dna barcoding of mycosphaerella species of quarantine ......cpc 15143 eugenia uniflora alfenas...

© 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures

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Persoonia 29, 2012: 101–115www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158512X661282RESEARCH ARTICLE

INTRODUCTION

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

1 CBS-KNAWFungalBiodiversityCentre,Uppsalalaan8,3584CTUtrecht,The Netherlands;

correspondingauthore-mail:[email protected],DepartmentofBiology,UtrechtUniversity,Padualaan8,3584CHUtrecht,TheNetherlands.

3 ColegiodePostgraduados,Km.36.5Carr,Mexico-Texcoco,Montecillo,Mpio.deTexcoco,Edo.deMexico56230,Mexico.

4WageningenUniversityandResearchCentre(WUR),LaboratoryofPhyto-pathology,Droevendaalsesteeg1,6708PBWageningen,TheNetherlands.

Key words

EPPOLecanostictaQ-bankQBOL

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.

102 Persoonia – Volume 29, 2012Ta

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CPC11181

Euc

alyp

tussp.

Indonesia

M.J.W

ingfield

JX902136

JX901571

JX901680

JX902258

JX902012

DQ302962

JX901889

CBS111189

Euc

alyp

tus

urop

hylla

–

M.J.W

ingfield

JX902137

JX901572

JX901681

JX902259

JX902013

DQ302960

JX901890

P. h

umul

ijap

onic

i CPC11315

Hum

ulus

japo

nicu

s RepublicofK

orea

H.D.S

hin

JX902138

JX901573

JX901682

JX902260

JX902014

JX901783

JX901891

CPC11462

Ple

ctra

nthu

s RepublicofK

orea

H.D.S

hin

JX902139

JX901574

JX901682

JX902261

JX902015

JX901784

JX901892

P. m

adag

asca

riens

is

CBS124155*

Euc

alyp

tus

cam

aldu

lens

is

Madagascar

M.J.W

ingfield

JX902140

–JX901683

JX902262

JX902016

FJ790268

JX901893

P. n

orch

iens

is

CBS120738*

Euc

alyp

tussp.

Italy

W.G

ams

JX902141

JX901575

JX901684

JX902263

JX902017

JX901785

JX901894

P. p

arag

uaye

nsis

CBS111286Δ

Euc

alyp

tus

nite

ns

Brazil

P.W.C

rous

JX902142

–JX901685

JX902264

JX902018

DQ267602

JX901895

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inid

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e CBS125139

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us th

unbe

rgii

Japan

Sung-OuiSuh

JX902143

–JX901686

JX902265

JX902019

JX901786

JX901896

C

BS

125

140

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us k

esiy

a Japan

Sung-OuiSuh

JX902144

–JX901687

JX902266

JX902020

JX901787

JX901897

CBS125138Δ

Pin

ussp.

Japan

Sung-OuiSuh

JX902145

–JX901688

JX902267

JX902021

JX901788

JX901898

P. p

seud

oeuc

alyp

toru

m

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Euc

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glob

ulus

Portugal

A.P

hillips

JX902146

JX901576

JX901689

JX902268

JX902022

GQ852759

JX901899

CPC13769

Euc

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tus

punc

tata

SouthAfrica

P.W.C

rous

JX902147

JX901577

JX901690

JX902269

JX902023

GQ852762

JX901900

CPC12568

Euc

alyp

tus

nite

ns

Tasm

ania

C.M

oham

med

JX902148

JX901578

JX901691

JX902270

JX902024

GQ852758

JX901901

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yrac

anth

igen

a CPC10808Δ

Pyr

acan

tha

angu

stifo

lia

RepublicofK

orea

H.D.S

hin

JX902149

–JX901692

JX902271

JX902025

JX901789

JX901902

P. r

hoin

a CPC11464

Rhu

s ch

inen

sis

RepublicofK

orea

H.D.S

hin

––

JX901693

JX902272

JX902026

JX901790

JX901903

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obus

ta

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ucal

yptu

s ro

bur

Malaysia

M.J.W

ingfield

JX902150

JX901579

JX901694

JX902273

JX902027

DQ303081

JX901904

P. s

chiz

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ii C

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cam

aldu

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Thailand

W.H

imam

an

JX902151

–JX901695

JX902274

JX902028

GQ852765

JX901905

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rulin

ae

CBS112621*

Euc

alyp

tussp.

–

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rous

–JX901580

JX901696

JX902275

JX902029

JX901791

JX901906

P. s

ubul

ata

CBS118489

Euc

alyp

tus

botry

oide

s New

Zealand

M.D

ick

JX902152

JX901581

JX901697

JX902276

JX902030

JX901792

JX901907

P. t

eret

icor

nis

CPC13008

Euc

alyp

tus

tere

ticor

nus

Australia

A.J.C

arnegie

JX902153

JX901582

JX901698

JX902277

JX902031

GQ852769

JX901908

CPC13315

Euc

alyp

tus

tere

ticor

nus

Australia

P.W.C

rous

JX902154

JX901583

JX901699

JX902278

JX902032

GQ852771

JX901909

CBS124996Δ

Euc

alyp

tus

nite

ns

Australia

A.J.C

arnegie

JX902155

JX901584

JX901700

JX902279

JX902033

GQ852768

JX901910

CPC13299*

Euc

alyp

tus

tere

ticor

nus

Australia

A.J.C

arnegie

JX902156

JX901585

JX901701

JX902280

JX902034

GQ852770

JX901911

P. v

itis

CP

C 1

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tis v

inife

ra

RepublicofK

orea

H.D.S

hin

JX902157

JX901586

JX901702

JX902281

JX902035

DQ073923

JX901912

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toria

abe

licea

e CBS128591

Zelk

ova

serr

ata

RepublicofK

orea

S.B.H

ong

JX902158

JX901587

JX901703

JX902282

JX902036

JX901793

JX901913

S. c

f. ch

rysa

nthe

mel

la

CBS483.63

Chr

ysan

them

umsp.

TheNetherlands

H.A.vanderAa

JX902159

JX901588

JX901704

JX902283

JX902037

JX901794

JX901914

CBS351.58

Chr

ysan

them

um in

dicu

m

Germany

R.S

chneider

JX902160

JX901589

JX901705

JX902284

JX902038

JX901795

JX901915

S. c

itri

CBS315.37Δ

–

–

L.L.Huillier

JX902161

JX901590

JX901706

JX902285

JX902039

DQ897650

JX901916

S. c

ucur

bita

cear

um

CBS178.77Δ

Cuc

urbi

ta m

axim

a –

H.J.B

oesewinkel

JX902162

JX901591

JX901707

JX902286

JX902040

JX901796

JX901917

S. l

ycop

ersi

ci

CBS353.49

–

–

S.P.D

oolittle

JX902163

JX901592

JX901708

JX902287

JX902041

DQ841155

JX901918

CBS128654Δ

Lyco

pers

icon

esc

ulen

tum

RepublicofK

orea

S.B.H

ong

JX902164

JX901593

JX901709

JX902288

JX902042

JX901797

JX901919

S. m

alag

utii

CBS106.80Δ

Sol

anum

sp.

Peru

L.J.Turkensteen

JX902165

JX901594

JX901710

JX902289

JX902043

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JX901920

S. m

atric

aria

e C

BS

109

000

Mat

ricar

ia d

isco

idea

TheNetherlands

G.V

erkley

JX902166

JX901595

JX901711

JX902290

JX902044

JX901798

JX901921

C

BS

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001

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ricar

ia d

isco

idea

TheNetherlands

G.V

erkley

JX902167

JX901596

JX901712

JX902291

JX902045

JX901799

JX901922

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,

GenBankAccessionno2

Spe

cies

Is

olat

e no

1 H

ost

Loca

tion

C

olle

cted

by

A

CT

CA

L

EF-

1α

Btub

RPB2

ITS

LSU

S. m

usiv

a CBS130559

Hybridpoplar

Canada

J.LeB

oldus

JX902168

JX901597

JX901713

JX902292

JX902046

JX901800

JX901923

D7L2#

P. d

elto

ides

× P

. bal

sam

ifera

Canada

J.LeB

oldus

JX902169

JX901598

JX901714

JX902293

JX902047

JX901801

JX901924

CBS130560

Hybridpoplar

Canada

J.LeB

oldus

JX902170

JX901599

JX901715

JX902294

JX902048

JX901802

JX901925

CBS130561

P. d

elto

ides

× P

. bal

sam

ifera

Canada

J.LeB

oldus

JX902171

JX901600

JX901716

JX902295

JX902049

JX901803

JX901926

CBS130562

Hybridpoplar

Canada

J.LeB

oldus

JX902172

JX901601

JX901717

JX902296

JX902050

JX901804

JX901927

CBS130563

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ides

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J.LeB

oldus

JX902173

JX901602

JX901718

JX902297

JX902051

JX901805

JX901928

CBS130564

Pop

ulus

del

toid

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Canada

J.LeB

oldus

JX902174

JX901603

JX901719

JX902298

JX902052

JX901806

JX901929

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Pop

ulus

del

toid

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Canada

J.LeB

oldus

JX902175

JX901604

JX901720

JX902299

JX902053

JX901807

JX901930

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Pop

ulus

del

toid

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Canada

J.LeB

oldus

JX902176

JX901605

JX901721

JX902300

JX902054

JX901808

JX901931

CBS130567

Pop

ulus

del

toid

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Canada

J.LeB

oldus

JX902177

JX901606

JX901722

JX902301

JX902055

JX901809

JX901932

CBS130568

Pop

ulus

del

toid

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Canada

J.LeB

oldus

JX902178

JX901607

JX901723

JX902302

JX902056

JX901810

JX901933

CBS130569

Pop

ulus

del

toid

es

Canada

J.LeB

oldus

JX902179

JX901608

JX901724

JX902303

JX902057

JX901811

JX901934

CBS130570

Pop

ulus

del

toid

es

Canada

J.LeB

oldus

JX902180

JX901609

JX901725

JX902304

JX902058

JX901812

JX901935

CBS130571

Pop

ulus

del

toid

es

Canada

J.LeB

oldus

JX902181

JX901610

JX901726

JX902305

JX902059

JX901813

JX901936

CBS130558Δ

P. d

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ides

× P

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sam

ifera

Canada

J.LeB

oldus

JX902182

JX901611

JX901727

JX902306

JX902060

JX901814

JX901937

S. o

besa

CBS354.58

Chr

ysan

them

um in

dicu

m

Germany

R.S

chneider

JX902183

JX901612

JX901728

JX902307

JX902061

AY489285

JX901938

CBS128759

Chr

ysan

them

um m

orifo

lium

RepublicofK

orea

S.B.H

ong

JX902184

JX901613

JX901729

JX902308

JX902062

JX901815

JX901939

CBS128623

Chr

ysan

them

um in

dicu

m

RepublicofK

orea

H.D.S

hin

JX902185

JX901614

JX901730

JX902309

JX902063

JX901816

JX901940

CBS128588

Arte

mis

ia la

vand

ulae

folia

RepublicofK

orea

S.B.H

ong

JX902186

JX901615

JX901731

JX902310

JX902064

JX901817

JX901941

S. p

opul

i CBS391.59Δ

Pop

ulus

pyr

amid

alis

Germany

R.S

chneider

JX902187

JX901616

JX901732

JX902311

JX902065

JX901818

JX901942

Ter

atos

phae

ria n

ubilo

sa

CPC12243Δ

Euc

alyp

tus

glob

ulus

Portugal

A.J.L.P

hillips

JX902188

JX901617

JX901733

JX902312

JX902066

JX901819

JX901943

1 CBS:C

entraalbureauvoorS

chimmelcultures,Utrecht,TheNetherlands;CPC:P

edroCrousworkingcollectionhousedatC

BS,LNPV:laboratoirenationaldelaprotectiondesvégétaux,A

ngers,France.MAFF

:MinistryofAgriculture,ForestryandFisheries,Tokyo,Japan.

2 ACT=Actin,B

tub=β-tubulin,C

AL=Calmodulin,LSU=28S

largesubunitofthenrRNAgene,R

PB2=RNApolymeraseIIsecondlargestsubunit,ITS=InternaltranscribedspacerandEF-1α

=Translationelongationfactor1-alpha.#isolateprovidedbyJ.LeB

oldus.

Tabl

e 1(cont.)

105W.Quaedvliegetal.:DNAbarcodingofMycosphaerella

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.

Kimura-2-parameter valuesInter-and intraspecificdistances foreach individualdatasetwerecalculatedusingMEGAv.4.0(Tamuraetal.2007)usingtheKimura-2-parameter(pair-wisedeletion)model.

RESULTS

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)

Actin ACT-512F ATGTGCAAGGCCGGTTTCGC 52 Forward Carbone&Kohn(1999)Actin ACT2Rd ARRTCRCGDCCRGCCATGTC 52 Reverse Groenewaldetal.(Inpress)Calmodulin CAL-235F TTCAAGGAGGCCTTCTCCCTCTT 50 Forward PresentstudyCalmodulin CAL2Rd TGRTCNGCCTCDCGGATCATCTC 50 Reverse Groenewaldetal.(Inpress)Translation elongation factor-1α EF1-728F CATCGAGAAGTTCGAGAAGG 52 Forward Carbone&Kohn(1999)Translation elongation factor-1α EF-2 GGARGTACCAGTSATCATGTT 52 Reverse O’Donnelletal.(1998)β-tubulin T1 AACATGCGTGAGATTGTAAGT 52 Forward O’Donnell&Cigelnik(1997)β-tubulin β-Sandy-R GCRCGNGGVACRTACTTGTT 52 Reverse Stukenbrocketal.(2012)RNApolymeraseIIsecondlargestsubunit fRPB2-5F GAYGAYMGWGATCAYTTYGG 49 Forward Liuetal.(1999)RNApolymeraseIIsecondlargestsubunit fRPB2-414R ACMANNCCCCARTGNGWRTTRTG 49 Reverse Quaedvliegetal.(2011)LSU LSU1Fd GRATCAGGTAGGRATACCCG 52 Forward Crousetal.(2009a)LSU LR5 TCCTGAGGGAAACTTCG 52 Reverse Vilgalys&Hester(1990)ITS ITS1 GAAGTAAAAGTCGTAACAAGG 52 Forward Whiteetal.(1990)ITS ITS4 TCCTCCGCTTATTGATATGC 52 Reverse Whiteetal.(1990)

Table 2Primersusedinthisstudyforgenericamplificationandsequencing.

Locus Act Cal EF1 RPB2 Btub ITS LSU

Amplificationsucces(%) 98 90 97 99 100 100 100Q-amplificationsucces(%) 100 86 100 100 100 100 100Numberofcharacters 615 385 800 337 430 658 751Uniquesitepatterns 235 228 551 165 290 214 120Sampledtrees 198 686 716 148 238 728 406Numberofgenerations(×1000) 150 642 857 123 168 433 272Substitutionmodelused GTR-I-gamma HKY-I-gamma HKY-I-gamma GTR-I-gamma HKY-I-gamma GTR-I-gamma GTR-I-gamma

Table 3Amplificationsuccess,phylogeneticdataandthesubstitutionmodelsusedinthephylogeneticanalysis,perlocus.


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

0.97

0.55

0.78

0.67

0.93

0.81

0.72

1

0.99

1

1

1

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

743/745 nt

744/745 nt

747/747 nt

747/747 nt

737/747 nt

LSU

0.78

0.73

0.58

0.97

1

1

1

0.96

1

1

0.74

0.83

1

1





M. endophytica CBS 111519




P. clematidis CPC 11657


D. pini CBS 116484

D. septosporum CBS 383.74





S. cucurbitacearum CBS 178.77





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

0.8

0.95

1

0.98

0.91

0.99

0.58

1

0.75

0.99

0.96 1 1


Ph. eugeniae CPC 15159







S. lycopersici CBS 354.49


S. populi CBS 391.59



D. pini CBS 116484






L. acicola LNPV 243



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



S. populi CBS 391.59

S. citri CBS 315.37









D. pini CBS 116484









L. acicola LNPV 243

0.1

1

453/462 nt

371/387 nt

275/316 nt

RPB2 - alpha

LSU ITS

RPB2 TEF1-alpha


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.

Kimura-2-parameter valuesTheKimura-2-parameterdistributiongraphs(Fig.2)visualisetheinter-andintraspecificdistancesperlocuscorrespondingtothebarcodinggap(Hebertetal.2003).Agoodbarcodinglocus

301/317 nt

300/307 nt

307/315 nt

305/322 nt

307/311 nt

0.8

1

0.95

1

0.9

1

1

1

1

0.52

1

0.98

1

1






Mycosphaerella sp. CBS 111166

D. pini CBS 116484

D. septosporum CBS 16798





P. vitis CPC 11595





P. paraguayensis CBS 111286



L. acicola LNPV 243

0.1

1

0.72

1

0.98

1

0.97

0.65

1

1

1

0.76


S. lycopersici CBS 128654





M. sumatrensis CPC 118501



P. tereticornis CPC 13315


L. acicola LNPV 243



M. ellipsoidea CPC 110843

M. populicola CPC 100042


D. pini CBS 116484


0.1

257/261 nt

282/303 nt

287/302 nt

294/304 nt

1

0.65

0.6

0.82

1

0.83

1

1

0.98

1

1

1





M. sumatrensis CPC 118501


L. acicola LNPV 243




D. pini CBS 116484





P. paraguayensis CBS 111286





0.1

0.6

535/537 nt

530/540 nt

536/545 nt

294/304 nt

- tubuli

Calmoduli 1

0.98

0.92

0.99




L. acicola LNPV 241




L. acicola LNPV243

L. acicola LNPV 242

L. acicola LNPV 247

L. acicola LNPV 250


L. acicola LNPV 248


L. acicola WPF 4.12

L. acicola WFP 73.12

L. acicola LNPV 246

L. acicola LNPV 249

L. acicola LNPV 252

L. acicola LNPV 253

L. acicola LA773B


0.1

2x

Lecanosticta guatemalensis

Lecanosticta acicola

Lecanosticta longispora

Lecanosticta brevisporaFig. 1(cont.)

β-tubulin Actin

Calmodulin

Lecanosticta7xmultigene


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.

0102030405060708090

100

Actin

Distance

Freq

uenc

y

05

1015202530354045

0.01

0.04

0.07

0.10

0.13

0.16

0.19

0.22

0.25

0.28

0.31

0.34

0.37

0.40

0.43

Calmodulin

intra

inter

Distance

0

20

40

60

80

100

120TEF 1-alpha

intra

inter

Distance

0

20

40

60

80

100

120

0.01

0.04

0.07 0.

1

0.13

0.16

0.19

0.22

0.25

0.28

0.31

0.34

0.37 0.

4

0.43

0.46

β-tubulin

intra

inter

Distance

0

20

40

60

80

100

120

0.02

0.06 0.

1

0.14

0.18

0.22

0.26 0.

3

0.34

0.38

0.42

0.46 0.

5

0.54

0.58

0.62

RPB2

intra

inter

Distance

0

10

20

30

40

50

60

70

80

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

0.13

0.14

0.15

0.16

0.17

0.18

0.19

0.20

ITS

intra

inter

Distance

010

30

60708090

100

0.00

0.01

0.01

0.02

0.02

0.03

0.03

0.04

0.04

0.05

0.05

0.06

0.06

0.07

0.07

0.08

0.08

0.09

0.09

0.10

LSU

intra

inter

Distance

5040

20

intra

inter

Freq

uenc

y

Freq

uenc

y

Freq

uenc

y

Freq

uenc

y

Freq

uenc

yFr

eque

ncy

. ........... ..

..... .. ...... ...

β-tubulin

TEF1-alpha

Actin Calmodulin

ITS

RPB2

LSU


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.

Taxonomy

Dothistroma septosporum (Dorog.)M.Morelet(as‘septospora’), Bull.Soc.Sci.Nat.Archéol.ToulonVar.177:9.1968

Basionym.Cytosporina septospora Dorog.,Bull.Trimestriel Soc.Mycol.France27:106.1911. ≡Septoriella septospora(Dorog.)Sacc.apudTrotter,Syll.Fung.25:480.1931. ≡Septoria septospora(Dorog.)Arx,Proc.Kon.Ned.Akad.Wetensch. C 86,1:33.1983. ≡Dothistroma septosporum var. keniense(M.H.Ivory)B.Sutton,inSut-ton,Thecoelomycetes.Fungiimperfectiwithpycnidiaacervuliandstromata(Kew):174.1980. = Actinothyrium marginatum Sacc., NuovoGiorn.Bot.Ital.27: 83.1920. = Dothistroma pinivar. lineare Thyr&C.G.Shaw, Mycologia 56:107.1964. = Dothistroma pinivar.kenienseM.H.Ivory(as‘keniensis’),Trans.Brit.Mycol.Soc.50:294.1967. = Mycosphaerella piniRostr., inMunk,DanskBot.Ark.17,1:312.1957. ≡Eruptio pini (Rostr.) M.E.Barr, Mycotaxon 60:438.1996. = Scirrhia pini A.Funk&A.K.Parker, Canad.J.Bot.44:1171.1966. ≡Mycosphaerella pini (A.Funk&A.K.Parker)Arx, Proc.Kon.Ned.Akad.Wetensch.C86,1:33.1983(nom.illegit.,Art.53).

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.

Lecanosticta acicola(Thüm.)Syd.,Ann.Mycol.22:400. 1924.—Fig.3

Basionym.Cryptosporium acicolaThüm.,Flora178.1878. ≡Septoria acicola(Thüm.)Sacc.,Syll.Fung.3:507.1884. ≡Dothistroma acicola (Thüm.)Schischkina&Tzanava,NovostiSist.Nizsh.Rast.1967:277.1967. = Lecanosticta piniSyd.,Ann.Mycol.20:211.1922. = Oligostroma acicolaDearn.,Mycologia18:251.1926. ≡Scirrhia acicola(Dearn.)Sigg.,Phytopathology29:1076.1939. = Systremma acicola(Dearn.)F.A.Wolf&Barbour,Phytopathology 31:70.1941. = Mycosphaerella dearnessiiM.E.Barr,Contr.Univ.MichiganHerb.9:587.1972.

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.

Lecanosticta brevispora Quaedvlieg & Crous, sp. nov. — MycoBankMB801940;Fig.4

Etymology.Namedafteritsrelativelyshortconidia.

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,


Fig. 3 Lecanosticta acicola.a–c.Needleswithascomata,asciandascospores(BPI643015);d–j.needleswithacervuli,conidiaandspermatia(BPI39329);k.colonyonPDA;l.colonyonSNA;m–p.conidiaformedonPNA(k–p=CPC12822).—Scalebars=10µm.

cb

d

a

g

h

i j

k m

n

l

e f

o p

Fig. 4 Lecanosticta brevispora(CPC18092).a,b.Conidiogenouscellsgivingrisetoconidia;c–e.conidia(notemucoidsheath).—Scalebars=10µm.

cb da e


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).

Lecanosticta guatemalensis Quaedvlieg & Crous, sp. nov. — MycoBankMB801941;Fig.5

Etymology.Namedafterthecountrywhereitwascollected,Guatemala.

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.

Lecanosticta longisporaMarm.,Mycotaxon76:395.2000.—Fig.6

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).

Mycosphaerella laricis-leptolepidis Kaz.Itô,K.Satô&M.Ota(as‘laricileptolepis’),Bull.Gov.ForestExp.Sta.96:84.1957

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.

Fig. 5 Lecanosticta guatemalensis(IMI281598).a.ColonysporulatingonPDA;b.colonysporulatingonSNA;c–e.conidiogenouscellsgivingrisetoconidia;f,g.conidia.—Scalebars=10µm.

cb

d

a

ge f


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.

Pseudocercospora angolensis(T.Carvalho&O.Mendes) Crous&U.Braun,Sydowia55:301.2003

Basionym.Cercospora angolensisT.Carvalho&O.Mendes,Bol.Soc.Brot.27:201.1953. ≡Phaeoramularia angolensis (T.Carvalho&O.Mendes)P.M.Kirk,Mycopathologia94:177.1986. ≡Pseudophaeoramularia angolensis(T.Carvalho&O.Mendes)U.Braun,Cryptog.Mycol.20:171.1999.

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.

Pseudocercospora pini-densiflorae (Hori&Nambu)Deigh-ton,Trans.Brit.Mycol.Soc. 88:390.1987

Basionym.Cercospora pinidensifloraeHori&Nambu,TokyoJ.PlantProtection 4:353.1917. ≡Cercoseptoria pinidensiflorae (Hori&Nambu)Deighton, Mycol.Pap. 140:167.1976. = Mycosphaerella gibsoniiH.C.Evans,Mycol.Pap.153:61.1984.

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

Specimen examined.pEru,Dep.Junin,Huasahuasi, fromaSolanum spp., 1975,L.J. Turkensteen,holotypeCBSH-18113,cultureex-typeCBS106.80.

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).

Septoria musiva Peck,Ann.Rep.NewYorkStateMus.Nat.Hist.35:138.1884

= Mycosphaerella populorumG.E.Thomps.,Phytopathology31:246.1941. ≡Davidiella populorum(G.E.Thomps.)Aptroot,inAptroot,Mycosphae-rellaanditsanamorphs:2.ConspectusofMycosphaerella:164.2006.

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.

Fig. 6 Lecanosticta longispora(CPC17940).a–d.Conidiogenouscellsgivingrisetoconidia;e.conidia.—Scalebars=10µm

cb da e


NameonEPPOA1andA2lists NameinEUCouncilDirective Validtaxonomicname EPPO-listedidentification Reference method

Mycosphaerella populorum / Mycosphaerella populorum Septoria musiva Fruitingbodymorphology Bier(1939),Peace(1962), Septoria musiva Waterman(1954)

Mycosphaerella gibsonii / Cercoseptoria pinidensiflorae Pseudocercospora pinidensiflorae Fruitingbodymorphology Deighton(1987) Cercoseptoria pinidensiflorae

Mycosphaerella laricisleptolepidis / Mycosphaerella laricileptolepis Mycosphaerella laricileptolepis Fruitingbodymorphology Peace(1962) Phyllosticta laricis

Phaeoramularia angolensis Cercospora angolensis Pseudocercospora angolensis Fruitingbodymorphology Kirk(1986)

Septoria lycopersici / Septoria lycopersici / Septoria malagutii Fruitingbodymorphology Cline&Rossman(2006) Spegazzinivar.malagutii Spegazzinivar. malagutii

Mycosphaerella dearnessii / Scirrhia acicola Lecanosticta acicola Fruitingbodymorphology/ Barnesetal.(2004) Lecanosticta acicola ITS-RFLP

Mycosphaerella pini / Scirrhia pini Dothistroma septosporum Fruitingbodymorphology/ Evans(1984),Barnesetal. Dothistroma septospora ITS-RFLP (2004)

Table 4EPPOandEUCouncilDirective-listedMycosphaerellaspeciesofquarantineimportance,theircurrentlyadvisedidentificationmethod(s)andtheirvalidtaxonomicnames.Taxonomicnamesmarkedingreyhaveyettoberesolved,thereforetheMycosphaerellanameforthisspeciesshouldstillbeused.

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.


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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