528 ISSN (p in ): 0031-9465 www. up ess.com/pm
ISSN (online): 1593-2095 © Fi enze Uni e si y P ess
Phy opa hologia Medi e anea (2012) 51, 3, 528−540
Co esponding au ho : J. Luque
Fax: +34 937533954
E-mail: [email p o ec ed]
Resea ch Pape s
Species o Dia ypaceae associa ed wi h g ape ine unk diseases in
Eas e n Spain
Jo di LUQUE1, F ancesc GARCIA-FIGUERES2, F ancisco Ja ie LEGORBURU3, a giñe MURUAMENDIARAZ3, Josep
ARMENGOL4 and Flo en p. TROUILLAS5
1 Ins i u de Rece ca i Tecnologia Ag oalimen à ies (IRTA), C a. de Cab ils km 2, E-08348 Cab ils, Spain
2 Labo a o i de Sani a Vege al, Via Ci culació No d, T am VI, Ca e 3, Zona F anca, E-08040 Ba celona, Spain
3 NEIKER-Tecnalia, Basque Ins i u e o Ag icul u e Resea ch and De elopmen , Apdo. 46, E-01080 Vi o ia/Gas eiz, Spain
4 Ins i u o Ag o o es al Medi e áneo, Uni e sidad Poli écnica de Valencia, Camino de Ve a s/n, E-46022 Valencia, Spain
5 Depa men o Plan Pa hology, Uni e si y o Cali o nia, Da is, Cali o nia 95616, USA
Summa y. The p esence and di e si y o Dia ypaceae species occu ing on g ape ines in Eas e n Spain we e
in es iga ed. Se e al species we e iden i ied on he basis o mo phological cha ac e s and phylogene ic analyses
o he comple e sequence o he in e nal ansc ibed space s o he ibosomal DNA and pa o he β- ubulin gene.
Fi e species o Dia ypaceae isola ed om he wood o diseased g ape ines, p uning deb is and/o pe i hecia
we e iden i ied, including An hos oma decipiens, C yp o alsa ampelina, Eu ypa la a, Eu ypella ci icola and Eu ypella
mic o heca. Addi ionally, ou axa could no be iden i ied o he species le el bu we e closely ela ed o Eu ypa
e agona based on phylogene ic analyses. Eu ypa la a was he mos p e alen species and showed he g ea es de-
g ee o gene ic di e si y. C yp o alsa ampelina and E. mic o heca anked second in he equency o isola ions, while
all he emaining species we e less equen ly isola ed. Eu ypella ci icola and E. mic o heca a e epo ed o he i s
ime as occu ing on g ape ine in Spain and his is he i s epo o A. decipiens occu ing on g ape ine.
Key wo ds: An hos oma, C yp o alsa, Eu ypa, Eu ypella, Vi is ini e a, Bayesian in e ence.
In oduc ion
Se e al ungi in he Dia ypaceae a e known o
occu on g ape ines (Vi is ini e a L.) in many g ape-
g owing coun ies, including species in he gene a
C yp osphae ia Ces. & De No ., C yp o alsa Ces. & De
No . ex Fuckel, Dia ype F ., Dia ypella (Ces. & De
No .) De No ., Eu ypa Tul. & C. Tul. and Eu ypella
(Ni schke) Sacc. (Ca e , 1988, 1991; Mos e e al.,
2004; T ouillas and Guble , 2004; Ca al e al., 2007;
Pi e al., 2009; Ú bez-To es e al., 2009; T ouillas and
Guble , 2010a, 2010b; T ouillas e al., 2010, 2011; Ú -
bez-To es e al., 2011). To da e, 14 known species o
his amily ha e been epo ed om g ape ine, while
abou i e axa a e s ill no assigned o any known
species (Fa and Rossman, 2011). Eu ypa la a (Pe s.)
Tul. & C. Tul., he causal agen o Eu ypa dieback,
is he mos impo an g ape ine pa hogen known
wi hin his amily (Ca e , 1988, 1991). While iden i-
ica ion, pa hogenici y, epidemiology and con ol o
E. la a ha e been ho oughly s udied wo ldwide, he
same subjec s conce ning o he dia ypaceous ungi
s ill equi e u he in es iga ion. Howe e , ecen
epo s on he iden i ica ion and pa hogenici y o
o he dia ypaceous axa, including C yp o alsa am-
pelina (Ni schke) Fuckel (Mos e e al., 2004; Luque
e al., 2006; Ma ín e al., 2009; T ouillas and Guble ,
2010b), Eu ypa lep oplaca (Mon .) Rappaz (T ouillas
and Guble , 2004, 2010b), Eu ypella i is (Schwein.)
Ellis & E e h. (Ca al e al., 2007; Jo dan and Schil-
de , 2007; Ú bez-To es e al., 2009, 2011), and C yp o-
sphae ia pullmanensis Glawe (T ouillas and Guble ,
529
Vol. 51, No. 3, Decembe , 2012
Dia ypaceae species on g ape ine in Spain
2010b) a e con ibu ing o a be e knowledge abou
hese ungi and, speci ically, he ole hey play in he
g ape ine unk diseases.
Recen s udies in Cali o nia and Aus alia ha e
sugges ed a possible co ela ion be ween in ec ion
o g ape ines by dia ypaceous species occu ing on
na u al and o namen al hos plan s in he immedi-
a e p oximi y o he ineya ds (T ouillas and Guble ,
2010b; T ouillas e al., 2010, 2011). G ape ine in ec-
ion could be pa ly explained by a possible oppo -
unis ic li es yle o hese ungi, no mally sap obic on
hei na u al hos (s) bu occasionally pa hogenic o
o he unusual hos s (e.g. g ape ine) ende ed sus-
cep ible due o p edisposing ac o s (T ouillas e al.,
2010, 2011). Pa hogenici y expe imen s on V. ini e a
ines using a ious species o Dia ypaceae ha e
been conduc ed in Cali o nia ineya ds. Resul s
showed ha Dia ypaceae we e capable o coloniz-
ing g ape ine wood. Howe e , al hough some spe-
cies seemed o p oduce ascula discolo a ions, he
disease caused by hese ungi and hei i ulence
emained unclea (T ouillas and Guble , 2010b). As
sugges ed by T ouillas and Guble (2010b), ex ended
incuba ion pe iods while conduc ing pa hogenici y
es s should be necessa y o be e cha ac e ize dis-
ease exp ession and agg essi eness o hese ungi. In
ineya ds, symp oms caused by E. la a ea ly in he
season (i.e. s un ed shoo g ow h wi h small, cupped
and chlo o ic lea es) a e exp essed 3 o 8 yea s a e
in ec ion (Ca e , 1988). Howe e , in g eenhouse as-
says, symp oms ha e been epo ed o occu wi hin
4 weeks (Pé os and Be ge , 1994), 6 weeks (Jung e al.,
2010), 8 mon hs (Sosnowski e al., 2007), o e en du -
ing he second g owing season (Mu uamendia az
and Lego bu u, pe sonal communica ion).
The occu ence o Dia ypaceae species on g ape-
ines in Spain is s ill poo ly known. S udies pub-
lished in he las decade con i med he exis ence
o only E. la a and C. ampelina. Eu ypa dieback was
epo ed o he i s ime in la e 1970’s in Ex ema-
du a, Sou hwes e n Spain (A ias and Mo al, 1981).
Symp oms o his disease we e la e epo ed in he
mid 1990’s in he Rioja wine egion, No h Cen al
Spain (Ma eo, 1995). Se e al u he s udies ha e
shown E. la a o be widely dis ibu ed in he g ape-
ine g owing egions in Spain (A mengol e al., 2001;
Ú bez-To es and Peláez, 2001; Pé os and Be ge ,
2003; San iago e al., 2005; Ma ín and Cobos, 2007;
Luque e al., 2009; Mu uamendia az e al., 2009). I is
cu en ly accep ed ha E. la a is he mos common
Dia ypaceae species ound on g ape ine in Spain.
C yp o alsa ampelina was epo ed o he i s ime
in 2006 in Ca alonia, No heas e n Spain, and was
ound mainly on p uning deb is and a ely on s and-
ing ines showing symp oms o unk diseases such
as dieback and canke s (Luque e al., 2006). A mod-
e a e i ulence has been sugges ed o his ungus
(Mos e e al., 2004; Luque e al., 2006). Ma ín e al.
(2009) also epo ed C. ampelina om he cen al e-
gion o Spain. To da e, no o he Dia ypaceae species
ha e been epo ed om g ape ines in Spain. The e-
o e, he p esen s udy aimed o de e mine he p es-
ence and di e si y o o he dia ypaceous ungi oc-
cu ing on g ape ines in his coun y. Mo phological
cha ac e s, such as colony mo phology and conidial
dimensions, we e used in combina ion wi h phylo-
gene ic analyses o he in e nal ansc ibed space s
o he DNA and pa o he β- ubulin gene o aid
in he cha ac e iza ion and iden i ica ion o he axa.
Ma e ials and me hods
Fungal isola es and mo phological cha ac e s s udied
The isola es used in his s udy we e ob ained om
in ec ed shoo s, co dons and unks showing die-
back and/o in e nal wood nec oses, o di ec ly om
pe i hecia embedded in he ba k o g ape ines ha
we e su eyed in Spain du ing he yea s 2002‒2010
(Table 1). Wood chips ob ained om he nec o ic is-
sues we e su ace-s e ilized (3‒4 min in 70% e ha-
nol), blo ed on s e ile il e pape o emo e exces-
si e e hanol, and pla ed on o Po a o Dex ose Aga
(PDA, Di co Labo a o ies, De oi , MI, USA) amend-
ed wi h s ep omycin sulpha e (Sigma-Ald ich Co.,
S . Louis, MO, USA) a 100 uni s pe ml (PDA-s )
as desc ibed by Johns on and Boo h (1983). Pu e cul-
u es o ungi we e ob ained by isola ion o single
hyphal ips. Monospo ic isola ions om pe i hecia
we e ca ied ou as ollows. S oma a we e cu wi h
a s e ile blade o e eal he pe i hecial con en s and
a d op o s e ile wa e (abou 100 μL) was placed on
he cu su ace o he ui ing bodies. A wa e d op
con aining masses o ascospo es was hen collec ed
wi h a pipe e, pla ed on o PDA-s and spo es we e
dispe sed wi h a Dig alsky sp eade . Twen y ou
hou s a e pla ing, a single ge mina ing spo e was
selec ed unde he mic oscope and ans e ed o a
esh PDA Pe i dish. Isola es we e main ained a
4°C in s e ile dis illed wa e o long- e m s o age.
Phy opa hologia Medi e anea
530
J. Luque e al.
Table 1. Isola es o se e al Dia ypaceae species ob ained om g ape ine in di e en loca ions o Spain, used in his s udy.
Species Isola e Loca ion P o ince Isola ion yea G ape ine a ie y Sou ce de ails
An hos oma decipiens JL567 False Ta agona 2004 Temp anillo Wood nec osis, a m
C yp o alsa ampelina JL413 Vimbodí Ta agona 2003 Red G enache Pe i hecia, on p uning deb is
C yp o alsa ampelina JL424 Capçanes Ta agona 2003 Ca ignane Pe i hecia, on p uning deb is
C yp o alsa ampelina JL476 Bo Ta agona 2003 Macabeo Canke , a m
C yp o alsa ampelina JL717 El Pla del Penedès Ba celona 2009 Cabe ne Sau ignon Wood nec osis, li ing shoo
Eu ypa la a JL355 Caldes de Mon bui Ba celona 2002 Cha donnay V-shaped nec osis, a m
Eu ypa la a JL399 Pacs del Penedès Ba celona 2003 Cabe ne Sau ignon Canke , a m
Eu ypa la a JL407 Mediona Ba celona 2003 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL411 Vimbodí Ta agona 2003 Red G enache V-shaped nec osis, a m
Eu ypa la a JL427 La Vilella Baixa Ta agona 2003 Cabe ne Sau ignon Canke , a m
Eu ypa la a JL431 Capçanes Ta agona 2003 Ca ignane V-shaped nec osis, a m
Eu ypa la a JL432 Òdena Ba celona 2003 Macabeo V-shaped nec osis, a m
Eu ypa la a JL479 Ba ea Ta agona 2003 Red G enache Canke , a m
Eu ypa la a JL600 Vilajuïga Gi ona 2005 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL677 Ba bas o Huesca 2007 Cabe ne Sau ignon Canke , a m
Eu ypa la a JL720 Ba iobus o Ála a 2006 Temp anillo Wood nec osis, a m
Eu ypa la a JL721 Lanciego Ála a 2006 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL723 Lagua dia Ála a 2008 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL725 Ba go a Na a a 2008 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL726 Labas ida Ála a 2008 Temp anillo V-shaped nec osis, a m
Eu ypa la a JL727 Na a idas Ála a 2007 Temp anillo Pe i hecia, on a m
Eu ypa la a JL731 La Mo a Bu gos 2002 Temp anillo Wood nec osis, unknown pa
Eu ypa la a JL732 Malagón Ciudad Real Unknown Red G enache Wood nec osis, unknown pa
Eu ypa la a JL739 Albace e Albace e 2010 Unknown Wood nec osis, unknown pa
Eu ypa la a JL740 Yecla Mu cia 2009 Unknown Wood nec osis, unknown pa
Eu ypa la a JL741 Villena Alican e 2009 Unknown Wood nec osis, unknown pa
Eu ypa la a JL743 La Cañada La Rioja 2010 Temp anillo Wood nec osis, unknown pa
Eu ypa la a JL744 Azo a La Rioja 2010 Temp anillo Wood nec osis, unknown pa
(Con inued)
531
Vol. 51, No. 3, Decembe , 2012
Dia ypaceae species on g ape ine in Spain
Spo ula ion was enhanced by cul u ing isola es
on PDA a 25°C wi h a 12/12 hou pho ope iod,
unde nea UV (Philips TLD 18W/08; Philips Elec-
onics N.V., Ams e dam, Ne he lands) and whi e
luo escen ligh (Os am L 18W/840; Os am GmbH,
Munich, Ge many). Isola es ha did no spo ula e
wi hin 3 mon hs o incuba ion in he abo e condi-
ions we e no used o he measu emen s o conid-
ia. The mean, s anda d de ia ion, 95% con idence
in e als and minimum and maximum alues we e
calcula ed om he measu emen s made wi h he
×100 mic oscope objec i e o 50 conidia moun ed in
wa e . Colony cha ac e s o isola es we e eco ded
a e g owing he ungi on PDA a he abo e ci ed
condi ions o 4 weeks. When possible, ungi we e
en a i ely iden i ied om hei colony and conidial
mo phology by compa ing wi h p e ious s udies
(T ouillas and Guble , 2010b; T ouillas e al., 2010;
T ouillas e al., 2011).
DNA ex ac ion and sequencing
DNA was ex ac ed om he ungal mycelium
as desc ibed by Al es e al. (2004). Ampli ica ion o
he ITS1 and ITS2 egions lanking he 5.8S iboso-
mal RNA gene was ca ied ou using he uni e sal
p ime s ITS1 and ITS4 (Whi e e al., 1990). Pa o he
β- ubulin gene was ampli ied by using he p ime s
B 2a and B 2b (Mos e e al., 2006). All eac ions we e
pe o med on a GeneAmp® PCR Sys em 9700 he -
mal cycle (PE Applied Biosys ems, Fos e Ci y, CA,
USA), ollowing he empe a u e p o iles desc ibed
in Luque e al. (2005) o he ITS egion, and Mos e e
al. (2006) o he β- ubulin gene. Pu i ica ion o PCR
p oduc s was acco ding o he me hods desc ibed in
Luque e al. (2005). The pu i ied amplicons we e se-
quenced in bo h di ec ions using he a o emen ioned
p ime s and he BigDye™ Te mina o 1.1 Cycle Se-
quencing Ki (Applied Biosys ems, Fos e Ci y, CA,
USA). The esul ing agmen s we e analyzed on an
ABI P ism 377 au oma ed DNA sequence (Pe kin
Elme , No walk, CT, USA). Sequences we e ead and
edi ed wi h BioEdi Sequence Alignmen Edi o Ve -
sion 7.0.8 (Hall, 1999). All sequences we e checked
manually, and nucleo ide a angemen s a ambigu-
ous posi ions we e cla i ied using sequences om
bo h s ands. Nucleo ide sequences ob ained in his
s udy we e deposi ed in GenBank (Table 2). Iden-
i ica ion o isola es was con i med by compa ing
he DNA sequences o he abo e men ioned egions
Species Isola e Loca ion P o ince Isola ion yea G ape ine a ie y Sou ce de ails
Eu ypa sp. JL488 Ba ea Ta agona 2003 Red G enache V-shaped nec osis, a m
Eu ypa sp. JL688 Ba bas o Huesca 2007 Me lo V-shaped nec osis, a m
Eu ypa sp.JL690 Ba bas o Huesca 2007 Cha donnay V-shaped nec osis, a m
Eu ypa sp. JL742 Albace e Albace e 2010 Unknown Wood nec osis, unknown pa
Eu ypella ci icola JL583 Olè dola Ba celona 2004 Chenin Blanc Wood nec osis, unk
Eu ypella ci icola JL734 Mu cia Mu cia 2009 Unknown Wood nec osis, unknown pa
Eu ypella mic o heca JL609 Vilajuïga Gi ona 2005 Temp anillo Pe i hecia, on p uning deb is
Eu ypella mic o heca JL625 Pe alada Gi ona 2005 Don Ma iano Wood nec osis, li ing shoo
Eu ypella mic o heca JL735 Villanue a de Alcolea Cas ellón 2009 Unknown Wood nec osis, unknown pa
Eu ypella mic o heca JL738 No elda Alican e 2009 Unknown Wood nec osis, unknown pa
All isola es a ailable h ough IRTA (J. Luque). All isola es ob ained om in ec ed wood excep o C yp o alsa ampelina JL413 (monospo ic), C. ampelina JL424 (monospo ic),
Eu ypa la a JL727 (mul ispo ic), and Eu ypella mic o heca JL609 (monospo ic), which we e all ob ained om pe i hecia. O he accession numbe o selec ed isola es deposi ed
a Cen aalbu eau oo Schimmelcul u es (CBS), U ech , The Ne he lands a e: JL411, CBS 121487; JL413, CBS 117484; JL424,CBS 117485; JL476, CBS 117486.
Table 1. Con inues.
Phy opa hologia Medi e anea
532
J. Luque e al.
wi h hose deposi ed a GenBank, and by combining
hese sequences in he phylogene ic analyses.
Phylogene ic analyses
Thi y se en isola es ob ained in his s udy we e
used in he phylogene ic analyses. Addi ional se-
quences co esponding o o he Dia ypaceae (26
o he ITS and 25 o he β- ubulin da ase s) we e
ob ained om GenBank (Figu es 1 and 2) o be in-
cluded in he analyses. Daldinia concen ica (Bol on)
Ces. & De No . (Xyla iaceae) was used as he ou -
g oup (wi h GenBank accession numbe s FJ185300
o ITS and FJ185285 o β- ubulin). Taxa om Gen-
Bank we e selec ed o include dia ypaceous spe-
cies p e iously epo ed om g ape ine as well as
species showing high simila i y wi h ou que y se-
quences. DNA sequences o bo h ITS and β- ubulin
da ase s we e aligned using Clus alW (Thompson
e al., 1994). Alignmen s we e e i ied and adjus ed
manually using BioEdi . ITS and b- ubulin da ase s
we e combined in o a single da ase , which did no
include An hos oma decipiens (DC.) Ni schke because
no β- ubulin sequence was a ailable o his species
in GenBank. The incong uence-leng h di e ence es
(ILD) (Fa is e al., 1995) o pa i ion homogenei y
es (HomPa ) was pe o med in PAUP* e . 4.0b10
(Swo o d, 2002) o de e mine whe he he ITS and b-
ubulin da ase s could be combined. P io o he ILD,
unin o ma i e cha ac e s we e emo ed and he ILD
es was un using a heu is ic sea ch and simple ad-
di ion o axa o 1,000 andom pa i ions o he da a.
P io o Bayesian in e ence (BI) analyses, he
mos app op ia e nucleo ide subs i u ion models
we e chosen using jModel es e sion 0.1.1 (Posada,
2008). BI analyses we e ca ied ou using M . Bayes
e sion 3.2 (Ronquis and Huelsenbeck, 2003). Fou
Ma ko chains we e un simul aneously o 1×106
gene a ions, and hese we e sampled e e y 100 gen-
e a ions. Da a om he i s 1,000 gene a ions we e
disca ded as he bu n-in pe iod, and a e con i m-
ing ha likelihood alues we e s abilized p io o he
1,000 h gene a ion. The 50% majo i y ule consensus
ee and pos e io p obabili y o he ee nodes we e
calcula ed om he pooled samples.
Resul s
A o al o 38 isola es o pu a i e Dia ypaceae
species om Spain we e used in his s udy (Table
1). Mos o hese isola es we e ob ained om in-
ec ed wood o diseased ines, whe eas wo isola es
o C. ampelina and one isola e o Eu ypella mic o heca
T ouillas, W.M. Pi & Guble we e ob ained om
pe i hecia occu ing on g ape ine p uning deb is.
One isola e o E. la a (JL727) was also ob ained
om pe i hecia, bu ound on old dead wood. Iso-
la es we e ob ained om ypical V-shaped canke s
as well as om i egula -shaped nec oses. Shapes
o canke s associa ed wi h he a ious isola es col-
lec ed a e summa ized in Table 1. While 37 isola es
we e used in he phylogene ic analyses, conidial
measu emen s we e ob ained o 21 isola es only.
These included one isola e o A. decipiens, h ee o C.
ampelina, 12 o E. la a, wo o Eu ypella ci icola Speg.,
and h ee o E. mic o heca. P oduc ion o conidia was
no obse ed o Eu ypa sp. isola es JL488, JL688,
JL690 and JL742.
PCR ampli ica ion o he ITS egion ga e p oduc s
o app oxima ely 0.6 kb while hose o he β- ubulin
we e abou 0.4 kb. The ITS and β- ubulin da ase con-
ained 64 and 63 sequences, espec i ely, including
he ou g oup. The ITS ma ix consis ed o 604 aligned
cha ac e s and he β- ubulin ma ix consis ed o 403
cha ac e s, including gaps. Resul s o he ILD es
(P<0.05) showed ha he ITS and β- ubulin da a we e
incong uen and hus could no be combined. Fo his
eason, wo sepa a ed BI analyses we e ca ied ou
and he esul ing phylogene ic ees a e p esen ed in
Figu es 1 and 2. The Akaike In o ma ion C i e ion
(AIC) implemen ed in jModel es was used o de e -
mine he bes i ing models o he ITS and β- ubulin
da ase s, and esul ed as GTR+G o he ITS da ase
and HKI+I+G o he β- ubulin da ase .
The phylogene ic ee o ITS showed i e main
clades ha we e suppo ed by pos e io p obabili ies
o e 95%. These g oups included ungi in C yp o alsa
(g oup 1), Dia ype and Dia ypella (g oup 2), Eu ypella
and An hos oma (g oup 3), and Eu ypa (g oups 4 and
5) (Figu e 1). On he o he hand, he phylogeny o he
β- ubulin gene showed ou la ge clades, coinciding
wi h he gene a C yp o alsa (g oup 1), Dia ype and
Dia ypella (g oup 2), Eu ypella (g oup 3), and Eu ypa
(g oup 4), al hough Eu ypa (g oup 4) only ecei ed a
pos e io p obabili y alue o 0.81 (Figu e 2). An hos-
oma decipiens JL567 was also included in his clade.
In addi ion o C. ampelina and E. la a, al eady
known om g ape ine in Spain, phylogene ic analy-
ses also de ec ed E. ci icola, E. mic o heca and A. de-
cipiens. Species iden i ica ion o hese i e axa was

533
Vol. 51, No. 3, Decembe , 2012
Dia ypaceae species on g ape ine in Spain
s ongly suppo ed by he high sequence simila i-
ies wi h e e ence sequences om GenBank (99 o
100%) and by he pos e io p obabili ies ob ained
in he BI analyses (0.97 o 1). The ITS and β- ubulin
analyses also dis inguished a sepa a e clade o an-
o he pu a i e Eu ypa sp. ha emained uniden i ied
bu showed a close ela edness o Eu ypa e agona
(Duby) Sacc. (Figu es 1 and 2).
Se e al isola es o each species we e ob ained
om di e en geog aphic egions (Table 1). Eu ypa
la a was he dia ypaceous species mos ly isola ed (23
isola es) and showed he g ea es phylogene ic di-
e si y in bo h genes (Figu es 1 and 2). Howe e , he
in aspeci ic di e si y o E. la a did no co espond
o a ela ionship o geog aphical p oximi y, and in-
aspeci ic g oups we e no consis en ac oss he wo
DNA phylogenies. ITS and β- ubulin sequences o
C. ampelina (4), E. ci icola (2), and E. mic o heca (4)
showed no gene ic a ia ion among isola es o each
species despi e hei di e en geog aphical o igins.
Colonies on PDA o he a ious ungal species
we e o e all qui e simila in mo phology. Howe e ,
sligh di e ences could be obse ed. Colonies o C.
ampelina on PDA we e whi e o c eam-whi e, wool-
ly, wi h di use ma gins, and apid g ow h (9 cm in
diam. in 4 days a 25°C). The e e se o colonies was
i s pale-yellow, la e (>20 days) de eloping i egu-
la , mos ly cen al, da k a eas. The spo odochium-
like conidioma a consis ed o conidiopho es agg e-
ga ed on blackened mycelial c us s, and p oduced
c eam colou ed conidial masses wi hin 4 weeks.
Cul u es o E. la a on PDA we e a iable in mo phol-
ogy: hey we e whi e when young (<1 week a 25°C),
some imes u ning c eam-whi e wi h age (4 weeks),
and wi h di use ma gins. Occasionally, p oduc ion
o b ownish exuda es was de ec ed in some isola es
Table 2. Nucleo ide sequences o he Spanish isola es o Dia ypaceae deposi ed in GenBank.
Species Isola e ITS β- ubulin
An hos oma decipiens JL567 JN975370 JN975407
C yp o alsa ampelina JL413 JN975335 JN975371
C yp o alsa ampelina JL424 AY920391 JN975372
C yp o alsa ampelina JL476 JN975336 JN975373
C yp o alsa ampelina JL717 JN975337 JN975374
Eu ypa la a JL355 JN975338 JN975375
Eu ypa la a JL399 JN975339 JN975376
Eu ypa la a JL407 JN975340 JN975377
Eu ypa la a JL411 JN975341 JN975378
Eu ypa la a JL427 JN975342 JN975379
Eu ypa la a JL432 JN975343 JN975380
Eu ypa la a JL479 JN975344 JN975381
Eu ypa la a JL600 JN975345 JN975382
Eu ypa la a JL677 JN975346 JN975383
Eu ypa la a JL720 JN975347 JN975384
Eu ypa la a JL721 JN975348 JN975385
Eu ypa la a JL723 JN975349 JN975386
Eu ypa la a JL725 JN975350 JN975387
Eu ypa la a JL726 JN975351 JN975388
Species Isola e ITS β- ubulin
Eu ypa la a JL727 JN975352 JN975389
Eu ypa la a JL731 JN975353 JN975390
Eu ypa la a JL732 JN975354 JN975391
Eu ypa la a JL739 JN975355 JN975392
Eu ypa la a JL740 JN975356 JN975393
Eu ypa la a JL741 JN975357 JN975394
Eu ypa la a JL743 JN975358 JN975395
Eu ypa la a JL744 JN975359 JN975396
Eu ypa sp. JL488 JN975360 JN975397
Eu ypa sp. JL688 JN975361 JN975398
Eu ypa sp. JL690 JN975362 JN975399
Eu ypa sp. JL742 JN975363 JN975400
Eu ypella ci icola JL583 JN975364 JN975401
Eu ypella ci icola JL734 JN975365 JN975402
Eu ypella mic o heca JL609 JN975366 JN975403
Eu ypella mic o heca JL625 JN975367 JN975404
Eu ypella mic o heca JL735 JN975368 JN975405
Eu ypella mic o heca JL738 JN975369 JN975406
Phy opa hologia Medi e anea
534
J. Luque e al.

0.06
JL488Eu ypa sp.
DQ006923Eu ypa e agona
DQ006924Eu ypalep oplaca
JL609Eu ypellamic o heca
JL720Eu ypala a
JL741Eu ypala a
DQ006935Eu ypala a
JL732Eu ypala a
HQ692540C yp o alsaampelina
JL407Eu ypala a
HM164735Eu ypape akii a .pe akii
HQ692593Dia ypella ulga is
JL734Eu ypellaci icola
DQ006928Eu ypala a
JL479Eu ypala a
JL476C yp o alsaampelina
HM164726Eu ypala a
JL726Eu ypala a
DQ006943Eu ypella i is
AY684223Eu ypa e agona
HQ692620C yp o alsa abenho s ii
DQ006926Eu ypamau a
JL583Eu ypellaci icola
JL740Eu ypala a
HM164718Eu ypala a
JL725Eu ypala a
JL739Eu ypala a
HQ692572Eu ypellamic o heca
JL731Eu ypala a
HQ692568Eu ypellamic o heca
FJ185300Daldiniaconcen ica
JL413C yp o alsaampelina
GQ293938Dia ypeo egonensis
JL399Eu ypala a
GQ293926Dia ypella e uci o mis
JL744Eu ypala a
DQ006922Eu ypalejoplaca
JL677Eu ypala a
JL567An hos omadecipiens
AM399021An hos omadecipiens
GQ293954Dia ypewhi manensis
HM164737 Eu ypalae a a
JL355Eu ypala a
JL721Eu ypala a
AJ302458Eu ypaas oidea
JL432Eu ypala a
JL600Eu ypala a
JL717C yp o alsaampelina
JL743Eu ypala a
DQ006944Eu ypala a
JL411Eu ypala a
JL723Eu ypala a
JL690 Eu ypa sp.
JL742Eu ypa sp.
AJ302457Dia ype la o i ens
JL424C yp o alsaampelina
AY684237Eu ypalep oplaca
JL738Eu ypellamic o heca
HQ692580Eu ypellaci icola
JL427Eu ypala a
JL625Eu ypellamic o heca
JL735Eu ypellamic o heca
JL727Eu ypala a
JL688Eu ypa sp.
1
0.96
0.91
0.99
0.96
0.87
0.86
0.83
0.97
1
0.96
0.66
1
1
1
1
0.51
1
1
1
0.83
1
1
1
1
0.92
0.88
1
2
4
3
5
Figu e 1. Majo i y ule consensus ee esul ing om he Bayesian analysis o he ITS sequence da a, wi h pos e io p ob-
abili ies epo ed a he nodes. Sequences ob ained om GenBank a e indica ed by hei accession numbe s while iso-
la es ob ained in his s udy a e indica ed by hei code numbe s. Clades a e numbe ed in ci cles. Ba s ep esen expec ed
changes pe si e.
535
Vol. 51, No. 3, Decembe , 2012
Dia ypaceae species on g ape ine in Spain

0.07
JL738Eu ypellamic o heca
HQ692470C yp o alsaampelina
JL734Eu ypellaci icola
JL355Eu ypala a
HM164760Eu ypala a
JL742Eu ypa sp.
JL413C yp o alsaampelina
JL583Eu ypellaci icola
FJ185285Daldiniaconcen ica
JL717C yp o alsaampelina
DQ006992Eu ypala a
JL424C yp o alsaampelina
HQ692520Eu ypellaci icola
JL688Eu ypa sp.
HM164769Eu ypape akii a .pe akii
GQ293991Dia ypella e uci o mis
JL677Eu ypala a
JL740Eu ypala a
JL411Eu ypala a
HQ692526Eu ypellamic o heca
DQ006999Eu ypella i is
JL732Eu ypala a
JL739Eu ypala a
JL720Eu ypala a
JL479Eu ypala a
AY684202Eu ypa e agona
JL723Eu ypala a
JL731Eu ypala a
DQ006960Eu ypa e agona
DQ007000Eu ypala a
JL600Eu ypala a
AY684212Eu ypalep oplaca
GQ293997Dia ypeo egonensis
JL609Eu ypellamic o heca
JL726Eu ypala a
GQ294010Dia ypewhi manensis
JL625Eu ypellamic o heca
DQ006966Eu ypaas oidea
HM164771Eu ypalae a a
JL690Eu ypa sp.
DQ006974Eu ypalejoplaca
DQ006961Eu ypalep oplaca
JL407Eu ypala a
JL399Eu ypala a
JL427Eu ypala a
JL725Eu ypala a
DQ006959Dia ype la o i ens
AY684198Eu ypamau a
JL741Eu ypala a
JL744Eu ypala a
JL743Eu ypala a
JL567An hos omadecipiens
HQ692504Dia ypella ulga is
JL727Eu ypala a
HM164752Eu ypala a
HQ692522C yp o alsa abenho s ii
JL432Eu ypala a
DQ006973Eu ypala a
JL721Eu ypala a
JL476C yp o alsaampelina
JL735Eu ypellamic o heca
JL488Eu ypa sp.
HQ692536Eu ypellamic o heca
0.63
0.99
0.71
1
0.99
0.86
0.91
1
0.98
1
0.82
0.93
1
0.81
0.89
1
1
0.87
1
0.88
1
1
1
0.59
1
1
0.95
0.96
1
0.71
1
1
2
3
4
Figu e 2. Majo i y ule consensus ee esul ing om he Bayesian analysis o he β- ubulin sequence da a, wi h pos e io
p obabili ies epo ed a he nodes. Sequences ob ained om GenBank a e indica ed by hei accession numbe s while iso-
la es ob ained in his s udy a e indica ed by hei code numbe s. Clades a e numbe ed in ci cles. Ba s ep esen s expec ed
changes pe si e.
Phy opa hologia Medi e anea
536
J. Luque e al.
(JL720, JL726, JL741). The e e se o colonies was i s
whi e, la e (>20 days) pale-yellow o c eamy in mos
isola es, and cul u es p oducing exuda es each ex-
hibi ed a b own e e se a e 4 weeks. Mos isola es
de eloped blackened, ci cula a eas, which coalesced
wi h ime (o e 4 weeks) o each show a blackish
colony e e se o some isola es. P oduc ion o con-
idioma a and conidia we e only obse ed in hal o
he s udied isola es. Cul u es o E. ci icola on PDA
we e whi e, wi h low dense ae ial mycelium and
woolly mycelium agg ega es, some imes u ning o
pale c eam (3 weeks). Blackened, ci cula a eas bea -
ing spo odochia we e seen occasionally on he colo-
ny su aces. Cul u es o E. mic o heca on PDA we e
simila o hose o E. ci icola bu de eloping a ligh
pinkish-o ange colou in he cen al pa s o he colo-
nies in some isola es (JL625, JL735), as epo ed by
T ouillas e al. (2011). The cul u e o A. decipiens JL567
on PDA was whi e o c eam-whi e and woolly, wi h
a mode a e g ow h (9 cm in diam. in 7 days a 25°C).
The e e se o he colony was i s pale yellow, la e
(>2 weeks) c eamy, wi h i egula blackened a eas.
Old colonies (4 weeks) u ned o da k g ey. Colonies
o isola es JL488, JL688, JL690 and JL742 on PDA we e
o e all simila , howe e sligh di e ences o mo -
phology could be obse ed. All colonies appea ed
whi e ini ially, wi h li le ae ial mycelium, bu apidly
(abou 3 days) u ned in o a pale-c eam colou . A e
one week, isola es JL688 and JL690 de eloped dense,
co ony mycelium wi h mode a e ae ial g ow h,
while mycelium in JL488 and JL742 emained almos
app essed o he cul u e medium. Re e se colou o
he colonies we e i s whi e (<7 d), u ning pale yel-
low in isola es JL488 and JL742 a e 10 d while iso-
la es JL688 and JL690 emained whi e. Ne e heless,
4-week-old cul u es we e simila in colou o all iso-
la es, mos ly pale-c eam in bo h sides. Isola es JL688
and 690 (app ox. 9 cm in diam. a e 7 d) g ew a li le
as e han JL488 and JL742 (app ox. 9 cm in diam.
a e 12 d). P oduc ion o conidioma a and conidia
we e no obse ed in any o hese Eu ypa sp. isola es
in wo independen assays.
Conidial dimensions o ep esen a i e isola es o
all species, excep o he unknown Eu ypa sp., which
did no spo ula e, a e shown in Table 3. Conidia o
A. decipiens we e luna e, (7)‒9‒ (11) × (1) ‒1.05‒ (1.1)
μm, and we e he smalles conidia o he s udied iso-
la es. Conidia o he emaining species we e simila
in o m (cu ed a he ob use end and s aigh a he
unca e base) and wi h o e lapping leng hs, which
did no allow o species di e en ia ion based on co-
nidial o m. Mean leng h o conidia o C. ampelina
anged om 21.5 μm o 24.2 μm, while a g ea phe-
no ypic a ia ion o mean conidium leng h was de-
ec ed among E. la a isola es, which anged om 21.9
μm (isola e JL723) o g ea e han 34 μm (JL427 and
JL 431) (Table 3). Mean leng h o conidia o E. ci icola
anged om 14.7 μm o 15.6 μm, which o e lapped
wi h he co esponding ange in E. mic o heca (15.2
μm o 15.9 μm). Mean wid hs o conidia o nea ly all
isola es anged om 1.0 μm o 1.5 μm (Table 3).
Discussion
This s udy has con i med he occu ence o i e
species o Dia ypaceae associa ed wi h g ape ines
in Eas e n Spain, namely An hos oma decipiens, C yp-
o alsa ampelina, Eu ypa la a, Eu ypella ci icola and
Eu ypella mic o heca. Addi ionally, ou uniden i ied
isola es o a Eu ypa closely ela ed o E. e agona we e
de ec ed on he basis o he phylogene ic analyses.
Howe e , we did no conside in oducing new spe-
cies names o hese isola es as none o hem (JL488,
JL688, JL690 and JL742) p oduced ei he conidia in
i o o sexual ep oduc i e s uc u es equi ed o
he desc ip ion o new ungal species. Fu he mo e,
he ITS and β- ubulin phylogene ic analyses showed
incong uen b anch opologies o hese isola es,
which did no allow us o de ini ely conclude i mul-
iple Eu ypa spp. occu ed wi hin his clade.
Despi e he incong uence ound be ween he ITS
and β- ubulin phylogenies, bo h ees in e ed h ough
Bayesian me hods showed ha species iden i ica ion
o ou isola es was consis en among he es ima ed
phylogenies. Howe e , he posi ion o A. decipiens s ill
emains unclea . While his axon was a sis e clade o
Eu ypella in he ITS ee, i was included in he la ge
clade o Eu ypa in he β- ubulin ee. Rega ding he
new pu a i e Eu ypa sp. isola es, mo phological ob-
se a ions we e in acco dance wi h he g oupings ob-
se ed in he ITS and he β- ubulin ees; hus, JL688
and JL690 showed simila cha ac e is ics (i.e. colony
colou and g ow h) o JL488 and JL742. In addi ion,
hese isola es ha we e closely ela ed o E. e agona
could in u n be dis an ela i es o E. lep oplaca.
While C. ampelina and E. la a we e al eady known
in Spain om p e ious s udies (A ias and Mo al,
1981; A mengol e al., 2001; San iago e al., 2005;
Luque e al., 2006; Ma ín and Cobos, 2007; Ma ín
e al., 2009; Mu uamendia az e al., 2009), A. decipi-