Viability of Botryosphaeriaceae species pathogenic to grapevine after hot water treatment

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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Jas- pe s, 2013a. Inoculum sou ces o Bo yosphae iaceae species