nitrogen deficiency and pathogen infection in winter wheat Use and of blue–green chlorophyll fluorescence measurements for differentiationbetween Journal of Plant Physiology

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Journal of Plant Physiology

j o ur na l hom e p a g e :w w w . e l s e v i e r . d e / j p l p h

Use of blue–green and chlorophyll fluorescence measurements for differentiation between nitrogen deficiency and pathogen infection in winter wheat

Kathrin Bürling, Mauricio Hunsche

, Georg Noga

UniversityofBonn,InstituteofCropScienceandResourceConservation(INRES)-HorticulturalScience,AufdemHügel6,D-53121Bonn,Germany

a r t i c l e i n f o

Articlehistory:

Received10November2010

Receivedinrevisedform26March2011 Accepted28March2011

Keywords:

Leafrust Mineralnutrition Powderymildew Sensorapplication Stressdifferentiation

a b s t r a c t

Inrecentyears,severalsensor-basedapproacheshavebeenestablishedtoearlydetectsingle plant stresses,butthechallengeofdiscriminatingbetweensimultaneouslyoccurringstressorsstillremains.

Earlierstudiesonwheatplantsstronglyaffectedbypathogensandnitrogendeficiencyindicatedthat chlorophyllfluorescencemightbesuitedtodistinguishbetweenthetwostressors.Nevertheless,thereis lackofinformationonthepre-symptomaticdetectionofsynchronizedoccurrenceofslightN-deficiency andtheearlystagesofpathogeninfection.Theusefulnessoftheblue,green,andyellowfluorescence signalsinthiscontexthasnotyetbeenexplored.Wehypothesizedthatdifferentiationbetweenwheat plants’physiologicalreactionduetoN-deficiencyandleafrust(Pucciniatriticina)aswellasN-deficiency andpowderymildew(Blumeriagraminisf.sp.tritici)mightbeaccomplishedbymeansofUVlaser-induced fluorescencespectralmeasurementsbetween370and620nminadditiontochlorophyllfluorescence (640–800nm).PlantswereprovidedwitheitheranormaloramodifiedHoaglandnutrientsolutionin ordertoinduceaslightNdeficit.Pathogeninoculationwascarriedoutonthesecondfullydeveloped leaf.Fourexperimentalgroupswereevaluated:(a)N-full-supply[N+];(b)N-deficiency[N−];(c)N- full-supply+pathogen[N+/LR]or[N+/PM];(d)N-deficiency+pathogen[N−/LR]or[N−/PM].Theresults revealedthat,inadditiontotheamplituderatioofR/FRfluorescence,B/Gfluorescencealsofacilitated reliableandrobustdiscriminationamongthefourexperimentalgroups.Thediscriminationamongthe experimentalgroupswasaccomplishedasearlyasoneandtwodaysafterinoculationforpowdery mildewandleafrustinfection,respectively.Duringthe3daysevaluationperiod,thedifferencesamong thetreatmentgroupsbecamemoreevident.Moreover,severalotheramplituderatiosandhalf-bandwidth ratiosprovedtobesuitedtoearlydetectfungalinfection,irrespectiveofthenitrogenstatusoftheplant.

© 2011 Elsevier GmbH. All rights reserved.

Introduction

Theincidenceofdiseaseanddeficiencyofnutrientsrepresent- ingbioticandabioticstresses,respectively,arethelimitingfactors forcropproductionworldwide.Asestimated,thepotentialofyield lossofwheatduetofungalpathogensmightamountto15%under specificconditions(OerkeandDehne,2004).Duringwheat’slife cycle,wheatplantsareofteninfectedbythebiotrophicfungiPuc- ciniatriticinaandBlumeriagraminisf.sp.tritici,causingleafrustand powderymildew,respectively.Ontheotherhand,nitrogenisakey

Abbreviations:A,absorbance;AOTF,acusto-optictunablefilter;B,blue;B.grami- nis,Blumeriagraminis;Chlt,totalchlorophyll;dai,dayafterinoculation;DMSO, dimethylsulfoxide;F,fluorescence;FR,far-red;FW,freshweight;G,green;hai, hoursafterinoculation;hbw,half-bandwidth;LR,leafrust;N−,N-deficiency;N+, Nfull-supply;P.triticina,Pucciniatriticina;PAR,photosyntheticactiveradiation;

PM,powderymildew;PMT,photomultiplier;R,red;RD,resistancedegree;SVM, SupportVectorMachines;UV,ultra-violet;UV–VIS,ultra-violet–visible.

∗Correspondingauthor.Tel.:+49228736540;fax:+49228735764.

E-mailaddress:MHunsche@uni-bonn.de(M.Hunsche).

elementinplantnutrition(Marschner,2005),anditsadequatesup- plyisthemostimportantnutritionalprocessafarmercanmanage incultivatedcrops(McMurtreyetal.,1994).

Inrecentyears,withadvancedtechnology,sensingofstress- induced alterations of metabolism and crop physiology have becomeincreasingly ofinterest todetectmodificationsat early stagesbeforeextensiveplantdamageoccurs.Forthispurpose,sev- eralnon-destructiveapproaches,e.g.fluorescence,reflectance,and thermal-imagingmeasurements,havebeenevaluatedandadopted for thefast and early detection of individual stresses, suchas diseases(e.g.Bodriaet al.,2002;Bravoetal.,2003;Frankeand Menz,2003;Lindenthaletal.,2005;Kuckenbergetal.,2009b)and mineraldeficiency, and especially the nitrogenstatus of plants (e.g.Bredemeieretal.,2003;Schächtletal.,2005;Subhashand Mohanan,1994;TartachnykandRademacher,2003;Buschmann, 2007).Inadditiontopromisingresults,thespecificityofthemea- suring system and theparticularities of the experiments must beconsideredwhenevaluatingorcomparingsuitabletechniques.

As recently shown, the chlorophyll fluorescence imaging tech- niquesappeartobemoresensitivethanthermalimagingforearly 0176-1617/$–seefrontmatter© 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.jplph.2011.03.016

detectionofpathogeninfectionandnutrientdeficiency(Chaerle etal.,2007a).Bothnitrogendeficiencyandpathogeninfectionare accompaniedbyadecreaseinchlorophyllcontent(Tartachnykand Rademacher,2003).

Ingeneral,reliabledetectionofstresscanbeachievedwhen evaluatingbioticandabioticstressesassinglefactors.However,it isnotunusualthatseveralstressesinfluencetheplantphysiology simultaneously.Despiteconsiderableadvances,reliablediscrimi- nationbetweenbioticandabioticstressesusingnon-destructive techniquesremainsachallenge.Tartachnyketal.(2006).showed that discrimination betweenstrong N-deficiency and pathogen infectionatadvancedstagescanbeaccomplishedonthebasisof fluorescencepeakratioF690/F730.However,asshownforacross- validation analysis of chlorophyll fluorescence, diseased leaves couldbemisidentified as N-deficiency and vice versa, whereas theclassificationwasimprovedwhenthestandard deviationof themeanwasalsoconsideredasaparameterfordiscrimination (Kuckenbergetal.,2009a).Unfortunately,theseconclusionswere basedonpathogeninfectionandN-deficiencyevaluatedondiffer- entleaves,whereasresearchstudyingbothstressorsconcomitantly onthesameplantsisscarce.

When exposed to stresses, specific pigments and other moleculesmightbesynthesized,accumulatedordegraded,hav- inganindirectordirectinfluenceonthefluorescencesignature.In general,nitrogendeficiencyleadstolesschlorophyllinthetissues (Ciompietal.,1996).Furthermore,nitrateavailabilityinfluences notonlythechlorophyllconcentrationandthereforechlorophyll fluorescence,but also phenol and lignin production, which are reducedinwheatshootsbyhighnitratelevels(Brownetal.,1984).

Wheninfected withfungalpathogens,plants mightaccumulate specificsubstances, suchas salicylicacid and phenylpropanoid compounds(e.g.cinnamicacid,stilbens,coumarinsandflavonoids) as the most important substances in plant disease resistance (Chaerle et al.,2007b; Lenket al.,2007).Accordingly,the fluo- rescenceintheblue–greenspectralrangemightyieldpromising results, since it hasbeen proven to bevery sensitive tosingle stresseventsreflecting,amongothers,accumulationofsecondary metabolites(LichtenthalerandMiehé,1997;Cerovicetal.,1999;

Buschmannetal.,2009).However,thesuitabilityofthefluores- cenceoutcomeintheblue,green,andyellowspectral rangefor discriminatingstressorsisnotyetproven.Therefore,wehypoth- esized that differentiation betweenwheat plants’ physiological reactionsduetoN-deficiencyandleafrust(Pucciniatriticina)as wellasN-deficiencyandpowderymildew(Blumeriagraminisf.sp.

tritici)mightbeaccomplishedbymeansofUVlaser-inducedflu- orescencespectral measurementsintheblue,greenandyellow range(370–620nm)in additiontothechlorophyllfluorescence (640–800nm).WefocusedonaslightN-deficiencyandtheearly stages of pathogen infection, based on the need of sensors to detectpre-symptomaticstresssignals.Ofprimaryinterestwasthe basicsuitabilityofcombinedspectralinformationbyevaluationof severalfluorescenceratiosforconsiderationinfuturefieldexperi- mentsrequiringmorecomplexanddevelopeddetectionsystems.

Materialsandmethods

Plantmaterial

Experiments were conducted in a controlled-environment cabinetsimulatinga14-hphotoperiodwith200␮Mm⁻²s⁻¹pho- tosyntheticactive radiation (PAR;Philips PL-L36W fluorescent lamps,Hamburg,Germany),day/nighttemperatureof20/15±2^◦C andrelativehumidityof75/80±10%.Winterwheat(Triticumaes- tivum L. emend. Fiori.et Paol.)seeds of the leaf rust(LR) and powderymildew(PM)susceptiblecultivarRitmoweresownin individualpots(5seedsperpot)filledwithperlite.Accordingto

thedescriptive varietylistof theGermanFederal Plant Variety Office(2008),Ritmoisclassifiedwitharesistancedegree(RD)of 8forleafrustandRD=5forpowderymildew,inaclassification rangefromone(resistant)tonine(susceptible).Inoculationofsin- gleleaveswitheitherleafrustorpowdery mildewwascarried outonthesecond fullydeveloped leaf,twentydays aftersow- ing. Experiments with combined nitrogen supply and leafrust orpowderymildewinoculationwereconductedseparately and repeatedatleasttwice.Accordingly,theexperimentalsetupwas asfollows:(a)N-full-supply[N+];(b)N-deficiency[N−];(c)N-full- supply+pathogen[N+/LR]or[N+/PM];(d)N-deficiency+pathogen [N−/LR]or[N−/PM].Ineachexperiment,thenumberofreplications wasn=12forthenitrogentreatments,and n=16forthenitro- gen+pathogentreatments.Pathogeninoculationwasperformed ontwoplantsperpot.

Fertilizationandchlorophylldetermination

Emerging plants were provided with either a standard or a modified Hoagland nutrient solution; the first contained all mineralnutrientsforoptimalplantgrowthanddevelopment,and thesecond wasadjusted toinducenitrogen deficiency. Several pre-experiments with defined amounts of nitrogen were con- ductedinordertodeterminetheappropriateNconcentrationto induceaslightNdeficitthatisnotevidentbyvisualobservation.

The full nitrogen supply solution (N+) contained 236.16gL⁻¹ Ca(NO₃)₂·4H₂O, 57.54gL⁻¹ NH₄H₂PO₄, and 67.74gL⁻¹ KNO₃ whereastheN-deficiencysolutioncontained68.05gL⁻¹KH2PO4, 74.55gL⁻¹ KCl, 126.12gL⁻¹ Ca(NO₃)₂·4H₂O, and 13.2gL⁻¹ (NH₄)₂SO₄.Consequently,thedeficiencysolution(N−)contained 40% of the N-amount of the standard solution. The content of micronutrientswassimilarinbothfertilizationsolutions.

Leavesatthesamedevelopmentalstageasthoseusedinthe main experiments were sampled and the nitrogen status was evaluatednon-destructivelywithachlorophyllmeter(SPAD502, KonikaMinolta, Langenhage, Germany) on theadaxial sides of theleavesbymeasuringred(∼650nm)andinfra-red(∼940nm) lighttransmission.Fromthesameleaves,chlorophyllcontentwas extractedfrom1cm²leafpieceswithdimethylsulfoxide(DMSO) andanalyticallydeterminedasdescribedelsewhere(Blanke,1992).

Theabsorbanceofextractswasevaluatedat665nm(A665)and 647nm(A647)withaUV–VISspectrophotometer(Perkin-Elmer, Lambda5,Massachusetts,USA).Totalchlorophyll(Chlt)concen- tration was calculated on fresh-weight basis according to the equation:Chlt=17.9×A647+8.08×A665.

Pathogeninoculation

InoculationofPucciniatriticina

Inoculationwascarriedoutwithanon-specificmixtureofPuc- ciniatriticinasporesproducedonwheatwithoutknownresistance genes(INRES-Phytomedicine,Universityof Bonn).Beforeeach experiment, fresh P.triticinaspores were suspended in a solu- tionofdistilledwater+Tween20(0.01%,w/v;Merck-Schuchardt, Hohenbrunn,Germany). Thesporeconcentrationwasestimated microscopically with a Fuchs-Rosenthal counting chamber and adjustedto1×10⁴sporesmL⁻¹.Oneach leaf,themiddleofthe leaflengthwasmarkedontheadaxialsidewithafelttippen,and seven6-␮Ldropletsofsporesuspensionwereappliedinarowon oneleafhalf(Fig.3).Priortotheapplication,leaveswerefixedhor- izontallyonasampleholdertopreventdropletrun-off.Duringthe inoculationperiod(22h),plantsweremaintainedintheclimate chamberatalmostwatervaporsaturatedatmosphereensuredby aplasticcover.Thereafter,theplasticcoverwasremovedandthe leaveswerereleasedfromtheirhorizontalfixation.Plantsofthe groupswithoutpathogeninoculationwerehandledsimilarlybut

treatedwithwaterdroplets+Tween20(0.01%,w/v).Fluorescence measurementswereperformedonthecentralofthesevendroplet applicationsites.Thedevelopmentofdiseasespotswasevaluated visuallyovertheexperimentalperiodinsituandondigitalpho- tographstakeninparalleltothefluorescencereadings.

InoculationofBlumeriagraminis

Similarto themethods described for theinoculation ofleaf rust,thetargetleaveswereselectedandthemiddleofleaflength wasmarkedwithafelttippen.Markedleaveswerehorizontally fixedbeforeinoculationwithconidiaofanon-specificmixtureof Blumeriagraminisf.sp.triticiproducedonwheatwithoutknown resistancegenes(INRES-Phytomedicine,UniversityofBonn).Stock plantsinoculatedwiththepathogenensuredthesupplyoffresh conidiawhenneeded.Forinoculationofexperimentalplants,coni- diawerecarefullyremovedfromthestockplantswithafinebrush anddirectlyappliedontheleafsurfaceofthetargetplants.Appli- cationsite(3×5mm)waslocatedattheleaflengthmiddleinthe centreofaleafhalf.Twenty-twohoursafterinoculation(hai),visi- bleconidiawereremovedbygentlyblowingandbrushingoverleaf surface.LeavesoftheplantgroupsN+andN−werehandledina similarwaywithoutconidia.

Fluorescencemeasurements

Fluorescence measurements were carried out using a com- pactfiber-opticfluorescencespectrometerwithnanosecondtime resolution and employing the boxcar technique (IOM GmbH, Berlin,Germany). ApulsedN₂ laser(MNL100, LTBLasertechnik BerlinGmbH,Berlin,Germany) withanemissionwavelengthof 337nm and a repetition rateof 20Hz served asthe excitation source.The fiber-optic probe for detection of fluorescence sig- nals consistedof a central excitationfiber and six surrounding emissionfibers,eachwitha200-␮mdiameter.Thepulseenergy attheprobe exitwasadjusted tobein therangeof 1.5–3.0␮J witha pulselengthof approximately2.5ns resultingin a den- sityof7.5–15×10¹⁵photonspercm²andpulse.Fluorescencewas recordedwithanacousto-optictunablefilter(AOTF)monochroma- tor,whichenablesaminimalstepwidthof1nm.Aphotomultiplier (PMT,H5783-01,Hamamatsu,HamamatsuCity,Japan)wasusedas detector.ThesensitivityofthePMTwasadjustedtooptimizethe signalintensityduringthespectralmeasurements.Timeresolution wasaccomplishedusingagatedintegratorwitha2-nshalf-width;

positioningofthegateallowedanaccuracyof0.1ns.

Detectionof fluorescence spectra wascarried out on leaves fixedhorizontallyonaplatewithintegratedsampleholder.The fiber-opticprobe was positionedat a 90^◦ angleto theleaf. By employing a laser-based rangefinder (OptoNCDT 1300; Micro- EpsilonMesstechnikGmbH&Co.KG,Ortenburg,Germany)fixed besidetheprobe,thedistancebetweenleafandprobesurfacewas adjustedto3.95mmatthepointofmeasurement.Thestandard distanceenabledfluorescenceintensitiesinanarrowdatarange, providingaminimumofsignalintensityandavoidingsignalsat- uration.Spectraweremeasuredat21–23^◦Cunderambientlight conditions(about 18␮Mm⁻²s⁻¹ PAR) attwo tofourdaysafter pathogeninoculation(dai)forleafrustexperiments,and oneto threedaiforpowderymildewexperiments.Priortofluorescence measurements,plantswereadaptedfor0.5htoroomconditions.

Foroptimizationoffluorescencesignals,equipmentsettingswere adjustedasfollows.Spectralanalysiswasaccomplishedatawave- lengthintervalof2nmbetween370and800nmwithagateposi- tionat5ns(inthetemporalsignalmaximum).Measurementswere donewithapulsecountof32,whichisthenumberoflaserpulses averagedforeachsingledatapoint.ThePMTsensitivitywassetto 600Volt.Fluorescencepeaksweredeterminedat451nm(blue,B), 522nm(green,G),689nm(red,R),and737nm(far-red,FR).

Fig.1. Exampleoffluorescencespectra(370–800nm)recordedfromahealthy wheatleafoftheN-full-supplytreatmentgroupwithafluorescencespectrometer withnanosecondtimeresolutionusingapulsednitrogenlaser(337nm)asexcita- tionsource.Thetrianglesindicatethemeasuredfluorescenceemissionspectrum, thedottedlinesdisplaytheindividualGaussianspectralcomponentsofthefitted spectrum,andthesolidlineshowsthefittedspectrum.

Dataprocessingandstatistics

The measured laser-induced fluorescence spectra were pro- cessed by Gaussian curve fitting using the freeware Gnuplot (version4.2patchlevel4,http://www.gnuplot.info,GeeknetInc., MountainView,CA,USA),asindicatedinFig.1.Position,ampli- tudesaswellashalf-bandwidths(hbw)ofpeaksweredetermined tocalculatetheratiosbetweenamplitudes,half-bandwidths,and amplitudes-to-half-bandwidths(*)forindividualpeaks.Thepro- cessed experimentaldata were subjected to statistical analysis usingtheSPSSpackage(SPSSInc.,Chicago,USA)version18.0.The relationbetweenSPADandchlorophyllcontentwasestablished withalinearregression.Foreachdayandevaluationgroup,the meanswerecomparedbyANOVA(p≤0.05)andmeansseparated withtheDuncan’smultiplerangetest.Graphs(mean±SD)were drawnwithSigmaPlot 8.02(SystatSoftwareInc.,Richmond,CA, USA).

Results

ValidationofN-deficiency

ThechlorophyllcontentofplanttreatmentswithN+andN− wasevaluatedtoshowthattheN− treatmentgroupwasnitro- gendeficientevenifvisualsymptomswerenotevident.On the basisofSPAD-valuesandchlorophyllextraction,alinearfunction expressedasChl[␮gg⁻¹FW]=53.34×SPAD−248.024(r²=0.93) wasestablished(Fig.2).However,theusefulnessoftheproposed linearfunctionmightbelimitedtoourexperimentalconditions (hydroponiccultivationofthewheatcultivarRitmoreceivingeither fullN-supplyor40%ofN),whereas forawide rangeofchloro- phyll concentrationsand SPADvalues, non-linear curves seems tobe more appropriate (Uddling et al., 2008).On average, leaf chlorophyll concentrations of N+ plants were 2311␮gg⁻¹FW, whereas the leavesof the N− treatmentgroup had a mean of 1698␮gg⁻¹FW.Asidefromthissignificantdifference,thevisual assessmentofN-deficiencyleavesrevealednodistinctstresssymp- toms(Fig.3A).

Fig.2.CorrelationbetweenSPADvaluesandchlorophyllcontentofwheatleaves asaffectedbytwolevelsofnitrogensupply(n≥6).

Fig.3.Digitalphotographsofwheatleavesaffectedbyabioticorbioticstressfactors:

(A)influenceofnitrogenfertilization,N-full-supply(N+)andN-deficiency(N−);(B) leavesinfectedbyleafrust,fourandeightdaysafterinoculation(dai);(C)infection ofpowderymildewatfourandeightdaysafterinoculation.

Combinednitrogendeficiencyandleafrustinfection

Visualevaluationsofleafrustdevelopmentindicatedsmalland loomchloroticspots4dai(daysafterinoculation)ontheadaxialleaf laminainbothnitrogenfullsupply(N+)andnitrogendeficient(N−) leaves(Fig.3B).Twodayslater(6dai),smallred-brownpustules appearedontheleafsurfaceandbecamelargerandmoredistinct inthefollowingdays.After8dai,diseasesymptomswereevident (Fig.3B).

The spectrally resolved fluorescence measurements and the identificationofpeakmaximaat451nm(B),522nm(G),689nm (R),and737nm(FR)(Fig.1)allowedustocalculatesixamplitude

Table1

Influenceofnitrogensupply(N+,full-supply;N−,40%offull-supply)andleafrust (LR)inoculationonselectedfluorescenceratios,determinedfromtwotofourdays afterinoculation(dai).

Fluorescenceratio Experimentalgroup 2dai 3dai 4dai

B/R

N+ 2.98a 2.55a 2.24a

N+/LR 3.53b 3.09b 2.91b

N− 3.01a 2.55a 2.29a

N−/LR 3.37b 3.04b 3.00b

B/FR

N+ 2.81a 2.34a 2.06a

N+/LR 3.50b 3.22b 3.38b

N− 2.90a 2.44a 2.20a

N−/LR 3.49b 3.37b 3.80b

G/R

N+ 0.77a 0.68a 0.62a

N+/LR 0.96b 0.93b 1.00b

N− 0.80a 0.71a 0.65a

N−/LR 0.96b 0.97b 1.09b

G/FR

N+ 0.72a 0.62a 0.57a

N+/LR 0.95b 0.97b 1.17b

N− 0.77a 0.67a 0.63a

N−/LR 1.00b 1.07b 1.39b

B/Ghbw

N+ 1.07b 1.06b 1.05b

N+/LR 1.06a 1.03a 1.01a

N− 1.08b 1.06b 1.06b

N−/LR 1.06a 1.03a 1.01a

G/FRhbw

N+ 1.52a 1.52a 1.53a

N+/LR 1.55b 1.58b 1.59b

N− 1.51a 1.52a 1.52a

N−/LR 1.54b 1.56b 1.58b

R/FRhbw

N+ 0.79a 0.78a 0.78a

N+/LR 0.80b 0.79b 0.79b

N− 0.79a 0.79a 0.78a

N−/LR 0.79b 0.79b 0.79b

FR*

N+ 923b 956b 975b

N+/LR 796a 784a 739a

N− 920b 933b 941b

N−/LR 788a 753a 694a

Meansofthefluorescenceparametersforeachevaluationdayfollowedbythe sameletterdonotdiffersignificantlyaccordingtoDuncan’smultiplerangetest (p≤0.05;n=12forN+andN−;n=16forN+/LRandN−/LR).hbw=half-bandwidth,

*=amplitude-to-half-bandwidthratio.

and sixhalf-bandwidths ratios(B/G, B/R, B/FR,G/R,G/FR, R/FR) aswellasfouramplitudes-to-half-bandwidthratios(B,G,R,FR).

However,notallratiosareappropriatetodetectN-deficiencyand pathogeninfectiononthesameleaves.Wethereforefocusedon themostpromisingones.AsshowninTable1,severaloftheexam- inedfluorescenceratiosfacilitatedreliablediscriminationbetween healthyandinoculatedleavesfrom2to4dai,irrespectiveofnitro- genfertilization.AmplituderatiosofB/R,B/FR,G/RandG/FRwere significantlyhigherininoculated thaninnon-inoculatedleaves.

Twodai,valuesforB/Rwere2.98and3.01forN+andN−,and3.53 and3.37forN+/LRandN−/LR,respectively(Table1).Inasimilar trend,valuesoftheG/Rratiowere0.77and0.80forN+andN− and0.96forbothnitrogenvariantsinoculatedwiththeleafrust pathogen.Inaddition,thepathogeninoculationreducedtheB/G andincreasedtheG/FRhbwandR/FRhbwratios(Table1).Ofall theevaluatedamplitude-to-half-bandwidthratios,acleardiffer- encebetweenthetreatmentgroupswasobservedfortheFR*peak, showingvaluesof923(N+),920(N−),796(N+/LR)and788(N−/LR) attwo daysafter inoculation(Table1).The differencebetween healthyandinoculatedleavesbecameslightlygreaterduring3days ofmeasurements,asindicatedbyvaluesofB/R(Table1).

Todistinguishamongthefourexperimentalgroups,twofluo- rescenceratioswereconsideredaspromisingparameters.Onall thethree measuringdays(2–4dai)theamplituderatioB/Gwas significantlydifferentamongthefourgroups(Fig.4A).Atthe2nd dai,leavesofN+plantshadthehighestvalues(3.89),followedby N−(3.75),N+/LR(3.68),andN−/LR(3.50)(Fig.4A).Inalltreat- ments,valuesdecreased overtime due toleafaging andatthe 4th dai, one daybeforefirstvisible symptoms appeared, ratios

Fig.4. Influenceofnitrogensupply(N+,full-supply;N−,40%offull-supply)anddiseasedevelopmentontheratiosoffluorescenceamplitudesB/GandR/FR.(AandB)Leaf rust(LR)infectionmeasuredfromtwotofourdaysafterinoculation(left);(CandD)powderymildew(PM)infection,measuredfromonetothreedaysafterinoculation (right).Means(±SD)oftheexperimentalgroups(withineachevaluationday)followedbythesameletterdonotdifferstatisticallyaccordingtoDuncan’smultiplerangetest (p≤0.05;n=12forN+andN−;n=16fortheothertreatmentgroups).

reached3.64forN+,3.50forN−,2.93forN+/LRand2.77forN−/LR (Fig.4A).Consequently,thedifferencebetweenN+andN−aswell asbetweenN+/LRandN−/LRremainedthesame,butthedifference betweeninoculated and non-inoculatedleavesbecame greater.

MeasurementsofthechlorophyllfluorescenceintheRandFRpeaks indicatedslightlyhigheramplituderatiosR/FRininoculated(N+, 0.94;N−,0.96)thaninnon-inoculated(N+/LR,0.99;N−/LR,1.04) (Fig.4B)leaves.ValuesforN+andN−remainedalmostconstant, whiletheinoculatedleavesshowedastrongincrease(Fig.4B).

Combinednitrogendeficiencyandpowderymildewinfection Visual evaluations of powdery mildew development first revealedsmallpatchesof whitishmyceliumontheleafsurface atfourdaysafterinoculation(Fig.3C).Duringthefollowingdays the patches increased in size, and new mycelia were formed.

Aswithleafrust,alargenumberofamplitude,half-bandwidth, andamplitude-to-half-bandwidthratioswerecalculated,butwe focused onthose parameters showing robustness for detection anddifferentiationoftheevaluatedstressfactors.Astheresults of the fluorescence measurements show, irrespective of nitro- gen level, only a small number of the evaluated fluorescence ratiosweresuitedtodiscriminatebetweenhealthyandinoculated

leaves(Table2).Duringthe3 dayperiod ofmeasurements,the half-bandwidthratiosB/G,G/RandG/FRrevealedconstantdiffer- encesbetweeninoculated andnon-inoculatedleaves.Asshown for theB/Ghbwat1dai,powdery mildewloweredtheratio to

Table2

Impactofnitrogensupply(N+,full-supply;N−,40%offull-supply)andpowdery mildew(PM)inoculationonselectedfluorescencehalf-bandwidthratios,deter- minedfromonetothreedaysafterinoculation(dai).

Fluorescenceratio Experimentalgroup 1dai 2dai 3dai

B/Ghbw

N+ 1.07b 1.06b 1.05b

N+/PM 1.00a 0.95a 0.94a

N− 1.07b 1.05b 1.04b

N−/PM 0.99a 0.96a 0.94a

G/Rhbw

N+ 1.95a 1.97a 1.97a

N+/PM 2.08b 2.16b 2.19b

N− 1.94a 1.97a 1.97a

N−/PM 2.09b 2.14b 2.16b

G/FRhbw

N+ 1.51a 1.52a 1.52a

N+/PM 1.60b 1.68b 1.70b

N− 1.51a 1.52a 1.53a

N−/PM 1.61b 1.67b 1.69b

Meansofthefluorescenceratiosforeachevaluationdayfollowedbythesameletter donotdiffersignificantlyaccordingtoDuncan’smultiplerangetest(p≤0.05;n=12 forN+andN−;n=16forN+/LRandN−/LR).hbw=half-bandwidth.

1.00(N+/PM)and0.99(N−/PM)ascomparedto1.07inthenon- inoculated leaves (Table 2). In contrast, ratios of G/Rhbw and G/FRhbwwerehigherinB.graminisinoculatedleavescompared tonon-inoculatedleaves.TheG/RhbwratioforN+andN−leaves at1daiwereof1.95and1.94,respectively,contrastingto2.08and 2.09intheinoculatedleaves(Table2).Duringthefollowingtwo days,thedifferencebetweenhealthyandinoculatedleavesbecame morepronounced,andonedayaheadoffirstvisibleinfectionsymp- toms(3dai),theG/Rhbwratioincreasedto1.97forbothN+and N−,2.19forN+/PMand2.16forN−/PM(Table2).

Fig.4CandDdisplaysthetime-coursedevelopmentoftheB/G andR/FRamplituderatiosofthefourexperimentalgroups(N+,N−, N+/PM,N−/PM).Duringthewholeevaluatedperiod,plantssup- pliedwithadequatenitrogenshowedthehighestvaluesforthe B/Gratio,followedbytheN−group,N+/PM,andfinallyN−/PM.

Onthefirstmeasuringday(1dai)amplituderatiosofB/GforN+

andN−wereof3.73and3.55respectively,whereasN+/PMand N−/PM leaves indicated significantly lower values of 3.19 and 3.06,respectively(Fig.4C).Duringthefollowingtwodaysvalues decreasedinallexperimentalgroupsbutthedifferencebetween inoculatedandnon-inoculatedleavesremainedalmostconstant.

Onthelastevaluationday(3dai)valuesonN+andN−were3.48 and3.31andforinoculatedones,N+/PMandN−/PM,2.76and2.66, respectively(Fig.4D).Similarly,valuesoftheratioR/FRonhealthy leavesremainedat acomparable level,whereas infectedleaves showedasignificantincrease.Alreadyat1dai,significantdiffer- encesbetweentreatmentgroupswerenoted,andvalueswereof 0.98and1.02 forN+andN−,and1.07 and1.15forN+/PMand N−/PM(Fig.4D).At3daivaluesforN+andN−werestillat0.96 and1.00,whereasthegroupsN+/PMandN−/PMindicatedratios of1.09and1.19,respectively(Fig.4D).

Discussion

Theobjectiveofthecurrentstudywastoevaluatethefeasibil- ityofspectralresolvedfluorescenceforsimultaneousdetectionof slightN-deficiencyandpathogeninfectiononthesameleavesat apre-symptomaticstage.Basedonthefluorescencepeaksinthe blue,green,redandfar-redregionsasdisplayedinFig.1,ampli- tude, half-bandwidth, and amplitude-to-half-bandwidth ratios wereestablished.Theoutcomewasthatseveralfluorescenceratios mightbeconsideredfordetectionanddifferentiationbetweenthe stressors.However,incontrasttothedifferentiationbetweenN- deficiencyandleafrust(Table1andFig.4A,B),onlyafewratios are suitable to differentiate N-deficiency and powdery mildew (Table2andFig.4C,D).Inadditiontotheearlydetectionofleaf rustandpowderymildewinfectionandslightnitrogendeficiency withthechlorophyllfluorescence,theamplituderatioR/FRisalso suited for simultaneous detection of both factorson the same leaves. Moreover, the blue–green fluorescence amplitude ratio (B/G)yieldsmorepreciseresultswhendistinguishingamongthe fourexperimentalgroupsN+,N−,N+/pathogen,andN−/pathogen.

Asobservedinbothpathosystems,leafrustandpowderymildew, thedifferencebetweenvaluesofthehealthyandinoculatedplants became morepronounced in the time-courseof the infection’s development.

Itis wellknownthatwheatplants grownunderreduced N- supplyexhibitlowerchlorophylllevelscomparedtoplantsgrown underfullNsupply(Cartelatetal.,2005),whereasthechlorophyll contentcanbeestimatedbyhandheldchlorophyllmeters(Uddling etal.,2008).Ourresultsrevealedareductionofchlorophyllcon- tent(Fig.2)and anassociated increaseoftheR/FRfluorescence ratio(Fig.4),whichisaninverseindicatorofthechlorophyllcon- tent(Buschmann,2007).Astrongnegativecorrelationcoefficient (r²=−0.86) betweenthechlorophyllcontentandtheamplitude

ratioR/FRwasestablished(datanotshown).Moreover,ouranal- yses showed a strong positive correlation coefficient (r²=0.90) betweenthechlorophyllcontentandtheB/Gamplituderatio(data not shown).Lichtenthaleret al. (1997)associated a decrease in theF440/F520ratiowiththechangeofchlorophyllcontentper leafarea,whereastheincreaseofbluefluorescencewithdecreas- ingchlorophyllandcarotenoidcontentwasdiscussedintermsof reducedre-absorptioneffects.

ExperimentswithbarleyandwheatgrownunderN-deficiency revealed close relations between the accumulationof phenolic metabolitesand changes of chlorophyll content, and modifica- tionsinUV-inducedfluorescencesignature(Mercureetal.,2004;

Cartelatetal.,2005).AsaconsequenceoflowerNsupplyinbar- ley, the amount of total soluble phenolic compounds and the blue–greenfluorescenceincreased(Mercureetal.,2004).In our study,theB/Gfluorescenceamplituderatiodecreasedwithless Nsupply(Fig.4).Thisisconsistentwiththefindingsof(Belanger etal.,2006)who pointedoutthattheratioF440/F520revealed differencesbetweenpotatoplantsfertilizedwithseveralnitrogen levels.Inourstudy,thedecreaseofB/Gvaluesforplantsgrown underN-deficientconditionsisexplainedbya combinationofa smallincreaseinbluefluorescence(2–3%)andamorepronounced increaseingreenfluorescence(6–7%).

Generalassociationsoftherelationshipbetweennitrogensta- tusandfungal infectionsuggestthat higherN supplyincreases the susceptibility of cereals to pathogens such as mildew and rusts(WaltersandBingham,2007).Alternatively,lownitrogenlev- elswereassociatedwithhigheramountsofphenolsandreduced disease intensity (Cartelat et al., 2005), as phenols are known toplay animportantrole in diseaseresistance(e.g. Hermsand Mattson,1992;NicholsonandHammerschmidt,1992;Vermerris and Nicholson,2006).In addition,thesynthesis and accumula- tion of suchcompounds depends onthe time scale. As shown previously,slightlyincreasedlevelsofboundandunboundhydrox- ycinnamicacidduetopowderymildewinfectionunderlowand mediumN-supplyweremeasuredalready20hafterinoculation (Sander and Heitefuss, 1998). In the present studies with leaf rustandpowderymildew, thedifferences inB/Gratiobetween healthy and infected leavesbecame larger when infection was furtherdeveloped (Fig.4).Nevertheless,thedifferencebetween N−/pathogenandN+/pathogenleavesremainedatthesamelevel.

Asobservedin ourstudies,absoluteintensitiesof rustinfected leavesincreasedonaveragefrom4%(dai2)to23%(dai4)forthe blueandfrom11%(dai2)to55%(dai4)forthegreenfluorescence ascomparedtothenon-inoculatedtissue,irrespectiveofnitrogen supply.

Alternatively,re-absorption effects ofblue fluorescencelight bychlorophyll(Langetal.,1991),aswellasapossibleshielding effectoftheexcitationlightbyphenolicslocatedintheepider- mis(ChaerleandVanDerStraeten,2000)shouldbeconsidered.

TheobservedincreaseoftheR/FRratioinourexperiments(Fig.4) indicatesadecreasein chlorophyllcontentinplants inoculated withleafrustorpowderymildew(Buschmann,2007),which is inaccordancewithLorentzenandJensen(1989)andOweraetal.

(1981).

Asreportedbyseveralauthors(McMurtreyetal.,1994;Heisel et al., 1996; Mercure et al., 2004), several computed fluores- cenceratiostheF440/F685,F440/F740,F525/F685andF740/F685 revealeddifferencesbetweenmaizeplantsfertilizedwith100%or with75%or lessnitrogen. TheratiosF440/F690and F440/F740 proved to be more sensitive to nutrient deficiencies than the F690/F740ratio(Heiseletal.,1996;Lichtenthaleretal.,1997).Our resultsdidnotconfirmthisforplantsgrownunderN-deficiency foracomparativelyveryshortperiod.Thesefluorescenceparam- etersseemtobemoresuitedtorevealearlypathogeninfection irrespectiveofthenitrogenstatus oftheplants,asobservedfor

amplitudeandhalf-bandwidthratiosintheleafrustexperiment (Table1),andofhalf-bandwidthratiosinthepowderymildewtrials (Table2).Intheexperimentwithleafrust,especiallytheamplitude ratiosG/RandG/FRaswellastheFR*amplitudetohalf-bandwidth ratioshowedastrongeffectofpathogeninfectionduringtwoto fourdaysafterinoculation(Table2).Nevertheless,changesinthese ratiosinresponsetonitrogendeficiencyareexpectedunderpro- gressedandmorepronouncedlimitationsascomparedtotheslight deficiencyconditionsinourexperiment.

Comparingbothleafrustandpowderymildewpathosystems, thedifferencebetweeninfectedandhealthyleavesfortheparam- etersB/GandR/FRamplituderatioweresmallerforleafrustthan forpowderymildew(Fig.4).Inaddition,thetime-coursedevel- opmentofthefluorescencesignalswasmorepronouncedforleaf rust.Nevertheless,both fluorescenceparameters alloweddiffer- entiationamong theexperimentalgroupsN+,N−,N+/LRorPM, andN−/LRorPM.Goingforward,improvedstatisticsandrawdata analysisunderconsiderationofclassificationalgorithmssuchas DecisionTrees,NaiveBayes,ArtificialNeural Networks,Logistic RegressionandSupportVectorMachines(SVMs)mightrendera morepreciseclassification.PreliminaryresultsindicatethatSVMs yieldaprecisediscriminationofhealthyandinfectedleaves(Römer etal.,2010).Ongoingstudieswillclarifywhethersuchdataevalua- tiontoolscansupportandimproverobustdifferentiationbetween simultaneousoccurringofbioticandabioticstresses.

Conclusion

Thefluorescencesignaturemeasuredbetween370and800nm isausefultoolwhenaddressingthechallengeofdiscrimination betweenbioticandabioticstressfactors.Thefocusonblue–green fluorescenceyieldsimportantadditionalinformationfor amore precisediscriminationascomparedtopreviousapproacheswith chlorophyllfluorescence.TheamplituderatioB/GaswellasR/FR revealedtobewellsuitedtodistinguishamongN-full-supply,N- deficiency,N-full-supply+pathogen,andN-deficiency+pathogen.

Inaddition,severalfluorescenceratiosfacilitatedearlydetection ofleafrustorpowderymildewinfectionirrespectiveoftheplants’

nitrogenstatus.

Acknowledgements

We acknowledge the German Research Foundation (DFG- research training group 722) for financial support, Dr. U.

Steiner-StenzelandDr.E.-C.Oerke(INRES-Phytomedicine,Uni- versityof Bonn) forthe Blumeriagraminisand Pucciniatriticina inoculum,andProf.Dr.H.Scherer(INRES-PlantNutrition)forthe kindsupportinadjustingthenutritionsolution.

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