Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. associated with black foot of grapevines

37 www. up ess.com/pm ISSN (p in ): 0031-9465 © Fi enze Uni e si y P ess ISSN (online): 1593-2095 Phy opa hologia Medi e anea (2012) 51, 1, 37−50 Co esponding au ho : J. A mengol Fax: +34 963879269 E-mail add ess: [email p o ec ed].es Resea ch Pape s E ec s o empe a u e, pH and wa e po en ial on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion in cul u e o Cylind oca pon spp. associa ed wi h black oo o g ape ines Ca los AGUSTÍ-BRISACH and Josep ARMENGOL 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 Summa y. The e ec s o empe a u e, pH and wa e po en ial (Ψs) on mycelial g ow h, spo ula ion and chlamydo- spo e p oduc ion o Cylind oca pon li iodend i, C. mac odidymum and C. paucisep a um isola ed om g ape ines was s udied. Th ee isola es pe species we e incuba ed on po a o dex ose aga (PDA) unde di e en empe a u e, pH, and Ψs condi ions. All isola es we e able o g ow o e a ange o empe a u es om 5 o 30ºC, wi h an op imum empe a u e be ween 20 o 25ºC, bu hey did no g ow a 35ºC. Ac i e mycelial g ow h was obse ed o e a ange o pHs, om 4 o 8. Rega ding he e ec o Ψs, in gene al, mycelial g ow h was g ea e on amended media a -0.5, -1.0 o /and -2.0 MPa compa ed wi h ha ob ained on nonamended PDA (-0.3 MPa), and was educed a Ψs alues lowe han -2.0 MPa. Mos o he Cylind oca pon spp. isola es we e spo ula ed a all empe a u es, pHs and wa e po en ials es ed. In all s udied condi ions, C. li iodend i had he g ea es spo ula ion capaci y compa ed wi h C. mac odidymum and C. paucisep a um. In gene al, chlamydospo e p oduc ion was no much a ec ed by empe a u e, pH and Ψs. Chlamydospo es we e obse ed in PDA cul u es o all isola es a all pH alues s udied, while some iso- la es did no p oduce chlamydospo es a 5 and 10ºC o -4.0 and/o -5.0 MPa. These esul s imp o e unde s anding o he biology o hese impo an g ape ine pa hogens. Key wo ds: Cylind oca pon li iodend i, Cylind oca pon mac odidymum, Cylind oca pon paucisep a um, soilbo ne diseases. In oduc ion Black oo disease o g ape ines, caused by Cy- lind oca pon spp., is a se ious disease in mos wine and g ape-p oducing egions o he wo ld, pa icu- la ly in nu se ies and young ineya ds (Halleen e al., 2006a). I was i s desc ibed in 1961 (G asso and Magnano Di San Lio, 1975), and o e he las decade, black oo disease has been epo ed in mos g ape- ine p oduc ion a eas o he wo ld, including Po u- gal (Rego e al., 2000), A gen ina (Ga ica e al., 2001), Ge many (Fische and Kassemeye , 2003), New Zea- land and Sou h A ica (Halleen e al., 2004), B azil (Ga ido e al., 2004), Cali o nia (Pe i and Guble , 2005), Chile (Auge e al., 2007), Aus alia (Whi el- aw-Wecke e al., 2007), Spain (Alaniz e al., 2007), I an (Mohammadi e al., 2009), U uguay (Ab eo e al., 2010), no heas e n Uni ed S a es and sou heas e n Canada (Pe i e al., 2011). Vines a ec ed by Cylind oca pon spp. o en show sunken nec o ic oo lesions wi h a educ ion in oo biomass and oo hai s. Remo al o oo s ock ba k e eals black discolo a ion and nec osis o wood is- sues which de elops om he base o he oo s ock. Mo eo e , a ec ed ines show low igou wi h small unks and sho in e nodes, a educ ion in o al oli- age and lea size, wi h lea es depic ing in e einal chlo osis and nec osis, equen ly leading o dea h o he plan s (Halleen e al., 2006a; Alaniz e al., 2007, 2009; Ab eo e al., 2010). Phy opa hologia Medi e anea 38 C. Agus í-B isach and J. A mengol Black oo disease o g ape ine is caused by Cylin- d oca pon spp. (Cylind oca pon des uc ans [Zinnsm.] Schol en, C. li iodend i J.D. MacDonald & E.E. Bu le , C. mac odidymum Sch oe s, Halleen & C ous and C. paucisep a um Sch oe s & C ous) and Campyloca - pon spp. (Campyloca pon ascicula e Sch oe s, Hal- leen & C ous and Campyl. pseudo ascicula e Halleen, Sch oe s & C ous) (Halleen e al., 2004; Halleen e al., 2006a, 2006b; Sch oe s e al., 2008). In Spain, su eys ca ied ou in ecen yea s in g ape ine nu se ies and young ineya ds ha e con i med he impo ance o Cylind oca pon spp. a ec ing his c op. These pa hogens we e ound in g ape ine nu se ies e y ea ly in he plan ing ma e- ial p oduc ion p ocess, in g ape ine plan s eady o be plan ed and in young ineya ds showing decline symp oms (A oca e al., 2006; Giménez-Jaime e al., 2006; Alaniz e al., 2007; G amaje e al., 2010). In all cases, Cylind oca pon spp. we e mos ly isola ed om oo s ocks, especially om he basal ends. To da e, C. li iodend i, C. mac odidymum and C. paucisep a um a e he species which ha e been iden i ied associ- a ed wi h young ines showing symp oms o black oo disease in Spain (Alaniz e al., 2007; Ma in e al., 2011). Species o Cylind oca pon a e common and may be isola ed as soil inhabi an s, sap obes on dead plan ma e ial, oo colonize s o pa hogens, o weak pa hogens o a ious he baceous and woody plan s (B ay o d, 1993). The p oduc ion o chlamydospo es may allow Cylind oca pon spp. o su i e o ex end- ed pe iods in soil (Halleen e al., 2004). Gi en hese indings, i could be assumed ha black oo disease pa hogens could su i e in he soil o in es g ape- ine plan s. Howe e , e y li le in o ma ion is cu - en ly a ailable ega ding he basic biology o hese pa hogens, such as mycelial g ow h, spo ula ion and chlamydospo e p oduc ion unde a ious en i on- men al condi ions, and he ole ha chlamydospo es migh play du ing he in ec ion p ocesses (Halleen e al., 2006a). In common wi h all mic oo ganisms, ungi a e p o oundly a ec ed by physical and physicochemi- cal ac o s, such as empe a u e, ae a ion, pH, wa e po en ial (Ψs), and ligh . These ac o s no only a ec he g ow h a e o ungi bu can also ac as igge s in de elopmen al pa hways (Deacon, 2006). These ac- o s a e known o in luence hos -pa hogen in e ac- ions in C. des uc ans on Ginseng (Rahman and Pun- ja, 2005) and in a numbe o o he soilbo ne pa ho- gens such as Monospo ascus cannonballus Pollack & Uecke on muskmelon and wa e melon (Fe in and S anghellini, 2006; A mengol e al., 2011), Rhizoc o- nia solani J.G. Kühn on lupin and po a o (Kuma e al., 1999; Ri chie e al., 2006; Ri chie e al., 2009), and diseases caused by Py hium and Phy oph ho a spp. (Somme s e al., 1970; Abdelzahe e al., 1997). In Cylind oca pon spp. associa ed wi h black oo disease o g ape ines, only g ow h empe a u e ex- pe imen s ha e been conduc ed so a . These s udies we e pe o med when hese pa hogens we e ecen ly desc ibed and/o cha ac e ized (Halleen e al., 2004; Pe i and Guble , 2005; Halleen e al., 2006b; Alaniz e al., 2007; Sch oe s e al., 2008). The aim o he p e- sen s udy was o expand knowledge o he e ec s o empe a u e, pH and Ψs on mycelial g ow h, spo u- la ion and chlamydospo e p oduc ion o C. li ioden- d i, C. mac odidymum and C. paucisep a um isola ed om g ape ines. Ma e ials and me hods Fungal isola es Th ee isola es o C. li iodend i (Cy59, Cy89 and Cy100), h ee isola es o C. mac odidymum (Cy47, Cy14 and Cy81) and one isola e o C. paucisep a um (Cy593), ob ained om oo s o he basal ends o oo s ocks om g ape ines exhibi ing symp oms o black oo in Spain we e used in his s udy. Addi ion- ally, wo isola es o C. paucisep a um om oo s o g ape ines in Slo enia (CBS120171 and CBS120173) we e ob ained om he collec ion o he Cen aal- bu eau oo Schimmelcul u es (CBS, U ech , he Ne he lands) (Table 1). Single spo e isola es we e s o ed in 15% glyce ol solu ion a –80°C in c yo ials (1.5 mL capaci y). P io o use, a small plug o he colonized aga om each c yo ial was ans e ed o po a o dex ose aga (PDA) (Bioka -Diagnos ics, Zac de The , F ance) pla es and allowed o g ow a 25ºC in da kness o 14 d. E ec s o empe a u e on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. To de e mine he e ec o empe a u e on myce- lial g ow h, all isola es we e main ained and g own on PDA pla es a 25°C. Aga plugs (8 mm diam.) we e cu om he leading edges o 14-d-old colonies 39 Vol. 51, No. 1, Ap il, 2012 G ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. and placed in he cen e o PDA pla es (one plug pe pla e) ha we e hen incuba ed in he da k a 5, 10, 15, 20, 25, 30 o 35°C. The e we e ou pseudo epli- ca es o each isola e and empe a u e combina ion. A e 10 d, colony diame e was measu ed along wo pe pendicula axes, and da a we e con e ed o a- dial g ow h (mm d-1). Colonies g own on PDA we e u he incuba ed o e 20 days o e alua e spo ula ion and de e mine he p esence/absence o chlamydospo es. The num- be o conidia p oduced on mycelia om aga plugs was measu ed ollowing he me hod desc ibed by Whi ing e al. (2001) and Alaniz e al. (2007). A e 30 d o incuba ion, wo plugs o aga (4 mm diam.) wi h mycelia and spo es, we e cu om he g owing edge o each colony, and each was placed in an Eppen- do ial (1.5 mL capaci y) con aining 1 mL o s e ile wa e . Vials wi h plugs we e o exed o 5 s, and he numbe o conidia pe mL was coun ed using a haemocy ome e . To obse e chlamydospo es, a small quan i y o ungal ma e ial was emo ed om each colony su ace wi h a s e ile needle aking ca e o minimize dis u bance o he ungal s uc u es, and placed in a d op o dis illed wa e in he cen e o a clean slide, and a co e slide was ca e ully lowe ed on o he d op. Two p epa a ions pe Pe i dish we e obse ed mic oscopically a ×100 and ×400 magni i- ca ion. The expe imen was epea ed. E ec s o pH on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. The e ec s o pH on mycelial g ow h and spo - ula ion o Cylind oca pon spp. in cul u e was de- e mined on PDA. Mycelial plugs (8 mm diam.) ob ained om he g owing edges o colonies we e ans e ed o he cen e o PDA pla es (one plug pe pla e) which we e adjus ed o pH 4, 5, 6, 7 and 8 wi h he addi ion o 50 mM ci a e phospha e bu e (pH 4‒7) o 50 mM T is-HCl bu e (pH 8) (Gomo i, 1955). Pla es we e incuba ed in he da k a 25ºC. The e we e wo eplica es o each isola e and pH combina ion. Table 1. Sou ces o isola es o Cylind oca pon spp. associa ed wi h black oo disease and hei op imum g ow h empe a- u es. Species/Isola e Yea Geog aphical o igin Scion/ oo s ock Op imum g ow h (°C)b Town P o ince C. li iodend i Cy59 2003 Ta azona de la Mancha Albace e Cencibel/1103 P 21.9 Cy89 2004 Aielo de Mal e i Valencia Ga nacha/110R 23.7 Cy100 2004 Alesanco La Rioja Ga nacha/110R 24.3 C. mac odidymum Cy14 2002 n.d.cBu gos Temp anillo/110R 24.5 Cy47 2003 Mollina Málaga Ped o Ximénez/1103 P 23.3 Cy81 2003 Beneixama Alican e Temp anillo/161-49C 24.1 C. paucisep a um Cy593 2009 Toba a Albace e Sy ah/S04 24.4 CBS120171a2005 K sko (Eslo enia) n.d. n.d. 24.0 CBS120173a2005 Doljenska (Eslo enia) n.d. n.d. 22.9 a Re e ence isola es o C. paucisep a um (CBS 120171 and CBS 120173) we e ob ained om he collec ion o Cen albu eau oo Schimmel- cul u es inU ech , he Ne he lands (CBS). b Fo each Cylind oca pon spp. isola e, empe a u e a e age g ow h a es we e adjus ed o a eg ession cu e o es ima e he op imum g ow h empe a u e. c n.d., No de e mined. Phy opa hologia Medi e anea 40 C. Agus í-B isach and J. A mengol Mean mycelial g ow h a es, he numbe o conidia p oduced on mycelia and he p esence/absence o chlamydospo es we e e alua ed as desc ibed abo e. The expe imen was epea ed. E ec s o wa e po en ial (Ψs) on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. The e ec o Ψs on mycelial g ow h and spo u- la ion o Cylind oca pon spp. in cul u e was de e - mined on PDA. Mycelial plugs (8 mm in diam.) ob ained om he g owing edges o colonies we e ans e ed o he cen e o PDA pla es (one plug pe pla e) amended wi h KCl o NaCl p io o s e iliza- ion o ob ain six Ψs alues: -0.5, -1.0, -2.0, -3.0, -4.0 and -5.0 MPa, acco ding o Robinson and S okes (1959). Non-amended PDA (-0.3 MPa) was used as and expe imen al con ol. Pla es we e incuba ed in he da k a 25ºC. The e we e wo eplica es o each isola e, ype o solu e and Ψs combina ion. Mean mycelia g ow h a es, he numbe o conidia p o- duced on mycelia and he p esence/absence o chla- mydospo es we e e alua ed as desc ibed abo e. The expe imen was epea ed. S a is ical analyses Da a om each Cylind oca pon spp. we e analyzed sepa a ely. Two way analyses o a iance (ANOVA) we e conduc ed wi h adial g ow h and spo ula ion da a ob ained om empe a u e and pH expe i- men s, and a h ee way ANOVA was conduc ed o Ψs expe imen s using S a g aphics Plus 5.1 so wa e (Manugis ics Inc., Rock ille, MD, USA). Fo all Cy- lind oca pon spp., ANOVA analyses indica ed ha he adial g ow h and spo ula ion da a be ween he wo epe i ions in empe a u e, pH and Ψs expe i- men s we e simila (P>0.05). Thus, in all cases, da a om bo h expe imen s we e combined. Fo each Cylind oca pon spp. isola e, empe a u e, pH o Ψs a e age mycelial g ow h a es we e adjus - ed o a eg ession cu e using S a g aphics Plus 5.1 so wa e, and he bes polynomial model was chosen based on pa ame e signi icance (P<0.05) and coe - icien o de e mina ion (R2). P e ious o his analy- sis, da a o mycelial g ow h om he Ψs expe imen we e con e ed o ela i e g ow h a e as a pe cen - age o he expe imen al con ols. Addi ionally, he polynomial models in he empe a u e expe imen s we e used o es ima e he op imum g ow h empe a- u e o each isola e. Resul s E ec s o empe a u e on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. S a is ically signi ican e ec s o he isola e on adial g ow h we e obse ed o C. mac odidymum (P=0.0019) and C. paucisep a um (P=0.0073), bu no o C. li iodend i (P=0.4999). The e ec o isola e × ex- pe imen was also no signi ican in all o he species s udied (P>0.05) (Table 2). The e ec s o empe a u e on mycelial adial g ow h and spo ula ion o he nine Cylind oca pon spp. isola es a e shown in Figu e 1. All isola es we e able o g ow on PDA o e a ange o empe a u es om 5 o 30ºC, and no g ow h was ob ained a 35ºC. A 5ºC, C. li iodend i isola es showed g ow h a es be ween 0.038 cm day-1 o isola e Cy89 and 0.043 cm day-1 o isola e Cy59, while he g ow h a es o he C. mac odidymum and C. paucisep a um isola es we e almos negligible a his empe a u e. Op imum g ow h empe a u es o all isola es anged be ween 21.9ºC o isola e Cy59 (C. li iodend i) and 24.5ºC o isola e Cy14 (C. mac odidymum) (Table 1). Rega ding spo ula ion, signi ican e ec s o he isola e on spo ula ion we e obse ed o C. mac o- didymum (P<0.001) and C. paucisep a um (P<0.001), bu no o C. li iodend i (P=0.9328). The e ec o iso- la e × expe imen was also no signi ican in all spe- cies s udied (P>0.05) (Table 2). Mos o he Cylind oca pon spp. isola es p oduced conidia a all empe a u es, showing b oad a ia- ion. In gene al, he h ee C. li iodend i isola es spo - ula ed mo e abundan ly han C. mac odidymum and C. paucisep a um isola es in all s udied empe a u es, wi h alues g ea e han 104 conidia mm-2. In C. mac- odidymum, he e was mo e a iabili y among he isola es, isola e Cy14 being he only one o which alues g ea e han 104 conidia mm-2 we e ob ained a 15, 20, 25 and 30ºC. Isola e Cy47 only spo ula ed a 15, 20 and 25ºC. Spo ula ion o C. paucisep a um iso- la es was also a iable among isola es, isola e Cy593 being he only one in which alues g ea e han 104 conidia mm-2 we e ob ained a 5, 20, 25 and 30ºC. Isola e CBS120173 did no spo ula e a 5ºC. Chlamydospo es we e obse ed in PDA cul u es o all isola es om 15 o 30ºC. No chlamydospo es we e obse ed a 5 o 10ºC. (Con inued) 41 Vol. 51, No. 1, Ap il, 2012 G ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. Table 2. Analysis o a iance o he e ec s o empe a u e, pH and Ψs on adial g ow h and spo ula ion o Cylind oca pon li iodend i, C. mac odidymum and C. paucisep a um isola es. Pa ame e C. li iodend i C. mac odidymum C. paucisep a um d. .aMSbP < Fcd. . MS P < Fd. . MS P < F Tempe a u e Radial g ow h Expe imen (A) 1 0.00012 0.8911 1 <0.0001 0.9894 1 0.00007 0.9199 Isola e (B) 2 0.00427 0.4999 2 0.04492 0.0019 2 0.03480 0.0073 A × B 2 0.00019 0.9688 2 0.00011 0.9839 2 0.00001 0.9980 Residual 326 0.00615 329 0.00703 330 0.00697 Spo ula ion Expe imen (A) 1 17.5208 0.1418 1 17.2798 0.0629 1 5.17642 0.3420 Isola e (B) 2 0.56193 0.9328 2 379.699 <0.001 2 195.172 <0.001 A × B 2 4.69997 0.5596 2 2.11654 0.6532 2 4.39406 0.4645 Residual 330 8.08063 330 4.96344 330 5.71707 pH Radial g ow h Expe imen (A) 1 <0.0001 0.9790 1 0.00005 0.8122 1 <0.0001 0.9793 Isola e (B) 2 0.00318 0.0895 2 0.04015 <0.001 2 0.00557 0.0006 A × B 2 0.00017 0.8787 2 0.00005 0.9459 2 <0.0001 0.9987 Residual 226 0.00131 221 0.00089 225 0.00073 Spo ula ion Expe imen (A) 1 0.02486 0.8548 1 <0.0001 0.9132 1 <0.0001 0.2186 Isola e (B) 2 2.26406 0.0492 2 <0.0001 <0.001 2 <0.0001 <0.001 A × B 2 0.09818 0.8759 2 <0.0001 0.9882 2 <0.0001 0.2346 Residual 212 0.74085 210 <0.0001 208 <0.0001 Ψs Radial g ow h Expe imen (A) 1 0.00004 0.8891 1 0.00002 0.9411 1 0.00002 0.9329 Isola e (B) 2 0.02469 <0.001 2 0.05107 <0.001 2 0.05301 <0.001 Sal ype (C) 1 0.05410 <0.001 1 0.05925 <0.001 1 0.16224 <0.001 A × B 2 0.00020 0.8982 2 0.00016 0.9547 2 <0.0001 0.9992 A × C 2 0.00247 0.2742 2 0.02014 0.0026 2 0.00127 0.6735 B × C 1 0.00182 0.3290 1 0.00021 0.8009 1 0.00005 0.9011 A × B × C 2 0.00042 0.8029 2 0.00019 0.9449 2 0.00006 0.9817 Residual 639 0.00191 658 0.00335 we e used o es ima e he op imum g ow h empe a- u e o each isola e. Resul s E ec s o empe a u e on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. S a is ically signi ican e ec s o he isola e on adial g ow h we e obse ed o C. mac odidymum (P=0.0019) and C. paucisep a um (P=0.0073), bu no o C. li iodend i (P=0.4999). The e ec o isola e × ex- pe imen was also no signi ican in all o he species s udied (P>0.05) (Table 2). The e ec s o empe a u e on mycelial adial g ow h and spo ula ion o he nine Cylind oca pon spp. isola es a e shown in Figu e 1. All isola es we e able o g ow on PDA o e a ange o empe a u es om 5 o 30ºC, and no g ow h was ob ained a 35ºC. A 5ºC, C. li iodend i isola es showed g ow h a es be ween 0.038 cm day-1 o isola e Cy89 and 0.043 cm day-1 o isola e Cy59, while he g ow h a es o he C. mac odidymum and C. paucisep a um isola es we e almos negligible a his empe a u e. Op imum g ow h empe a u es o all isola es anged be ween 21.9ºC o isola e Cy59 (C. li iodend i) and 24.5ºC o isola e Cy14 (C. mac odidymum) (Table 1). Rega ding spo ula ion, signi ican e ec s o he isola e on spo ula ion we e obse ed o C. mac o- didymum (P<0.001) and C. paucisep a um (P<0.001), bu no o C. li iodend i (P=0.9328). The e ec o iso- la e × expe imen was also no signi ican in all spe- cies s udied (P>0.05) (Table 2). Mos o he Cylind oca pon spp. isola es p oduced conidia a all empe a u es, showing b oad a ia- ion. In gene al, he h ee C. li iodend i isola es spo - ula ed mo e abundan ly han C. mac odidymum and C. paucisep a um isola es in all s udied empe a u es, wi h alues g ea e han 104 conidia mm-2. In C. mac- odidymum, he e was mo e a iabili y among he isola es, isola e Cy14 being he only one o which alues g ea e han 104 conidia mm-2 we e ob ained a 15, 20, 25 and 30ºC. Isola e Cy47 only spo ula ed a 15, 20 and 25ºC. Spo ula ion o C. paucisep a um iso- la es was also a iable among isola es, isola e Cy593 being he only one in which alues g ea e han 104 conidia mm-2 we e ob ained a 5, 20, 25 and 30ºC. Isola e CBS120173 did no spo ula e a 5ºC. Chlamydospo es we e obse ed in PDA cul u es o all isola es om 15 o 30ºC. No chlamydospo es we e obse ed a 5 o 10ºC. (Con inued) Phy opa hologia Medi e anea 42 C. Agus í-B isach and J. A mengol E ec s o pH on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. S a is ically signi ican e ec s o he isola es on adial g ow h we e obse ed o C. mac odidymum (P<0.001) and C. paucisep a um (P=0.0006), bu no o C. li iodend i (P=0.0895). The e ec o isola e × ex- pe imen was also no signi ican in he h ee species s udied (P>0.05) (Table 2). The e ec s o pH on mycelial adial g ow h and spo ula ion o he nine Cylind oca pon spp. isola es a e shown in Figu e 2. All isola es we e able o g ow on pH-adjus ed PDA a all pH alues s udied. In gen- e al, o each Cylind oca pon spp. all isola es showed simila g ow h a es om pH 4 o pH 8, al hough in C. li iodend i and C. mac odidymum adial g ow h inc eased sligh ly as pH inc eased. In C. li iodend i, mycelial adial g ow h was g ea es a pH 8 o all isola es, and in C. mac odidymum, mycelial g ow h o Cy47 and Cy81 isola es was also g ea es a pH 8, while o Cy14 his was g ea es a pH 6. In C. pauci- sep a um, mycelial g ow h o isola es CBS120171 and CBS120173 was g ea es a pH 6, while o isola e Cy593 his was g ea es a pH 8. Rega ding spo ula ion, signi ican e ec s o he isola e on spo ula ion we e obse ed o C. li ioden- d i (P=0.0492), C. mac odidymum (P<0.001) and C. paucisep a um (P<0.001). The e ec o isola e × expe - imen was no signi ican o all Cylind oca pon spp. (P>0.05) (Table 2). Mos o he isola es p oduced conidia a all pH alues, showing a b oad ange o a ia ion among Cylind oca pon spp. In gene al, he h ee C. li iodend i isola es spo ula ed mo e abundan ly han C. mac- odidymum and C. paucisep a um a all s udied pH alues, p oducing mo e han 105 conidia mm-2. In C. mac odidymum, he e was a iabili y among he isola es, isola e Cy14 being he only one o which alues g ea e han 105 conidia mm-2 we e ob ained a pH 6 and 8. Isola es Cy47 did no spo ula e a pH 5 and Cy81 did no spo ula e a pH 6. Spo ula ion o C. paucisep a um isola es was also a iable among isola es, isola e Cy593 being he only one o which alues g ea e han 105 conidia mm-2 we e eco ded a pH 6, bu his isola e did no spo ula e a pH 4. Isola e CBS120171 only spo ula ed a pH 5, 6 and 7, and isola e CBS120173 did no spo ula e a pH 5. Chlamydospo es we e obse ed in PDA cul u es o all isola es a all pH alues s udied. E ec s o wa e po en ial (Ψs) on mycelial g ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. S a is ically signi ican e ec s o he isola e and sal ype on mycelial g ow h we e obse ed o all h ee species (P<0.001). All he in e ac ions we e no Pa ame e C. li iodend i C. mac odidymum C. paucisep a um d. .aMSbP < Fcd. . MS P < Fd. . MS P < F Spo ula ion Expe imen (A) 1 4.06421 0.1092 1 2.37550 0.5960 1 0.52946 0.8002 Isola e (B) 2 0.88494 0.5723 2 68.3923 0.0003 2 91.4964 <0.001 Sal ype (C) 1 0.67427 0.0391 1 1.26907 0.6983 1 118.754 0.0002 A × B 2 1.70771 0.3409 2 1.70329 0.8175 2 3.05828 0.6908 A × C 2 10.0494 0.0019 2 297.890 <0.001 2 52.2602 0.0019 B × C 1 3.36179 0.1452 1 0.62988 0.7848 1 15.4059 0.1721 Residual 2 0.06835 0.9578 2 7.15412 0.4293 2 8.13652 0.3742 A × B × C 660 1.58416 660 8.44962 660 8.26433 a Deg ees o eedom. b Mean squa e. c P obabili ies associa ed wi h indi idual F- es s. Table 2. Con inues. 43 Vol. 51, No. 1, Ap il, 2012 G ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. Tempe a u es (°C) Radial g ow h (cm day-1) Spo ula ion [log 10 (conidia mm-2 + 1)] CP CM CL CL CL ! (Cy59) y= -0.00002x3-0.0005x2 + 0.0107x - 0.0331, R2=0.8925 ! (Cy89) y= -0.00004x3+0.0016x2 + 0.0084x - 0.0423, R2=0.9409 ! (Cy100) y= -0.00005x3 - 0.002x2 + 0.0159x - 0.0687, R2=0.9871 ! (Cy14) y= -0.00004x3 + 0.0018x2 - 0.0151x - 0.0446, R2=0.7615 ! (Cy47) y= -0.00003x3 + 0.0011x2 + 0.0033x - 0.0379, R2=0.9494 ! (Cy81) y= -0.00004x 3 + 0.0017x 2 - 0.0081x - 0.0042, R 2 =0.8302 CM ! (Cy593) y= -0.00004x3 + 0.0015x2 - 0.0084x - 0.0076, R2=0.9179 ! (CBS120171) y= -0.00005x3 + 0.0018x2 + 0.0059x - 0.0112, R2=0.9719 ! (CBS120173) y= -0.00003x3+ 0.0008x2 - 0.0082x - 0.0569, R2=0.9656 CP A B Figu e 1. A, mean colony adial g ow h a es (cm day-1) o h ee isola es o C. li iodendi (CL), C. mac odidymum (CM) and C. paucisep a um (CP), a e 10 days o incuba ion in da kness on PDA a 5, 10, 15, 20, 25, 30 o 35ºC; B, mean amoun s o spo u- la ion [log10(conidia mm-2 + 1)] o h ee isola es o C. li iodend i (CL), C. mac odidymum (CM) and C. paucisep a um (CP), a e 30 days o incuba ion in da kness on PDA a 5, 10, 15, 20, 25, 30 o 35ºC. Resul s a e he mean o wo independen se s o ou pseudo eplica es o each empe a u e. Ve ical ba s a e he s anda d e o o he means. Phy opa hologia Medi e anea 44 C. Agus í-B isach and J. A mengol Figu e 2. A, mean colony adial g ow h a es (cm day-1) o h ee isola es o C. li iodendi (CL), C. mac odidymum (CM) and C. paucisep a um (CP), a e 10 days o incuba ion in da kness on PDA a pHs 4, 5, 6, 7 o 8; B, mean amoun s o spo ula ion [log10(conidia mm-2 + 1)] o h ee isola es o C. li iodend i (CL), C. mac odidymum (CM) and C. paucisep a um (CP), a e 30 days o incuba ion in da kness on PDA a pHs 4, 5, 6, 7 o 8. Resul s a e he mean o wo independen se s o ou pseudo - eplica es in each pH.Ve ical ba s a e he s anda d e o o he means. B A pH Radial g ow h (cm day-1) Spo ula ion [log 10 (conidia mm-2 + 1)] ! (Cy593) y= - 0.0027x2 + 0.0399x + 0.0794, R2=0.3507 ! (CBS120171) y= - 0.0087x2 + 0.1048x - 0.0624, R2=0.3632 ! (CBS120173) y= - 0.0085x 2 + 0.1031x - 0.0729, R 2 =0.4469 CP ! (Cy14) y= - 0.0094x2 - 0.1295x - 0.1454, R2=0.5203 ! (Cy47) y= - 0.0002x2 + 0.0156x - 0.1487, R2=0.7905 ! (Cy81) y= - 0.002x2 - 0.0366x - 0.0934, R2=0.958 CM ! (Cy59) y= 0.0135x2 - 0.1423x + 0.5737, R2=0.8288 ! (Cy89) y= 0.0076x2 - 0.0817x + 0.4365, R2=0.6599 ! (Cy100) y= 0.0086x2 - 0.0836x - 0.3978, R2=0.8066 CL CL CM CP 45 Vol. 51, No. 1, Ap il, 2012 G ow h, spo ula ion and chlamydospo e p oduc ion o Cylind oca pon spp. signi ican wi h he excep ion o expe imen × sal ype o C. mac odidymum (P<0.0026) (Table 2). The e ec o Ψs on mycelial g ow h o he nine Cylind oca pon spp. isola es is shown in Figu e 3, and on spo ula ion is shown in Figu e 4. The pa e ns o he mycelial adial g ow h esponses o he isola es o dec easing Ψs we e simila o he wo osmo ica es ed, bu , in gene al, Cylind oca pon spp. we e mo e ole an o NaCl han KCl. Mycelial g ow h gene - ally inc eased compa ed wi h mycelial g ow h on nonamended PDA (-0.3 MPa) a -0.5 MPa and -1.0 Mpa by he addi ion o KCl and NaCl. The excep ion was isola e Cy89 which showed he g ea es mycelial g ow h a -2.0 MPa. A lowe wa e po en ials, my- celial g ow h dec eased as Ψs educed, showing he lowes pe cen ages a -5.0 MPa. Rega ding spo ula ion, a signi ican e ec o he isola e on spo ula ion was obse ed o C. mac o- didymum (P=0.0003) and C. paucisep a um (P<0.001), bu no o C. li iodend i (P=0.5723). The e ec o sal ype was signi ican o C. li iodend i (P=0.0391) and C. paucisep a um (P=0.0002), bu no o C. ma- c odidymum (P=0.6983). All he in e ac ions we e no signi ican wi h he excep ion o expe imen × sal ype o all Cylind oca pon spp. (Table 2). Mos o he isola es we e able o p oduce conidia a all Ψs alues, showing a b oad ange o a ia ion. In gene al, he h ee C. li iodend i isola es spo ula ed mo e abundan ly han C. mac odidymum and C. pau- cisep a um isola es in all s udied Ψs alues o each sal es ed, wi h alues g ea e han 105 conidia mm-2 in KCl and 104 conidia mm-2 in NaCl. In C. mac o- didymum, he e was mo e a iabili y among he iso- la es, and isola es Cy47 and Cy81 did no spo ula e a -5.0 MPa in NaCl. In C. paucisep a um, spo ula ion was also a iable among isola es. Isola e CBS120171 did no spo ula e a -1.0 MPa in KCl and a -0.5 in NaCl, and isola e Cy593 did no spo ula e a -5.0 MPa in NaCl. Chlamydospo es we e obse ed in PDA cul u es o all isola es a almos all Ψs alues in bo h sal s. No chlamydospo es we e obse ed in isola e Cy593 (C. paucisep a um) a -4.0 MPa in NaCl and a -5.0 MPa bo h in NaCl and KCl, and in Cy59 (C. li iodend i) a -4.0 and -5.0 MPa in KCl. Discussion This s udy has iden i ied di e ences in he e ec s o empe a u e, pH and Ψs on mycelial g ow h, spo - ula ion and chlamydospo e p oduc ion o C. li ioden- d i, C. mac odidymum and C. paucisep a um. In gene al, hese Cylind oca pon spp. we e able o g ow o e a ange o empe a u es om 5 o 30ºC, wi h op imum empe a u es o g ow h be ween 20 o 25ºC, bu hey did no g ow a 35ºC. These esul s a e in ag eemen wi h hose ob ained in p e ious s udies (Halleen e al., 2004; Pe i and Guble , 2005; Halleen e al., 2006b; Alaniz e al., 2007; Sch oe s e al., 2008), and indica e ha Cylind oca pon spp. asso- cia ed o black oo disease o g ape ine a e meso- philic, as mos ungi, which commonly g ow wi hin he ange 10‒40ºC (Deacon, 2006). In addi ion, ou s udy showed di e ences among Cylind oca pon spp. in he e ec o low empe a u es on mycelial g ow h. G ow h o C. mac odidymum and C. paucisep a um a 5 and 10ºC was almos negligible compa ed wi h ha o C. li iodend i. This is in ag eemen wi h he esul s ob ained by Alaniz e al. (2007), who demons a ed ha C. mac odidymum can be di e en ia ed om C. li iodend i by g owing mo e slowly a 5 and 10ºC. P e ious esea ch has sugges ed ha species be- longing o he genus Cylind oca pon a e calcicolous, wi h op imal pH a ound 7.0 and being poo ly ep- esen ed in acid soils (Ma u i and S en on, 1964a). Ne e heless, in ou s udy Cylind oca pon spp. as- socia ed wi h black oo o g ape ine showed b oad pH ole ance o mycelial g ow h, al hough wi h sligh di e ences among isola es. All isola es we e able o g ow be ween pH 4.0 o 8.0. This co esponds wi h he op imum pH ange indica ed by Deacon (2006) o mycelial g ow h o mos ungi. Rega ding he e ec o Ψs, he esponse o Cylin- d oca pon spp. isola es was educed mycelial g ow h as Ψs dec eased. Ou esul s indica e ha he iso- la es may ha e bene i ed om small o modes ad- di ions o solu es. In gene al, mycelial g ow h was g ea e on amended media a -0.5, -1.0 and/o -2.0 MPa compa ed wi h ha on nonamended PDA (-0.3 MPa), and was educed a Ψs alues less han -2.0 MPa. Mo eo e , he e ec o Ψs on mycelial g ow h was simila whe he KCl o NaCl was used as he osmo icum, indica ing ha he obse ed esponses we e caused by changes in Ψs a he han by oxic- i y o he osmo ic. These esul s, oge he wi h hose ob ained in he pH expe imen s, sugges ha Cylin- d oca pon spp. pa hogenic o g ape ine a e likely o p oli e a e in mos ineya d soils. In all s udied condi ions, C. li iodend i was he species wi h he g ea es capaci y o spo ula ion