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
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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
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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