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In order to manage phytosanitary risks in an ever growing and increasingly dynamic import andexportmarket, theEU7thFramework Program funded the Quarantine Barcoding of Life projecttodevelopaquick,reliableandaccurateDNAbarcode-based diagnostic tool for selected species on the EPPO A1/A2 listsandEUCouncilDirective2000/29/EC(www.QBOL.org).There are currently almost 350pest andquarantine organ-isms, covering bacteria, phytoplasmas, fungi, parasitic plants, insects and mites, nematodes, virus and virus-like organisms ontheEPPOA1(currentlyabsentfromtheEPPOregion)andA2(locallypresentbutcontrolledintheEPPOregion)listsoforganismsthatrequirestandardisedprotocolsagainstintroduc-tioninto,andspreadwithin,theEPPOregion.UnderQBOL,informativelocifromtheselectedquarantinespeciesandtheirtaxonomicallyrelatedspeciesweresubjectedtoDNAbarcoding fromvoucherspecimensinordertoproducereliableDNAbar-codesequencesthataremadepubliclyavailablethroughanonlineandsearchabledatabasecalledQ-bank(www.q-bank.eu)(Bonantsetal.2010).WithintheQBOLproject,theCBS-KNAWFungalBiodiversityCentre(Utrecht,TheNetherlands),was tasked with barcoding the Mycosphaerellacomplex(orderCapnodiales, class Dothideomycetes)ontheEPPOA1/A2listsandtheirtaxonomicallyrelatedclosestsisterspecies(Table1).
AmajorproblemwithcorrectlyidentifyingmanyoftheEPPOA1/A2-listed fungi is the fact that individual species are often named for their particularmorphs in separate publications.Dual nomenclaturemakes effective cooperation betweenscientistsandtheindividualquarantineauthoritiesverycon-fusedandcomplicated.Thedualnomenclaturalsystemwasrecently abandoned at the International Botanical Congress in Melbourne(Hawksworthetal.2011,Wingfieldetal.2012).Inaccordance with this decision, the concept ‘one fungus = one name’willbeappliedinthispaper.The Mycosphaerella generic complex comprises one of the largest families within the phylum Ascomycota, whose spe-cies have evolved as either endophytes, saprophytes and symbionts.Mostly,Mycosphaerella s.l. consists of foliicolous plantpathogenswhicharethecauseofsignificanteconomicallossesinbothtemperateandtropicalcropsworldwide(Crousetal.2001).TheMycosphaerella teleomorph morphology is relativelyconserved,butislinkedtomorethan30anamorphgenera(Crous2009).Althoughoriginallyassumedtobemono-phyletic(Crousetal.2001),phylogeneticanalysesofnumerousMycosphaerella species and their anamorphs by Hunter et al.(2006)andCrousetal.(2007)haveshownthattheMycosphaerella complexisinfactpolyphyletic.Thishassinceledto taxonomic redistribution of most of the phylogenetic clades within the complex, although several clades remain unresolved duetolimitedsampling(Crous2009,Crousetal.2009a,c).Duringthe2011FungalDNABarcodingWorkshopinAmster-dam, The Netherlands, it was decided that the internal tran-scribed spacers region (ITS) of the nrDNAoperonwas tobecometheofficialprimaryfungalbarcodinggene(Schochetal.2012).TheITSlocusiseasilyamplifiedandgivesagoodspeciesresolutioninmanyfungalgroups.LackofsufficientITSinterspecies variation within some genera of Mycosphaerella-likefungi(e.g.Septoria, Cercospora and Pseudocercospora)
DNA barcoding of Mycosphaerella species of quarantine importance to EuropeW.Quaedvlieg1,2,J.Z.Groenewald1,M.deJesúsYáñez-Morales3,P.W.Crous1,2,4
Abstract TheEU7thFrameworkProgramprovidedfundsforQuarantineBarcodingofLife(QBOL)todevelopaquick,reliableandaccurateDNAbarcode-baseddiagnostictoolforselectedspeciesontheEuropeanandMediter-raneanPlantProtectionOrganization(EPPO)A1/A2quarantinelists.Sevennucleargenomiclociwereevaluatedto determine those best suited for identifying species of Mycosphaerellaand/oritsassociatedanamorphs.Thesegenes included β-tubulin(Btub),internaltranscribedspacerregionsofthenrDNAoperon(ITS),28SnrDNA(LSU),Actin(Act),Calmodulin(Cal),Translationelongationfactor1-alpha(EF-1α)andRNApolymeraseIIsecondlarg-estsubunit(RPB2).LociweretestedontheirKimura-2-parameter-basedinter-andintraspecificvariation,PCRamplificationsuccessrateandabilitytodistinguishbetweenquarantinespeciesandcloselyrelatedtaxa.Resultsshowedthatnoneoftheselociwassolelysuitedasareliablebarcodinglocusforthetestedfungi.Acombinationofa primary and secondary barcoding locus was found to compensate for individual weaknesses and provide reliable identification.AcombinationofITSwitheitherEF-1α or Btub was reliable as barcoding loci for EPPO A1/A2-listed Mycosphaerellaspecies.Furthermore,Lecanosticta acicola was shown to represent a species complex, revealing two novel species described here, namely L. brevisporasp.nov.onPinussp.fromMexicoandL. guatemalensis sp.nov.onPinus oocarpafromGuatemala.EpitypeswerealsodesignatedforL. acicola and L. longispora to resolve thegeneticapplicationofthesenames.
Article info Received:8October2012;Accepted:2November2012;Published:13December2012.
103W.Quaedvliegetal.:DNAbarcodingofMycosphaerella M
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104 Persoonia – Volume 29, 2012
might make this locus less than ideal for resolving some an-amorph genera or cryptic species complexes within these genera(Verkleyetal.2004,Hunteretal.2006,Schochetal.2012).Tocompensateforthisperceivedlackofresolutionwithinthe ITS locus of Mycosphaerella-like species, seven loci were screened, which have individually or in combination been used in the past to successfully identify Mycosphaerella-likespecies.These include β-tubulin(Btub)(Feau etal. (2006)),internaltran-scribedspacer(ITS),Actin(Act)(Schubertetal.2007,Crousetal.Inpress),Translationelongationfactor1-alpha(EF-1α)(Schubertetal.2007,Crousetal.Inpress)and28SnrDNA(LSU)(Hunter etal. 2006),Calmodulin(Cal)(Groenewaldetal.2005)andRNApolymeraseIIsecondlargestsubunit(RPB2)(Quaedvlieg etal. (2011)).Theaimsofthisstudywereto1)identifytheclosestneighboursof seven Mycosphaerella-likespeciesofquarantineimportanceusingsequencesofboththeinternaltranscribedspacerregionsand5.8SnrRNAgeneofthenrDNAoperon(ITS).Theseisolateswerethen2)screenedwiththesevenpreviouslymentionedtestlocitodeterminethemostoptimalDNAbarcoderegion(s)basedonPCRefficiency,thesizeoftheK2Pbarcodegapsandthemolecularphylogeneticresolutionoftheindividualloci.Basedontheobtainedresultsandexistingliterature,3)thetaxonomicstatusofthesequarantinespecieswasthenrevisedemployingthe one fungus one name principle as stated by Hawksworth etal.(2011).
MATERIALS AND METHODS
Isolates and morphologyMostoftheDNAusedduringthisstudywereisolatedfrompurecultures that were either available at, or were made available to, theCBS-KNAWFungalBiodiversityCentre,Utrecht, theNetherlands(CBS).Referencestrainswereeithermaintainedin the culture collection of CBS, the Ministry of Agriculture, For-estryandFisheriesofJapanculturecollection(MAFF)and/orattheLNPV–Mycologie,Malzéville,France(LNPV)(Table1).Fresh collections were made from leaves of diverse hosts by placingmaterialindampchambersfor1–2d.Singleconidialcolonies were established from sporulating conidiomata on Petri dishescontaining2%maltextractagar(MEA)asdescribedearlierbyCrousetal.(1991).Coloniesweresub-culturedontopotato-dextroseagar(PDA),oatmealagar(OA),MEA(Crousetal.2009b),andpineneedleagar(PNA)(Lewis1998),andincubated at 25 °C under continuous near-ultraviolet light to promotesporulation.Morphologicaldescriptionsarebasedonslide preparations mounted in clear lactic acid from colonies sporulatingonPNA.ObservationsweremadewithaZeissV20Discoverystereo-microscope,andwithaZeissAxioImager2lightmicroscopeusingdifferentialinterferencecontrast(DIC)il-luminationandanAxioCamMRc5cameraandsoftware.Colonycharacters and pigment production were noted after 1 mo of growthonMEA,PDAandOA(Crousetal.2009b)incubatedat 25°C.Colony colours (surface and reverse)were ratedaccordingtothecolourchartsofRayner(1970).SequencesderivedinthisstudywerelodgedwithGenBank,thealignmentsinTreeBASE(www.treebase.org),andtaxonomicnoveltiesinMycoBank(www.MycoBank.org)(Crousetal.2004a).
Multi-locus DNA screeningGenomicDNAwasextractedfrommyceliumgrowingonMEA(Table1),usingtheUltraClean®MicrobialDNAIsolationKit(MoBioLaboratories,Inc.,SolanaBeach,CA,USA).Thesestrainswerescreenedforsevenloci(ITS,LSU,Act,Cal,EF-1α,RPB2andBtub)usingtheprimersetsandconditionslistedinTable2.ThePCRamplificationswereperformedinatotalvolumeof 12.5 µL solution containing 10–20ng of templateDNA,
1 ×PCRbuffer,0.7µLDMSO(99.9%),2mMMgCl2,0.4µMofeachprimer,25µMofeachdNTPand1.0UBioTaqDNApolymerase (BiolineGmbH, Luckenwalde,Germany).PCRamplificationconditionsweresetasfollows:aninitialdenatura-tiontemperatureof96°Cfor2min,followedby40cyclesofdenaturationtemperatureof96°Cfor45s,primerannealingatthetemperaturestipulatedinTable3,primerextensionat72°Cfor90sandafinalextensionstepat72°Cfor2min.TheresultingfragmentsweresequencedusingthePCRprimerstogetherwithaBigDyeTerminatorCycleSequencingKitv.3.1(AppliedBiosystems,FosterCity,CA).Sequencingreactionswere performed as described by Cheewangkoon etal.(2008).
Phylogenetic analysisAbasicalignmentoftheobtainedsequencedatawasfirstdoneusingMAFFTv.6(http://mafft.cbrc.jp/alignment/server/index.html(Katohetal.2002)andifnecessary,manuallyimprovedinBioEdit v.7.0.5.2 (Hall 1999).Bayesiananalyses (criticalvalueforthetopologicalconvergencediagnosticsetto0.01)wereperformedontheindividuallociusingMrBayesv.3.2.1(Huelsenbeck&Ronquist2001)asdescribedbyCrous etal.(2006b).SuitablemodelswerefirstselectedusingModelsofnucleotide substitution for each gene as determined using MrModeltest (Nylander 2004), and included for each genepartition.ThesubstitutionmodelsforeachlocusareshowninTable3.Teratosphaeria nubilosa(CPC12243)wasusedasoutgroupforallphylogeneticanalyses.
Identification of the ideal DNA barcodeThe dataset of the seven test loci was individually tested for threefactors,namelyamplificationsuccess,Kimura-2-parame-tervalues(barcodegap)andmolecularphylogeneticresolution.
Amplification successTheamplificationsuccessscoresoftheseventestlocionthe118strainsvariedfrom100%amplificationsuccessforbothITSandLSUtoonly90%forCal.Theotherfourtestloci(EF-1α,Act,RPB2andBtub)gaveamplificationsuccessscoresofrespectively97,98,99and100%(Table3).ThetestedCalprimersfailedtoamplifythequarantinespeciesPseudocercospora pinidensiflorae and several other associated Pseudocercosporaspecies.Consequently,Calisconsideredunsuitableasabarcodinglocusforthisquarantinedataset.Althoughithadaveryhighoverallamplificationsuccessrate(99%),RPB2 failed to amplify inM. populicola.AlthoughM. populicola is not a quarantine species, it is very closelyrelated andmorphologically similar to the quarantine spe-cies Septoria musiva.Thisdeficit,combinedwiththefactthatRPB2amplificationwithin thedatasetwasnot robust (oftenmultiplePCRand/or sequencing runswere needed to getgoodsequencingreads),makesRPB2unsuitabletoserveasabarcodinglocusforthequarantinedataset.Theremainingfivetestlocisuccessfullyamplifiedallquarantinespecies.
Molecular phylogeniesGeneral informationper locus for theanalysis, suchas thenumber of characters used per dataset and the selected model aredisplayedinTable3.ThetreesresultingfromtheBayesiananalyses of the seven individual loci showed that most loci have difficultydiscriminatingbetweencloselyrelatedSeptoria and Pseudocercosporaspecies.Decidingthesequencedifference
Locus Primer Primersequence5’to3’: Annealing Orientation Reference temperature (°C)
Fig. 1SubsetofBayesian50%majorityruleconsensustreesoftheindividualtestlociincorporatingallMycosphaerellaceaequarantinespecies(markedingrey)andtheirclosestneighbourspeciesasdeterminedfromthefull-scaleindividuallocitreescontainingthecompletedataset(availableassupplementarydatainTreeBASE).Thefollowingabbreviationswereusedforthegenera:T = Teratosphaeria, M = Mycosphaerella, Ph = Phaeophleospora, P = Pseudocercospora, D = Dothistroma and S = Septoria.Astoprule(setto0.01)forthecriticalvalueforthetopologicalconvergencediagnosticwasusedfortheBayesiananalyses.ThetreeswereallrootedtoTeratosphaeria nubilosa(CPC12243).Thescalebarindicates0.1expectedchangespersite.
TEF1
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Teratosphaeria nubilosa CPC 12243
L. guatemalensis IMI 281598
Mycosphaerella sp. CBS 111166
L. acicola CBS 871.95
L. acicola CBS 133789
L. acicola LNPV 243
L. longispora CPC 17940
L. longispora CBS 133602
L. brevispora CBS 133601
D. septosporum CPC 16798
D. pini CBS 116484
M. ellipsoidea CBS 110843
M. laricis-leptolepidis MAFF 410633
M. sumatrensis CBS 118501
P. angolensis CBS 149.53
P. sphaerulinae CBS 112621
S. citri CBS 315.37
S. malagutii CBS 106.80
M. populicola CBS 100042
S. musiva CBS 130558
P. pini-densiflorae CBS 125138
P. pyracanthigena CPC 10808
0.1
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LSU
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Teratosphaeria nubilosa CPC 12243
P. angolensis CBS 149.53
P. pini-densiflorae CBS 125139
P. pyracanthigena CPC 10808
M. endophytica CBS 111519
M. ellipsoidea CBS 110843
M. sumatrensis CBS 118501
M. laricis-leptolepidis MAFF 410633
P. clematidis CPC 11657
L. brevispora CBS 133601
D. pini CBS 116484
D. septosporum CBS 383.74
L. guatemalensis IMI 281598
L. longispora CPC 17940
L. longispora CBS 133602
S. malagutii CBS 106.80
S. cucurbitacearum CBS 178.77
S. musiva CBS 130558
M. populicola CBS 100042
L. acicola CBS 133789
L. acicola CBS 871.95
L. acicola LNPV 243
0.1
488/495 nt
465/471 nt
411/436 nt
473/474 nt
462/466 nt
ITS
1
0.92
0.75
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0.58
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Teratosphaeria nubilosa CPC 12243
Ph. eugeniae CPC 15159
L. brevispora CBS 133601
P. angolensis CBS 149.53
M. sumatrensis CBS 118502
M. laricis-leptolepidis MAFF 410234
M. ellipsoidea CBS 110843
S. malagutii CBS 106.80
S. lycopersici CBS 354.49
S. musiva CBS 130556
S. populi CBS 391.59
P. clematidis CPC 11657
D. septosporum CBS 543.74
D. pini CBS 116484
L. acicola CBS 871.95
L. longispora CPC 17940
P. pini-densiflorae CBS 125138
P. pyracanthigena CPC 10808
L. acicola CBS 133789
L. acicola LNPV 243
L. guatemalensis IMI 281598
L. longispora CBS 133602
0.1
310/322 nt
336/337 nt
326/337 nt
1
1
0.99
0.6
0.73
1
0.77
1
1
0.71
1
Teratosphaeria nubilosa CPC 12243
M. laricis-leptolepidis MAFF 410234
S. populi CBS 391.59
S. citri CBS 315.37
M. ellipsoidea CBS 110843
P. clematidis CPC 11657
P. angolensis CBS 149.53
S. musiva CBS 130556
M. populicola CBS 100042
S. lycopersici CBS 354.49
S. malagutii CBS 106.80
D. septosporum CBS 383.74
D. pini CBS 116484
L. brevispora CBS 133601
P. pyracanthigena CPC 10808
P. pini-densiflorae CBS 125138
L. guatemalensis IMI 281598
L. acicola CBS 133789
L. longispora CPC 17940
L. longispora CBS 133602
L. acicola CBS 871.95
L. acicola LNPV 243
0.1
1
453/462 nt
371/387 nt
275/316 nt
RPB2 - alpha
LSU ITS
RPB2 TEF1-alpha
107W.Quaedvliegetal.:DNAbarcodingofMycosphaerella
that constitutes a positive discrimination threshold between spe-ciesisarbitrary.Ifathresholdvalueofatleastfivebasepairsdifference is accepted as successfully discriminating between species, then only EF-1α discriminated between all tested Q-species(Fig.1).Ifwesetthethresholdvaluetofourbasepairs difference, then Cal, EF-1α and Btub successfully discrimi-natedbetweenalltestedspecies(Fig.1).The ITS, LSU, Act and
RPB2 lociwere unable to discriminate among the various Q-speciesandcloselyrelatedneighbours.
shouldnot overlapbetween inter- and intraspecificKimura-2-parameterdistances.The individual test loci showed varying degrees of overlap in theirKimura-2-parameterdistributiongraphs.Forexample,Act,ITSandLSUhadmuchhigheroverlapthanRPB2,EF-1α, Cal andBtub,whichhadminimaloverlap.Theprimarycauseforthe existing Kimura-2-parameter overlap within the test loci is thelowinterspecificvariationbetweenthePseudocercospora speciesusedinthisdataset.ExcludingthePseudocercospora
speciesfromtheanalyses(datanotshown)removedtheexist-ingKimura-2overlapforRPB2,EF-1α and Btub, while reduc-ingitsignificantlyinAct.ExcludingthesePseudocercospora species had only negligible effect on the ITS and LSU Kimura-2-parameteroverlap(i.e.theirlackofvariationismoreuniver-sal).BecauseCalhadavery lowamplificationsuccessratewithin the negatively affecting Pseudocercospora species used inthisdataset,itsKimura-2-parametergraphissubsequentlymuch less negatively affected (i.e. noKimura-2-parameter
Fig. 2Frequency distribution of Kimura-2-parameter distances for theseventestloci.
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109W.Quaedvliegetal.:DNAbarcodingofMycosphaerella
overlap)thantheotherfourprotein-codingtestloci.TheITSand LSU loci, either with or without the Pseudocercospora dataset, showed a generally large Kimura-2-parameter over-lap.BasedonKimura-2-parametervalues, theRPB2,Btub,Act, Cal and EF-1α loci are not ideally suited for identifying Pseudocercospora species, but havea sufficient barcodinggap to successfully serve as the barcoding locus for the other speciesinthisdataset.BothITSandLSUarenotsuitabletoserveasbarcodinglociforthisdataset.
Specimens examined.Brazil, São Paulo, Santo Antonio do Pinhal, on needles of Pinus pinaster, 1974,T. Namekata,CBS543.74. –Ecuador, on needles of P. radiata,CPC3779=CBS112498.–FrancE, Meurthe et Moselle,Arboretumd’Amance,onneedlesofP. coulteri, 27Feb.1970,CBS383.74.–ThE nEThErlands, Lunteren,PinetumDennenhorst, onneedlesof Pinus mugo ‘Rostrata’,1June2009,W. Quaedvlieg,CPC16799,CPC16798=CBS128782.
Notes — Dothistroma septosporum is the causal agent of Dothistromaneedleblight (Redbanddiseaseofpine).Thisdisease is endemic to virtually all continents and occurs on a small number of Pinus and Larixspp.where itcancausevarying degrees of needle blight depending on humidity and temperature.Periodsofhigherhumidityandtemperatureleadtomoreseveresymptoms(Evans1984,Barnesetal.2004,EPPO2012).BasedonLSUdata,isolatesofM. pini cluster with D. pini and M. africana(Crousetal.2009c,2011b)anda large number of Passalora-likespecies (Videiraetal.un-publ.data).Because thegenusMycosphaerella is linked to Ramularia(Verkleyetal.2004,Crousetal.2009c),thenameDothistroma should be used for this clade, and D. septosporum forthisspecies.
On PNA: Conidiomataacervular,erumpent,brown,upto600µmdiam,openingbymeansoflongitudinalslit.Conidiophores subcylindrical, densely aggregated, dark brown, verruculose, unbranchedorbranchedatbase,1–3-septate,20–60×4–6µm.Conidiogenous cells terminal, integrated, subcylindrical, brown, verruculose, 8–20× 3–4.5 µm; proliferating sev-eraltimespercurrentlynearapex.Conidia solitary, straight to curved, subcylindrical with obtusely rounded apex, base trun-cate,brown,guttulate,verruculose,(0–)3(–8)-septate,base2.5–3.5µmdiam,withminutemarginalfrill,(17–)30–45(–55)×(3–)4(–4.5)µm. Culture characteristics — Colonies erumpent, spreading, with sparse aerial mycelium, surface folded, with smooth, lobatemargin; colonies reaching 7mmdiamafter 2wk at25°C.OnMEAsurfaceolivaceous-greytoiron-grey,reverseolivaceous-grey.OnPDAsurfaceolivaceous-greywithdiffuseumberpigmentinagar,reversepaleolivaceous-grey.OnOAsurfaceolivaceous-greywithdiffuseumberpigment.
Specimens examined.FrancE,Gironde,LeTeich,onneedlesofPinus radiata,Apr.1995,M. Morelet,CBSH-21114,cultureCBS871.95.–liThu-ania, on needles of Pinus mugo, 2009, S. Markovskaja, A. Kačergius & A. Treigienė,CBSH-21109, cultures LA773A& LA773B=CBS133790. –MExico, on needles of a Pinussp.,30Nov.2009,M. de Jesús YáñezMorales, CBSH-21112,culturesCPC17822=CBS133789.–USA,SouthCarolina,Aiken, needles of Pinus caribaea,1876,H.W. Ravenel,IMI91340,isotypeofCryptosporium aciculaexPadovaNo1484;Arkansas,PikeCity,alt.700ft,needles of Pinus(palustris or taeda),24Apr.1918,coll.J.A. Hughes,det.Sydow, syntype of Lecanostricta pini,BPI393329,BPI393331;Florida,SilverSpring, needles of Pinus palustris,27Feb.1919,coll.Geo G. Hedgcock, det.J. Dearness, type of Oligostroma acicola,BPI643015;Maine,Bethel,on needles of P. strobus,14June2011,coll.B. Ostrofsky,det.K. Broders, WPF4.12;ibid.,onneedlesofP. strobus,15June2011,coll.B. Ostrofsky, det.K. Broders,WPF13.12;NewHampshire,Blackwater, onneedles of P. strobus,25June2011,coll.J. Weimer,det.K. Broders,WPF13.12,epitypedesignatedhereCBSH-21113,cultureex-epitypeCBS133791.
Notes — Lecanosticta acicola is the causal agent of brown spot needle blight on Pinus spp.This disease is endemicto North and Central America, the central EPPO region and Eastern Asia where it causes yellowish, resin-soaked lesions withaprominentorangeborderon infectedneedles.As thedisease progresses, lesions coalesce and cause defoliation and dieback.Overseveralyearsthismayleadtobranchandtreedeath(Evans1984,Barnesetal.2004,EPPO2012).Basedon LSU data, L. acicolaclustersinauniquecladewithintheMycosphaerellaceae, for which Crous etal.(2009c)chosethegeneric name Lecanosticta(basedonL. acicola).ThenameMycosphaerella dearnessii is no longer applicable, as Mycosphaerella s.str.islinkedtothegenusRamularia(Verkleyetal.2004,Crousetal.2009c).Thecorrectnameforthisspeciesshould therefore be Lecanosticta acicola.
On PNA: Conidiomata acervular, erumpent, brown, up to 500 µmdiam,openingbymeansoflongitudinalslit.Conidiophores subcylindrical, densely aggregated, dark brown, verruculose, unbranched or branched at base, 0–2-septate, 10–25 ×3–4µm.Conidiogenous cells terminal, integrated, subcylindrical, brown,verruculose,5–8×2–3µm;proliferatingseveraltimespercurrentlynearapex.Conidia solitary, subcylindrical to nar-rowly fusoid-ellipsoidal, with subobtusely rounded apex, base truncate,brown,verruculose,frequentlywithmucoidsheath,(0–)1-septate, base 2 µmdiam,withminutemarginal frill,(11–)13–15(–18)×3(–4)µm. Culture characteristics — Colonies flat to somewhat erum-pent, spreading, with sparse aerial mycelium, surface folded,
with smooth, lobate margin; colonies reaching 15 mm diam after 2wkat25°C.OnMEAsurfacedirtywhitewithpatchesofpaleolivaceous-grey, reverse olivaceous-grey in centre, luteous in outerregion.OnPDAsurfacedirtywhiteincentre,isabellineinouterregion,andisabellineinreverse.OnOAsurfacedirtywhitewithdiffuseumberouterregion.
Specimen examined.MExico, on needles of a Pinussp.,24Oct.2009,M. de Jesús YáñezMorales, holotype CBS H-21110, cultures ex-type CPC 18092=CBS133601.
Notes — Lecanosticta brevispora is distinguished from the other taxa within the genus by either Btub or EF-1α.Mor-phologically it is distinct in having much smaller conidia than L. acicola; with narrower and less septate conidia than L. cinereum (1–3-septate,(12–)14–18(–20)×(3.5–)4–5µm,withobtuseapices),andL. gloeospora(1–3-septate,(9.5–)10.5–14.5(–17)×3.5–4.5µm,withobtuseapices)(Evans1984).
On PNA: Conidiomata acervular, erumpent, brown, up to 500 µmdiam,openingbymeansoflongitudinalslit.Conidiophores subcylindrical, densely aggregated, brown, verruculose, un-branchedorbranchedatbase,0–3-septate,15–25×3–4µm.Conidiogenous cells terminal, integrated, pale brown, finelyverruculose, subcylindrical to narrowly ampulliform, 6–15× 2.5–3.5µm;proliferatingseveraltimespercurrentlynearapex.Conidia solitary, straight to curved, subcylindrical with subob-tusely rounded apex, tapering towards truncate base, pale brown,finelyverruculose,(0–)1(–2)-septate,base2–2.5µmdiam,withminutemarginalfrill,(12–)15–20(–23)×3(–3.5)µm. Culture characteristics — Colonies erumpent, spreading, with sparse aerial mycelium, surface folded, with smooth, lobate margin, exceptonPDA,wheremargin is feathery; coloniesreaching30mmdiamafter2wkat25°C.OnMEAsurfacedirtywhite, reverse cinnamon with patches of isabelline, olivaceous-greytoiron-grey,reverseolivaceous-grey.OnPDAsurfaceandreverseolivaceous-grey.OnOAsurfacebuff.
Specimen examined.GuaTEMala, on needles of Pinus oocarpa,28Apr.1983,H.C. Evans,holotypeCBSH-21108,cultureex-typeIMI281598.
Notes — Lecanosticta guatemalensis can easily be distin-guished from the other taxa presently known within the genus by either Btub or EF-1α.Morphologicallyitisdistinguishedbyhaving conidia that are smaller than those of L. acicola, but larger than those of L. brevispora.
On PNA: Conidiomataacervular,erumpent,brown,upto600µmdiam,openingbymeansoflongitudinalslit.Conidiophores subcylindrical, densely aggregated, brown, verruculose, un-branched or branched at base, 0–4-septate, 15–55 × 3–4µm.Conidiogenous cells terminal, integrated, subcylindrical, brown, verruculose, 10–15 ×2–3.5µm;proliferatingseveraltimespercurrentlynearapex.Conidia solitary, subcylindrical with subobtusely rounded apex, base truncate, brown, gut-tulate,verruculose,1–3-septate,base2µmdiam,withminutemarginalfrill,(16–)30–45(–50)×3(–4)µm. Culture characteristics — Colonies flat, somewhat erum-pent, spreading, with sparse aerial mycelium, surface folded, withsmooth,lobatemarginonMEA,butfeatheryonPDAandOA;coloniesreaching20mmdiamafter2wkat25°C.OnMEAsurfacepaleolivaceous-greywithpatchesofolivaceous-grey.OnPDAsurfaceolivaceous-grey, reverse iron-grey.OnOAsurface dirty white in centre, with patches of pale olivaceous-greyandolivaceous-grey.
Specimens examined.MExico,NuevoLeón,Galeana,CerrodelPotosí,on Pinus culminicola, J.G. Marmolejo, 6June1993,holotypeCFNL;Mi-choacanState,Zinapecuaroarea,onneedlesofaPinussp.,24Oct.2009, M. de Jesús Yáñez-Morales & C. Méndez-Inocencio, epitype designated here CBSH-21111,culturesex-epitypeCPC17941,CPC17940=CBS133602.
Notes — Lecanosticta longispora is distinguished from the other taxa within the genus by either Btub or EF-1α.Morpho-logically it is similar to L. acicola in conidial length, but distinct inthatconidiahave1–3septa(Marmolejo2000).
Specimens examined. Japan, Yamagata, on needles of Larix leptolepis, 1954–1955, K. Itô,MAFF410081;Hokkaidou,onneedlesofL. leptolepis, 1954–1955, T. Yokota,MAFF410632,MAFF410633;Yamagata,onneedlesof L. leptolepis, May 1954, N. Ota,MAFF410234.
Notes — Mycosphaerella laricisleptolepidis is the causal agentofneedlecastofJapanese larch.Thisdisease isen-demic to East Asia and Japan where it occurs on indigenous Larixspecies.Itcausesbrownnecroticlesionsontheneedlesthat coalesce, leading to defoliation, stunted growth and even host plant death (Kobayashi 1980,EPPO2012).BasedonLSU data, M. laricisleptolepidis clusters in a clade described as ‘Polythrincium’byCrous etal.(2009c).AlthoughthegenusMycosphaerellas.str.isdistinctfromthe‘Polythrincium’clade,the name M. laricisleptolepidis is retained until more data becomesavailable.
Specimens examined.anGola,Bié,fromCitrus sinensis,Dec.1953,T. de Carvalho & O. Mendes, holotype IMI56597,ex-typeCBS149.53.–ziMBaBwE, from Citrussp.,March1993,P.W. Crous,CPC751=CBS244.94;ibid., from Citrussp.,2002,P.W. Crous,CPC4111=CBS112748;ibid., from Citrus sp.,Sept.2002,M.C. Pretorius,CBSH-20851,CPC4118=CBS112933;ibid., from Citrussp.,2002,P.W. Crous,CPC4117=CBS115645.
Notes — Pseudocercospora angolensis is the causal agent ofCitrus leafspot (Citrus fruitspot)and isendemic tosub-SaharanAfrica,whereitoccursonallmajorCitrusspecies.Itcauses greenish yellow lesions on leaves and fruit that coalesce and turn necrotic, leading to defoliation or abscission of young fruit(Timmeretal.2000,Crous&Braun2003,EPPO2012).Based on LSU data, P. angolensis clusters within the Pseudocercosporaclade(Pretoriusetal.2003,Crousetal.2009c,Inpress).As thegenusPseudocercospora is taxonomically correct and in current use, Pseudocercospora angolensis is thecorrectnameforthecausalagentofCitrusfruitleafspot.
Specimens examined.Japan, from needles of Pinus thunbergii,1971,SungOui Suh,CBS125139;fromneedlesofPinus kesiya,1971,SungOui Suh, CBS 125140; from needles of a Pinussp.,1971,SungOui Suh, CBS 125138.
Notes — Pseudocercospora pinidensiflorae is the causal agentofbrownneedleblightofpine(Cercosporapineblight).This disease is mostly endemic to the tropics and subtropics inBrazil,sub-SaharanAfrica,India,SoutheastandEastAsia,where it may infect indigenous Pinus spp. It causesbrownnecrotic lesions on the needles leading to defoliation and is especially damaging on young saplings, on which defoliation leadstostuntedgrowthandhostplantdeath(Deighton1987,Lewis 1998,EPPO2012).Based on LSUdata, isolates of P. pinidensiflorae cluster within the Pseudocercospora clade (Crousetal.Inpress),confirmingitsgenericplacementasre-portedbyDeighton(1987).ThegenericnameMycosphaerella is considered a synonym of the genus Ramularia(Verkleyetal.2004,Crousetal.2009c),andthereforeMycosphaerella should not be used for the pathogen associated with brown needle blightofpine.TheapplicationofthenamePseudocercospora pinidensifloraeisthereforecorrect.
Septoria malagutii E.T.Cline,Mycotaxon98:132.2006
= Septoria lycopersici var. malagutiiCiccar.&Boerema,Phytopathol.Medit. 17:87.1978;nom.inval.,Art.37.1
Notes — Septoria malagutii is the causal agent of Septoria leafspot(angularleafspot)ofpotato,andisendemictoCentraland South America, where it occurs on leaves of potato and other tuber-bearing Solanumspecies.Itcausesleaflesionsthatcoalesce until the leaves turn necrotic, leading to defoliation andseverelossesincropproduction(Stevenson2001,EPPO2012).BasedonLSUdata,S. malagutii clusters within Septoria s.str.asdefinedbyQuaedvlieg etal. (2011).Thecorrectnamefor this species is therefore Septoria malagutii(Cline&Ross-man2006).
Specimens examined.canada, Quebec City, from leaf of Populus deltoides, J. LeBoldus,MAC=CBS130564,LP3=CBS130565,PPP=CBS130566,PP=CBS130567,LPR=CBS130568,RCL=CBS130569,SA=CBS130570,RPN=CBS130571,D2L2=CBS130558;Alberta,fromleavesof P. deltoides × P. balsamifera, J. LeBoldus,D2L2=CBS130558,NW3L1=CBS130563,NW2L2=CBS130561,D7L2;Alberta,fromleavesofhybridPopulusspp.,J. LeBoldus,APC=CBS130559,APH1=CBS130560,APH3=CBS130562.
Notes — Septoria musiva is the causal agent of Septoria canker of poplar and is endemic to North America and Argen-tina, where it occurs on all native Populusspp.Itcausesseverecankering and die-back and is especially damaging to hybrid Populus species (Bier 1939,Waterman 1954,Ostry 1987,Dickmann2001,EPPO2012).BasedonLSUdata,S. musiva clusters within Septoria s.str.asdefinedbyQuaedvlieg etal. (2011).However, ongoingwork byQuaedvlieg andVerkley(unpubl.data)revealedthatS. musiva is located in a cryptic phylogenetic lineage sister to Septorias.str.,andthereforethegenusnameofthisclademightchangeinthefuture.
DISCUSSION
Current EPPO protocols for identifying A1/A2 listed Mycosphaerella speciesarebasedeitheron ITS-RFLPor fungalmorphology(Table4).Theseapproacheseachhavelimitationsthatmakethemill-suitedasidentificationtoolsforplantprotec-tionpolicyenforcementofficers.Morphology-basedtechniquesareheavilydependentonhigh- ly skilled personnel that need to perform time-consuming iden- tificationsofmature,sporulatingculturesthatoftenneedtobegrownonspecificmediaandunderspecificconditions.Therapid advanceofmolecular techniques in recent years hasunderlined the limitations of identifications based solely onmorphologyand/orITSsequencing.Examplesofthisarethenew Lecanosticta species that have been described during this study.These isolateshadpreviously been identifiedasLecanosticta acicola based both on morphology and limited ITSsequencing.Thesequencingofadditionallocirevealedthat L. acicola actually represented a species complex rather than a singlespecies.ThisisyetanotherexampleofthetenetofCrous&Groenewald(2005)whichstates“Showmeaplantpathogen,andIwillshowyouaspeciescomplex”.Anotherexamplewasthe Cercospora apii complex, which was considered to be a singlespeciesbasedonmorphology(Crous&Braun2003),butwhichwasfoundtorepresentseveralspecieswhenDNAsequencingtechniqueswhereemployed(Crousetal.2004b,2006a,Inpress,Groenewaldetal.2005,Inpress).Thisinabilityto discriminate between cryptic species and their dependency on mature, sporulating cultures make morphology-based tech-niquespoorlysuitedfortherapidandreliableidentificationofMycosphaerellaspeciesontradegoods.PCR-RFLP-basedmethodsworkona ‘hitormiss’principle,and work well for identifying small groups of well-characterised fungalspecieswithlittlegeneticvariation.Unfortunatelythesemethods lack the inherent ability to cope with expanding natural variation.Pointmutations,insertionordeletioneventscanleadtothelossofrestrictionsites,makingisolatesunrecognizable
forPCR-RFLPbasedmethods(Majeretal.1996).SpeciesofMycosphaerellaalsoco-colonizelesions,increasingthechanceofhavingamixedDNAsampleifsingle-sporedorhyphal-tippedcoloniesarenotusedintheassay(Crous&Groenewald2005).TheuseofaDNAbarcodeorthecombinationofsequencedata from twoormore discriminatory loci (multi-locus sequencetyping),fortherecognitionofspeciesofquarantineimportancehasnumerousadvantagesoverpreviouslyusedtechniques.Itdoesnotrequirefruitingbodiesoramaturelifestage,itisfast,(relatively)cheap,andcanbeperformedbymoderatelyskilledpersonnel and has a high probability of yielding a result, even withunknownspecies.ButthesinglemostimportantaspectofDNAbarcodingisitsabilitytoidentifyspecies(evencrypticspecies)withalmostnomarginoferror,onconditionthatalarge,validated,referencedatabaselibraryisavailable.Oneof themaingoals of this projectwas to determine themost suitable barcoding locus/loci by which to identify Mycosphaerella-like spp. on theEPPOA1/A2 lists.Hebert et al.(2003)proposedthatagoodbarcodinglocusshouldshowaclear separation between the distributions of the mean intra- andinterspecificdistances(theso-called‘Kimura-2-parameterbarcodinggap’).Theauthorsproposedthatalocusshouldhaveameaninter-/intraspecificdistanceratioofatleast10,tobesuitableasabarcodinglocus.Thelocitestedinthisstudyallhadmeaninter-,intraspecificdistanceratiosthatweremuchhigher than10.Meandistribution ratiosvaried from486 forLSUto69forITS(Fig.2).Bythesecriteriaalone,theselocishouldallbesuitablebarcodingloci.AlmostalllocishowedaKimura-2-parameter overlap between their absolute inter- and intraspecific distribution frequencies.When thePseudocercosporaisolateswereincludedinthedataset,thesizeofthisabsoluteinter-andintraspecificdistributionfrequenciesdataoverlap varied from 12% (LSU), 16% (ITS), 3.4% (Act),1.2% (EF-1α), 0.6% (RPB2), 0.5% (Btub) and0% (Cal),respectively.Calmodulindidnotoverlapsimplybecausethislocus failed to amplify most of the Pseudocercosporaspp.thatare mostly responsible for this Kimura-2-parameter inter- and intraspecificdistributionoverlapintheotherloci.The relatively high Kimura-2-parameter distribution overlap in thetwonuclearribosomalDNAloci(ITSandLSU)iscausedby the low natural variation that exists within these loci between speciesofcertaingenera (in thisdatasetSeptoriaspp.andPseudocercosporaspp.hadverylowvariabilitybetweenspe-cies).Thisdifferencewithinthenaturalvariationpresentwithinthe different genera in the complete dataset can clearly be seen in the ITS and LSU Kimura-2-parameter distribution graphs (Fig.2).Thesetwographsclearlyshowmultiple‘peaks’thatrepresent the difference in natural variation within the varying generausedinthisdataset.
114 Persoonia – Volume 29, 2012
From the three independent barcode suitability tests we can concludethat,basedonathresholdofatleastfivebasepairsdifference, EF-1αisthebestlocustouseforDNAbarcodingoftheisolateswithinthisdataset.Ifweuseathresholdoffourbasepairs,thenBtubisalsosuitedtoserveasDNAbarcodinglocusforthisdataset.Theothertestedlocieitherhaveaclearamplificationproblem(Cal)ordonothavesufficientresolution(Δ≥4nt)(ITS,LSU,ActandRPB2)todiscriminatebetweensomeofthequarantinespeciesandtheirclosestrelativespe-cies(Fig.1).Although the EF-1α and Btub loci have the highest species discrimination levels for the species used in this dataset, these loci have the disadvantage that there is not much reference data concerning these loci available in online databases which can helpidentifyisolatesnotusedinthisdataset.Tocompensatefor this lack of reference data, we recommend using a combina-tion of a primary and a secondary locus to give more reliable identificationresults.The ITS locus is theprimecandidate for theprimary locus.ITS has recently been proposed as one of the primary fungal barcodingloci(Schochetal.2012).ITSsequencingdataiseasily obtained and a good starting point to rapidly identify generaandsometimesspecies.Ifanunknowngenusorspe-cies is not represented in a curated database such as Q-bank, aGenBankblastcouldbeusedtosupplementthesecurateddatabases.MycologyhasalonghistoryofusingITSdatatoidentify fungalspeciesandGenBankwould thusbeagoodsupplementary (althoughnotcompletelycurated)database.The use of ITS as the primary locus, and if necessary using a secondary locus following a molecular decision protocol, would bethemoststableapproachforareliableidentification.ThisisalsotheidentificationprotocolasitiscurrentlyimplementedinQ-bank.As a secondary barcoding locus to supplement the ITS se-quencedata,eitherBtuborEF-1αwouldsufficeforthisdataset.Bothlociareeasilyamplifiableandhaveahighamplificationrate(100%and97%,respectively),possesonlyminimalKimura- 2-parameterinter-andintraspecificdistributionoverlap(0.5%and1.2%,respectively)andbothhave100%speciesdiscri-minationsuccessratewithinthetesteddataset(Δ≥4nt).Theuse of either Btub or EF-1α may complement each other if amplificationproblemswith either locusoccur, thus leadingtoasuccessful identificationofanunknownMycosphaerella speciesofpossiblequarantineimportance.
Acknowledgements Wethankthetechnicalstaff,ArienvanIperen(cul-tures)andMarjanVermaas(photographicplates)fortheirinvaluableassis-tance.SpecialthanksgotoProf.KirkD.Broders(DepartmentofBiologicalSciences,UniversityofNewHampshire,USA),Dr.IsabelleMunck(USDAForestService,NewHampshire,USA),JenniferWeimer(NewHampshireDivisionofForests&LandsForestProtection,Blackwater,USA),Dr.JaredLeBoldus(DepartmentofPlantPathology,NorthDakotaStateUniversity,USA)andWilliamOstrofsky(MaineForestService,Bethel,USA)forcollectingand providing fresh material of Lecanosticta acicola.WewouldalsoliketothankDr.RenaudIoos(LaboratoiredelaSantédesVégétaux,Anses,France)forprovidingDNAsamplesofLecanosticta acicolaandDr.SarahL.Boyer(DepartmentofBiology,MacalesterCollege,SaintPaul,USA)forprovidingsupportwiththeKimura-2-parameterdistributiongraphs.Theresearchlead-ingtotheseresultshasreceivedfundingfromtheEuropeanCommunity’sSeventhFrameworkProgram(FP7/2007–2013)/grantagreementno.226482(Project:QBOL–DevelopmentofanewdiagnostictoolusingDNAbarcodingtoidentifyquarantineorganismsinsupportofplanthealth)bytheEuropeanCommissionunderthetheme‘DevelopmentofnewdiagnosticmethodsinsupportofPlantHealthpolicy’(no.KBBE-2008-1-4-01).
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