The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

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The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

Arne Schwelm, Cédric Berney, Christina Dixelius, David Bass, Sigrid Neuhauser

To cite this version:

Arne Schwelm, Cédric Berney, Christina Dixelius, David Bass, Sigrid Neuhauser. The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism. Protist, Elsevier, 2016, 167 (6), pp.544 - 554. �10.1016/j.protis.2016.08.008�. �hal-01433101�

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http://www.elsevier.de/protis

Publishedonlinedate9September2016

ORIGINALPAPER

The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

ArneSchwelm^a^,^b, CédricBerney^c^,^d, ChristinaDixelius^a, DavidBass^c,e,and SigridNeuhauser^b,c,1

aSwedishUniversityofAgriculturalSciences,DepartmentofPlantBiology,Uppsala BioCenter,LinneanCentreforPlantBiology,P.O.Box7080,SE-75007Uppsala,Sweden

bUniversityofInnsbruck,InstituteofMicrobiology,Technikerstraße25,6020Innsbruck, Austria

cDivisionofGenomicsandMicrobialDiversity,DeptofLifeSciences,NaturalHistory MuseumLondon,CromwellRoad,SW75BD,UK

dGroupeEvolutiondesProtistesetEcosystèmesPélagiques,UMR7144,CNRS&

UniversitéPierreetMarieCurie,StationBiologiquedeRoscoff,PlaceGeorgesTeissier, 29680Roscoff,France

eCentreforEnvironment,FisheriesandAquacultureScience(Cefas),BarrackRoad,The Nothe,Weymouth,DT48UB,UK

SubmittedApril19,2016;AcceptedAugust28,2016 MonitoringEditor:DavidMoreira

Clubroot disease caused by Plasmodiophora brassicae is one of the most important diseases of cultivated brassicas. P. brassicae occurs in pathotypeswhich differ in the aggressiveness towards their Brassicahostplants.To dateno DNAbased methodtodistinguishthesepathotypeshasbeen described.In2011polymorphismwithinthe28SrDNAofP.brassicaewasreportedwhichpotentially couldallowtodistinguishpathotypeswithouttheneedoftime-consumingbioassays.However,isolates ofP.brassicaefromaroundtheworldanalysedinthisstudydonotshowpolymorphismintheirLSU rDNAsequences.ThepreviouslydescribedpolymorphismmostlikelyderivedfromsoilinhabitingCer- cozoamorespeciﬁcallyNeoheteromita-likeglissomonads.HerewecorrecttheLSUrDNAsequenceof P.brassicae.ByusingFISHwedemonstratethatournewlygeneratedsequencebelongstothecausal agentofclubrootdisease.

Keywords: Clubroot; FISH (ﬂuorescencein-situhybridisation); LSU rDNA;pathotyping; phytomyxea; plas- modiophorids.

Introduction

Plasmodiophora brassicae is a soil-borne plant pathogenthatcausesclubrootdiseaseincrucifers.

1Correspondingauthor.Fax:+435125072928 e-mail [email protected](S.Neuhauser).

Clubrootdiseaseisaneconomicallyimportantdis- ease of oilseed rape and Brassica spp. food and feed crops worldwide (Dixon 2009; Hwang et al.

2012). Phylogenetically, P. brassicae belongs to the Plasmodiophorida, a plant pathogenic group of protists in the Phytomyxea within the eukary- otic supergroup Rhizaria (Bass et al. 2009; Burki

http://dx.doi.org/10.1016/j.protis.2016.08.008

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et al. 2010; Neuhauser et al. 2014). P. brassicae isolates differ in preferences and aggressiveness towards different Brassica hosts (Buczacki et al.

1975). Because of the huge yield losses caused by P.brassicae,toolsforarapididentification and characterisation of the different pathotypes are importantforphytopathologists,plantbreedersand plant growers alike. But despite various efforts, to date pathotypes of P. brassicae can still only be classified usingtime-consuming, labour inten- sivebioassays(Buczackietal.1975;Hatakeyama etal.2004;Someetal.1996;Williams1966).Cur- rently no conclusive DNA marker suitable for a rapidpathotypeidentificationcouldbeidentified.A numberofdifferentDNAmethodssuchasrandom amplifiedpolymorphicDNA(RAPD)andamplified fragmentlength polymorphism (AFLP)have been used to characterise P. brassicae isolates (Möller andHarling 1996;Strehlowetal.2010).Although thesestudiesrevealedhighpolymorphismsamong as well as within P. brassicae populations, so far no correlations to their pathogenicity was found (Manzanares-Dauleux et al. 2001; Osaki et al.

2008; Strehlow et al. 2014).A report of polymorphism in the large subunit (LSU) of the rDNA in differentJapaneseisolatessuggestedconsiderable variationacrosshostsandgeographicareas(Niwa etal.2011).Thisresultheldgreatpotentialforthe molecular distinction of P. brassicae isolates and pathotypes.

While analysing LSU rDNA sequences from P.

brassicae isolates of cosmopolitan origin, we did notretrievetheintraspeciﬁcmoleculardiversitypat- tern reported by Niwa et al. (2011). After further analyses involving the LSU of other phytomyxids and related Rhizaria species, it became clear that the second half of the sequence published by Niwa and colleagues (2011) did not corre- spond to the LSU sequences we had obtained.

The aim of this study was to identify the correct LSU sequence of P. brassicae. By using speciﬁc ﬂuorescencein-situhybridisation(FISH)wetested whichLSUsequencebelongstotheclubrootcaus- ing species. In addition, we analysed the LSU sequences in a wider context of rhizarian LSU sequencestoverifythetaxonomicpositionofthese sequences.

Results

PhylogeneticAnalyses

Weobtainedthenearlycompletesequenceofthe full rDNA operon (18S+ITS1+5.8S+ITS2+28S)

fromP.brassicae(isolateATfromKematen,Austria –thesamesitewherethesamplefortheFISHanal- yseswascollected),W.pythii(isolatePD0236),and M.ectocarpii(strainCCAP1538/1).Becauseofthe variable natureofthe3-end ofthe28Sregionwe didnotgeneratesequencedataforapproximately thelast230bpoutofatotallengthofca.3600bp.

A second nearlycomplete sequenceof therDNA operonofP.brassicae(isolatee3)wasderivedfrom the genomic data. In W. pythii and M. ectocarpii thelengthofthe18Sand28SregionsoftherDNA operoncorrespondedtowhatistypicallyobserved inmosteukaryotes(about1800bpforthecomplete 18S and about 3200bp for the nearly complete 28S).InP.brassicae,therDNAoperonprovedtobe signiﬁcantlylonger(3094bpforthecomplete18S, and4354bpforthenearlycomplete28S).In18S, thisisduetothepresenceofthreeintrons(of374, 393,and479bp.,SupplementaryMaterialFig.S1) which are also present in the sequence from the JapaneseP.brassicaeisolateNGY(AB526243).In 28S, this was due to introns (of 501 and 485bp, respectively) present in thelast thirdof the gene, downstreamfromwhereintronscanbefoundinthe sequencesobtainedbyNiwaetal.(2011).

ThesequencesoftherDNAoperonsofourtwo isolates – AT obtained by direct sequencing of PCRamplicons,ande3obtainedfromthegenomic assembly(Schwelmetal.2015)–differonlyinthree positions: (1)atposition 58of theﬁrst18Sintron, there is a 3bp insertion (’ATT’) in isolate e3; (2) position 342of thethird18Sintron consistsofan

‘A’ in isolate AT and a‘G’ in isolate e3;(3) at the end of theITS2, we found a 2bp. difference in a regionconsistingofan‘AC’motifrepeatedﬁve(isolate AT) or six (isolate e3) times (Supplementary Material Fig. S1). The 18S, ITS1, 5.8S, and 28S regionsare100%identical,andsoarethesecond 18S intron and the two 28S introns. By contrast, thesequencefromtheJapaneseP.brassicaeiso- lateNGY(AB526243)onlymatchesoursequences up to position 4422of sequence KX01115, about one fourth (850bp) within 28S. Upstream of that position, there are only a few differences in the three 18S introns, while the sequences are identical in 18S, ITS1, 5.8S, ITS2, and beginning of 28S. Downstream from position 4422, sequence AB526243becomessigniﬁcantlydistinctfromours;

it differs in many positions in conserved regions, exhibitsnumeroussizeandprimarysequencedif- ferencesinvariableregions,andlacksbothofthe introns we found in the last third of the 28S in our isolates. When blastingthis part of sequence AB526243 against GenBank, we found the clos- est related hit to be sequence FJ973380 from

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the glissomonad Neoheteromitaglobosa, and not that of a previously obtained sequence from an environmental Spongospora-like plasmodiophorid (KJ150290) from a soil sample collected in War- wick,UK(Hartikainenetal.2014).

We then investigated the possible chimeric origin of sequence AB526243 by phylogenetic analysis. Thefour full-lengthsequences of phytomyxids obtained in this study were aligned with the sequence from the Japanese P. brassicae isolate NGY (AB526243), the sequence of the environmental Spongospora-like plasmodiophorid (KJ150290), and a selection of Stramenopiles, Alveolates and Rhizaria from GenBank. We focusedontheregionsoftherDNAoperonencod- ingthe largesubunitof ribosomalRNA (5.8Sand 28S), and performed phylogenetic analyses on two separate datasets consisting of the regions upstream and downstream from position 4422 of sequenceAB526843,respectively(Supplementary Material Fig. S2). The overall topology of the trees we obtained from these two datasets was stable,highlysimilarandwellsupported.Thephy- tomyxidsequencesgeneratedforthisstudyformed a well-supported clade in both phylogenies, and the branching pattern within the Rhizaria corre- spondedtowhatwasexpectedfromearlierstudies based on 18S rDNA (Fig. 1A).However, compar- ing the two tree topologies it became clear that whereas the ﬁrst half of sequence AB526843 is identical to that of our isolates, its second half doesnotgroupwiththeotherPhytomyxeabutnext toNeoheteromitaglobosa,withintheglissomonad Cercozoa(Fig.1B).

Intraspeciﬁc Biodiversityof Plasmodiophora brassicae

We generated 21 new sequences from P. bras- sicae isolates from different continents using primersLPR4, NDL22f,NDL22, 28s3r and 28s4r.

These primers bind at the region of the LSU rDNA of P. brassicae which is located in the 3 part of the alignment (Supplementary Mate- rial Fig. S2). From this position the “P. brassicae”

NGY-typeLSUsequences(AB588900–AB588918;

AB526843–AB526848) group within Glissomona- dida and with Neoheteromita globosa (sequence similaritiesbetween99%and92%).Incontrast,the P.brassicaeLSUsequencesobtainedinthisstudy doformawell-supportedcladewiththesequences ofotherphytomyxids(Fig.2).Nointraspeciﬁcvaria- tionbetweentheP.brassicaesequencesgenerated inthisstudycouldbedetected,allsequencesofP.

brassicae were100% identicalinthis region. The

diversity pattern of the previously assigned non- phytomyxid“P. brassicae”sequencesis similaras described in theoriginalstudy(Niwaetal.2011).

This diversity pattern is high enough in hyper- variableregionstobecompatiblewithinter-speciﬁc differences.

FISHAnalyses

Plasmodiophora brassicaeshows aweak autofluorescence at 488nm (Fig. 3B) and a very weak autofluorescenceat514/561nm(Fig.3D).Butthis autofluorescence is considerably weaker than a true signalobtainedfrom usingFISHprobes(e.g.

Fig. 3F). The FISH probes binding the region in which the sequence of the Japanese isolate NGY (AB526843) and our sequences are identical (Pl_LSU_2313, Pl_LSU_3690) did work well, returningaclearpositivesignalat488nmonboth the lobose plasmodia (Fig. 3F) and the resting spores(Fig.4F)ofP.brassicae.Intheregionwhere sequence AB526843 differs from ours, the two FISH probes matchingthe sequencefrom isolate e3butnotisolateNGYdidbothreturnaclearpos- itivesignal(Figs3J,4J). However,thetwoprobes designed to matchonly isolate NGYdid not yield any positive results in FISH (Figs 3P, 4P). These resultsstronglysupportthemolecularphylogenetic results that thesequenceof theJapanese isolate NGYischimericfromposition4400onwards.

Discussion

Our results demonstrate that there is no genetic diversity in the LSU sequence of P. brassicae in isolatesfromaroundtheworld.Therefore,theLSU isnotsuitabletodistinguishbetweendifferentiso- latesorpathotypesofP.brassicae.FISHanalyses conﬁrmed that the LSU sequences we are pre- senting here do belong to the causal agent of clubroot disease, P. brassicae. Our phylogenetic analyses performed on different partsof theLSU showthatdownstreamofthebindingsiteofprimers NDL22/NDL22F, the sequences of the NGY-type (AB525843)describingdifferencesinthe“P.brassi- cae”LSUsequencesNiwaetal.(2011)mostlikely originatedfromaglissomonadcercozoan.Thelack of overlap with the sequence they had obtained upstream ofthatbindingsitepreventedthemfrom identifying the different biological origin of these sequences.ThespeciﬁcprimersdesignedbyNiwa etal.(2011)fortheirdiversitystudyaresituatedin thedownstreamregionofthatpointandasaresult insteadof amplifyingthe LSUof P.brassicae iso-

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Figure1. CladogramoftreesgeneratedfromtheposterioroutputtreefromBayesiananalysesof the5 end (Fig. 1Aleft) andthe 3 end(Fig. 1Bright) of the LSUrDNA. Thisshows that sequenceP. brassicaeNGY (AB526843) is a chimeric sequence of P. brassicae and a glissmonad sequence similar to Neoheteromita globosa. Fig. 1A was generated using an alignment of LSU sequences up to position 4400 of sequence (AB526843),theright treewasgeneratedstartingfromposition 4500. Cercozoansequencesareshadedin grey,phytomyxidsequencesareshadedinlightblue.Treesweregeneratedusingphytomyxidsequencesplus aselectionofRhizaria,alveolatesandstramenopilesfromGenBankusing.29sequences,A:5999positions, B:4358positions.TreesshowingdistancesaregivenintheSupplementaryMaterialFiguresS3andS4.

lates, theLSUofglissomonads thatwerepresent in the rhizosphere of the clubroot-infected plants was ampliﬁed. Glissomonad Cercozoa are highly abundantanddiverseorganismsinsoil(Howeetal.

2009),itistherefore notsurprisingthattheycould be found in most/all rhizosphere samples. Glis- somonadsarealso–like phytomyxids–knownto haveintronsintheirribosomalgeneclusters(Howe etal.2009).Thepresenceofanintroninthedown- streamfragmentobtainedbyNiwaetal.(2011)was therefore in concordance with a plasmodiophorid originofthesequence.Wedidfindonelongintron in the downstream sequence of P. brassicae that made amplification and sequencing of that frag- mentdifficult, but notinthe sameposition asthat present intheglissomonadsequence.Atthetime whenthestudyofNiwaetal.(2011)waspublished onlyasmallnumberofLSUsequenceswasavail- ableforCercozoa.Giventhislimitationandthefact that P. brassicae and glissomonads both belong to Cercozoa, thechimeric nature of theP. brassi-

caesequencedidresultinaplausiblephylogenetic tree and did not arouse suspicion. However, with an enlarged dataset as the one presented here, it became possible to highlight this. This empha- sises the need to generate more sequence data for lessstudiedeukaryoticgroups toincrease the accuracyofphylogeneticanalyses.Ourresultsalso suggestthat thedescribeddifferences intheLSU sequences of the Japanese “P. brassicae” NGY- typeisolatesdoactuallyrepresentgeneticdiversity intheLSUbetweendifferentglissomonadspecies.

Bycontrast,wecouldnotidentifyanydifferencesin theLSUsequencesofP.brassicaefromcosmopoli- tanisolatesandthereforethisgeneisnotsuitableto distinguishbetweendifferentisolatesorpathotypes ofP.brassicae.

Methods

PathogenmaterialandDNAextraction:DNAfromP.bras- sicae(AT)andWoroninapythiiwasextractedandsequenced

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Figure2. IntraspeciﬁcdiversityofP.brassicaeisolatesofworldwideorigin.Sequencesgeneratedinthisstudy (KX011115-KX011135) form a well-supported clade with sequences of other Phytomyxea from this study (KX011113, KX011114) and genbank (KJ150290) while the NGY-type sequences (AB526844–AB526849;

AB588900–AB588918) form a cladewithGlissomonadida sequences from genbank (FJ97377, FJ973380).

63Sequences,PhyMLtree,supportvaluesaregivenChi²supportvalues(%)/Posteriorprobabilities(%).

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Figure3. FISHimagesofP.brassicae,anasteriskisusedtohighlightthechannelwheretherespectiveprobe shouldbevisible.Allimageswhichwerecreatedusingthesameexcitationwavelengtharearrangedvertically, whilehorizontallyallsamplesweretreatedwiththeidenticalprobe.A–HandM–Pshowsporogenicplasmodia ofP.brassicae.ImagesI–Lshowboth,plasmodiaandrestingspores.Thenucleiofactivelygrowingplasmodia resemble doughnuts, which is caused by the characteristic cruciform nuclear division (e.g. Fig. 3C, G, K).

Bars=10␮m.

as describedbyNeuhauseretal.(2014).DNAfrom thesin- glesporeisolatee3grownintherootsofBrassicanapuswas extractedasdescribedbySchwelmetal.(2015).DNAfromthe Swedishisolateswasobtainedinthesameway.DNA-extracts forotherisolateswerekindlyprovidedbythesourceslistedin Table1,wherealsotheoriginofP.brasssicaeisolatesusedin thisstudyisgiven.

PCRampliﬁcation,cloning,andsequencing:Intraspeciﬁc variationwithinarangeofP.brassicaeisolateswasanalysed by PCR using primersLPR4 and primer 28s4r (Niwa etal.

2011). PCRampliﬁcation was performedin a50␮l reaction mixturewiththeExpandHigh-FidelityPLUSPCRSystem(New EnglandBiolabs)containing0.5␮Mprimersand5–10ngDNA

template.Allreactionswereperformedwithaninitialdenatura- tionstepof98^◦Cfor2minfollowedby10cyclesofdenaturation, annealing,andextensionasfollows:98^◦Cfor15s,initialanneal- ingtemperatureof60^◦Cfor 15swithreductionofannealing temperature of0.5^◦C percycle, and72^◦Cfor 90s,followed by20cyclesof98^◦Cfor15s,annealingtemperatureof54^◦C for15s,and72^◦Cfor90sandaﬁnalstepof5minat72^◦C.

ThePCRproductswereanalyzedona1.2%agarosegelin 0.5×TBEbufferandgelorPCRpuriﬁed.Productswerecloned into pJET1.2 vector and sequenced by Macrogen sequencing service(Netherland)using pJETsequencingprimer and primersLPR4,NDL22f,NDL22,28s4r,28s3r(Niwaetal.2011).

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Figure4. FISHimagesofP.brassicae,anasteriskisusedtohighlightthechannelwheretherespectiveprobe shouldbevisible.Allimageswhichwerecreatedusingthesameexcitationwavelengtharearrangedvertically, while horizontallyallsamples weretreatedwith theidenticalprobe. Fig.4M–Pshowsporogenic plasmodia of P. brassicae.Images E–Hshow restingspores.In Fig.4E–Ha germinating fungalspore canbeseen in between therestingsporesandtheplasmodium.Thedifference intheappearanceof thenucleiislinkedto thedifferentlifecycle stages:Nucleiarevery smallandcondensedinthe restingspores(Fig.4G, K)while thenucleiofactivelygrowingplasmodiaresembledoughnuts,whichiscausedbythecharacteristiccruciform nucleardivision(Fig.4C,O,seealsoFig.3G,K).DifferencesintheintensityoftheFISHsignalsuchasinin Fig.4ForJ,butalsolikeinFig.3ForJcanbeexplainedbytheagingofP.brassicae,withthestrongestsignal inthemostactivecells,thedividingplasmodiaandafadingsignalintherestingspores.Thecontrolimagesin Fig.4A–DareidenticalwithFig.3A–D.Bars=10␮m.

Thesequenceofthesinglesporeisolatee3wasconﬁrmedby thegenomesequence(Schwelmetal.2015).

Nearlycompletesequencesofthefull rDNAoperonwere obtained in two or three overlapping PCR fragments from P. brassicae (isolate AT), Woronina pythii (isolate PD0236), andMaulliniaectocarpii(strainCCAP1538/1),usinga(semi- )nestedPCRapproach.Thenearlycomplete18Spartofthe

operonwasamplifiedusingafirstroundofPCRsperformedwith universalprimerssA1n(forward)andsB1n(reverse),followed by (semi-)nestedPCRswithsA1nand semi-specificreverse primer sB2phy (P. brassicae); sA1n and Maullinia-specific reverse primer V9r-Mau (M. ectocarpii); and Phytomyxea- specificforwardprimersA6-phyandreverseprimerC9r-Phyt (W.pythii).Theremainingpartoftheoperon(fromthe3’end

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Table1. P.brassicaeisolatesanalysedinthisstudy.Isolatesmarkedwith*aresinglesporeisolates.

Accessionnumber P.brassicae isolate

Origin Source Reference

Germany KX011116(PCR)

KX011135 (genomic)

e3^* Germany J.Ludwig-Müller

(TUDresden, Germany)

Fahlingetal.(2004)

Sweden KX011120

KX011121

Påarp6 Påarp7

Påarp Påarp

A.C.

Wallenhammar (HSKonsultAB, Sweden)

Thisstudy

KX011119 H21 Hallsberg

KX011117 KX011118

H5 H16

Hallsberg Hallsberg

KX011123 V9 Veneberg

KX011122 PbBro Skara

NewZealand

KX011124 Lily-1 NZSouth

Island

S.Bulman(PFR Lincoln,New Zealand) Canada

KX011129 ON-2 Ontario S.Strelkov

(Universityof Alberta,Canada)

KX011128 MB-2 Manitoba Caoetal.(2009)

KX011131 P2 Quebec

KX011132 QB Quebec

KX011127 P5 Alberta

KX011130 P6 Ontario

KX011125 F290-07 Alberta Caoetal.(2009)

KX011126 Leduc-SS2* Alberta Xueetal.(2008)

Austria

KX011115 AT Kematen,

Tyrol

M.Kirchmair,S.

Neuhauser

Thisstudy SouthKorea

KX011133 KX011134

C1 C2

SouthKorea P.Y.Lim (Chungnam National

University,South Korea)

*singlesporeisolate.

of 18Sto the 3’ end of 28S)was obtained in twooverlap- ping fragments in P.brassicae and a single fragment inW.

pythiiandM.ectocarpii.ForP.brassicae,afirstPCRwasper- formedwithplasmodiophorid-specificforwardprimerV7f-Plas anduniversalreverseprimer22R(abouttwo-thirdsinside28S), followedby anestedPCRwithplasmodiophorid-specificfor- wardprimerC9f-Plasanduniversalreverseprimer21R.Then thelastthirdof28SwasobtainedusingafirstPCRperformed withPhytomyxea-specificforwardprimerD14f-phy1anduniver- salreverseprimernH4r(about250bp.beforethe3endof28S), followedbyasemi-nestedPCRwithPhytomyxea-specificfor-

wardprimerD14f-phy2andnH4r.ForW.pythii,aﬁrstPCRwas performedwithV7f-PlasandnH4r,followedbyanestedPCR withC9f-PlasanduniversalreverseprimernH2r(about330bp.

beforethe3endof28S).ForM.ectocarpii,aﬁrstPCRwas performedwithV7f-PhagandnH4r,followedbyasemi-nested PCRwithphagomyxid-speciﬁcforwardprimer C9f-Phagand nH4r.SupplementaryTableS1listsallthePCRprimerscited above.

PCRampliﬁcationsweredoneinatotalvolumeof30␮lin reaction mixtures containinga ﬁnal concentration of0.4mM dNTPs,0.5␮Mofeachprimer,1×PCR-reactionbuffer,2␮g/ml

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Table2. FISHprobesused.Probeswerelabelledonthe5 endusingeither6-FAMorTamra.Thetablealso indicateswhereontheLSUrDNAfragmenttheseprobesbindandforwhichsequence-typestheyarespeciﬁc.

Probe Probesequence Dye (KX011135)

Isolatee3, Germany

AB526843 IsolateNGY, Japan

Pl_LSU_3690 GCGGCAGTGATTTTCGGTTT 6-FAM bp3675 bp3684

Pl_LSU_2313 CCAGGCCTTTCAGCCAAGTA 6-FAM bp2313 bp2317

Pl_LSU_5721 CAGCGTCACCCAACCCTTAG 6-FAM bp5711 N

J_LSU_5721 TCCATCCGCTCAACCCTTAG Tamra N bp5723

Pl_LSU_6826 ATTCCAGAAGTCTGCCGCTC 6-FAM bp6819 N

J_LSU_6826 AGTCTAGAAACAAAGGCCTC Tamra N bp6336

Bovine SerumAlbumin, 2.5mM MgCl2,and 0.2U Phusion- Polymerase (Finnzymes), usingas template1␮lofgenomic DNA(firstroundPCRs)or2␮lfromthefirstroundPCRproduct (nestedPCRs).FirstroundPCRconditionswere:initialdenat- urationfor 2min at95^◦C, followedby 36cycles with30sec at 95^◦C, 30sec at58^◦C, and 2min at72^◦C, followed by a finalelongationstepof10minat72^◦C.NestedPCRconditions werethesame,butwith32cyclesandanannealingtemper- atureof63^◦C.NestedPCRproductswererunon1.5%TAE agarosegels;bandsoftheappropriatelengthswereexcised andcleanedfollowingtheprotocoloftheQIAquick^®GelExtrac- tionKit(Qiagen).DirectSangersequencingofallcleanedPCR amplicons was done using the PCR primers and appropri- ateinternalsequencingprimers(detailsofwhichareavailable from theauthors uponrequest). Sequencingwas performed withtheABI-PRISMBigDyeTerminatorCycleSequencingKit, andanalysedwithanABI-377DNAsequencer(Perkin-Elmer, Rotkreuz,Switzerland).

Sequence analysis: A primary alignment of the full lengthphytomyxidLSUsequenceswascreatedusingMAFFT (Katoh and Standley 2013) implemented in Geneious 9.0.5 (Drummondetal.2012)andthisalignmentwasimprovedman- uallyusingBioEdit(version7.0.5.3;Hall1999).Thisalignment wasthensplitintotwoatposition4400ofsequenceAB526843 (IsolateNGY,Japan)wherethesequenceAB526843andthe phytomyxidsequencesgeneratedinourstudystarttodiverge.

Bayesian consensustrees were constructedusing MrBayes v3.1.2inparallelmode(Ronquistetal.2012).Twoseparate MC3runswithrandomlygeneratedstartingtreeswerecarried outfor3Mgenerationseachwithonecoldandthreeheated chains. Theevolutionary modelincluded aGTR substitution matrix,afour-categoryauto-correlatedgammacorrectionand thecovarionmodel.All parameterswereestimated fromthe data.Treesweresampledevery1000generations.300000gen- erationswerediscardedas“burn-in”(treessampledbeforethe likelihoodplotsreachedaplateau)andconsensustreescon- structed from the returning sample. Thetwo returning trees wererearrangedvisuallybyrotatingbranchestomirroreach otherasmuchaspossible.Sequencesgeneratedtoanalyse intraspeciﬁcvariationwerealigned,thealignmenttrimmedand analysedasdescribedabove.

FISHanalyses:FreshclubrootswerecollectedatKematen (Tyrol,Austria)inSeptember2013.Therootswererinsedand ﬁxed in1% PFAsolution overnight and subsequently dehy- drated in an ascending Ethanol series (50%, 80%, 96%).

Clubroots were thencut by hand into thin sectionssuitable for lightmicroscopyusinga sterileblade.Thesectionswere rinsedinsterile,distilledwaterandsubsequentlyequilibrated for10minutesinhybridisationbuffer(900mMNaCl,20mMTris

HClpH7.5,35%formamide,0.01%SDS).Sectionsweretrans- ferredintofreshtubescontaininghybridisationbuffer;50ngof the respectiveprobe was added (Table2) and the samples wereincubatedovernightat46^◦C.Sampleswerethenwashed twicefor10min inwashingbuffer(900mMNaCl,20mMTris HClpH7.5,5mMofNaEDTApH8,0.01%SDS)andmounted inVectashieldcontaining0.05mgmL⁻¹ DAPI.Samples were analysedusingaLeicaTCSSP5IIconfocalmicroscopeusing excitation wavelength of 405nm (DAPI), 488nm(FAM) and 514/561nm(TAMRA).Controls containingonlyHybridisation buffer and DAPI were included to ensure no false positives causedbyautoﬂuorescenceofthesamples.

Probes were designed using the ARB software package (Ludwiget al. 2004) tobind totheregions of28SrDNA (i) wherethesequenceoftheJapaneseisolateNGY(AB526843) andoursequences(KX011115,KX011135)areidentical(i.e.

before position 4429of sequence AB526843) (ii) the region wherethesequenceAB526843differsfrom ournewlydeter- minedsequences(i.e.afterposition4400),butavoidingtheﬁrst 400bpup-anddownstreamfromthisposition.Downstreamof position4400,pairsofprobesweredesignedinthesametwo regions,onematchingsequenceAB526843,andonematching theLSUsequences wedetermined(SupplementaryMaterial Fig.S1).

Probes were synthesised and labelled with FAM (excitation 488nm, green) or TAMRA (excitation 514/561nm, red) at the 5 end by Biomers (Ulm, Germany). All slides were initially screened using the same settings of the LSM, but the energy settings of the laser were adjusted for each sample to avoid excessive over- or underexposure of fea- tures.MicroscopicimageswereprocessedusingGIMP2.8.16 (www.gimp.org). Imageswere trimmed and resized, overlay images ofthethree recordedchannels were produced, and contrast andcoloursettingswere adjustedtoensure ahigh quality of ﬁgures 1–28. All images of the same excitation wavelengthweremodiﬁedtogethertoensurethatdifferences between the FISH-probes were not under- or overstated.

Theoriginal,unprocessedimagesweredepositedatFigshare (http://dx.doi.org/10.6084/m9.ﬁgshare.3184399.v1).

Acknowledgements

The authors would like to thank S. Strelkov (Uni- versity of Alberta, Edmonton, Canada), S.Lim (Chungnam NationalUniversity, South Korea), S.

Bulman (Foodand Plant Research, Lincoln, New Zealand) Ludwig-Müller (TU Dresden, Germany)

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and A.C. Wallenhammar (HS Konsult AB, Swe- den) for kindly providing DNA samples and M.

Kirchmair (University of Innsbruck) for collecting clubrootsforFISHaswellasA.Sandbichler(Uni- versity of Innsbruck) for technical support using the confocal microscope. A. S. was ﬁnancially supported by BioSoM. S. N. was funded by the Austrian Science Fund (FWF): grant J3175-B20 (Erwin SchrödingerFellowship). We thank NERC for a Standard Research Grant (NE/H009426/1) supportingD.B.andC.B.andaNewInvestigator Grant(NE/H000887/1)supportingD.B.

AppendixA. SupplementaryData

Supplementary data associated with this arti- cle can be found, in the online version, at http://dx.doi.org/10.1016/j.protis.2016.08.008.

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The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

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The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

Arne Schwelm, Cédric Berney, Christina Dixelius, David Bass, Sigrid Neuhauser

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The Large Subunit rDNA Sequence of Plasmodiophora brassicae Does not Contain Intra-species Polymorphism

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