Characterization of Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and environmental samples from grapevine nurseries in Spain

D. G amaje e al. S112 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 S113 Vol. 50, Supplemen , 2011 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 S115 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 S117 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-