37
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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