D. G amaje e al.
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Phy opa hol. Medi e . (2011) 50 (Supplemen ), S112−S126
Co esponding au ho : D. G amaje
Fax: +34 963879269.
E-mail: [email p o ec ed]
In oduc ion
The genus Cadopho a (C.) Lage be g & Melin
was es ablished by Lage be g e al. (1927), wi h
he ype species Cadopho a as igia a Lage be g
& Melin, which causes s ains in so wood imbe .
Melin and Nann eld (1934) added i e new spe-
cies o he genus: C. ame icana Nann ., C. obscu a
Nann ., C. lage be gii Melin & Nann ., C. melinii
Nann . and C. icha dsiae Nann . Da idson (1935)
desc ibed wo mo e species, isola ed om s ained
wood o pulpwood p oduc s: C. b unnescens R.W.
Da idson and C. epens R.W. Da idson. Subse-
quen ly, Conan (1937) de e mined ha Phial-
opho a (P.) Medla and Cadopho a we e congene -
ic and ans e ed he eigh species o Cadopho a
o Phialopho a. This esea che also conside ed C.
ame icana o be a synonym o P. e ucosa Med-
la . Since hen a e y b oad concep o Phialopho-
a has been e ained, e en in he majo e ision by
Schol-Schwa z (1970).
Gams (2000) p oposed ha he phialopho a-like
anamo phs wi h a ini ies o he De ma eaceae in
he Helo iales, such as Mollisia (F .) P. Ka s . and
Py enopeziza Fuckel, should be accommoda ed in
he Cadopho a, he species o which ha e pale o
hyaline colla e es on op o hei phialides wi h
he ege a i e hyphae mo e o less pigmen ed.
This au ho sugges ed he ollowing binomials o
he genus Cadopho a: C. as igia a, C. melinii,
C. epens and C. malo um (Kidd & Beaum.) W.
Gams. Ha ing on and McNew (2003) used mo -
Key wo ds: Cadopho a species, g ape ine nu se ies, Vi is ini e a, young ineya ds.
Summa y. Fi y-eigh Cadopho a lu eo-oli acea and h ee C. melinii isola es we e eco e ed om g ape-
ines showing black ascula s eaking and decline symp oms cha ac e is ic o Pe i disease, and om di -
e en s ages o he g ape ine nu se y p ocess in Spain. The isola es we e s udied by means o pheno ypical
cha ac e iza ion, DNA analysis and pa hogenici y es s. The mo phological cha ac e s s udied included co-
nidiopho e, phialide and conidial mo phology. Colony cha ac e s and pigmen p oduc ion on MEA, PDA and
OA we e also examined. Pheno ypical da a we e subjec ed o clus e analysis, which clea ly sepa a ed C.
lu eo-oli acea isola es in o ou g oups. Ma ing es s we e pe o med on all possible combina ions o each
Cadopho a species bu no sexual ui ing bodies we e p oduced. Pa ial sequences o he nuclea ibosomal
in e nal ansc ibed space (ITS), be a- ubulin (BT) and he elonga ion ac o 1a (EF) we e analysed, bu no
gene ic a ia ion occu ed wi hin he C. lu eo-oli acea isola es o wi hin he C. melinii isola es in any o he
egions s udied. Pa hogenici y es s we e conduc ed on 1-yea -old g ape ine cu ings o ou di e en oo -
s ocks using ou C. lu eo-oli acea isola es and one isola e o C. melinii. All Cadopho a isola es excep he C.
melinii isola e caused signi ican ly longe lesions in he xylem o g ape ine oo s ocks han in he con ols.
Da iD GRaMaJE1, LizEL MOSTERT2 and JOSEp aRMENGOL1
1 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, 46022, Valencia, Spain
2 Depa men o Plan Pa hology, Uni e si y o S ellenbosch, P i a e Bag X1, S ellenbosch 7602, Sou h A ica
Cha ac e iza ion o Cadopho a lu eo-oli acea and C. melinii isola es ob ained
om g ape ines and en i onmen al samples om g ape ine nu se ies in Spain
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D. G amaje e al. Cadopho a lu eo-oli acea and C. melinii in Spain
phology and DNA sequences o de e mine which
phialopho a-like species migh ha e a ini ies o
he Helo iales, and o con i m he connec ion o
Cadopho a o he De ma eaceae. They ound ha
he pigmen a ion o he species s udied was o -
en qui e a iable be ween species, p oposed new
combina ions in Cadopho a and also e iewed he
axonomy o he ecognized species. The genus
Cadopho a hen comp ised C. as igia a ( he ype
species), C. malo um, C. melinii, C. lage be gii, C.
inlandia (C.J.K. Wang & H.E. Wilcox) T.C. Ha .
& McNew, C. g ega a (Alling on & D.W. Chamb.)
T.C. Ha . & McNew, and C. lu eo-oli acea (F.H.
Beyma) T.C. Ha . & McNew. The inclusion o P.
epens R.W. Da idson in he Cadopho a g oup was
no suppo ed by ITS sequence analysis. P. a a
F.H. Beyma and C. he e ode ae F.H. Beyma we e
synonymized wi h C. malo um, and P. goidanichii
Deli ala wi h C. lu eo-oli acea by Ha ing on and
McNew (2003).
The known Cadopho a species and hei ela-
i es occu in many habi a s such as decaying
wood (Nilsson, 1973; Mo ell and Zabel, 1985;
Blanche e e al., 2004; Held e al., 2005; A enz e
al., 2006), soil (Ke y, 1990; Aislabie e al., 2001;
A enz e al., 2006; Hujslo á e al., 2010) o plan s.
Cadopho a species on plan s a e mainly associa -
ed wi h decay symp oms in ines and ui - o ing
in kiwi [Ac inidia deliciosa (A. Che .) C.F. Liang
& A.R. Fe guson a . deliciosa] (Di Ma co e al.,
2004; Johns on e al., 2005; Haegi e al., 2006; Ric-
cioni e al., 2007; Di Ma co and Os i, 2008; P odi
e al., 2008). Species o Cadopho a ha e also been
eco e ed om g ape ines showing decline symp-
oms in Sou h A ica, Pennsyl ania, Cali o nia
and U uguay (Halleen e al., 2005; O e on e al.,
2005; Rooney-La ham, 2005; Ab eo e al., 2008).
Halleen e al. (2007) con i med ha C. lu eo-oli a-
cea is a ascula pa hogen o g ape ines in Sou h
A ica and can also be eco e ed om heal hy
nu se y cu ings. Recen ly, Manning and Mundy
(2009) ha e isola ed C. lu eo-oli acea and C. meli-
nii om esca-diseased g ape ines in New Zealand.
O e he las ew yea s, Cadopho a spp. ha e
equen ly been isola ed om nu se y g ape ines
and Pe i-diseased g ape ines in Spain. The spe-
cies iden i y o hese isola es was unknown p io
o his s udy. Limi ed in o ma ion is a ailable on
he pheno ypical and molecula a iabili y o Ca-
dopho a isola es ob ained om g ape ine and on
hei ole in he decline o young ines. The e o e,
he objec i e o his esea ch was o s udy hese
Cadopho a isola es by means o pheno ypical
cha ac e iza ion, DNA analysis and pa hogenici y
es s.
Ma e ials and me hods
Fungal isola es
In his s udy we used 58 C. lu eo-oli acea and
h ee C. melinii isola es ep esen a i e o di e en
loca ions in Spain and eco e ed in 2007 and 2008
(Table 1). Some o he isola es we e eco e ed om
ines in young ineya ds, o he s om plan ing
ma e ial showing black ascula s eaking and
decline symp oms cha ac e is ic o Pe i disease.
Sec ions we e cu om a ec ed a eas, washed un-
de unning ap wa e , su ace-disin ec ed o 1
min in a 1.5% sodium hypochlo i e solu ion, and
washed wice wi h s e ile dis illed wa e . Small
pieces o discolou ed o decayed issue we e pla ed
on mal ex ac aga (MEA) (Oxoid L d., Basing-
s oke, Han s, England) supplemen ed wi h 0.5
g L-1 s ep omycin sulpha e (MEAS) (Sigma-Al-
d ich, S . Louis, MO, USA). Pla es we e incuba ed
o 10–15 days a 25ºC in he da k.
Addi ionally, some isola es we e ob ained om
nu se y samples 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. App oxima ely 100 mL o wa e
was sampled om anks in which cu ings had
been soaked o 72 hou s. Scisso s and g a ing
ools we e washed wi h a 0.2% s e ile solu ion o
Tween-20, and 30 o 60 mL o e e y washing was
placed in a s e ile ube. Wa e samples om he
hyd a ion anks, washings om he scisso s and
washings om he g a ing ools we e il e ed as
desc ibed by Eskalen and Guble (2001). Wa e
samples (10 mL) we e o cibly passed h ough 5
µm and 0.45 µm po e-size sy inge il e s (Sa o-
ius S edium Bio ech, Gö ingen, Ge many) a -
ached in andem o s e ile 20 mL sy inges (BD
Biosciences, Mad id, Spain). The 5 µm il e s
apped deb is and la ge ungal spo es. The 0.45
µm il e s apped small pa icula es and smalle
spo es, including spo es o Cadopho a species. The
il e s we e insed wi h 4 mL o s e ile wa e o
backwash any apped spo es and collec ed in mi-
c o uge ubes. Aliquo s o 1 mL we e hen sp ead
Phy opa hologia Medi e anea
D. G amaje e al.
S114
Table 1. Isola ion de ails and GenBank accession numbe s o he Cadopho a isola es s udied.
Species/
Isola e
Geog aphical o igin GenBank accessionsb
Yea
Town P o ince Sou ce o collec ionaITS β- ubulin EF
C. lu eo-oli acea
Clo-1 2007 Ayelo Valencia Temp anillo/110 R HQ661084 HQ661054 HQ661069
Clo-2, CBS 128571 2007 Ciudad Real Ciudad Real Temp anillo/110 R HQ661085 HQ661055 HQ661070
Clo-3 2007 Ciudad Real Ciudad Real Temp anillo/110 R
Clo-4 2007 Ayelo Valencia G a ing machine HQ661086 HQ661056 HQ661071
Clo-5, CBS 128572 2007 Ayelo Valencia Hyd a ion ank
Clo-6 2007 Ciudad Real Ciudad Real Temp anillo/110 R
Clo-7, CBS 128573 2007 Ciudad Real Ciudad Real Temp anillo/110 R HQ661087 HQ661057 HQ661072
Clo-8 2007 Ayelo Valencia Hyd a ion ank
Clo-9 2007 Ayelo Valencia G a ing machine
Clo-10 2007 Ayelo Valencia P uning scisso s
Clo-11 2007 Ayelo Valencia G a ing machine
Clo-12 2007 Ayelo Valencia G a ing machine
Clo-13 2007 Ayelo Valencia G a ing machine
Clo-14 2007 Ayelo Valencia Hyd a ion ank
Clo-15, CBS 128574 2007 Ayelo Valencia Hyd a ion ank HQ661088 HQ661058 HQ661073
Clo-16 2007 Ayelo Valencia G a ing machine
Clo-17 2007 Ayelo Valencia G a ing machine
Clo-18, CBS 128575 2007 Ciudad Real Ciudad Real Temp anillo/110 R HQ661089 HQ661059 HQ661074
Clo-19 2007 Oli enza Badajoz Sy ah
Clo-20 2007 Requena Valencia Temp anillo/110 R
Clo-21 2007 Ayelo Valencia 1103 P
Clo-22 2007 Ayelo Valencia 1103 P HQ661090 HQ661060 HQ661075
Clo-23 2007 Ayelo Valencia 1103 P
Clo-24 2007 Ayelo Valencia 1103 P
Clo-25 2007 Ayelo Valencia 1103 P
Clo-26 2007 Ayelo Valencia 1103 P
Clo-27 2007 Ayelo Valencia 1103 P
Clo-28 2007 Ayelo Valencia 1103 P HQ661091 HQ661061 HQ661076
Clo-29 2007 Ayelo Valencia 1103 P
Clo-30 2007 Ayelo Valencia 1103 P
Clo-31 2007 Ayelo Valencia 1103 P
Clo-32 2007 Ayelo Valencia 110 R
Clo-33, CBS 128576 2007 Ayelo Valencia 110 R HQ661092 HQ661062 HQ661077
Clo-34, CBS 128577 2007 Ayelo Valencia 110 R
Clo-35 2007 Ayelo Valencia 110 R
Clo-36 2007 Ayelo Valencia 110 R
Clo-37 2007 Ayelo Valencia 110 R
Clo-38 2007 Ayelo Valencia 110 R
Clo-39 2007 Ayelo Valencia 110 R
Clo-40 2007 Socuéllamos Ciudad Real Ga nacha/110 R HQ661093 HQ661063 HQ661078
Clo-41 2007 Socuéllamos Ciudad Real Ai en/1103 P
Clo-42 2008 Ayelo Valencia 1103 P
Clo-43 2008 Ayelo Valencia 110 R
Clo-44 2008 Ayelo Valencia 110 R
Clo-45 2008 Ayelo Valencia 110 R
Clo-46, CBS 128578 2008 Ayelo Valencia 110 R HQ661094 HQ661064 HQ661079
Clo-47 2008 Ayelo Valencia 110 R
con inues
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Vol. 50, Supplemen , 2011
D. G amaje e al. Cadopho a lu eo-oli acea and C. melinii in Spain
on a semi-selec i e cul u e medium adap ed om
Tello e al. (2009) [2% MEA amended wi h 1.5 g L-1
s ep omycin sulpha e; 0.02 g L-1 Folpe (A agon-
esas Ag o, Mad id, Spain); and 0.015 g L-1 ampicil-
lin (Labo a o io Reig Jo é S.A., San Joan Despí,
Spain)]. Pla es we e ai d ied unde a lamina low
hood wi h he lids pa ially li ed o 30 min. Pla es
we e hen sealed wi h pa a ilm and incuba ed
a 25ºC in da kness o one mon h. Pe i dishes
we e inspec ed daily o mycelial g ow h and e-
isola ion. All isola es we e ans e ed o po a o
dex ose aga (2% PDA; Bioka -Diagnos ics, Beau-
ais, F ance). The isola es we e single-spo ed by
se ial dilu ion p io o use (Dhing a and Sinclai ,
1995) and we e s o ed in a 15% glyce ol solu ion
a -80ºC in 1.5 mL c yo ials. Re e ence isola es o
C. lu eo-oli acea (CBS 141.41) and C. melinii (CBS
268.33) we e ob ained om he Cen aalbu eau
oo Schimmelcul u es in he Ne he lands (CBS)
(Table 1).
Mo phological and cul u al cha ac e iza ion
Mo phological cha ac e s used in his s udy
included conidiopho e, phialide and conidial mo -
phology. Colony cha ac e s and pigmen p oduc-
ion we e de e mined on MEA, PDA (2% PDA;
Biolab, Mid and, Sou h A ica) and oa meal aga
(OA; 60 g oa meal; 12.5 g aga ; Di co, Osi, Mau e-
pas, F ance) (Gams e al. 2007) incuba ed a 25ºC
o 8 and 16 d. Colony colou s we e de e mined
using he colou cha s o Rayne (1970). Ca dinal
empe a u es o g ow h we e de e mined by in-
cuba ing h ee MEA pla es pe isola e in he da k
a empe a u es anging om 5 o 40ºC a 5ºC in-
e als. Radial g ow h was measu ed a e 8 d a
25ºC on MEA.
Ae ial mycelium o colonies cul i a ed
on MEA we e inspec ed unde he mic oscope
o by a slide cul u e echnique (A zanlou e al.,
2007). Slide cul u es we e se up in Pe i dishes
con aining 2 mL o s e ile wa e , in o which a
U-shaped glass od was placed, ex ending abo e
he wa e su ace. A block o an ac i ely g owing
ungal colony, app ox. 1 cm squa e, was placed on
a s e ile mic oscope slide, co e ed wi h a some-
wha la ge , s e ile glass co e ing slip, and in-
cuba ed in a mois chambe . Fungal spo ula ion
was moni o ed, and when i was op imal, images
Clo-48 2008 Ayelo Valencia 110 R
Clo-49 2008 Ayelo Valencia 110 R
Clo-50 2008 Ayelo Valencia 110 R
Clo-51 2008 Ayelo Valencia 110 R
Clo-52 2008 Ayelo Valencia 110 R
Clo-53 2008 Ayelo Valencia 110 R
Clo-54 2008 Ca iñena Za agoza Sy ah/110 R
Clo-55 2008 Sa Pun a Mallo ca Me lo /110 R HQ661095 HQ661065 HQ661080
Clo-56 2008 Sa Pun a Mallo ca Me lo /110 R
Clo-57 2008 Pe a Mallo ca Man o Neg o
Clo-58 2008 Pe a Mallo ca Man o Neg o
CBS 141.41 1939 Munksund Sweden Was e wa e o
Schlei e ei Byske
C. melinii
Cme-1 2007 Oli enza Badajoz Sy ah HQ661096 HQ661066 HQ661081
Cme-2 2007 Oli enza Badajoz Sy ah HQ661097 HQ661067 HQ661082
Cme-3 2007 Oli enza Badajoz Sy ah HQ661098 HQ661068 HQ661083
CBS 268.33 1933 - Sweden -
a
Isola es we e collec ed om Vi is iní e a plan s (indica ed as oo s ock o scion/ oo s ock combina ions) o om di e en s ages
in he g ape ine nu se y p ocess (hyd a ion anks, g a ing machines o p uning scisso s).
b The sequences o a selec ion o isola es ha e been deposi ed in GenBank.
Species Geog aphical o igin GenBank accessionsb
Isola e Yea
Town P o ince Sou ce o collec ionaITS β- ubulin EF
Table 1. Con inued
Phy opa hologia Medi e anea
D. G amaje e al.
S116
o he spo ula ing ungus we e pho og aphed wi h
a Nikon came a sys em (Digi al Sigh DXM 1200,
Nikon Co po a ion, Japan). Slides we e moun ed
in lac ic acid, and 30 measu emen s (×1000 mag-
ni ica ion) o he ele an s uc u es we e made.
The 5 h and 95 h pe cen iles we e de ined o all
measu emen s wi h he ex emes gi en in pa en-
heses.
Ma ing es s wi h all C. lu eo-oli acea and C.
melinii isola es we e pe o med as desc ibed by
Mos e e al. (2003). Cadopho a spp. isola es
we e g own on MEA pla es o 2 weeks, using 5
pla es pe isola e. Conidia we e dislodged om
he aga su ace wi h a glass od, and suspensions
we e p epa ed in 5 mL s e ile dis illed wa e . Two
aliquo s o 100 mL each, ep esen ing wo di e -
en isola es, we e pipe ed on o wice-au ocla ed
pieces o g ape ine canes placed on 2% wa e aga
pla es (GWA) (Di co, F ance). Each Cadopho a
species was ma ed in all possible combina ions.
Con ols consis ed o a 200 mL aliquo o one iso-
la e only. Pla es we e incuba ed a 20ºC and 25ºC
unde con inuous whi e ligh (Philips TLD18W/33)
and mic oscopically examined a weekly in e als
o ui ing bodies. Each pai o isola es was epli-
ca ed h ee imes. The expe imen was epea ed.
To g oup C. lu eo-oli acea isola es by hei
pheno ypical cha ac e s, clus e analysis was con-
duc ed on he Euclidean dis ance ma ix wi h he
unweigh ed pai g oup me hod based on a i hme-
ic a e ages (UPGMA). The mo phological cha ac-
e s used in he analysis we e: colony colou on
MEA and PDA as nominal a iables; he maxi-
mum numbe o gu ules in conidia and he op-
imum g ow h empe a u es as symme ic-bina y
a iables; conidopho es, phialides, colla e es
and conidia measu emen s and adial g ow h as
in e al a iables; and yellow pigmen a ion on
PDA and OA as asymme ic-bina y a iables. The
clus e ing me hod and simila i y coe icien we e
es ed using he NCSS 2007 p ocedu e (S a is ical
Solu ions L d, Co k, I eland).
Molecula analysis
Fungal mycelium and conidia we e sc aped
om pu e cul u es g own on PDA o 2 weeks a
25ºC in he da k and g ound o a ine powde wi h
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, No c oss, GA, USA)
ollowing manu ac u e ’s ins uc ions. DNA was
isualized on 0.7% aga ose gels (aga ose D-1 Low
EEO, Conda, Mad id, Spain) s ained wi h e hidi-
um b omide. The DNA was s o ed a –20ºC.
The in e nal ansc ibed space s 1 and 2 as well
as he 5.8S ibosomal RNA gene o app oxima ely
595 bp o C. lu eo-oli acea isola es and 555 bp o
C. melinii isola es was ampli ied wi h he p ime s
ITS1 and ITS4 (Whi e e al., 1990). Oligonucleo-
ide p ime s BT1a and BT1b we e used o ampli y
he pa ial β- ubulin (BT) gene o app oxima ely
480 bp o he C. lu eo-oli acea isola es and 300
bp o he C. melinii isola es (Pe i and Guble ,
2005). Ampli ica ion o he pa ial ansla ion
elonga ion- ac o 1a (EF) gene o app oxima ely
555 bp o C. lu eo-oli acea isola es and 525 bp o
C. melinii isola es was done wi h he p ime s EF1-
688F and EF1-1251R (Al es e al., 2008).
Each PCR eac ion con ained 1× PCR bu e ,
2.5 mM MgCl2, 200 µM each dNTP, 0.4 µM o
each p ime , 1 U o DNA Taq polyme ase (Domin-
ion MBL, Có doba, Spain), and 1 µL o empla e
DNA. The PCR eac ion mix was adjus ed o a i-
nal olume o 25 µL wi h wa e (Ch omasol Plus,
Sigma-Ald ich, S einheim, Ge many). PCR am-
pli ica ions we e pe o med on a Pel ie The mal
Cycle -200 (MJ Resea ch, Wal ham, USA). The
p og am consis ed o an ini ial s ep o 4 min a
94°C, ollowed by 30 cycles (35 cycles o BT) o
dena u a ion a 94°C o 1 min, annealing a 58°C
o 1 min, and an elonga ion a 72°C o 1.5 min. A
inal ex ension was pe o med a 72°C o 10 min.
The PCR p oduc s we e isualized in 1.5% aga-
ose gels. A 100 bp ladde was used as a molecula
weigh ma ke (Dominion MBL). PCR p oduc s
we e pu i ied wi h he High Pu e PCR p oduc
pu i ica ion ki (Roche Diagnos ics, Ge many) and
sequenced in bo h di ec ions by he DNA Sequenc-
ing Se ice o he Uni e sidad Poli écnica de Va-
lencia-CSIC.
The sequences we e aligned using he MAFFT
sequence alignmen p og am e sion 6 (Ka oh
and Toh, 2008) ollowed by manual adjus men s o
he alignmen s in he Sequence Alignmen Edi o
. 2.0a11 (Rambau , 2002). Re e ence sequences
o he ex- ype cul u es o he ele an Cadopho-
a species and e e ence sequences used by Ha -
ing on and McNew (2003) we e ob ained om
GenBank. Phylogene ic analysis o he aligned

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Vol. 50, Supplemen , 2011
D. G amaje e al. Cadopho a lu eo-oli acea and C. melinii in Spain
sequence da a was pe o med wi h Phylogene ic
analysis using pa simony (PAUP) . 4.0b10 (Swo -
o d, 2003). Alignmen gaps we e ea ed as miss-
ing da a and all cha ac e s we e uno de ed and
o equal weigh . Any ies we e b oken andomly
when encoun e ed. Fo pa simony analysis, align-
men gaps we e ea ed as missing da a and all
cha ac e s we e uno de ed and o equal weigh .
Maximum pa simony analysis was pe o med
using he heu is ic sea ch op ion wi h 10 andom
simple axon addi ions and ee bisec ion and e-
cons uc ion (TBR) as he b anch-swapping algo-
i hm wi h he op ion o sa ing no mo e han 10
ees wi h a sco e g ea e han o equal o 5 (Ha -
ison and Langdale, 2006). B anches o ze o leng h
we e collapsed and all mul iple, equally pa si-
monious ees we e sa ed. The obus ness o he
ees ob ained was e alua ed by 1000 boo s ap
eplica ions. T ee leng h (TL), he consis ency in-
dex (CI), he e en ion index (RI) and he escaled
consis ency index (RC) we e calcula ed. Sequences
de i ed in his s udy we e lodged a GenBank (Ta-
ble 1) and he alignmen and phylogene ic ee in
T eeBASE (numbe S10849).
Pa hogenici y es s
One isola e o C. lu eo-oli acea o each o he
ou mo phological g oups de ined by clus e
analysis and one isola e o C. melinii we e used.
Pa hogenici y es s we e conduc ed on 1-yea -old
g ape ine cu ings o ou oo s ocks (140 Rugge i,
161-49 Coude c, 1103 Paulsen and 110 Rich e ). In
o al 96 do man cu ings o each oo s ock we e
cu in o equal leng hs con aining ou o i e buds,
and hen ho -wa e ea ed a 53ºC o 30 min o
elimina e any ungal unk pa hogens (G amaje e
al., 2009). In o de o enhance callusing and oo -
ing, do man cu ings we e bu ied in s e ilized pea
moss in plas ic boxes, and placed in a callusing
oom a 25ºC and 100% humidi y o 4 weeks. A e
callusing and oo ing, he 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-week-
old cul u e was placed in he wound. Wounds we e
w apped wi h pa a ilm. Eigh cu ings pe ungal
isola e we e used o each oo s ock. Eigh cu ings
o each oo s ock we e inocula ed wi h 5 mm non-
colonized PDA aga plugs om wo di e en pla es
o 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 25ºC and wa e ed e e y 3 days o
as needed. Plan s we e a anged in a comple ely
andomized design. The expe imen was epea ed.
Cu ings we e collec ed a e 14 weeks and in-
spec ed o lesion de elopmen . The ex en o as-
cula discolou a ion was measu ed upwa ds and
downwa ds om he inocula ion poin . Addi ion-
ally, shoo d y weigh was e alua ed o he shoo s
abo e he inocula ion poin . Small pieces (0.5 o
1 cm) o nec o ic issue om he edge o each le-
sion we e cu and placed on MEAS in an a emp
o eco e he inocula ed ungi and ul ill Koch’s
pos ula es. Fungi we e iden i ied as p e iously de-
sc ibed.
Lesion leng h and shoo d y weigh da a we e
subjec ed o analysis o a iance using SAS . 8.1
(SAS Ins i u e, Ca y, No h Ca olina, USA) and
S uden ’s - es o Leas Signi ican Di e ence
was calcula ed a he 5% signi icance le el o com-
pa e he ea men means o he di e en ungal
species and g ape ine oo s ocks.
Resul s
Mo phological cha ac e iza ion
The isola es we e iden i ied as C. lu eo-oli acea
o C. melinii based on mo phological cha ac e s
(Cole and Kend ick, 1969; Schol-Schwa z, 1970;
Gams, 2000; Ha ing on and McNew, 2003). Con-
idiopho e, phialide and conidial mo phology we e
simila in all C. lu eo-oli acea isola es (Figu e 1).
Ae ial mycelium on MEA consis ed o b anched,
sep a e hyphae occu ing singly o in bundles o
up o 5; hyphae ube cula e wi h wa s up o 3 µm
diam, e uculose o smoo h, medium b own and
2.5-4 µm wide. Conidiopho es we e mos ly sho ,
usually unb anched, a ising om ae ial o sub-
me ged hyphae, e ec o lexuous, up o 7-sep a e,
pale b own, (9–)11.5–65(–69) (a .=26) µm long and
2–2.5 (a .=2) µm wide. Phialides we e e minal o
la e al, mos ly monophialidic, smoo h o e ucu-
lose, hyaline, wi h colla e es 2.5–3 µm long and
2–2.5 µm wide, mos ly cylind ical, some elonga e-
ampulli o m, a enua ed a he base o na icula ,
(4–)8.5–26(–31) × 1.5–3 (a .=5×2) µm. Conidia
we e hyaline, wi h up o 3 gu ules, o oid o ob-
long ellipsoidal, (3–)3.5–7.5×2–3 (a .=4.5×2) µm,
L/W=2.1.
Some di e ences we e ound in colony cha -
ac e s and pigmen p oduc ion o he C. lu eo-
Phy opa hologia Medi e anea
D. G amaje e al.
S118
oli acea isola es s udied (Figu e 2 and Table 2).
Colonies on MEA we e la , el y, wi h e en edge
and a ying in colou om whi e o g eenish-oli a-
ceous (23’’’b). Colonies on PDA we e la , el y and
co ony in he middle, wi h an e en edge and a y-
ing in colou om whi e o g ey-oli aceous (21’’’’i).
Colonies on OA we e la , el y and co ony in he
middle, wi h an e en edge and a ying in colou
om whi e o oli aceous-bu (21’’’d). Fo all iso-
la es, colonies eached a adius o 15.5–17.2 mm
a e 8 d a 25ºC. The minimum empe a u e o
g ow h was 5ºC, op imum 20–25ºC and maximum
30ºC. Some C. lu eo-oli acea isola es p oduced yel-
low pigmen a ion on PDA and/o MEA pla es.
The C. lu eo-oli acea isola es we e di e en
om he ype desc ip ion since hey had p edomi-
nan ly monophialidic phialides, which we e hin-
ne . The conidial shape and he di e si y o he
colony colou s on he media s udied also di e ed.
Van Beyma (1940) desc ibed colonies o C. lu eo-
oli acea as ha ing hick phialides o en in bunch-
es, conidia ellipsoid o elonga e, and colonies oli e-
yellow wi h whi e mycelium in he middle.
A compa ison based on he mo phological and
colony cha ac e s o he 58 C. lu eo-oli acea iso-
la es s udied a e shown in a dend og am (Figu e
3). Analysis e ealed ha isola es we e dis ibu ed
in wo clus e s a a Euclidean dis ance wi h a cu
o alue o 1.26. Fou dis inc g oups we e o med.
Clus e A comp ised g oup I. Clus e B was di id-
ed in o h ee g oups, II, III and IV. The cophene ic
co ela ion alue was 0.9419. G oup I con ained 6
isola es. This g oup was he mos di e se, wi h a
dissimila i y anging om 0 o 0.92. G oup II con-
ained 21 isola es and had a dissimila i y anging
om 0 o 0.88. Isola e CBS 141.41 was included
in his g oup and joined he o he isola es o he
same g oup a a dissimila i y o 0.88. G oup III
con ained 23 isola es wi h a dissimila i y anging
om 0 o 0.81. G oup IV con ained 9 isola es and
was he mos homogeneous (dissimila i y anging
om 0 o 0.51). Colony cha ac e s and pigmen
p oduc ion we e he mos use ul pa ame e s dis-
inguishing hese g oups (Figu e 2 and Table 2).
The majo i y o C. lu eo-oli acea isola es we e
om g ape ines. Se en isola es we e om he
g a ing machines, ou om he hyd a ion ank
and one om p uning scisso s.
The mo phological and colony cha ac e s o
C. melinii isola es a e shown in Figu e 4. Ae ial
mycelium on MEA consis ed o b anched, sep a e
hyphae occu ing singly o in bundles o up o
13; hyphae ube cula e wi h wa s up o 2.5 µm
diam, e uculose o smoo h, medium b own and
3–3.5 µm wide. Conidiopho es mos ly sho , usu-
ally b anched, a ising om ae ial o subme ged
Figu e 1. Cadopho a lu eo-oli acea. a– , Ae ial s uc u es on MEA; a-d, conidiopho es; e- , hyphal swellings; g-s,
conidiopho es and phialides; , conidia. Scale ba : a– =10 µm. Scale ba o a also applies o b– .
S119
Vol. 50, Supplemen , 2011
D. G amaje e al. Cadopho a lu eo-oli acea and C. melinii in Spain
hyphae, e ec o lexuous, up o 4-sep a e, pale
b own, (11–)11.5–50.5(–65) (a .=24.5) µm long
and 2–3 (a .=2.5) µm wide. Phialides e minal
o la e al, mos ly monophialidic, smoo h o e -
uculose, hyaline, wi h 1.5–3.5 µm long, 2–3 µm
wide, subcylind ical o na icula colla e es, (4–
)5–13.5(–15)×1.5–3(–4) (a .= 8×2.5) µm. Conid-
ia hyaline, wi h up o 2 gu ules, mos ly o oid,
(3–)4–5.5×1.5–2.5 (a .=4.5×2) µm, L/W=2.1.
Rega ding cul u al cha ac e is ics, colonies
eached a adius o 12–14.5 mm a e 8 d a 25ºC.
The minimum empe a u e o g ow h was 5–10ºC,
he op imum 20–25ºC and he maximum 30ºC.
Colonies on MEA we e la , el y, wi h an e en
edge; a e 16 d, oli aceous-black (27’’’’k) o g ey-
oli aceous (23’’’’i) om he op, oli aceous-black
(27’’’’k) om he bo om. Colonies on PDA we e
la , el y, wi h an e en edge; a e 16 d, whi e o
oli aceous-bu (21’’’ ) om he op and he bo om.
Colonies on OA we e aised wi h s ia ing u ows,
Figu e 2. Cadopho a lu eo-oli acea. G oup 1: a–c. Six een-day-old colonies on MEA (a), PDA (b) and OA (c). G oup 2:
d- , Six een-day-old colonies on MEA (d), PDA (e) and OA ( ). G oup 3: g-i. Six een-day-old colonies on MEA (g), PDA
(h) and OA (i). G oup 4: j-l. Six een-day-old colonies on MEA (j), PDA (k) and OA (l).
Phy opa hologia Medi e anea
D. G amaje e al.
S120
Table 2. Summa y o colony colou use ul o he iden i ica ion o each g oup o Cadopho a lu eo-oli acea.
C. lu eo-oli acea
g oup
Colony colou
on MEA
Colony colou
on PDA
Colony colou
on OA
Yellow
pigmen a ion
on PDA
Yellow
pigmen a ion
on OA
G oup 1 G eenish oli aceous (23’’’b)
o oli aceous bu (21’’’b)
abo e and in e e se
Oli aceous black (27’’’’k)
o g ey oli aceous (21’’’’i)
abo e and in e e se
Oli aceous (21’’k) o
oli aceous bu (21’’’d)
abo e
No No
G oup 2 Oli aceous black (25’’’’k)
o whi e owa ds he ma gin
abo e and in e e se
Whi e o g eenish oli a-
ceous (21’’’i) close o he
cen e
Whi e o oli aceous
bu (21’’’d) close o he
cen e abo e
No No
G oup 3 Whi e o g ey oli aceous
(21’’’’i) close o he cen e
abo e and in e e se
Whi e o s aw (19’ )
abo e, s aw (19’ ) opu e
yellow (21’b) in e e se
Oli aceous bu (21’’’d)
o s aw (21’d) abo e
Yes Yes
G oup 4 Whi e o oli eaceous black
(25’’’’k) close o he cen e
abo e and in e e se
Whi e o oli aceous bu
(23’’’b) close o he cen e
abo e and in e e se
G eenish oli aceous
(23’’’b) o s aw (21’ )
abo e
No Yes
woolly when close o he cen e, wi h an e en edge;
a e 16 d, hey we e pale oli aceous-g ey (21’’’’’d)
o oli aceous-black (27’’’’k) om he op.
The C. melinii isola es di e ed om he ype
desc ip ion in hyphal colou and in phialide shape.
Cole and Kend ick (1969) desc ibed colonies o C.
melinii as ha ing hyphae ligh -b own o hyaline,
wi h he phialides subcylind ical o lexuous. A
use ul ea u e o C. melinii no obse ed in ha
wo k bu men ioned by Cole and Kend ick (1973)
and by Domsch e al., (1980) was ha he colla -
e es we e inwa dly cu ed.
The isola es did no co espond o he ype de-
sc ip ion o C. as igia a in ha e y ew o he
mul iple b anched o e icilla e conidiopho es
cha ac e is ic o C. as igia a we e seen. C. as igi-
a a also had globose and subglobose conidia. Such
conidia we e e y a ely ound in he C. lu eo-oli-
acea and C. melinii isola es.
A e 2 mon hs o e alua ion, no sexual ui ing
bodies we e p oduced by c ossing C. lu eo-oli acea
wi h each o he , o C. melinii isola es wi h each
o he .
Molecula analysis
The ITS phylogeny (Figu e 5) clea ly showed
ha he isola es we e ei he g ouped wi h he
e e ence sequences o C. lu eo-oli acea, wi h a
boo s ap suppo o 85%, o wi h he e e ence
sequences o C. melinii, wi h a boo s ap suppo
o 72%. No BT o EF sequences o any Cadopho a
species we e a ailable on GenBank, he e o e, no
analyses we e made wi h hese sequences. Mino
di e ences we e ound among he C. lu eo-oli acea
sequences wi h 3 nucleo ides a ying in he ITS
egion, 3 nucleo ides in he BT egion, and 2 nu-
cleo ides in he EF egion. The C. melinii isola es
om g ape ines we e e y simila , wi h 1 nucle-
o ide a ying in he ITS egion, 2 nucleo ides in
he BT egion, and 2 nucleo ides in he EF egion.
Sequences in he C. melinii clade exhibi ed mo e
a ia ion when he isola es came om Eu opean
sil e i o kiwi.
Pa hogenici y es s
The assays on lesion leng h and on shoo
d y weigh ga e simila esul s (P=0.4224 and
P=0.5180 espec i ely) so ha hese esul s we e
combined in a single analysis. Analysis o a iance
on he lesion leng h o he oo s ocks de ec ed a
signi ican ea men e ec (P<0.001; ANOVA no
shown). All Cadopho a isola es excep C. melinii
caused lesions in he oo s ock xylem ha we e
signi ican ly longe han he lesions in he con-