325
Phy opa hologia Medi e anea (2015) 54, 2, 325−334
www. up ess.com/pm ISSN (p in ): 0031-9465
© Fi enze Uni e si y P ess ISSN (online): 1593-2095
DOI: 10.14601/Phy opa hol_Medi e -15526
Co esponding au ho : J. Luque
E-mail: [email p o ec ed]
RESEARCH PAPERS - 9TH SPECIAL ISSUE ON GRAPEVINE TRUNK DISEASES
Viabili y o Bo yosphae iaceae species pa hogenic o g ape ine a e
ho wa e ea men
Geo Gina eLena1, Vi o io Di BeLLa2, Josep a MenGoL3 and Jo Di LUQUe1
1 IRTA Cab ils, C a. de Cab ils km 2, 08348 Cab ils, Spain
2 Uni e si à degli S udi di Pale mo, Viale delle Scienze, Edi icio 4 Ing . B, 90128 Pale mo, I aly
3 Ins i u o Ag o o es al Medi e áneo, Uni e sidad Poli écnica de Valencia (IAM–UPV), Camino de Ve a s/n, 46022 Valencia,
Spain
Summa y. The iabili y o eigh species o Bo yosphae iaceae pa hogenic o g ape ine was s udied a e a ho
wa e ea men (HWT) in o de o e alua e he easibili y o his echnique as a po en ial ool o con ol hese
species du ing he g ape ine p opaga ion p ocess. In a i s ial (in i o), mycelial plugs con ained in Eppendo
ubes wi h s e ile dis illed wa e we e subjec ed o di e en combina ions o empe a u e (50–54°C) and exposu e
ime (15, 30 and 45 min) in a ho wa e ba h. G ow h a es o ea ed mycelia we e compa ed o un ea ed con ols.
Signi ican di e ences in su i al and g ow h o all ac o s (species, empe a u e and ime) and hei 2-way in e -
ac ions we e obse ed. Fungal su i al and g ow h gene ally dec eased wi h inc easing empe a u es and expo-
su e imes. Diplodia se ia a, Neo usicoccum lu eum, N. pa um and Spence ma insia i icola we e he mos suscep ible
species o empe a u e, while Lasiodiplodia heob omae and N. i i usi o me we e he mos ole an . In a second ial
(in plan a), he ungi we e inocula ed in o g ape ine canes (Rich e 110 oo s ock). Inocula ed canes we e incuba ed
a 25°C o 3 weeks o allow he ungi o colonize he wood and hen subjec ed o HWT in he ange o 50–53°C o
30 min, and su i al o ungi a e HWT was assessed. Su i al o all species was sha ply educed a e HWT o
30 min a 51°C and highe empe a u es. A 50°C, Lasiodiplodia heob omae was he mos ole an species whe eas
N. lu eum was he mos suscep ible. Resul s ob ained in his s udy demons a e he easibili y o con olling hese
pa hogens by HWT in he nu se y g ape ine p opaga ion p ocess.
Key wo ds: Bo yosphae ia dieback, con ol, g ape ine unk diseases, nu se y, p opaga ion.
In oduc ion
Se e al species included in he Bo yosphae iace-
ae Theiss. & Syd. a e causal agen s o Bo yosphae ia
dieback o g ape ine (Vi is ini e a L.), and ha e been
epo ed as majo unk pa hogens o g ape ines
wo ldwide (Ú bez-To es, 2011). These pa hogens
cause wood nec osis in unks and a ms o in ec ed
ines, which dec ease he p oduc i i y and longe i y
o ineya ds (Ú bez-To es, 2011; Be sch e al., 2013).
Al hough he incidence o Bo yosphae ia dieback
is common in 8-yea and olde ineya ds (La ignon
and Dubos, 2001), Bo yosphae iaceae species ha e
also been epo ed om g ape ine nu se ies. These
pa hogens ha e been isola ed om g ape ine oo -
s ock mo he plan s, canes o scion and oo s ock
a ie ies, nu se y cu ings and young g a ed g ape-
ines in nu se ies (Fou ie and Halleen, 2002; 2004a;
Halleen e al., 2003; Giménez-Jaime e al., 2006; A oca
e al., 2010; Spagnolo e al., 2011; Billones-Baaijens e
al., 2013a; 2013b). These indings demons a e ha
Bo yosphae iaceae species a e p esen in g ape ine
p opaga ion ma e ial and in he g a ed plan s sup-
plied by nu se ies, indica ing ha some o he cu -
en ineya d in ec ions may ha e o igina ed in he
p opaga ion p ocess (Billones-Baaijens e al., 2013b).
Howe e , in some cases he p esence o Bo yospha-
e iaceae species in g ape ine p opaga ion ma e ial
Phy opa hologia Medi e anea
326
G. Elena e al.
was no ela ed o he obse ed ex e nal symp oms
(Fou ie and Halleen, 2004a; A oca e al., 2010; Bil-
lones-Baaijens e al., 2013b). P esence o hese pa ho-
gens in symp omless g ape ines could be explained
by published epo s ha indica e ha Bo yospha-
e iaceae species can beha e as endophy es o la en
pa hogens in asymp oma ic wood o symp omless
plan s (Slippe s and Wing ield, 2007; Phillips e al.,
2013). The e o e hese pa hogens could be ca ied
in o ineya ds in appa en ly heal hy plan s used o
ine es ablishmen o eplacemen (Billones-Baaijens
e al., 2013b).
Con ol o endogenous ungal pa hogens in
g ape ines is di icul and he e is an u gen need o
e ise he managemen s a egies applied in nu s-
e ies o imp o e he phy osani a y quali y o plan -
ing ma e ial (G amaje and A mengol, 2011). Se e al
esea ch s udies ha e e alua ed he in i o and in
i o e ec i eness o di e en ungicides o con ol
Bo yosphae iaceae spp. in ineya ds, mainly when
applied as p uning wound p o ec an s (Bes e e al.,
2007; Pi e al., 2010; 2012; Rolshausen e al., 2010;
Amponsah e al., 2012). Ne e heless, he applica ion
o ungicides o con ol ungal unk pa hogens in he
nu se y p ocess is di icul , because adi ional ech-
niques, such as chemical sp ays and dips used o he
con ol o su ace pa hogens, do no pene a e su -
icien ly in o do man g ape ine cu ings o con ol
in e nal in ec ions (G amaje and A mengol, 2011). In
addi ion, he ange o au ho ized p oduc s o his
pu pose is becoming limi ed in mos coun ies.
The use o ho wa e ea men (HWT) a di e en
empe a u e- ime combina ions has been epo ed as
a p omising managemen s a egy o he con ol o
black- oo and Pe i disease pa hogens in g ape ine
p opaga ion ma e ial (Fou ie and Halleen, 2004b;
G amaje e al., 2008; 2009; 2010; 2014; Bleach e al.,
2013). One o he main ad an ages o a HWT p o o-
col is ha he hea is able o comple ely pene a e he
wood, killing in e nal pa hogens, wi hou any de -
imen al e ec s on he ine issue (Wai e and Mo -
on, 2007). Recen ly, Billones-Baaijens e al. (2014)
epo ed ha HWT can e ec i ely educe he in ec-
ion incidence o Neo usicoccum lu eum (Pennycook
& Samuels) C ous, Slippe s & A.J.L. Phillips and
N. pa um (Pennycook & Samuels) C ous, Slippe s
& A.J.L. Phillips in a i icially in ec ed 5C oo s ock
cu ings, as well as any exis ing Bo yosphae iaceae
species in na u ally in ec ed cu ings. To he bes o
ou knowledge, no addi ional in o ma ion on he
suscep ibili y o Bo yosphae iaceae species o HWT
is a ailable o da e. The e o e, he objec i e o his
s udy was o assess he suscep ibili y o eigh pa ho-
genic Bo yosphae iaceae species o HWT and o
e alua e he e icacy o HWT as a con ol s a egy
o Bo yosphae iaceae species in g ape ine cu -
ings. The sensi i i y o Bo yosphae iaceae species
o HWT was assessed in wo complemen a y es s,
by 1) e alua ing he su i al and g ow h o ungal
mycelia a e HWT in i o, and 2) assessing ungal
su i al in a i icially inocula ed g ape ine cu ings
ha we e subjec ed o HWT.
Ma e ials and me hods
Fungal isola es
Eigh Bo yosphae iaceae axa we e es ed o
su i al and g ow h a e HWT, namely Bo yospha-
e ia do hidea (Moug.:F .) Ces. & De No ., Diplodia se-
ia a De No ., Lasiodiplodia heob omae (Pa .) G i on
& Maubl., N. lu eum, N. medi e aneum C ous, M.J.
Wing . & A.J.L. Phillips, N. pa um, N. i i usi o me
(Nieke k & C ous) C ous, Slippe s & A.J.L. Phil-
lips and Spence ma insia i icola (A.J.L. Phillips & J.
Luque) A.J.L.Phillips, A. Al es & C ous. Two isola es
pe axon we e used in his s udy excep o N. i i-
usi o me, wi h only one ep esen a i e isola e (Table
1). All isola es used in he expe imen s we e p e i-
ously hyphal ip subcul u ed. Isola es we e s o ed
a 4°C in s e ile dis illed wa e ubes wi h mycelial
plugs p e iously g own on po a o dex ose aga
(PDA; Di co). Fungi we e g own o 1 week on PDA
pla es a 25°C o ob ain su icien quan i ies o myce-
lium o he expe imen s.
E ec o ho wa e ea men on mycelial su i al and
g ow h in i o
An expe imen was ca ied ou o check o su -
i al and g ow h o isola es a e ea ing ungal my-
celia a di e en combina ions o empe a u e (50 o
54°C a 1°C in e al) and exposu e ime (15, 30 o 45
min) in a wa e ba h (JP SELECTA model Uni onic
Vai en; Ab e a, Spain; homogenei y 0.1°C, esolu-
ion 0.1°C). All h ee exposu e imes we e es ed ini-
ially a 50°C, and empe a u e was inc eased in 1°C
in e als as ungal g ow h a e HWT was obse ed
in a leas 10% o cases. Aga plugs wi h mycelium (5
mm diam.) we e ob ained om he ac i ely g owing
327
Vol. 54, No. 2, Augus , 2015
Ho -wa e ea men on Bo yosphae iaceae
edges o 1-week-old colonies g owing on PDA. Fou
aga plugs pe isola e we e placed in o a 1.5 mL Ep-
pendo ube con aining 1 mL o s e ile dis illed wa-
e , and ea ed a he abo e empe a u e- ime com-
bina ions in he wa e ba h. Fou Eppendo ubes
pe isola e we e used as pseudo eplica es in each
empe a u e- ime combina ion. A e HWT, Eppen-
do ubes we e cooled unde unning ap wa e o
5 min. The aga plugs we e emo ed om he ubes
and placed in he cen e o PDA pla es, one plug pe
pla e, wi h he mycelium acing he medium su ace.
Pla es we e incuba ed a 22°C in da kness o 3 d. In
addi ion, six non- ea ed mycelial plugs we e pla ed
on o PDA and incuba ed unde he same condi ions
as expe imen al con ols. A e he incuba ion pe i-
od, su i al o each mycelial plug was eco ded and
ou colony adii we e measu ed om he colony
cen e a pe pendicula angles. Radial g ow h da a
we e a e aged (0.25 cm sub ac ed om he mean
alue o accoun o he o iginal aga plug size), and
he ela i e g ow h compa ed o he con ols was cal-
cula ed o each isola e as
100 × (Tijk/Ck)
whe e Tij is he mean o he ou PDA pla es pe Ep-
pendo ube in each empe a u e- ime combina ion
i, pseudo eplica e j (j = 1–4), and expe imen k (k =
1–2), and Ck is he mean o he six con ol PDA pla es
pe expe imen k.
Mean mycelium su i al in each empe a u e-
ime combina ion was exp essed as a pe cen age (0,
25, 50, 75 o 100%) hus co esponding o he p opo -
ion o li ing mycelial plugs wi hin he same ube.
The expe imen was epea ed once.
E ec o ho wa e ea men on mycelial su i al in
plan a
The su i al o Bo yosphae iaceae species colo-
nizing g ape ine wood was e alua ed a e he in
i o es was comple ed. Ex eme exposu e imes o
HWT in he in plan a assay we e no es ed because 30
min is he mos common du a ion ime used o HWT
in g ape ine nu se ies. This allows empe a u e o
Table 1. Bo yosphae iaceae species and ep esen a i e isola es ob ained om g ape ine and used in his s udy. CBSa ac-
cession numbe s a e gi en o some isola es.
Species Cul u e No. CBS Accession No. Geog aphic o igin Collec o / Isola o
Bo yosphae ia do hidea JL353 CBS 121484 Caldes de Mon bui, Spain J. Luque
B. do hidea JL380 CBS 110302 Mon emo -o-No o, Po ugal A.J.L. Phillips
Diplodia se ia a JL354 Caldes de Mon bui, Spain J. Luque
D. se ia a JL398 CBS 121485 Pacs del Penedès, Spain J. Luque
Lasiodiplodia heob omae JL664 Fon ana s, Spain R. Raposo
L. heob omae JL819 CBS 124060 Ma sala, Sicily, I aly S. Bu uano
Neo usicoccum lu eum JL381 Sin a, Po ugal A.J.L. Phillips
N. lu eum JL519 CBS 121482 Gandesa, Spain J. Luque
Neo usicoccum medi e aneum JL562 Gines a , Spain J. Luque
N. medi e aneum JL763 Spain R. Raposo
Neo usicoccum pa um JL396 CBS 121486 L’A boç del Penedès, Spain J. Luque
N. pa um JL445 Pie a, Spain J. Luque
Neo usicoccum i i usi o me JL563 CBS 121481 Gandesa, Spain J. Luque
Spence ma insia i icola JL525 CBS 117007 Gandesa, Spain J. Luque
S. i icola JL571 CBS 117009 Vimbodí, Spain J. Luque
a Cen aalbu eau oo Schimmelcul u es, U ech , The Ne he lands.
Phy opa hologia Medi e anea
328
G. Elena e al.
uni o mly equilib a e in he in e nal wood issues o
oo s ock o scion cu ings (G amaje and A mengol,
2011). The in plan a es was he e o e conduc ed a
di e en empe a u es (50 o 53°C) and wi h a single
30 min ea men pe iod. Do man g ape ine cane
segmen s (20 cm long) o Rich e 110 oo s ock ob-
ained om a comme cial nu se y loca ed in Valencia
p o ince (Spain) we e su ace s e ilized o 10 min in
a sodium hypochlo i e solu ion (1.5% a ailable chlo-
ine), washed wice wi h s e ile dis illed wa e , and
allowed o d y on s e ile il e pape in a lamina low
cabine o 1 h. Two holes (4 mm diam.) we e d illed
on a cane in e node a 10 cm om each o he . The
d ill was s e ilized by laming be o e each use. A my-
celial plug (4 mm diam.) ob ained om he g owing
edge o a 1-week-old ungal PDA colony was placed
on he hole and he inocula ion si e was sealed wi h
Pa a ilm®. Fo each isola e and empe a u e combi-
na ion, ou cane segmen s we e used (eigh holes in
o al o each pseudo eplica e). A e inocula ion, he
ends o each cane segmen we e sealed wi h pa a in
wax o p e en wa e loss. Cane con ols we e inocu-
la ed wi h he ungi as explained abo e bu we e no
ho wa e ea ed. Cane segmen s co esponding o
each ime and empe a u e combina ion we e incu-
ba ed sepa a ely in closed plas ic boxes (50 × 35 × 15
cm; 26 L capaci y) a 25°C o 3 weeks in da kness, o
allow o ungal coloniza ion o he wood. The boxes
we e opened wice each week o p e en he onse o
e men a i e espi a ion ha could a ec he physi-
ology o he cu ings and he pa hogens. A e he
incuba ion pe iod, cane segmen s we e ea ed in a
wa e ba h (JP SELECTA Uni onic 320OR, s anda d
e o ± 0.1°C) illed wi h dis illed wa e and ollow-
ing he abo e desc ibed schedule. A e HWT, canes
we e cooled o 30 min in a ba h o ap wa e a oom
empe a u e, and ungal eco e y was pe o med as
ollows. Two equidis an poin s (15 mm) we e se a
each side om he inocula ion si e. Ba k a each ei-
sola ion si e was emo ed wi h a s e ile scalpel and
a wood chip (5 mm) was cu wi h a s e ile seca eu .
Each wood chip was hen subdi ided in o wo piec-
es, which we e su ace s e ilized (70% e hanol, 4 min)
and pla ed on mal ex ac aga (MEA) supplemen ed
wi h 0.5 g·L-1 s ep omycin sulpha e (Sigma-Ald ich).
Fungi we e eisola ed and iden i ied a e 4 d incuba-
ion a 25°C. Fungal su i al was anno a ed o each
indi idual wood chip and he pe cen age o eco e y
was calcula ed o each inocula ion si e (hole). The
expe imen was epea ed once.
S a is ical analyses
Mycelium su i al in he in i o and in plan a ex-
pe imen s, and ela i e colony g ow h a es in he in
i o expe imen we e logi ans o med p io o anal-
yses. The expe imen s used a mul i ac o ial design
wi h species, empe a u e and exposu e ime (when
applicable) conside ed as ixed ac o s. Expe imen
epe i ion and isola es (when applicable) we e used as
andom ac o s. The in i o expe imen s used a spli
plo design wi h empe a u e as he whole plo ac o
and ime as he subplo ac o . The in plan a expe i-
men s used a comple ely andomized design. Analy-
ses o a iance o he main ac o s and hei 2-way
in e ac ion e ec s on he dependen a iables we e
pe o med using SAS En e p ise Guide 4.2 unning
on SAS 9.2 (SAS Ins i u e Inc.), using he MIXED p o-
cedu e. The Residual Maximum Likelihood (REML)
me hod was used o es ima e he a iance compo-
nen s. T ans o med da a means we e compa ed using
a leas signi ican di e ence es (P = 0.05).
Resul s
E ec o ho wa e ea men on mycelial su i al and
g ow h in i o
A p elimina y s a is ical analysis o he whole
da a se s o bo h mycelial su i al and g ow h
showed ha all ixed ac o s and hei 2-way in e -
ac ions we e signi ican (P<0.05), wi h he Species
ac o and all 2-way in e ac ions being highly sig-
ni ican (P<0.001). The e o e he whole da a se was
spli in o di e en species g oups and he da a we e
e-analysed. A summa y o he s a is ical analyses
esul s is shown in Table 2. Rega ding mycelium
su i al, Tempe a u e was signi ican (P<0.05) o
B. do hidea, L. heob omae and N. medi e aneum. Ex-
posu e ime o ho wa e was signi ican only o N.
i i usi o me. Howe e , he in e ac ion Tempe a u e
× Time was signi ican o all species excep N. i i-
usi o me and S. i icola (Table 2).
The mean alues o mycelium su i al o all he
species a di e en empe a u e and ime combina-
ions a e shown in Figu e 1. Spence ma insia i icola
was he mos suscep ible ungus o HWT since su -
i al a 50°C o 15 min was only 1.6% and no g ow h
was eco ded a highe empe a u es and longe ex-
posu e imes (Figu e 1). In a second g oup, su i al
o D. se ia a, N. lu eum and N. pa um a 50°C, 15 min
anged be ween 26.6 and 56.3 %, bu was less han 15%
329
Vol. 54, No. 2, Augus , 2015
Ho -wa e ea men on Bo yosphae iaceae
o D. se ia a and N. pa um when he exposu e ime
was inc eased o 30 min, and no g ow h was obse ed
o N. lu eum. These h ee species we e no eco e ed
o e 4% a any ime a 51°C, and we e he e o e no
es ed a 52°C and highe empe a u es (Figu e 1). In
a hi d g oup, su i al o B. do hidea and N. medi e a-
neum om HWT a e 15 min was mo e han 67.2 and
90.6% espec i ely o empe a u es equal o o lowe
han 52°C. A HWT a 52°C o 45 min educed he i-
abili y o hese wo ungi down o 6.3% o B. do h-
idea and 1.6% o N. medi e aneum. Howe e , a la ge
educ ion in mycelium su i al was obse ed o B.
Table 2. Summa y o he s a is ical analyses o da a o su i al and g ow h o mycelia o eigh Bo yosphae iaceae spe-
cies a e ho wa e ea men in he in i o expe imen . Va iance componen s es ima ed h ough he Residual Maximum
Likelihood (REML) me hod no shown.
Species Fac o
Mycelium su i al Mycelium g ow h
Num DFaDen DFaF alue P>F Num DFaDen DFaF alue P>F
Bo yosphae ia
do hidea
Tempe a u e (A) 3 3 11.46 0.0377 3 3 12.97 0.0318
Time (B) 2 2 14.64 0.0640 2 2 11.68 0.0788
A × B 6167 13.78 <.0001 6 167 31.95 <0.0001
Diplodia se ia a Tempe a u e (A) 1 1 1.67 0.4197 1 1 1.39 0.4480
Time (B) 2 2 4.92 0.1690 2 2 1.81 0.3555
A × B 2 81 27.95 <.0001 2 81 40.13 <0.0001
Lasiodiplodia
heob omae
Tempe a u e (A) 4 4 14.69 0.0117 4 4 8.00 0.0343
Time (B) 2 2 7.07 0.1469 2 2 21.87 0.0437
A × B 8 210 5.91 <.0001 8 210 10.78 <0.0001
Neo usicoccum
lu eum
Tempe a u e (A) 1 1 1.43 0.4430 1 1 0.76 0.5436
Time (B) 2 2 1.22 0.4508 2 2 0.84 0.5444
A × B 2 66 23.16 <.0001 2 66 13.20 0.0005
Neo usicoccum
medi e aneum
Tempe a u e (A) 3 3 9.72 0.0470 3 3 5.81 0.0911
Time (B) 2 2 9.39 0.0962 2 2 23.41 0.0410
A × B 6167 5.63 <.0001 6 167 23.65 <0.0001
Neo usicoccum
pa um
Tempe a u e (A) 1 1 1.44 0.4422 1 1 1.43 0.4438
Time (B) 2 2 1.72 0.3670 2 2 1.39 0.4190
A × B 2 81 26.62 <.0001 2 81 35.15 <0.0001
Neo usicoccum
i i usi o me
Tempe a u e (A) 4 4 0.25 0.8976 4 4 0.31 0.8585
Time (B) 2 2 25.93 0.0371 2 2 8.77 0.1024
A × B 898 1.64 0.1227 898 4.75 <0.0001
Spence ma insia
i icola
Tempe a u e (A) 1 1 1.25 0.4646 1 1 1.25 0.4646
Time (B) 2 2 1.04 0.4898 2 2 1.04 0.4898
A × B 2 66 1.25 0.2676 2 66 1.25 0.2676
a Num DF, nume a o deg ees o eedom; Den DF, denomina o deg ees o eedom.

Phy opa hologia Medi e anea
330
G. Elena e al.
do hidea and N. medi e aneum a 53°C, wi h pe cen ag-
es lowe han 7% o bo h species a exposu e imes o
30 and 45 min (Figu e 1). Lasiodiplodia heob omae and
N. i i usi o me we e included in a ou h g oup ha
was he mos ole an o HWT (Figu e 1). Su i al o
L. heob omae was signi ican ly educed a 53°C, wi h
su i al pe cen ages o 75.0% a e 15 min, 29.7% a e
30 min and 3.1% a e 45 min, compa ed wi h he co -
esponding alues a lowe empe a u es. Su i al o
his ungus a 54°C was below 16% a e he 15 min
ea men . No mycelium su i al was obse ed a
exposu e imes longe han 15 min. In con as , su -
i al o N. i i usi o me p og essi ely dec eased wi h
empe a u e and ime, and mean pe cen ages a he
maximum es ed empe a u e anged om 40.6% (15
min) o 12.5% (45 min), as shown in Figu e 1.
Rega ding he e ec s o HWT on mycelial g ow h
a es, Tempe a u e was signi ican (P<0.05) only o
B. do hidea and L. heob omae (Table 2). Exposu e ime
o ho wa e was signi ican (P<0.05) o L. heob omae
and N. medi e aneum. In addi ion, he in e ac ion
Tempe a u e × Time was highly signi ican (P<0.001)
o all species excep S. i icola (Table 2).
The mean alues o he ela i e mycelium g ow h
o all he species a di e en empe a u e and ime
combina ions a e shown in Figu e 2. Neo usicoccum
lu eum and S. i icola we e he mos suscep ible ungi
o HWT, since g ow h a es o bo h ungi we e ne -
e g ea e han 1.0% o he co esponding con ols
ega dless o he ea men (Figu e 2). In a second
g oup, D. se ia a and N. pa um showed ela i e
g ow h a es o app ox. 20% o he con ols a 50°C
o 15 min, bu he g ow h a es a o he Tempe a-
u e × Time combina ions we e ne e g ea e han
2% (Figu e 2). In a hi d g oup, mycelial g ow h a es
o B. do hidea and N. medi e aneum a e HWT de-
FIGURE 1. Elena e al.
Mycelium su i al (%)
Tempe a u e (°C)
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Diplodia se ia a
a
b
bb
b b
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Spence ma insia i icola
aa a a a a
0
20
40
60
80
100
50
51
52
53
54
n. .
Bo yosphae ia do hidea
a
a
ab
abc
bcd
cde
de
e
0
20
40
60
80
100
50
51
52
53
54
Lasiodiplodia heob omae
a a ab
bc
cd
de
e
b
aa
ab
ab
ab
e e
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Neo usicoccum lu eum
a
b b b
b
b
0
20
40
60
80
100
50
51
52
53
54
n. .
Neo usicoccum medi e aneum
a
bcde
a
ab
a
bc
cde
bcd
de
e
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Neo usicoccum pa um
a
b
bbbb
0
20
40
60
80
100
50
51
52
53
54
Neo usicoccum i i usi o me
a
ab
cd
abc abc
abc
cd
ab
ab
abc
a
abc
bcd
cd
d
Figu e 1. Mean mycelium su i al o eigh Bo yosphae iaceae species a e ho wa e ea men in he in i o expe imen .
Su i al is exp essed as he pe cen age o li ing mycelial plugs a e ea men s (N = 16 in each empe a u e by ime com-
bina ion o all species, excep N. i i usi o me, whe e N = 8). Means o di e en exposu e imes a e exp essed in black (15
min), g ey (30 min) and whi e (45 min). Mean alues signi ican ly di e en (P < 0.05) acco ding o LSD es s a e indica ed
by di e en le e s. n. . = no es ed.
331
Vol. 54, No. 2, Augus , 2015
Ho -wa e ea men on Bo yosphae iaceae
c eased p og essi ely wi h inc easing empe a u es
and exposu e imes, and eached negligible alues
(< 2.5%) a 53°C (Figu e 2). Lasiodiplodia heob omae
and N. i i usi o me we e he species mos ole an
o highe empe a u es (Figu e 2). Mycelial g ow h
o bo h species was se e ely inhibi ed only a 54°C,
wi h mean ela i e g ow h a es below 6% a any
exposu e ime wi hin his empe a u e. Howe e ,
g ow h a es o N. i i usi o me we e ne e g ea e
han 50% whe eas a es o L. heob omae we e g ea e
han hose o N. i i usi o me especially a 52°C and
lowe empe a u es.
E ec o ho wa e ea men on mycelial su i al in
plan a
No signs o e men a ion we e no iced in he incu-
ba ion pe iod p io o HWT in he expe imen s. No
bo yosphae iaceous ungi o he han he inocula ed
species we e eisola ed om he un ea ed con ols.
Mean mycelial su i al in con ol, un ea ed canes
anged be ween 39.8% (D. se ia a) and 72.7% (L. heo-
b omae) (Table 3). Su i al o ungi a e HWT was
signi ican ly dependen on Species (P=0.004), Tem-
pe a u e (P=0.005) and hei in e ac ion (P<0.001).
The e o e, he whole da a se was e-analysed in wo
sepa a e 1-way analyses acco ding o each main ac-
o .
Neo usicoccum lu eum was no eco e ed om he
ea ed canes a any es ed empe a u e (Table 3),
showing his o be he species mos suscep ible o
HWT in colonised g ape ine wood. In gene al, ia-
bili y o ungi was educed as empe a u e inc eased,
wi h nil o e y low eco e y equencies (< 4%) o
all species a 51°C and highe empe a u es. Howe e ,
L. heob omae and N. i i usi o me we e eco e ed oc-
FIGURE 2. Elena e al.
Mycelium g ow h a e (%)
Tempe a u e (°C)
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Diplodia se ia a
a
bb
bbb
Con ol mean: 2.89 ±0.11 cm
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Spence ma insia i icola
aa a a a a
Con ol mean: 3.97 ±0.01 cm
0
20
40
60
80
100
50
51
52
53
54
n. .
Bo yosphae ia do hidea
a
a
ab
abc
bcd
cde
de
e
Con ol mean: 3.24 ±0.04 cm
0
20
40
60
80
100
50
51
52
53
54
Lasiodiplodia heob omae
ab a
cde
g
g
g
g
e
ab
bcd
abc
e
de
g g
Con ol mean: 4.00 ±0.00 cm
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Neo usicoccum lu eum
a
a a
a
a
a
Con ol mean: 3.97 ±0.01 cm
0
20
40
60
80
100
50
51
52
53
54
n. .
Neo usicoccum medi e aneum
a
de
bc
cd
ab
de
e
cde
e
e
e
Con ol mean: 2.32 ±0.06 cm
0
20
40
60
80
100
50
51
52
53
54
n. .
n. .n. .
Neo usicoccum pa um
a
b
bb
b
b
Con ol mean: 3.36 ±0.05 cm
0
20
40
60
80
100
50
51
52
53
54
Neo usicoccum i i usi o me
a
ab
b
ab
ab
b
b
ab
ab
ab
ab ab
b
bb
Con ol mean: 2.93 ±0.04 cm
Figu e 2. Mean ela i e mycelium g ow h o eigh Bo yosphae iaceae species a e ho wa e ea men in he in i o ex-
pe imen . G ow h a es a e exp essed as he ela i e pe cen age o he adial colony g ow h o ea ed mycelial plugs (N =
16 in each empe a u e by ime combina ion o all species, excep N. i i usi o me, whe e N = 8) compa ed o an un ea ed
con ol (N = 6). Means o di e en exposu e imes a e exp essed in black (15 min), g ey (30 min) and whi e (45 min). Mean
alues signi ican ly di e en (P < 0.05) acco ding o LSD es s a e indica ed by di e en le e s. The absolu e mean alues
+/- s anda d e o s o he mean o he con ol g oup is shown a he op igh co ne o each g aphic. n. . = no es ed.
Phy opa hologia Medi e anea
332
G. Elena e al.
casionally om ea ed canes unde hese condi ions
(Table 3). A 50°C, all ungi we e eco e ed a lowe
equencies han in he con ol ea men s, bu di e -
ences we e s a is ically signi ican only o N. lu eum
and N. i i usi o me (Table 3). Reduc ion in eco e y
pe cen ages o each species a 50°C we e as ollows:
B. do hidea 63.5%, D. se ia a 37.3%, L. heob omae 20.4%,
N. lu eum 100%, N. medi e aneum 45.3%, N. pa um
48.6%, N. i i usi o me 61.0% and S. i icola 76.3%. Re-
duc ion in eco e y pe cen ages a 51°C o L. heob o-
mae was 98.9%, and o N. i i usi o me was 95.1%.
Discussion
The esul s ob ained in his s udy show ha , de-
pending on ime and empe a u e, HWT can educe
he iabili y and mycelial g ow h o Bo yospha-
e iaceae pa hogens o g ape ines. In he in i o ex-
pe imen , esponses o ungal iabili y and g ow h
o di e en empe a u e and exposu e ime combina-
ions we e a iable and highly dependen on all he
expe imen al ac o s, including species, empe a u e
and exposu e ime. Diplodia se ia a, N. lu eum, N. pa -
um and S. i icola we e he mos suscep ible species
o empe a u e, while L. heob omae and N. i i usi-
o me we e he mos ole an . In gene al, he iabili y
anges o he eigh Bo yosphae iaceae species in-
cluded in he in i o expe imen s we e in acco dance
wi h p e ious published da a on he ange o ca di-
nal empe a u es o mycelial g ow h o hese spe-
cies, so ha he highe a e he op imal empe a u es
o g ow h he g ea e is he ole ance o HWT. Van
Nieke k e al. (2004) epo ed op imal empe a u e
o g ow h o N. i i usi o me o be 30°C. Ú bez-To -
es e al. (2006) s udied he op imum empe a u e o
in i o colony g ow h o se en Bo yosphae iaceae
species and es ima ed ha he op imum empe a u e
was 30.8°C o L. heob omae and B. do hidea, 29.4°C
o N. lu eum, 28.2°C o N. pa um, and 26.8°C o D.
se ia a. Ma os (2008) obse ed ha minimum em-
pe a u e o g ow h o se e al Bo yosphae iaceae
species (B. do hidea, D. se ia a, N. lu eum, N. pa um
and N. i i usi o me) in di e en cul u e media was
abou 15°C, whe eas maximum empe a u e was
abou 35°C. She also obse ed ha he minimum
empe a u e o g ow h o S. i icola was 10°C, and
his species also showed highe g ow h a es han
any o he species a 10 and 15°C. Howe e , g ow h
o his species was educed a empe a u es abo e
20°C. These da a sugges ha S. i icola is well
Table 3. Fungal eco e y o di e en Bo yosphae iaceae species om inocula ed g ape ine wood ea ed wi h ho wa e o 30 min a di e en empe a-
u es. Da a a e exp essed in pe cen ages as mean alues +/- s anda d e o s (SE) o he means (N = 16 o all species, excep N. i i usi o me, whe e N = 8).
Mean alues in he same column ollowed by di e en capi al le e s we e s a is ically di e en among species. Mean alues ac oss he same ow ollowed
by di e en lowe case le e s we e s a is ically di e en among empe a u e ea men s.
Species
Un ea ed 50°C 51°C 52°C 53°C
Mean ± SE G ouping Mean ± SE G ouping Mean ± SE G ouping Mean ± SE G ouping Mean ± SE G ouping
B. do hidea 49.22 ± 7.32 B a 17.97 ± 4.79 BC ab 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
D. se ia a 39.84 ± 6.13 B a 25.00 ± 5.83 B ab 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
L. heob omae 72.66 ± 6.58 A a 57.81 ± 5.88 A a 0.78 ± 0.78 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
N. lu eum 42.19 ± 6.29 B a 0.00 ± 0.00 C b 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
N. medi e aneum 50.00 ± 6.83 B a 27.34 ± 5.43 B ab 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
N. pa um 56.25 ± 6.55 AB a 28.91 ± 5.84 B ab 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
N. i i usi o me 64.06 ± 9.67 AB a 25.00 ± 7.91 B b 3.13 ± 2.13 A b 0.00 ± 0.00 A b 3.13 ± 2.13 A b
S. i icola 46.09 ± 6.16 B a 10.94 ± 3.54 BC ab 0.00 ± 0.00 B b 0.00 ± 0.00 A b 0.00 ± 0.00 A b
333
Vol. 54, No. 2, Augus , 2015
Ho -wa e ea men on Bo yosphae iaceae
adap ed o g ow h a coole empe a u es. In addi-
ion, Ma os (2008) epo ed ha g ow h a es o B.
do hidea and N. i i usi o me a 35°C we e g ea e han
hose o any o he Bo yosphae iaceae species, hus
showing hei capabili y o g ow h a high empe a-
u es. In a s udy on colony g ow h o eigh Bo y-
osphae iaceae species associa ed wi h g ape ine,
Pi e al. (2013) epo ed compa able esul s o hose
ci ed abo e, as hey obse ed ha S. i icola showed
high g ow h a es a low empe a u es (10°C), and
ha op imal g ow h o L. heob omae and B. do hidea
was a close o 30°C. In addi ion, he wo la e spe-
cies we e s ill able o g ow a 40°C, whe eas all o he
species did no . Pi e al. (2013) also epo ed op i-
mal empe a u es o D. se ia a (26.6°C) and N. pa -
um (26.8°C), which we e simila o hose epo ed
by Ú bez-To es e al. (2006).
In he in plan a expe imen , esponses o ungal
iabili y we e no as a iable as hose obse ed in
he in i o expe imen . Wi h he excep ion o N. lu-
eum, which was comple ely inhibi ed a 50°C, myce-
lium eco e y o all he o he species was p ac ically
supp essed a 51°C and highe empe a u es. E ec s
o HWT on he iabili y o N. lu eum and N. pa um
ha e been ecen ly s udied by Billones-Baaijens e al.
(2014). They ound ha 5C oo s ock cu ings a i i-
cially in ec ed wi h ei he pa hogen and ho wa e
ea ed a 50°C o 30 min esul ed in in e nal in ec-
ions indices o 100% o N. lu eum and 50% o N.
pa um. While he educ ion in iabili y o N. pa -
um was compa able o he esul s ob ained in he
p esen s udy, 100% su i al o N. lu eum obse ed
by Billones-Baaijens e al. (2014) is clea ly in con lic
wi h ou esul s. Howe e , bo h pa hogens we e e -
ec i ely con olled by ea men a 53°C (Billones-
Baaijens e al., 2014), in ag eemen wi h ou esul s.
Diplodia se ia a, N. lu eum, N. pa um and S. i i-
cola, showed simila esponses o HWT bo h in
i o and in plan a, as hey we e clea ly inhibi ed a
51°C in bo h ypes o expe imen s. Con e sely, he
o he ou species es ed, B. do hidea, L. heob omae,
N. medi e aneum and N. i i usi o me, showed di -
e en iabili y esponses among he expe imen s,
wi h a highe ole ance o empe a u e in he in i o
expe imen s han in plan a. We ha e no explana ion
o his di e en ial beha iou , bu i is clea ha he
condi ions in he in plan a expe imen , i.e. he use o
in ec ed g ape ine canes o check o ungal iabili y
a e HWT, a e simila o he condi ions in nu se ies,
whe e in ec ed wood coming om in ec ed mo he
ines is ho wa e ea ed o con ol la en wood
pa hogens.
Resul s ob ained in his s udy p o ide u he
e idence suppo ing he po en ial o HWT as an e -
ec i e con ol me hod o g ape ine ungal unk
pa hogens in p opaga ion ma e ial. In Spain, G ama-
je e al. (2008; 2009; 2010) de e mined ha HWT a
53°C o 30 min is needed o e ec i ely con ol Pe i
disease pa hogens, al hough he ea men s abo e
51–52°C d as ically educed conidial ge mina ion
and mycelial g ow h o he pa hogens, while HWT
p o ocols a 50°C o 30 min may be su icien o con-
ol black- oo pa hogens. Howe e , u he esea ch
is equi ed o enhance he de elopmen o e ec i e
HWT p o ocols o educe he incidence o ungal in-
ec ions and ensu e heal hy p opaga ion ma e ial is
eleased om g ape ine nu se ies.
Acknowledgemen s
D A.J.L. Phillips (Uni e sidade No a de Lisboa,
Po ugal), D R. Raposo (INIA, Spain), and P o D
L. Mugnai (Uni e si à degli S udy, Fi enze, I aly)
p o ided some o he isola es used in his s udy. This
esea ch was unded by he Spanish ‘Ins i u o Na-
cional de In es igación y Tecnología Ag a ia y Ali-
men a ia’ (INIA), p ojec RTA2010-00009-C03, wi h
ma ching unds om he Eu opean Regional De el-
opmen Fund (ERDF). Geo gina Elena was suppo -
ed by INIA wi h a p edoc o al g an .
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gicides as po en ial g ape ine p uning wound p o ec an s
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