Multilocus ISSR markers reveal two major genetic groups in Spanish and South African populations of the grapevine fungal pathogen Cadophora luteo-olivacea

Mul ilocus ISSR Ma ke s Re eal Two Majo Gene ic G oups in Spanish and Sou h A ican Popula ions o he G ape ine Fungal Pa hogen Cadopho a lu eo-oli acea Da id G amaje 1 *, Maela Leo ´n 1 , Ma cela San ana 1 , Ped o W. C ous 2 , Josep A mengol 1 1Ins i u o Ag o o es al Medi e a ´neo, Uni e sidad Poli e ´cnica de Valencia, Valencia, Spain, 2CBS-KNAW Fungal Biodi e si y Cen e, U ech , The Ne he lands Abs ac Cadopho a lu eo-oli acea is a lesse -known ungal unk pa hogen o g ape ine which has been ecen ly isola ed om ines showing decline symp oms in g ape g owing egions wo ldwide. In his s udy, 80 C. lu eo-oli acea isola es (65 om Spain and 15 om Sou h A ica) we e s udied. In e -simple-sequence epea -polyme ase chain eac ion (ISSR-PCR) gene a ed 55 polymo phic loci om ou ISSR p ime s selec ed om an ini ial sc een o 13 ISSR p ime s. The ISSR ma ke s e ealed 40 mul ilocus geno ypes (MLGs) in he global popula ion. Minimum spanning ne wo k analysis showed ha he MLGs om Sou h A ica clus e ed a ound he mos equen geno ype, while he geno ypes om Spain we e dis ibu ed all ac oss he ne wo k. P incipal componen analysis and dend og ams based on gene ic dis ance and boo s apping iden i ied wo highly di e en ia ed gene ic clus e s in he Spanish and Sou h A ican C. lu eo-oli acea popula ions, wi h no in e media e geno ypes be ween hese clus e s. Mo emen wi hin he Spanish p o inces may ha e occu ed epea edly gi en he equen e ie al o he same geno ype in dis an loca ions. The esul s ob ained in his s udy p o ide new insigh s in o he popula ion gene ic s uc u e o C. lu eo-oli acea in Spain and highligh s he need o p oduce heal hy and quali y plan ing ma e ial in g ape ine nu se ies o a oid he sp ead o his ungus h oughou di e en g ape g owing egions. Ci a ion: G amaje D, Leo ´n M, San ana M, C ous PW, A mengol J (2014) Mul ilocus ISSR Ma ke s Re eal Two Majo Gene ic G oups in Spanish and Sou h A ican Popula ions o he G ape ine Fungal Pa hogen Cadopho a lu eo-oli acea. PLoS ONE 9(10): e110417. doi:10.1371/jou nal.pone.0110417 Edi o : Niklaus J. G unwald, USDA ARS, Uni ed S a es o Ame ica Recei ed June 20, 2014; Accep ed Sep embe 19, 2014; Published Oc obe 13, 2014 Copy igh : ß2014 G amaje e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y: The au ho s con i m ha all da a unde lying he indings a e ully a ailable wi hou es ic ion. All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: The au ho s ecei ed no speci ic unding o his wo k. Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * Email: [email p o ec ed] In oduc ion G ape ines a e one o he mos widely g own ui c ops in he wo ld wi h signi ican plan ings in Eu ope, No h and Sou h Ame ica, Sou h A ica and Aus alasia. G apes a e used in he p oduc ion o wine, b andy, o non- e men ed d inks and a e ea en esh o d ied as aisins [1]. The his o y o Eu opean g ape- g owing was cha ac e ized by a oubled second hal o he 19 h cen u y, in which Eu opean g ape g owe s we e aced wi h he a i al o h ee majo phy osani a y p oblems: i s powde y mildew, hen phylloxe a and, inally, downy mildew. Powde y mildew caused by he ungus E isyphe neca o , was in oduced om No h Ame ica o England in 1845, and onwa ds o F ance in 1847. The oo - eeding aphid called phylloxe a was ound in wes e n F ance abou 1865 and apidly sp ead h oughou Eu ope. In 1878, esis an oo s ocks we e in oduced o Eu ope om No h Ame ica in an e o o con ol his pes , bu he downy mildew pa hogen (Plasmopa a i icola) was inad e - en ly in oduced in o F ance, p obably as oospo es on he impo ed oo s ocks, and sp ead widely h oughou Eu ope [2]. These pas epidemics illus a e he des uc i e e ec s o new o in oduced pa hogens and he diseases hey cause on g ape ine cul i a ion. Fungal unk diseases, namely esca, eu ypa and Bo yosphae ia diebacks, as well as black- oo and Pe i diseases, a e cu en ly h ea ening he i icul u e indus y wo ldwide causing signi ican economic losses due o educed yields, inc eased c op managemen cos s o cul u al and chemical p e en i e measu es, and sho ened li e span o he ines [3], hus being named as he ‘‘Phylloxe a o he 21 h cen u y’’ [4]. Among hem, Pe i disease has eme ged as one o he mos de as a ing diseases a ec ing young ines in newly plan ed ineya ds [5]. The ascomyce e ungus Cadopho a lu eo-oli acea was adi ionally conside ed as a mino ascula pa hogen associa ed wi h Pe i disease o g ape ine. This ungus has been ound in many habi a s, such as decaying wood [6] and wa e [7] in he An a c ica, saline and acidic soils in he Czech Republic [8] o associa ed wi h decay symp oms in ines and ui - o ing o kiwi in I aly [9]. Howe e , in ecen yea s C. lu eo-oli acea has been inc easingly epo ed in g ape ine nu se ies and young ineya ds wo ldwide, causing a majo conce n in bo h g ape g owe s and nu se ies [10–15]. Halleen e al. [11] isola ed 15 s ains o C. lu eo-oli acea, p e iously iden i ied as Phialopho a sp., om he g a union o appa en ly heal hy plan s in comme cial nu se ies in Sou h A ica. The pa hogenici y o one o hese isola es was u he demon- s a ed unde glasshouse and ield condi ions [12]. Rooney- La ham [14] indica ed ha C. lu eo-oli acea was qui e common in esca and Pe i-disease a ec ed g ape ines and isola ed 13 s ains h oughou Cali o nian ineya ds. G amaje e al. [10] eco e ed 58 C. lu eo-oli acea isola es om g ape ines showing black ascula s eaking and decline symp oms cha ac e is ic o Pe i PLOS ONE | www.plosone.o g 1 Oc obe 2014 | Volume 9 | Issue 10 | e110417 disease, and om en i onmen al samples collec ed a di e en g ow h s ages o g ape ines in Spanish nu se ies. The pa hoge- nici y o ep esen a i e Spanish isola es was con i med unde ield [16] and g eenhouse condi ions [10]. In U uguay, Na a e e e al. [13] e alua ed he pa hogenici y o one s ain o C. lu eo-oli acea ob ained om nu se y plan ing ma e ial showing ex e nal and in e nal symp oms o Pe i disease, bu esul s we e inconclusi e. Mo e ecen ly, U ´ bez-To es e al. [15] isola ed six s ains o C. lu eo-oli acea associa ed wi h young ine decline in B i ish Columbia (Canada), and ep oduced symp oms o da k ascula s eaking on inocula ed g ape ine wood unde g eenhouse condi ions. Despi e he clea implica ion o C. lu eo-oli acea in ea ly s ages o g ape ine wood disease, he biology, epidemiology and managemen o his ungus a ec ing g ape ines emain poo ly unde s ood. An unde s anding o i s mode o ep oduc ion and he sou ce o inoculum esponsible o C. lu eo-oli acea dispe sal is, howe e , essen ial o he e icien managemen o Pe i disease. To da e, no sexual ui ing bodies o C. lu eo-oli acea ha e e e been ound in ineya ds. Howe e , sexual ep oduc ion may be ansien in ineya ds o may occu on cu en ly uniden i ied al e na i e hos plan s. In Spain, his species was isola ed om asymp oma ic xylem issues o weeds collec ed in comme cial ineya ds [17], and i was also de ec ed in na u al soils om g ape ine nu se ies by using bai plan s [18]. G amaje e al. [19] in es iga ed in i o he sensi i i y o C. lu eo-oli acea o ho -wa e ea men s and ound ha conidial ge mina ion o his ungus was inhibi ed a empe a u es abo e 51uC, while ea men s o up o 54uC o 60 min we e necessa y o inhibi mycelial g ow h. Popula ion gene ics as applied o plan pa hogens hold eno mous p omise o unde s anding ce ain e olu iona y o ces con olling pa hogen popula ions (e.g., selec ion, mig a ion, and ecombina ion). Popula ion gene ic in e ence also p o ides insigh s in o whe he popula ions a e sexual o clonal, di e en ia ed o admixed, and whe he pheno ypes a e linked o geno ypes [20]. All o his knowledge is impo an o he de elopmen o imp o ed in eg a ed pes managemen s a egies and success ul esis ance b eeding p og ams. Con ol o endogenous pa hogens such as C. lu eo-oli acea in g ape ines is p oblema ic. In ineya ds, managemen s a egies ecommended o p e en ion and disease managemen mainly in ol e he p e en ion and/o co ec ion o p edisposing s ess si ua ions [21]. Nu se y ines ha e been iden i ied as a signi ican sou ce o Pe i disease in ineya ds a ound he wo ld. The e a e many oppo uni ies o in ec ion by ungal unk pa hogens du ing he p opaga ion p ocess: wounds made in he issue a e e y s age o p oduc ion, hyd a ion anks o callusing ooms. The e o e, an in eg a ed managemen p og am ha includes HWT, chemical, biological, o o he con ol measu es has been sugges ed o be he mos in e es ing p ocedu e o educe in ec ions by ungal unk pa hogens du ing he nu se y s ages [5]. Mul ilocus molecula ma ke echnology has been showed o be highly sui ed o assess he gene ic s uc u e o plan -pa hogenic ungi [22–24]. In con as o o he ma ke s such as i ulence and ungicide esis ance, molecula ma ke s a e p esumed o be selec i ely neu al and he e o e may be used o s udy e olu iona y p ocesses [25]. One app oach, in e -simple-sequence epea (ISSR)-PCR, is a disc imina o y, genome-wide DNA inge p in - ing me hod ha in ol es ampli ica ion o a DNA segmen lanked by wo iden ical mic osa elli e epea s bu o ien ed in opposi e di ec ions [26]. These simple-sequence epea s (SSRs) o mic o- sa elli es a e hype a iable and a e andomly dis ibu ed h ough- ou he genome; he numbe o hese epea ing mo i s will a y acco ding o he indi idual. The main ad an ages o ISSR-PCR a e ha hey do no equi e he knowledge o genome sequences, de ec ion o high le els o polymo phism, low cos , simple ope a ion, high s abili y and ep oducibili y, and apid u no e [27]. Disad an ages include he ac ha hese a e dominan ma ke s p ecluding he possibili y o de ec ing he e ozygosi y, lack o knowledge o allelic bands, in e p e ing alleles ha a e iden ical in s a e as being iden ical by descen , po en ial p oblems o con amina ed empla e DNA o can ha e ep oducibili y p oblems [28]. To da e, he only s udy o he biology and gene ic s uc u e o C. lu eo-oli acea popula ions om g ape ines was pe o med by G amaje e al. [10], who ob ained iden ical sequence p o ile among 58 Spanish C. lu eo-oli acea isola es by ampli ica ion o he highly conse ed ITS egion, he pa ial b- ubulin (BT) o elonga ion- ac o 1a(EF) genes, sugges ing ha he popula ion o his species was ep esen ed by a single clonal lineage. The Spanish and Sou h A ican C. lu eo-oli acea isola es ep esen he unique collec ion o his ungus a ailable o i s use in popula ion gene ic s udies. The e o e, he objec i es o he p esen s udy we e (i) o de e mine he gene ic s uc u e o C. lu eo-oli acea in Spain a a egional scale by using mul ilocus ISSR ma ke s, (ii) o compa e he gene ic s uc u e o C. lu eo-oli acea isola es om Spain and Sou h A ica and (iii) o assess he i ulence o ep esen a i e C. lu eo-oli acea isola es om he gene ic clus e s ob ained in his s udy. Ma e ials and Me hods E hics s a emen No speci ic pe mi s we e equi ed o he desc ibed ield s udies. Loca ion o g ape ine nu se ies and ineya d si es we e acili a ed by egional Plan Heal h se ices in Spain and by ARC In ui ec- Nie oo bij in Sou h A ica. Pe mission o sample he ineya ds and nu se ies we e g an ed by he landowne and nu se ymen, who a e collabo a ing wi h he Uni e sidad Poli e´cnica de Valencia (Valencia, Spain). The si es a e no p o ec ed in any way. The a eas s udied do no in ol e any species endange ed o p o ec ed in Spain and Sou h A ica. Isola es In his s udy 80 C. lu eo-oli acea isola es we e geno yped. The collec ion consis ed o 65 isola es om Spain and 15 isola es om Sou h A ica (Figu e 1, Table 1). Spanish popula ions we e sampled om oo s ock wood o young plan s showing black ascula s eaking and decline symp oms cha ac e is ic o Pe i disease (n = 55), and om nu se y samples and plan ing ma e ial a di e en s ages o he p opaga ion p ocess (n = 10). Vineya d isola es we e collec ed be ween 2007 and 2010 om six p o inces in Spain (Badajoz, Ciudad Real, G anada, Mallo ca, Valencia and Za agoza) (Figu e 1, Table 1). Addi ionally, 10 isola es we e collec ed in 2008 om ou nu se ies in Aielo de Mal e i (loca ed be ween Valencia and Alican e p o inces) and ea ed as a single popula ion. This is one o he main nu se y a eas in Spain and dis ibu es plan ing ma e ial o all Spanish g ape ine egions. Fi y-eigh Spanish isola es we e included in a p e ious s udy by G amaje e al. [10]. The Sou h A ican popula ion was sampled in 1999–2000 om appa en ly heal hy oo ed cu ings in g ape ine nu se ies in Malmesbu y (n = 1) and Welling on (n = 14) in he Wes e n Cape P o ince (Figu e 1, Table 1). These 15 isola es we e iden i ied p e iously as Phialopho a sp. and epo ed by Halleen e al. [11]. Cadopho a lu eo-oli acea isola es we e collec ed om oo s ock sec ions in Spain and oo ed cu ings in Sou h A ica as p e iously desc ibed by G amaje e al. [10] and Halleen e al. [11], Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 2 Oc obe 2014 | Volume 9 | Issue 10 | e110417 espec i ely. Samples om Spanish nu se ies we e aken a h ee s ages o he p opaga ion p ocess: samples om p e-g a ing hyd a ion anks, washings om scisso s and washings om g a ing ools. Wa e samples we e collec ed and il e ed as desc ibed by A oca e al. [29]. The isola es we e single-spo ed by se ial dilu ion [30] and iden i ied acco ding o hei mo phological cha ac e s as well as he sequence analyses o he ITS egion, BT and EF genes p io o use [10]. They we e hen s o ed in a 15% glyce ol solu ion a 280uC in 1.5 mL c yo ials. DNA isola ion and quan i ica ion F eeze-d ied ungal issue was g ound o a ine powde unde liquid ni ogen using a mo a and pes le. To al DNA was ex ac ed using he E.Z.N.A. Plan Minip ep Ki (Omega Bio ek, Do a ille, GA) ollowing he manu ac u e ’s ins uc ions. DNA was quan i ied using a spec opho ome e (ND-2000, NanoD op Technologies, Wilming on, DE) and adjus ed o a inal concen- a ion o genomic DNA a 15 ng ml –1 o PCR ampli ica ion. ISSR p o iling Fi een C. lu eo-oli acea isola es we e used in a p elimina y sc een o de e mine hose ISSR p ime s ha enabled gene a ion o polymo phic, ep oducible ma ke s ha could be used o gene a e polymo phic DNA inge p in s o all C. lu eo-oli acea isola es. This sc een es ed 13 ISSR p ime s (Table 2), which we e p e iously epo ed o p oduce polymo phic and ep oducible DNA inge p in s om o he ungi and plan species. Each PCR eac ion con ained 1X PCR bu e , 2.5 mM MgCl 2 , dNTPs a 100 mM each, p ime s a 0.4 mM each, 0.5 U DNA Taq polyme ase and 0.5–5 ng empla e DNA. The PCR eac ion mix was adjus ed o a inal olume o 25 mL wi h wa e (Ch omasol Plus, Sigma-Ald ich). PCR ampli ica ions we e pe o med in a Pel ie The mal Cycle -200. Condi ions included an ini ial s ep o 5 min a 95uC, ollowed by 34 cycles o dena u a ion a 95uC o 1 min, annealing a p ime -speci ic empe a u e (Table 2) o 1 min, and elonga ion a 72uC o 2 min. A inal ex ension was pe o med a 72uC o 10 min. PCR p oduc s we e isualized in 1.5% aga ose gels (aga ose D-1 Low EEO, Conda). The Gene Rule 100-bp DNA ladde plus was used as a molecula weigh ma ke (Fe men as Inc., Hano e , MD, USA). Analysis was eplica ed a leas h ee imes o he subse o isola es, wi h independen DNA ex ac ions, PCR, and sizing o agmen s o con i m ep oducibili y o esul s. Those p ime s ha gene a ed ep oducible, clea ly disce nible, polymo phic bands in epea ed expe imen s we e selec ed o subsequen ly ampli y genomic DNA om all isola es. Ampli ica ion p o iles o he 80 C. lu eo-oli acea isola es gene a ed by he ou selec ed ISSR p ime s we e compa ed and he DNA agmen s we e sco ed compu a ionally using he GelAnalyze 2010a so wa e (h p://www.gelanalyze .com). Only bands (size: 100 o 2,500 bases) ha could be sco ed consis en ly o all samples we e used, wi h he assump ion ha each band wi h di e en molecula weigh ep esen ed a dis inc locus and amplicons sha ing he same molecula weigh we e conside ed o be he same allele a a speci ic locus. The absence o amplicons was conside ed as an al e na e allele. Da a analysis Ampli ied DNA agmen s we e ans o med in o a bina y cha ac e ma ix (1 = p esence, 0 = absence). A mul ilocus geno ype (MLG) was cons uc ed o each isola e by combining da a o single ISSR inge p in s by using he p ocedu e a ailable in he package POPPR [31] o R e sion 3.0.3 (The R Founda ion o S a is ical Compu ing) [32]. Isola es wi h he same MLG we e conside ed clones, and some analyses we e conduc ed o he global and clone-co ec ed da a se . To assess he possible e olu iona y ela ionships among MLGs, minimum spanning ne wo ks we e cons uc ed, i s om geno- ypes o di e en p o inces o nu se ies in Spain (nu se y is conside ed as a single popula ion sepa a e om he ineya d subpopula ions by p o inces) and also including he geno ypes om Sou h A ica. Rela i e dissimila i y dis ances we e calcula ed acco ding o he index o associa ion [33]. I e u ns a dis ance e lec ing a a io o he numbe o obse ed di e ences by he Figu e 1. Geog aphic dis ibu ion o he Spanish and Sou h A ican Cadopho a lu eo-oli acea isola es analyzed wi h ou ISSR p ime s. The esul s o he PCA and dend og am analyses o he Spanish and Sou h A ican mul i-locus geno ypes (MLGs) a e epo ed on he map: in whi e, MLGs assigned o clus e 1, in g ay, MLGs assigned o clus e 2. The numbe o isola es o each popula ion is indica ed in b acke s. Spanish popula ions wi h less han i e indi iduals we e no conside ed o all he analyses, and a e indica ed by an as e isk. doi:10.1371/jou nal.pone.0110417.g001 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 3 Oc obe 2014 | Volume 9 | Issue 10 | e110417 numbe o possible di e ences. The numbe o possible di e ences is he numbe o loci mul iplied by ploidy. The R package POPPR [31] was used o calcula e he dissimila i y dis ance ma ices and o gene a e minimum spanning ne wo ks om he ma ices. We es ed o he exis ence o di e gen gene ic pools o C. lu eo-oli acea in Spain using a gene ic mul i a ia e analysis [34] and a dend og am including boo s ap suppo o clades o de ec gene ically di e en ia ed g oups. These me hods a oid he clus e ing o indi iduals on a p io i knowledge such as geog aph- ical loca ions ha may mix di e gen gene ic lineages in oduced in he same a ea and may hinde he de ec ion o admix u e e en s among hese lineages. Fi s , we used a p incipal componen analysis (PCA) o in es iga e he gene ic s uc u e o he C. lu eo- oli acea popula ion in Spain, and he global popula ion in Spain and Sou h A ica. PCA is independen o any gene ic hypo heses and i is sui able o he analysis o pa ially clonal species. PCA analysis was pe o med using he R package ADEGENET [35]. Fo his analysis, only single copies o he di e en geno ypes we e used o gi e iden ical weigh o MLGs. Popula ions wi h less han i e indi iduals (Badajoz, G anada, Mallo ca and Za agoza) we e no conside ed o he analyses. UPGMA dend og ams we e also in e ed om he dis ance ma ices and isualized using Molecula E olu iona y Gene ic Analysis (MEGA) so wa e, e sion 6 o Windows [36]. Boo s apping was pe o med wi h he R package P clus wi h 10,000 boo s ap esamplings [37]. Popula ions wi h less han i e indi iduals (Badajoz, G anada, Mallo ca and Za agoza) we e no conside ed o all he analyses. The gene ic ichness (R) and he e enness index adap ed om he Simpson index o geno ypic di e si y (ED*) we e calcula ed o each popula ion using he R package POPPR [31]. Geno ypic di e si y (D) was calcula ed using D = n/(n–1)(1–Spi 2 ) as imple- men ed in MULTILOCUS [38], whe e pi is he equency o he i h geno ype and n is he numbe o indi iduals sampled. D ep esen s he p obabili y ha wo indi iduals sampled a andom wi h eplacemen ha e he same geno ype, and is simila o he di e si y measu e o Pielou [39]. Ra e ac ion cu es ep esen ing species ichness we e calcula ed o de e mine i he sampling in ensi y was adequa e o de ec he majo i y o C. lu eo-oli acea s ains p esen in each popula ion. Because sample size a ied among popula ions we employed a e ac ion o explo e he e ec o sample size on obse ed species ichness [40]. The unc ion ‘ a ecu e’ om he R package egan [41] was used o gene a e a e ac ion cu es. To s udy he con ibu ion o possible sexual ep oduc ion o he geno ypic di e si y obse ed o each popula ion, he p obabili y ha a geno ype was ob ained by chance h oughou a sexual e en (P gen ) was calcula ed using GENCLONE [42], as well as he p obabili y ha an isola e sha ed he same MLG as ano he in he sampled popula ion (P sex ), assuming ha he wo isola es de i e om sexual ep oduc ion [43]. Linkage disequilib ium as an Table 1. Cadopho a lu eo-oli acea isola es ob ained om g ape ine in Spain and Sou h A ica included in his s udy a . O igin Coun y, p o ince Vineya d Nu se y Numbe o isola es Yea Spain b Badajoz 1 … 1 2007 Ciudad Real 10 … 10 2007–2010 G anada 2 … 2 2009 Mallo ca 4 … 4 2008 Valencia 37 10 47 2008–2010 Za agoza 1 … 1 2008 Sub o al 55 10 65 Sou h A ica … 15 15 1999–2000 To al 55 25 80 a Isola es om Spain we e collec ed by D. G amaje. Sou h A ican isola es we e collec ed by F. Halleen om g ape ine nu se ies in Malmesbu y (1 isola e) and Welling on (14 isola es). b Fi y-eigh Spanish isola es we e included in a p e ious s udy by G amaje e al. [10]. doi:10.1371/jou nal.pone.0110417. 001 Table 2. Band cha ac e is ics o he ou in e -simple-sequence epea (ISSR) p ime s ha we e selec ed and ul ima ely used in epea ed expe imen s a . ISSR p ime Ta(uC) b Nuo ampli ied bands Polymo phism (%) P ime desc ip ion (GAC) 5 46.0 20 80 Ancho less i-nucleo ide (ACTG) 4 48.0 12 91.7 Ancho less e a-nucleo ide (GACA) 4 46.0 22 86.4 Ancho less e a-nucleo ide DDB(CCA) 5 61.0 13 84.6 T i-nucleo ide, 59ancho p esen a ISSR p ime s ha we e unable o gene a e polymo phic and ep oducible ma ke s: (GAA) 6 , (GGAT) 4 , (GATA) 4 , DBDA(CA) 7 , YHY(GT) 5 G, HBH(AG) 7 A, BDB(ACA) 5 , DHB(CGA) 5 and DBH(TCG) 5 . b Annealing empe a u e. doi:10.1371/jou nal.pone.0110417. 002 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 4 Oc obe 2014 | Volume 9 | Issue 10 | e110417 indica ion o andom ma ing was calcula ed and es ed o signi icance wi h 1,000 andomiza ions using he R package POPPR [31]; measu es o game ic disequilib ium es ed we e he index o associa ion (I A ), and a s anda dized al e na i e o he I A ( d ) [38]. The null hypo hesis o his es is ha he e is andom associa ion among alleles a di e en loci and I A = 0; he null hypo hesis o andom ma ing is ejec ed whe e I A .0. These es s we e no pe o med on samples sizes ,10 due o lack o s a is ical powe [44]. Popula ion gene ic s uc u e was analyzed by conduc ing an analysis o molecula a iance (AMOVA) [45] on clone-co ec ed da a using ARLEQUIN e sion 3.5 [46]. The ixa ion index (F ST ) was also calcula ed using ARLEQUIN. Signi icance o F ST was es ed using 1,000 pe mu a ions. Due o low sample sizes, popula ions om Badajoz, G anada, Mallo ca and Za agoza we e excluded om he analysis. Vi ulence assays Rep esen a i e isola es o C. lu eo-oli acea we e andomly selec ed om each o he wo gene ic clus e s (6 isola es ep esen ing 4 MLGs om clus e 1 and 8 isola es ep esen ing 5 MLGs om clus e 2) in e ed by he Bayesian clus e ing me hod and he gene ic mul i a ia e analysis. Vi ulence assays we e conduc ed on 1-yea -old g ape ine cu ings o 110 Rich e (R) oo s ock. In o al 168 do man cu ings we e cu in o uni o m leng hs con aining ou o i e buds, and hen ho -wa e ea ed a 53uC o 30 min o elimina e he possible incidence o ungal unk pa hogens [47]. In o de o enhance callusing and oo ing, do man cu ings we e bu ied in o s e ilized pea moss in plas ic boxes, and placed in a callusing oom a 25uC and 100% humidi y o 4 weeks. A e callusing and oo ing, cu ings we e wounded be ween he wo uppe in e nodes wi h a 5 mm co k bo e . A 5 mm mycelium aga plug om a 2-weeks-old cul u e was placed in he wound. Wounds we e w apped wi h Pa a ilm. Six cu ings pe ungal isola e we e inocula ed wi h 5 mm uncolonized PDA plugs om wo di e en pla es as nega i e con ols. Inocula ed cu ings we e plan ed immedia ely in indi idual po s, placed in a g eenhouse a 25uC and wa e ed e e y 3 d o as needed. Plan s we e a anged in a comple ely andomized design. The expe i- men was epea ed. Cu ings we e collec ed a e 4 mon hs and inspec ed o lesion de elopmen . Ex en o ascula discolo a ion was measu ed upwa d and downwa d om he inocula ion poin . Addi ionally, shoo d y weigh was e alua ed o sp ou s o med abo e he inocula ion poin . Small pieces (0.5 o 1 cm) o nec o ic issue om he edge o each lesion we e cu and pla ed on o mal ex ac aga (MEA) (Oxoid L d., Basings oke, Han s, England) supplemen ed wi h 0.5 g L 21 s ep omycin sulpha e (MEAS) (Sigma-Ald ich, S . Louis, MO, USA) in an a emp o eco e he inocula ed ungi and con i m Koch’s pos ula es. Fungi we e iden i ied as p e iously desc ibed. Analyses o a iance (ANOVA) we e conduc ed o analyze lesion leng h and shoo d y weigh da a. Homogenei y o a iance was es ed using Le ene’s es . Residuals we e isually inspec ed o each ea men , and when necessa y a log 10 ans o ma ion was used o imp o e homogenei y o a iance. T ea men means among indi iduals and gene ic clus e s we e compa ed using S uden ’s leas signi ican di e ence (LSD) es a P,0.05. In all cases he S a is ix 10 so wa e (Analy ical So wa e, FL, USA) was used. Resul s ISSR ma ke s In o al, 55 polymo phic loci we e gene a ed om ISSR-PCR using he ou ISSR p ime s selec ed om a sc een o 13 ISSR p ime s (Table 2). The highes numbe o polymo phic bands (PBs) was p oduced o p ime (GACA) 4 (an ancho less e a- nucleo ide, 19 PBs), ollowed by p ime (GAC) 5 (an ancho less i- nucleo ide, 16 PBs), p ime (ACTG) 4 (an ancho less e a- nucleo ide) and p ime DDB(CCA) 5 (a 59-ancho ed i-nucleo ide), bo h wi h 11 PBs. The mean pe cen age o polymo phic loci was 85.65%. Geno ypic di e si y In gene al, Spanish subpopula ions showed high geno ypic di e si y. The ou ISSR p ime s gene a ed a o al o 31 MLGs among he 65 Spanish C. lu eo-oli acea isola es (Table 3). The mos equen MLG was MLG31 (18.4%), which was p esen in subpopula ions om nu se y, Mallo ca and Valencia, ollowed by MLG14 (12.3%) which was p esen in nu se y subpopula ion and Ciudad Real and Valencia p o inces, and MLG29 (12.3%), con aining only isola es om Valencia p o ince. MLG13 (10.8%) was also equen and was de ec ed in Ciudad Real and Valencia p o inces. O he 31 MLGs, wo we e obse ed wice (MLG3 in Mallo ca egion and MLG26 in Valencia p o ince) and he emaining MLGs we e obse ed once (80.6% o he MLGs). When including he 15 isola es om Sou h A ica in he analysis, he ou ISSR p ime s gene a ed a o al o 40 MLGs. O he 10 MLGs obse ed in he Sou h A ican subpopula ion, he mos equen MLG was MLG13 (40.0%). The emaining 9 MLGs we e obse ed once. O e all, gene ic ichness (R) a ied ac oss subpopula ions de ined by di e en p o inces in Spain and Sou h A ica, om e y high in Ciudad Real o Sou h A ica (0.64 o 0.78), o high in Valencia o he nu se y subpopula ion (0.44) (Table 3). E enness (ED*) alues we e highe in he Ciudad Real subpopula ion (0.93) compa ed wi h he egional popula ions in Spain and Sou h A ica (0.62–0.65). Di e si y (D) alues a ied om e y high (0.95) in he Ciudad Real subpopula ion, whe e nea ly all indi iduals we e a dis inc geno ype, and in Valencia and Sou h A ica (0.86 o 0.89); o high o he nu se y subpopula ion, whe e i e geno ypes we e de ec ed wi hin he 10 isola es sampled. Acco ding o he a e ac ion cu es (Figu e 2), Valencia p o ince had a highe numbe o samples and obse ed axa ela i e o he o he subpopula ions. A sample size o 10 ( he sample size o Ciudad Real p o ince and nu se y subpopula ion) was common o all popula ions. Compa ison o a e ac ion cu es showed signi ican di e ences in di e si y among hese popula ions a his common sample size (Figu e 2) wi h Ciudad Real p o ince being mo e di e se han he o he s, and wi h Sou h A ica and Valencia p o ince ha ing compa able amoun s o di e si y as well as being mo e di e se han nu se y subpopula ion. Rela ionship among geno ypes The minimum spanning ne wo k o he Spanish popula ion dis inguished he mos equen MLGs in Spain, MLG13, MLG14, MLG29 and MLG31 (Figu e 3A). The global MLG ne wo k showed ha he MLGs om Sou h A ica clus e ed a ound he mos equen geno ype (MLG13), while he geno ypes om Spain we e dis ibu ed all ac oss he ne wo k (Figu e 3B). Popula ion s a i ica ion P incipal componen analysis e ealed wo clus e s o MLGs (Figu e 4a). Axis 1 and 2 o he PCA accoun ed o 33.1% and Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 5 Oc obe 2014 | Volume 9 | Issue 10 | e110417 12.8% o o al gene ic a iabili y. Rega ding he analysis o he da ase om Spain and Sou h A ica, 12 MLGs we e g ouped in one clus e , con aining wo MLGs om Sou h A ica, namely MLG13 and MLG36 (Figu e 4b). A second clus e was composed o 23 MLGs, and included eigh MLGs om Sou h A ica. P incipal componen analysis also e ealed wo clus e s o MLGs (Figu e 4b). Axis 1 and 2 o he PCA accoun ed o 25.3% and 12.1% o o al gene ic a iabili y. Da a om he gene ic dis ance ma ices we e analyzed using an UPGMA algo i hm; only he op imal UPGMA ees a e p esen ed (Figu e 5). The wo gene ic clus e s we e sepa a ed by la ge gene ic dis ances, wi h obus boo s ap (BS) suppo o sepa a ion among clus e s (BS.70%). In bo h analyses, he clus e s disc imina ed using he dend og am analysis we e in ag eemen wi h he clus e s in e ed using mul i a ia e app oach (Figu e 4). The AMOVA esul s based on Spanish popula ions (Table 4), showed 6.3% o he a ia ion among ineya d popula ions conside ed oge he ega dless o geog aphical o igin and nu se y subpopula ions, 5.5% among popula ions wi hin ineya d and nu se y en i onmen s, and 88.1% wi hin popula ions. The o e all F ST was 0.062 and he pe mu a ion P alue was 0.03. The AMOVA esul s based on popula ions de ined by isola es’ coun y o o igin (Table 4) showed 13.3% o he a ia ion among popula ions and 86.7% wi hin popula ions. The o e all F ST was 0.135 and he pe mu a ion P alue was ,0.00001. The wo clus e s iden i ied in mul i a ia e and dend og am clus e ing analyses in he Spanish C. lu eo-oli acea popula ion we e also es ed o signi icance and appo ionmen o a ia ion wi h hie a chical AMOVA; whe ein 23.8% o he a ia ion was appo ioned among he wo clus e s and 76.2% wi hin clus e s (Table 4). The o e all F ST was 0.238 and he pe mu a ion P alue was ,0.00001. Sel ing and clonali y in popula ions The linkage disequilib ium es s (I A and d ) on all indi iduals and he clone-co ec ed da a ejec ed he null hypo hesis o ecombina ion (P,0.01) (Table S1). The p obabili ies ha he mos equen geno ypes de ec ed in Spain and Sou h A ica e ol e by chance h ough sexual ep oduc ion (P gen ) and ha wo isola es sha e hese geno ypes in he Spanish and Sou h A ican popula ions (P sex ) we e e y low (Table S2). Vi ulence assays Da a o he wo i ulence assays we e combined because o he lack o signi ican di e ences be ween he wo es s and among he s udied a iables (P.0.05). All isola es o C. lu eo-oli acea used in his s udy we e pa hogenic o g ape ine cu ings o 110 R oo s ock (Figu e 5). Symp oms de eloped 4 mon hs a e inocu- la ion consis ed o lea es wi h in e einal chlo osis and nec osis, and nec o ic xylem lesions. The s a is ical analysis indica ed signi ican (P,0.05) di e ences in i ulence among ea men s. Values o lesion leng h anged be ween 3.6 and 6.0 cm, and he alues o shoo d y weigh om 1.5 o 4.6 g. All he C. lu eo- oli acea isola es caused lesions in he xylem o g ape ine oo s ock ha we e signi ican ly longe han in he con ol. The a e age shoo d y weigh o he isola es CR2, SA11, V23, N5, V13 and CR10 was signi ican ly di e en compa ed o he con ol (Figu e 6). T ea men means among isola es belonging o he wo gene ic clus e s showed no signi ican di e ences when e alua ing he lesion leng h (P= 0.2628; Clus e 1: 4.3860.35; Clus e 2: 4.7760.38) and he shoo weigh (P= 0.9894; Clus e 1: 3.1960.28; Clus e 2: 3.1960.23). Discussion This s udy is he i s o employ gene ic ma ke s o analyze he popula ion s uc u e o a collec ion o Cadopho a lu eo-oli acea isola es ob ained om ineya ds and g ape ine nu se ies om Table 3. Gene ic di e si y o he Spanish and Sou h A ican popula ions o Cadopho a lu eo-oli acea con as ing egional ineya d popula ions (Ciudad Real and Valencia), he nu se y popula ion om Valencia egion and popula ions be ween coun ies a . Pa ame e s b To al Spain Ciudad Real Valencia Nu se y Sou h A ica Sample Size (N) 80 65 10 37 10 15 Geno ypes (G) 40 31 8 17 5 10 Geno ypic ichness (R) 0.49 0.47 0.78 0.44 0.44 0.64 E enness (ED*) 0.55 0.60 0.93 0.65 0.62 0.63 Di e si y (D) 0.93** 0.92** 0.95** 0.89** 0.67** 0.86** a The non edundan indices o geno ypic di e si y ecommended by A naud-Haond e al. [43] we e calcula ed o each popula ion on he o al da a se . b R, geno ypic ichness, R = (G–1)/(N–1) whe e G is he numbe o mul ilocus geno ypes disc imina ed; ED*, e enness index adap ed om Simpson di e si y; D, geno ypic di e si y. ** = P,0.01. doi:10.1371/jou nal.pone.0110417. 003 Figu e 2. Ra e ac ion cu es o sampling Cadopho a lu eo- oli acea isola es in Ciudad Real and Valencia p o inces, in nu se y subpopula ion and in Sou h A ica. doi:10.1371/jou nal.pone.0110417.g002 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 6 Oc obe 2014 | Volume 9 | Issue 10 | e110417 di e en p o inces in Spain and Sou h A ica. Clus e ing analysis (PCA and dend og ams based on gene ic dis ance and boo - s apping) iden i ied wo highly di e en ia ed gene ic clus e s in he Spanish C. lu eo-oli acea popula ion wi h no in e media e geno ypes among hese clus e s, one including a common MLG, namely MLG13, and he second including he o he h ee dominan geno ypes, namely MLG14, MLG29 and MLG31. Mo emen o MLGs wi hin he Spanish p o inces may ha e occu ed epea edly, gi en he low le el o geog aphic gene ic s uc u e and he equen e ie al o he same geno ype in dis an loca ions. Highly simila , bu no iden ical, clonal geno ypes a e unlikely o ha e a isen independen ly in sexual ep oduc ion and can hen be in e ed o ep esen he same clonal lineage [48,49]. Figu e 3. Minimum Spanning Ne wo k showing he ela ionship among he indi idual mul i-locus geno ypes (MLGs) obse ed in A, he Spanish popula ion o Cadopho a lu eo-oli acea and B, he popula ions om Spain and Sou h A ica. Each node ep esen s a di e en MLG. Node sizes and colo s co espond o he numbe o indi iduals and popula ion membe ship, espec i ely. Edge hickness and colo a e p opo ional o absolu e gene ic dis ance. Edge leng hs a e a bi a y. The ou mos common MLGs (13, 14, 29 and 31) a e ou lined in he g aphs. doi:10.1371/jou nal.pone.0110417.g003 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 7 Oc obe 2014 | Volume 9 | Issue 10 | e110417 The wo gene ic g oups displayed no speci ic geog aphic dis ibu ion, hus sugges ing clonal di e gence and admix u e be ween he Spanish C. lu eo-oli acea geno ypes. In ac , AMOVA analyses showed ha mos gene ic di e si y (88.1%) was ound wi hin popula ions and only 5.5–6.3% among popula ions. The wo de ined clus e s would hen co espond o an es ablished asexual lineage in e mingled wi h indi iduals de i ed om ano he asexual lineage ha we e ecen ly admixed. P o iding ha sexual ep oduc ion is a a e and episodic e en in C. lu eo-oli acea [10], such admix u e p ocesses a e expec ed o main ain hei signa u e in popula ions o se e al gene a ions. The e a e se e al o he documen ed examples o asexual plan Figu e 4. Coo dina es o A, he 26 di e en Cadopho a lu eo-oli acea geno ypes sampled in Spain and B, he 35 geno ypes sampled in Spain and Sou h A ica on he wo main axes o he PCA. The ou mos common MLGs (13, 14, 29 and 31) a e ou lined in he g aphs. As e isk (*) indica es he obse ed MLGs in Sou h A ica. MLG13 was obse ed in Spain and Sou h A ica. Spanish popula ions wi h less han i e indi iduals we e no conside ed o he analysis. doi:10.1371/jou nal.pone.0110417.g004 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 8 Oc obe 2014 | Volume 9 | Issue 10 | e110417 pa hogens consis ing o gene ically di e en ia ed clus e s, such as Phy oph ho a amo um [50], Puccinia s ii o mis [51], Fusa ium ci cina um [52], o he main ungal species associa ed wi h Pe i disease and esca o g ape ine, Phaeomoniella chlamydospo a [53]. Figu e 5. UPGMA dend og am o gene ic dis ance among A, 26 obse ed mul ilocus geno ypes (MLGs) in Spain and B, 26 Spanish MLGs and 10 obse ed MLGs in Sou h A ica. Clus e s a e colo ed acco ding o PCA analyses esul s: in black, MLGs assigned o clus e 1, in g ay, MLGs assigned o clus e 2. Suppo alues g ea e han 70% using 1,000 boo s ap samples a e shown. As e isk (*) indica es he obse ed MLGs in Sou h A ica. MLG13 was obse ed in Spain and Sou h A ica. Spanish popula ions wi h less han i e indi iduals we e no conside ed o he analysis. doi:10.1371/jou nal.pone.0110417.g005 Gene ic Di e si y o Cadopho a lu eo-oli acea PLOS ONE | www.plosone.o g 9 Oc obe 2014 | Volume 9 | Issue 10 | e110417