymeristemwasprotectedbyscales(Fig.4B).Itisimpor- tanttonotethatat49DAEonlyonenewfixationwasdetected, whileyoungandoldtuberclesweremuchmorenumerousthan buds(Table1).Fromthatpointonwardthebudsgrewverticallyand formedyoungundergroundstemswhichmadetheirwaythrough thesoiltowardsthesurface.Thefirstundergroundstems(Fig.4C) wereobserved70DAE(Fig.1,rosettestage).Atthattimefixations hadalldisappearedwhereasallotherstagesexceptthebudstage wereinconstantlyincreasingnumbers(Table1).Atthefollow- ingsampling date(97DAE, rosettestage), youngtubercleshad disappeared.Till145DAE(C1,i.e.boltingstage),thenumbersof oldtubercles,budsandundergroundstemsincreasedconsiderably.

Conversely,nonewﬁxationoryoungtuberclewasdetected.During thatperiod,oilseedrapestoppeditsvegetativedevelopment,and nonewP.ramosaseedgerminationleadingtothedevelopmentof newyoungparasiticdevelopmentalstagesappearedtohavebeen induced.At194DAE(E-stage,i.e.separate-ﬂower-budstage),old tubercleswerenotvisibleanymorewhileeachrootsystemcarried morethan50budsandundergroundstems(Table1).

AerialphaseofP.ramosa

Phelipancheramosaemergedatthesoilsurface205daysafter oilseedrapeemergence(Fig.1,F1,i.e.earlyfloweringstage).The short, frail, rather hairy, branchedstem bulged out at its base (Fig.4D). It had leavesthat containedno chlorophyllpigments andwerereducedto6-to12mmlongthick,acuminate,alternate scales.Thisnon-chlorophyllic,scalyaxisthenturnedintoafloral scape244DAE(G1/G2,i.e.earlyfructificationstage).Theflowers were1–1.5cmlong.Theywereinsertedindividuallyattheaxils ofscalybracts.Theyhadanirregular,curvedshape,andablue- tintedmauvecolour.Theydidnothavedistinctpedunclesandthey weregroupedinratherlongfloralscapeswhenfloweringwaswell advanced(Fig.5AandB).Itisimportanttonotethatatthatlast time-pointbudscouldnolongerbefoundwhilelargenumbers ofundergroundstemswithsurroundingbunchesofadventitious rootsremainedreadytoemerge(Table1).Oncefecundationhad takenplace,eachpollinatedflowerturnedintoacapsulecontaining approximately600–800seedseach (Fig.5C).Phelipancheramosa started fructifying 273DAE (Fig.1, G4,i.e. end-of-fructification stage)andthefloralscapesofbroomrapewereripeafewdaysafter thoseofoilseedrape(Fig.5D).

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Fig.3. (A)LongitudinalsectionofanearlypenetrationstageofP.ramosa.Theoilseedraperootiscuttransversely.(B)Xylemlinks(arrows)inalongitudinalsectionofthe baseofaP.ramosatubercle.hlr,hostlateralroot;hx,host’sxylem;ic,intrusivecells;p,parasite;pc,parenchymacells;px,parasite’sxylem.

Fig.4. (A)Oldtuberclewithyoungroots(arrows)whichhidethecaulinarymeristeminSEM,inthefield49DAE.(B)Phelipancheramosabudwithnumerouslongadventitious roots(arrows)inthefield49DAE.(C)BunchofP.ramosaundergroundstems,inthefield70DAE.(D)Phelipancheramosaemergence,inthefield205DAE.b,bud;hlr,host lateralroot;us,undergroundstem.

Phenotypicandagronomicalconsequencesonoilseedrape

Duringtheautumn–winterphaseofoilseedrape,theshootpart ofthehostplantshowednovisibleparasitism-relatedsymptoms.

Itwasonlybydiggingoutrootsystemsandexaminingthemclosely

thatwedetectedthepresenceofP.ramosa.Butinthespringphase, thefirstsymptomsbecamevisiblewhenoilseedrapefloralpedun- clesstartedgrowing(Fig.1,Estage).Parasiticattackinducedoilseed rapedwarfism,whichwasvisibleasadepressivezoneatthefield level(Fig.6A,arrows)andleafchlorosis(Fig.6B,arrows).OnceP.

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Fig.5.(A)Phelipancheramosaflowering,inthefield244DAE.(B)DetailofaP.ramosaflower.(C)YoungP.ramosacapsule.Immatureseedsformayellowcompactbulk (arrow).(D)FructifiedP.ramosa,inthefield273DAE.

ramosahademerged,itsbranchedfloralscapeswereeasilyiden- tifiable.Themostseverelyinfestedpartswereusuallythose on theoutskirtsof thefields(Fig.6C). Inthe case ofsevere infes- tations,eachoilseedrapeplantcouldharbourasmanyas15–20 floralscapes;theinfestedzoneappearedthentobefullycovered byP.ramosa(Fig.6D).Whenoilseedraperipened,thesizeofthe depressivezoneincreasedwhilethelowdensityofthecropmadeit possibleforweedstodevelop.Infestedoilseedrapesproducedfew siliquesandmanyofthemaborted(Fig.6E,arrows).Yieldlosses weresignificant:fromanaverage38qha⁻¹whenoilseedrapewas healthy,theywentdownto4qha⁻¹inthemostseverelyinfested fields.Afteroilseedrapeharvest,onlywiltedP.ramosafloralscapes remained(Fig.6F),perfectlyalignedwiththesowinglines.

Discussion

Acombinationofﬁeldandcontrolledexperimentalapproaches allowedustocharacterizeprecisely thedifferentstepsoftheP.

ramosacycle,atthecelllevelaswellasatthewholeplantlevel.To ourknowledge,thisistheﬁrstsuchstudytobecarriedoutabout thispathosystem.

BiologicaltraitsofP.ramosacontributingtothesuccessofits biologicalcycle

Phelipanche ramosa as a parasite of oilseed rape possesses differentspeciﬁctraitsthatcontributetothesuccessofitscycle.

Theﬁrst traitconcerns itsdispersion: itproduces light, minute seedsin huge numbers. Thus its seedsare easily dispersed by

wind, waters, animals, farming machinery, and contaminated seedtransport(Berneretal.,1994;ParkerandRiches,1993).The secondtraitisthetoughnessofitsseedcoat.Thistraitiscommon toOrobancheandStrigaseedsandcouldexplainwhytheirseeds canretaintheirgerminatingcapacityinthesoilforseveralyears (Parker and Riches, 1993;Worsham, 1987).The third traitlies in theneed forseedgerminationtobestimulatedby hostroot exudates.WeobservedthatP.ramosaseedgerminationcouldonly occurwithinashortdistancefromoilseedraperoots.Wefound thatdistancetobe4mm.Oilseedraperootexudatesthusappear tohaveastimulatingeffectwithinalimitedzonearoundtheroots comparedtothe10mmwiththefababean/O.crenatapathosystem (Salléetal.,1984).Suchasystemcouldpreventtheseedsfrom germinating inthe absenceof ahost andavoid P.ramosaseed germinationattoogreatadistancefromoilseedraperoots.

ThethreebiologicaltraitsofP.ramosaasaparasiteofoilseed rapementionedabovecompleteothertraitsalreadymentionedin theliterature,namely:theabsenceofaprimarydormancy,oravery earlybreakofprimarydormancy,asP.ramosaseedscangerminate asearlyasharvesttime.Apartfromtheindispensablepresence ofasusceptible hostplant,P.ramosaseedpre-conditioningand germinationdonotrequireanyspeciﬁcenvironmentalconditions (Gibot-Leclercetal.,2004,2006).

EarlyefﬁcacyofrootexudatesonP.ramosagermination

Ourresultsshowthatwhicheverco-cultivationtechniquewas used,P.ramosaseedgerminationwasmassivelyinduced21DAE, whileoilseedrapehadonlyreachedthe6-leafstage.Oilseedrape

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Fig.6.(A)Depressivezone(arrows)duetoslowedgrowthofinfestedoilseedrape.(B)Chlorosisintheleavesofinfestedoilseedrape(arrows).(C)Phelipancheramosaﬂoral scapeslocatedontheoutskirtofanoilseedrapeplot.(D)VeryhighP.ramosadensityinaseverelyinfestedoilseedrapeplot.(E)Toppartofaninfestedoilseedrapeﬂower bunchwithabortedsiliques(arrows).(F)Harvestedinfestedoilseedrapeplot.

rootexudatesthereforeappeartobeeffectiveintheearlystages ofitsgrowth.ForP.ramosa,suchearlinesshasbeenshownwith tobacco(Labrada,1994).Itisnoteworthythatnonewﬁxationor youngtuberclewasobservedwhileoilseedrapewasbetweenthe

rosettestage(97DAE)andtheshootingstage(145DAE).Taken together, theseresults suggest a tight correlation betweenthe developmentalstagereachedbythehostand theefﬁcacyofits rootexudates.

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Fixation,afundamentalprocessforresourceacquisitionbyP.

ramosa

Onceithasgerminated,P.ramosaveryquicklyﬁxesitself(28 days)onoilseedrapehostroots.Asimilarprecocitywaspreviously reportedonhempandtobaccoroots(Braultetal.,2007;Labrada, 1994).Broomrapeﬁxationhastobecompletedfortheparasite tobeabletopenetrate hosttissueslater and drawthecarbon, mineralandwaterresourcesneededforitsgrowth.Acontinuum betweenoilseedrapeandP.ramosaxylemswashighlighted.Such xylem-to-xylemcontactshavealsobeenevidencedinthecaseof theP.ramosa/carrotpathosystem(Zehharetal.,2003).Ourlight- microscopyobservationsdidnotallowustodeterminewhether hostandparasitexylemvesselsweresimplyadjacentorhadmade directlumencontact(HibberdandJeschke,2001).

Xylem-mediatedsolutesupplytotheparasiteislikelytobelow duetothelowtranspirationrateoftheparasitethatgeneratesonly aweakdrivingforceforthesoluteﬂuxwithinthexylem(Hibberd etal.,1999).Yetwedidnotobserveanyphloem-to-phloemconnex- ions.Butphloemtissuesarefoundinbroomrapehaustoria(Dörr andKollmann,1975;HibberdandJeschke,2001).Thehostphloem isknowntoprovidealmostallsugars(>99%)andmostminerals suchasnitrogen,magnesiumandpotassium(Abbesetal.,2009;

HibberdandJeschke,2001;Hibberdetal.,1999).

Tuningofthehostandparasitedevelopmentcycles

Phelipancheramosa tightlytunes theduration of its biologicalcycle toits host’s(273 days).Such tuning wasfoundfor P.

ramosaparasitingdifferentspecies(Braultetal.,2007;Kogan,1994;

Neumannand Sallé,2000), witha biological cyclethat canlast 45–150days accordingto thespeciesconsidered. Inourstudy, P.ramosaemerged,i.e.startedtheaerialphaseofitscycle,when oilseedrapestartedﬂowering,inaccordancewithcommonobser- vationsondifferenthostplants,suchashemp,eggplant,tobacco, tomatoorwatermelon(Braultetal.,2007;Kogan,1994;Labrada, 1994).AnotheraspectofcycletuningwaswhenP.ramosaﬂoral scapesstartedgrowingatthesametime asoilseedrapestarted formingitssiliques.SuchtuningisbelievedtoenableP.ramosato deprivethehostofnutrientsasoptimallyaspossiblebydiverting waterandnutrientsfromthehostforitsownuse.

Consequencesofnutrientdiversiononthegrowthand agronomicalperformancesofoilseedrape

The broomrape biological cycle phase that occurs between seedgerminationandhaustoriumpenetrationtakesplaceexclu- sivelyunderground,soitcannotbedetectedbyoutsiders.Besides, thesymptoms of P.ramosa parasitism onoilseedrapebecome detectablelateintheoilseedrapelifecycle,i.e.onlyonceitsﬂo- ralpedunclesstartelongating.Anoverallslowergrowthofoilseed rapecouldbeobserved,alongwithleafchlorosis,lowsiliquepro- duction,andnumerousabortions.Suchsymptomsareoftenseen inhost/parasiterelationships(Salléetal.,1995).

Bydevelopingonhostroots,theparasitecompeteswiththehost forwater,mineralsandsugars.Sometimes,thedecreaseinhost biomassisnotfullycompensatedforbyparasitebiomass(Grenz etal.,2008).Inthiscase,photosyntheticproteindiversionislikely topreventthehostfrommaintainingitsphotosyntheticlevel.This phenomenoncouldbeattheoriginoftheoilseedrapeleafchloro- sisandbiomassdropobservedinourstudy.Besides,followingthe source-sinkconcept,Manschadietal.(2001)proposedthatthepar- asitecouldbeaprioritysinkcomparedtohostvegetativeorgans andnewlyformedpods,whereaspodsintheﬁllingstagecouldbe prioritysinkscomparedtotheparasite.Thiscouldaccountforthe lownumbersofsiliquesandthenumerousabortionsobservedin

ourstudy.Thehighyieldlossesofoilseedrapethatweobservedare inaccordancewithotherstudies(Bernhardetal.,1998;Manschadi etal.,2001;Mesa-GarcíaandGarcía-Torres,1986).

Lastofall,hormonalimbalance,characterizedbyadecreasein gibberellinandcytokininecontentandanincreaseinabscisicacid content,couldcausethegrowthdelayattheoriginoftheinfested plants’dwarﬁsm.Theimbalancecouldalsoresultfromwoundsand waterstresscausedbytheparasite’ssettingin(WatlingandPress, 1997).

Conclusion

The highly specialized parasitic way of life set up by Pheli- pancheramosaonoilseedrapehighlightsdifferentadaptivetraits thatfavouritssuccess.ThesetraitsconcernP.ramosagermination andfixation,withthedeterminationofa4-mminfluencezoneof oilseedraperootexudatesonparasiticseedgerminationandan earlyeffectof exudatesfollowedbyavery rapidfixationofthe parasiteonhostroots.Consequently,anever-increasingparasitic seedbankismaintained.Thesetraitsaremoregloballyrelatedto thewholebiologicalcycleofP.ramosasincethedifferentstepsof itscycledependlittleonweather conditionsbutcompletelyon thepresenceofahost.Ourstudyevidencedafinetuningbetween oilseedrapeandP.ramosacyclephases,whichledtoasynchro- nizationoftheirfructificationstages.AlthoughthewayP.ramosa germinatesandfixesitselfonhostrootsiswellcharacterized,the physiologicalresponsesthatinducethefinetuningofthepara- site’scyclephasestoitshost’sremaintobeelucidated.Besides,the fulfilmentofP.ramosa’sbiologicalcycleinducesconsequenceson oilseedrape,atthephenotypicandtheagronomicallevels.

Ourstudycanconstitutearelevantbasisforfutureexperimental studies.KnowingthenumberofdaysneededforP.ramosatoreach itsearlydevelopmentalstages,whichtakeplaceundergroundand arethusinvisibletooutsiders,willmakeitpossibletobetterplan oilseedraperootsamplinginordertogetP.ramosaplantsatthe wantedstage.ConcerningP.ramosaemergence,itwillbepossi- bletoplanobservationsfromoilseedrapephenologyonceithas reachedtheearlyﬂoweringstage.Asfortheparasite’slaterdevel- opmentalstages,theyarevisibletothenakedeye,whichsolvesthe problem.Inadditiontothesepracticalapplications,ourworkopens ontonewresearchperspectivesthatcanfocusonkey-processesof thehost–parasiterelationship.Inparticular,thetrophicrelation- shipsconcerningcarbonassimilatesandmineralssuchasnitrogen needtobefurtherinvestigatedinordertobetterunderstandthe competitionthat takes placebetween hostand parasitein this pathosystem.

Acknowledgements

ThepresentworkwasﬁnancedjointlybytheCETIOMandthe Poitou-Charentesregion.WethanktheCETIOM(experimentalsta- tionofSurgères,France)fortechnicalsupportatharvesttime.

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