Levels of phytoalexins in vine leaves with different degrees of grapevine leaf stripe disease symptoms (Esca complex of diseases)

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1 Phytopthologi Mediterrne (2017) 56, 3, DOI: /Phytopthol_Mediterr SHORT NOTES Levels of phytolexins in vine leves with different degrees of grpevine lef stripe disese symptoms (Es omplex of diseses) Frneso CALZARANO 1, Fio OSTI 2, Vinenzo D AGOSTINO 1, Alessi PEPE 1, Flvio DELLA PELLE 1, Mirko DE ROSSO 3, Rirdo FLAMINI 3 nd Stefno DI MARCO 2 1 Università degli Studi di Termo, Foltà di BioSienze e Tenologie Agro-Alimentri ed Amientli, Vi Rento Blzrini, 1, Termo, Itly 2 CNR, IBIMET, Vi Goetti 101, Bologn, Itly 3 CREA-VIT, Vile XXVIII Aprile 26, Coneglino (TV), Itly Summry. Grpevine lef stripe disese (GLSD) is one of the most ommon diseses in the es omplex. Losses in grpe yields nd qulity used y GLSD re orrelted with the severity of the lef symptoms. The time ourse of phytolexin levels ws exmined in vine leves of two vineyrds, in leves of helthy, symptomti/disesed nd symptomti vines. Symptomti leves were further divided into four tegories ording to symptom severity: 1, 5% hlorosis; 2, 20% hlorosis; 3, 40% lef surfe overed with tiger stripes; or 4, 65% lef surfe overed with tiger stripes. Leves were smpled t three growth stges: erries eginning to touh ; erries developing olour ; nd erries ripe for hrvesting. The phytolexins trns-resvertrol, trns-ε-viniferin; trns-δ-viniferin; nd trnspterostilene were deteted nd ssyed. Ptterns of eh of the phytolexins were similr t eh growth stge to those found erlier for trns-resvertrol, with n inresed phytolexin levels with inresing lef symptom severity on the lef lde, espeilly t the stges erries eginning to touh nd erries ripe for hrvest. A lortory test ws lso rried out. Petioles of helthy grpevine leves were immersed in ulture filtrtes of Pheomoniell hlmydospor nd then dipped in solutions of trns-resvertrol or trns-pterostilene to ssess the effets of these sustnes on the lef ldes. Adding these phytolexins did not influene the effets of P. hlmydospor. These results indited tht phytolexins inresed in the leves fter GLSD symptoms ppered, nd not efore host response (phytolexin synthesis) to ontrol symptom development. Key words: phytolexins, GLSD, lef symptoms. Introdution Grpevine lef stripe disese (GLSD) is vine wood disese, nd one of the Es omplex of diseses. GLSD is widespred in ll vine-growing res, ut despite onsiderle experimentl dvnes mde in reent yers (Di Mro et l., 2011; 2011), it is still diffiult to ontrol. Initilly, GLSD ws ssoited with white rot used y the sidiomyete Fomitipori mediterrne (Fisher, 2002; Surio et l., 2006) nd ws nmed Corresponding uthor: F. Clzrno E-mil: flzrno@unite.it es proper, ut experimentl findings hve mde it ler tht the three fungi minly involved in es proper, Pheomoniell hlmydospor (Ph) (Crous nd Gms, 2000), Pheoremonium minimum (syn. P. leophilum, Grmje et l., 2015) nd F. mediterrne, n eh olonise vines without ny of the others, nd in no prtiulr hronologil order (Sprpno et l. 2000). Es proper hs now een onfirmed to derive from t lest two distint diseses; trheomyosis, used y P. hlmydospor, P. minimum nd proly some other speies of Pheoremonium; nd white wood rot, used y F. mediterrne. It ws susequently found tht white rot used y F. mediterrne ws not neessry for the expression of lef symptoms ttriuted to P. hlmydospor nd 494 ISSN (print): ISSN (online): Firenze University Press 2017 Author(s). This is n open ess rtile distriuted under the terms of the Cretive Commons Attriution Liense (CC-BY-4.0), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl uthor nd soure re redited.

2 Phytolexins in vine leves with GLSD symptoms P. minimum (Clzrno nd Di Mro, 2007; 2008). Consequently, the term GLSD ws proposed to designte speifilly the disese tht uses the typil lef symptoms on infeted grpevines (Surio, 2009). The fungi minly ssoited with GLSD re P. hlmydospor nd P. minimum. These fungi olonise the grpevine wood nd produe rown nerosis nd drk streking of the vessels (Mrhi et l., 2001; Clzrno nd Di Mro, 2007; Surio, 2009). The toxi metolites produed y these fungi in olonised portions of vines re trnsloted to the rowns y the sp strem, nd the GLSD symptoms develop on leves (Sprpno et l., 1998; Mugni et l., 1999; Evidente et l., 2000; Thi et l., 2000; Surio, 2009). These symptoms re: lef hlorosis nd nerosis (tiger stripes), purple spots, nd more or less extensive withering of the grpe unhes tht result in losses in yield nd qulity proportionl to the severity of the lef symptoms (Clzrno et l., 2001; 2004; Bertsh et l., 2013). Despite the dvnes of knowledge on these diseses, the ide tht the lef symptoms of GLSD re due to trnsloted toxi metolites is still only hypothesis. Tests undertken with ulture filtrtes or with the onstitutent fungl metolites involved hve produed unertin results (Sprpno et l., 1998, 2000; Aou-Mnsour et l., 2004). Only Sprpno et l. (2001) nd Feliino et l. (2004) were le to indue lef symptoms, ut these were different from typil GLSD symptoms. The well-known phenomenon of the prtil remission or totl dispperne of the folir symptoms (ut not of the disese itself) on ertin plnts in ertin yers suggests tht other physiologil or environmentl ftors my lso e involved in symptom expression. These my inlude rinfll erly in the growing seson (Mrhi et l., 2006; Clzrno nd Di Mro, 2007; 2008). More reently, vritions in the levels of the phytolexin trns-resvertrol, the min phytolexin in grpevine (Jendet et l., 2002), hve een exmined in leves of vines infeted y GLSD in some vineyrds t different growth stges (Clzrno et l., 2008). In following study, trns-resvertrol levels were mesured in leves differing for GLSD symptoms severity (Clzrno et l., 2013). Furthermore, lortory tests with helthy vine leves dipped y petiole, first in P. hlmydospor ulture filtrte nd then in trns-resvertrol suspension, showed tht trns-resvertrol did not influene the effets of Ph filtrtes on the withering of the leves (Clzrno et l., 2013). The vilility of new nlytil tehnique for the extrtion nd identifition of trnsresvertrol nd other derived phytolexins, nmely viniferins nd trns-pterostilene, hs mde it possile to etter ssess the levels of trns-resvertrol, nd to mesure the mounts of the other stilene phytolexins in helthy nd disesed leves (Clzrno et l. 2016). The im of the present study ws to dvne understnding of the roles of these phytolexins in the formtion of lef symptoms in the rowns of grpevines infeted with GLSD. In prtiulr, we determined the levels of vrious phytolexins in leves with different GLSD symptom severity t different vine growth stges. The lortory test with the Ph ulture filtrtes ws repeted mesuring the effets of trns-resvertrol nd trns-pterostilene. Mterils nd methods Folir symptom survey, smpling of leves nd phytolexin extrtion The study ws rried out in 2014 in two 36-yerold vineyrds, plnted with the v. Treino d Aruzzo on 420A rootstok, mnged with IPM strtegies, nd loted t Controguerr (Genev Doule Curtin system) nd Giulinov (Tendone system) in the provine of Termo, Aruzzo, Itly. Both vineyrds were on ly-lreous soils. The v. Treino d Aruzzo is usully severely ffeted y GLSD. The snitry sttus of eh vine (helthy or GLSDffeted) in oth vineyrds ws known euse the vines hd een monitored for GLSD symptoms sine In the 2014 test yer, the vines ould e positively identified s either helthy, disesed/symptomti or disesed/symptomti. Inidene nd severity of GLSD folir symptoms were evluted in 2014 on 20 Septemer in Controguerr nd 22 Septemer in Giulinov, during the mximum of symptom expression, reorded nd nlyzed using the methods desried in Clzrno et l. (2014). Lef smples were olleted from eh of the three groups of vines, designted: H (helthy), AS (disesed/symptomti) or T (disesed/symptomti). Leves from T vines were ritrrily divided into four sugroups: T1, exhiiting 5% hlorosis; T2, 20% hlorosis; T3, 40% lef surfe overed with ti- Vol. 56, No. 3, Deemer,

3 F. Clzrno et l. ger-stripes (hlorosis + nerosis); or T4, 65% lef surfe overed with tiger-stripes (hlorosis + nerosis). This gve totl of six tegories of leves. Leves were smpled t three growth stges, ording to BBCH-sle for grpevine (Lorenz et l., 1995): i, erries eginning to touh (); ii, erries developing olour (); or iii, erries ripe for hrvest (). From eh of the three groups of vines, six vines were seleted, nd from eh of H nd AS vines eight leves, nd from eh of T vines 32 leves (eight leves for eh symptom sugroup), t eh growth stge, were smpled from the entrl portion of vine shoot, loted in position opposite grpe unh, to minimise nturl vritions etween leves due to lef position long eh shoot. The leves smpled t the three growth stges were lwys tken from the sme vines in eh groups. Eh smple then onsisted of eight leves. The 8 leves of eh smple were omined nd treted s one replite, from whih phytolexins were extrted nd identified ording to the proedure desried in Clzrno et l. (2016). stge, from positions opposite to grpe unhes to minimise nturl vritions long the shoots. Petioles of leves from helthy vines were individully immersed in 50 ml of Ph ulture filtrte (for totl of 18 leves) or in sterile liquid growth medium s ontrols (dditionl 18 leves) for 3 h, nd were then evluted using n ritrry lef withering severity sle; where 1 ws mximum withering nd 5 ws sene of withering (fresh green lef). Leves from Ph ulture filtrte tretment were then rndomly sudivided into three groups of six leves eh. Leves in these groups were individully immersed in 50 ml of distilled wter or in distilled wter ontining 80 mg L -1 of trns-resvertrol, or 15 mg L -1 of trns-pterostilene, orresponding to the mximum onentrtions found previously in GLSD-ffeted Treino d Aruzzo grpevine leves in oth vineyrds (Clzrno et l., 2001; 2004; 2008; 2013; 2014). The ontrol ws group of six leves immersed in sterile growth medium nd in distilled wter. All leves were evluted for lef withering t 15 h, 58 h, or 106 h of immersion. Lortory ssy A lortory ssy ws rried out to determine the effet of the intertions mong phytolexins - grpevine lef - Ph filtrte. To otin Ph filtrtes, n dequte volume of Czpek Dox medium, modified y dding 0.5% yest nd 0.5% mlt extrt (ph 6.8), ws utolved, nd 150 ml ws dded to eh of 1 L pity Roux flsks. Hlf of the prepred flsks were inoulted with of gr/ myelium otined from the edges of 2-week-old Ph olonies grown on potto dextrose gr. The resulting liquid ultures were inuted in sttionry phse t 25 C in drkness. After 28 d, myelium ws removed y filtrtion through Whtmn No. 4 filter pper (Mrtos et l., 2008; Osti nd Di Mro, 2010). Filtrtes form the liquid ultures were pooled, mixed nd oserved with light mirosope to ensure tht fungl strutures hd een exluded y the filtrtion. The remining hlf of the flsks were sujeted to the sme proedure exept tht they were not inoulted, to provide sterile liquid growth medium. The petioles of helthy grpevine leves were immersed in Ph ulture filtrte, nd then in solutions of trns-resvertrol or trns-pterostilene. The leves were tken from the middle portions of the primry grpevine shoots t the growth Sttistil nlyses Dt of the onentrtions of eh phytolexin in the different GLSD tegories of leves were ompred seprtely t eh growth stge using Tukey s honest signifint differene (HSD) test t P = In the lortory test for eh time of immersion, tretments were ompred nd dt were sttistilly nlyzed using Tukey s honest signifint differene (HSD). Sttistil nlyses were rried out using SAS version 9.3 (SAS Institute In.). Results Phytolexins determintions In 2014, the men mounts of trns-resvertrol levels in T4 leves, reorded t the vine growth stge, were in Controguerr nd in Giulinov. These levels were mong the gretest mounts of trnsresvertrol reorded in those vineyrds (Clzrno et l., 2016). These high levels were onsistent with the high inidene (30.4%) nd severity (11.8%) of GLSD lef symptoms reorded in the Controguerr vineyrd, nd 9.2% inidene nd 5.8 % severity reorded in the Giulinov vineyrd. 496 Phytopthologi Mediterrne

4 Phytolexins in vine leves with GLSD symptoms A 45, , , ,00 1 5,00 25/07/2014 erries eginning to touh trns-resvertrol Controguerr Vineyrd 25/08/2014 erries developing olours d 23/09/2014 erries ripe for hrvest B 5 45, , , ,00 1 5,00 d d 26/07/2014 erries eginning to touh trns-resvertrol Giulinov Vineyrd 26/08/2014 erries developing olours 24/09/2014 erries ripe for hrvest helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe C 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 25/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe trns-ε viniferin Controguerr Vineyrd 25/08/2014 erries developing olours 23/09/2014 erries ripe for hrvest symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe D 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 26/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe trns-ε viniferin Giulinov Vineyrd 26/08/2014 erries developing olours symptomti vine leves 24/09/2014 erries ripe for hrvest symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe Figure 1. Levels of trns-resvertrol (A, B) nd trns-ε-viniferin (C, D) in the leves of vines infeted with GLSD with vrying degrees of hlorosis or tiger-stripes nd in the leves of disesed/symptomti nd helthy vines t vrious growth stges in the 2014 growing seson in the Controguerr nd Giulinov vineyrds. Sttistil nlysis ws performed ording to Tukey s honest signifint differenes (HSD) test. Eh of the six grpevine tegory (4 sugroups of disesed/ symptomti, 1 group of disesed/symptomti nd 1 group of helthy) hd 6 replitions for eh tegory of leves in eh growth stge nd in eh vineyrd. Different letters represent signifint differenes t P=0.05. In oth vineyrds, trns-resvertrol levels were prtiulrly high in symptomti leves t the BBCH 77 nd BCCH 89 growth stges, ompred with helthy nd symptomti/disesed leves. At the stge, trns-resvertrol levels delined showing similr vlues for the three groups of vines (Figure 1A 1B). Levels of ε-viniferin nd δ-viniferin in symptomti leves exhiited similr ptterns over the growth stges, s ompred with helthy nd disesed/symptomti levels, exept tht they were higher thn in helthy nd disesed/symptomti leves, lso t the erries developing olour stge. This ws despite the generlly lower mounts of the phytolexins t this stge (Figures 1C 1D nd 2A 2B). Conentrtions of trns-resvertrol, ε-viniferin nd δ-viniferin, inresed in proportion to the hlorosis nd nerosis severity on symptomti leves (Figures 1A 1D nd 2A 2B), s oserved for trnsresvertrol t nd BCCH 89 stges, nd for ε-viniferin nd δ-viniferin t ll of the growth stges (Figures 1 nd 2). These findings gree with those for trns-resvertrol reported y Clzrno et l. (2013). Trns-pterostilene levels were lwys very low nd similr in ll types of leves nd t ll growth stges (Figure 2C 2D). Lortory ssy Trns-resvertrol or trns-pterostilene filed to improve the turgor of the helthy grpevine leves Vol. 56, No. 3, Deemer,

5 F. Clzrno et l. A 0,80 0,70 0,60 0,40 0,30 0,20 0,10 d d d 25/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe trns-δ viniferin Controguerr Vineyrd 25/08/2014 erries developing olours 23/09/2014 erries ripe for hrvest symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe B 1,40 1,20 1,00 0,80 0,60 0,40 0,20 26/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe trns-δ viniferin Giulinov Vineyrd 26/08/2014 erries developing olours 24/09/2014 erries ripe for hrvest symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe C trns-pterostilene Controguerr Vineyrd D trns-pterostilene Giulinov Vineyrd 0,40 0,30 0,20 0,40 0,30 0,20 0,10 0,10 25/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe 25/08/2014 erries developing olours 23/09/2014 erries ripe for hrvest symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe 26/07/2014 erries eginning to touh helthy vine leves symptomti vine leves with 5 % hlorosis symptomti vine leves with 40 % tiger-stripe 26/08/2014 erries developing olours 24/09/2014 erries ripe for hrvest symptomti vine leves symptomti vine leves with 20 % hlorosis symptomti vine leves with 65 % tiger-stripe Figure 2. Levels of trns-δ-viniferin (A, B) nd trns-pterostilene (C, D) in the leves of vines infeted with GLSD with vrying degrees of hlorosis or tiger-stripes nd in the leves of disesed/symptomti nd helthy vines t vrious growth stges in the 2014 growing seson in the Controguerr nd Giulinov vineyrds. Sttistil nlysis ws performed ording to Tukey s honest signifint differenes (HSD) test. Eh of the six grpevine tegory (4 sugroups of disesed/ symptomti, 1 group of disesed/symptomti nd 1 group of helthy) hd 6 replitions for eh tegory of leves in eh growth stge nd in eh vineyrd. Different letters represent signifint differenes t P=0.05. olleted t growth stge nd immersed in Ph ulture filtrte nd then in phytolexin suspensions (Figure 3). In the first prt of the lortory ssy, results showed sttistilly signifint differenes ompring the 3 hours Ph filtrte tretment (men lef withering sore = 1.39) with the 3 h sterile growth medium tretment (men lef withering sore = 5) (Q = , d.f. = 1, P<0.0001). The three groups of six leves eh treted with Ph ulture filtrte did not differ signifintly for lef turgor, with men lef withering sores rnging from 1.3 to 1.5 (Q = 0.2, d.f: = 2, P=0.8209). These leves immersed for 15 or 58 h in suspensions of trns-resvertrol, trns-pterostilene or distilled wter fter immersion in Ph filtrtes, showed similr levels of withering fter 15 or 58 h, ut these were signifintly different from the leves immersed in distilled wter fter immersion in sterile growth medium s ontrols (Q = , d.f: = 3, P< fter 15 h; nd Q = d.f: = 3, P<0.0001, fter 58 h) (Figure 3). At these times, ontrol leves showed no withering. At 106 h, ll leves dried up nd no signifint differenes in withering sores ould e deteted mong tretments, ording to Tukey s HSD test (Q = , d.f. = 3; P=0.9138) (Figure 3). Disussion Results of this study highlighted the differenes in phytolexin levels in GLSD-symptomti grpe- 498 Phytopthologi Mediterrne

6 Phytolexins in vine leves with GLSD symptoms Ph filtrtes (3 hours) then dipped in. Sterile growth medium (3 hours) then dipped in distilled wter trns-pterostilene trns-resvertrol distilled wter 15 h 58 h 106 h 15 h 58 h 106 h 15 h 58 h 106 h 15 h 58 h 106 h Figure 3. Effet on leves of the immersion of petioles in Ph filtrtes or sterile growth medium then dipped in suspensions of trns-resvertrol or trns-pterostilene in distilled wter, or in distilled wter lone. Results were expressed using n ritrry 1-5 sle of lef withering severity where 1 is the mximum withering nd 5 is the sene of withering (fresh green lef). The sttistil nlysis ws rried out t eh time of immersion using Tukey s honest signifint differene (HSD). vine leves, depending on different symptom severities, t different plnt growth stges. This study hs lso identified in vitro tivity of trns-resvertrol nd trns-pterostilene towrds the phytotoxi effets of Ph ulture filtrtes on grpevine leves. In the Controguerr vineyrd, the high trns-resvertrol levels in leves orresponding to the mximum symptom severity were onsistent with the high inidene nd severity of GLSD lef symptoms reorded in In Giulinov, the inidene nd severity of lef symptoms were less thn in Controguerr, ut not so low s to led to dereses in phytolexin levels, even though lower severity of lef symptoms re lwys ssoited with lower levels of phytolexins (Clzrno et l., 2016). A sustntil differene etween the levels of trns-resvertrol, trns-ε-viniferin, trns-δ-viniferin nd trns-pterostilene reorded in the present study nd those previously noted in the sme vineyrds (Clzrno et l., 2016) ws oserved. Thus, in the grpevines nlysed in this study, the dimeristion nd demethyltion of trns-resvertrol ws muh less; onsequently, mounts of viniferin, nd espeilly trnspterostilene, were muh less in 2014 thn in previous growing sesons (Clzrno et l., 2016). Amounts of phytolexins in leves t the 2014 growth stges exhiited the sme flututions s those seen in previous yers (Clzrno et l., 2008; 2013; 2016). These findings suggest tht the timeourse of phytolexin mounts my lso depend on vritions in vine physiology during the growing seson. The renewl of rohydrte reserves in the vine wood t the erries developing olour stge (Leon et l., 2008), nd the need to trnslote energy for erry ripening, ould explin the redued synthesis of the phytolexins t tht stge. Leves showing the sme extension of tiger-stripe symptoms synthesised different mounts of phytolexins in different yers. The mounts of the phytolexins in leves with the gretest severity of tigerstripe symptoms vried onsiderly etween growing sesons (Clzrno et l., 2016). For exmple, mounts of trns-pterostilene in 2014 ( mg kg -1 d wt) were onsiderly less thn those reorded in previous yers ( ; Clzrno et l., 2016). Similrly, trns-resvertrol mounts flututed from 20 to 50 in the yers exmined (Clzrno et l., 2016). These flututions proly indite tht environmentl ftors lso ffet the mounts of phytolexins produed in symptomti leves, i.e., the rinfll mount during the growing seson. These ftors ould inrese the inidene of GLSD symptoms (Mrhi et l., 2006), nd ould lso determine the mounts of phytolexins produed in grpevine leves. The ft tht levels of trns-resvertrol, ε-viniferin nd δ-viniferin inresed with inresing severity of GLSD symptoms on the lef, onfirmed tht ll these sustnes re lrgely synthesised s retion to lesions suffered y leves, similrly to ny ioti or ioti events tht dmge the leves (Kuć, 1995; Smith, 1996; Bvreso nd Fregoni, 2001). Toxi metolites produed y fungi in wood (Andolfi et l., 2011) nd trnsloted to the leves my trigger plnt responses tht inlude the formtion of neroti lesions on leves, s hypersensitive retion. These retions re usully followed y formtion of ntimiroil ompounds suh s stilene derivtives (Heth, 2000). In the green portions of symptomti leves, gene expression of phenyllnine mmoni lyse nd stilene synthse, involved in the Vol. 56, No. 3, Deemer,

7 F. Clzrno et l. trns-resvertrol synthesis pthwy, did not our. These enzymes re found only in the hloroti res of leves where symptoms hve lredy formed (Mgnin-Roert et l., 2011). Moreover, in symptomti grpevine leves efore the erries eginning to touh growth stge (when ny GLSD symptoms tht my pper lter re not yet visile), levels of trns-resvertrol nd ε-viniferin were low nd levels of δ-viniferin nd trns-pterostilene were undetetle (Clzrno et l., 2016). The mounts of ll these sustnes in symptomti/disesed leves then inresed t the erries eginning to touh stge, when GLSD symptoms egin to pper, s reported in other studies (Mgnin-Roert et l., 2011; Vltud et l., 2011; Lmert et l., 2013), nd levels of phytolexins were espeilly high in symptomti leves. All these results indite tht the phytolexins do not prevent lef symptoms to form until the erries eginning to touh growth stge (Clzrno et l., 2016). From tht stge onwrd, the differenes in the levels of the phytolexins etween helthy nd symptomti/disesed leves were so smll tht it is unlikely tht these phytolexins hve ny roles in the mehnisms tht inhiit symptom formtion. An lterntive hypothesis ould e tht symptoms sometimes do not pper in disesed vines during n entire growing seson is due to environmentl ftors, or to the prtiulr onditions of individul vines, or to intertions etween environmentl ftors nd the onditions of vines. Tht phytolexins hve no role in prevention of lef symptom development seems more likely sine oth trns-resvertrol nd trns-pterostilene filed to improve the turgor of the leves, when these leves hd previously een immersed in Ph ulture filtrtes, ny more thn did immersion in distilled wter. Nevertheless, it is yet to e determined if inresed mounts of the phytolexins indued in symptomti/disesed vine leves efore lef symptom pperne (i.e. prior to the erries eginning to touh growth stge) ould ontriute vines remining symptomti during prtiulr growing sesons. Repeted pplitions of lef fertilising mix ontining lium, mgnesium nd extrts of lge during vegettive growth strongly redued the inidene of GLSD symptoms, nd used high phytolexins levels t the fruit-set nd the pe-sized erries stges (Clzrno et l., 2014; 2017). In onlusion, this study hs onfirmed tht the growth stge of grpevines plys n importnt role in determining the mount of phytolexins ourring in leves. The study lso reveled tht these phytolexins re not involved, under nturl onditions, in the mehnisms tht inhiit development of GLSD lef symptoms. Literture ited Aou-Mnsour E., E. Couhè nd R. Thi, 2004: Do fungl nphthlenones hve role in the development of es symptoms? Phytopthologi Mediterrne 43, Andolfi A., L. Mugni, J. Luque, G. Surio, A. Cimmino nd A. Evidente, Phytotoxins produed y fungi ssoited with grpevine trunk diseses. Toxins, 3 (12), Bvreso L. nd C. Fregoni, 2001: Physiologil role nd moleulr spets of grpevine stileni ompounds. In: Moleulr Biology nd Biotenology of the grpevine (K.A. Rouelkis-Angelkis, ed.), Kluwer Ad. Pul., Dordreht, The Netherlnds, Bertsh C., M. Rmírez-Suero, M. Mgnin-Roert, P. Lrignon, J. Chong, E. Aou-Mnsour, A. Spgnolo, C. Clèment nd F. Fontine, Grpevine trunk diseses: omplex nd still poorly understood. Plnt Pthology 62(2), Clzrno F., A. Cihelli nd M. Odordi, Preliminry evlution of vritions in omposition indued y es on v. Treino D Aruzzo grpes nd wines. Phytopthologi Mediterrne 40, Supplement, S443 S448. Clzrno F., L. Seghetti, M. Del Crlo nd A. Cihelli, Effet of es on the qulity of erries, musts nd wines. Phytopthologi Mediterrne 43, Clzrno F. nd S. Di Mro, Wood disolortion nd dey in grpevines with es proper nd their reltionship with folir symptoms. Phytopthologi Mediterrne, 46, Clzrno F. nd S. Di Mro, Approfondimenti sull dinmi di olonizzzione e sull mnifestzione dei sintomi fogliri nell es propri. Miologi Itlin, 2, Clzrno F., V. D Agostino nd M. Del Crlo, Trns-resvertrol extrtion from grpevine: pplition to erries nd leves from vines ffeted y es proper. Anlytil Letters 41, Clzrno F., V. D Agostino, F. Osti nd S. Di Mro, Levels of trns-resvertrol in leves of vines ffeted with es. In: Protetion nd Prodution in Vitiulture, IOBC/WPRS Bulletin Vol. 85, Clzrno F., S. Di Mro, V. D Agostino, S. Shiff nd L. Mugni, Grpevine lef stripe disese (es omplex) re redued y nutrients nd seweed mixture. Phytopthologi Mediterrne 53(3), Clzrno F., V. D Agostino, A. Pepe, F. Osti, F. Dell Pelle, M. De Rosso, R. Flmini nd S. Di Mro, Ptterns of phytolexins in the grpevine lef stripe disese (es omplex)/grpevine pthosystem. Phytopthologi Mediterrne 55(3), Clzrno F., F. Osti, V. D Agostino, A. Pepe, nd S. Di Mro, Mixture of lium, mgnesium nd seweed ffets lef phytolexin ontents nd grpe ripening on vines 500 Phytopthologi Mediterrne

8 Phytolexins in vine leves with GLSD symptoms with grpevine lef stripe disese. Phytopthologi Mediterrne 56, Di Mro S., F. Osti, F. Clzrno, R. Roerti, A. Veronesi, nd C. Amlfitno, Effets of grpevine pplitions of fosetyl-luminium formultions for downy mildew ontrol on es nd ssoited fungi. Phytopthologi Mediterrne 50, S285 S299. Di Mro S., F. Osti, nd L. Mugni, First studies on the potentil of opper formultion for the ontrol of lef stripe disese within es omplex in grpevine. Phytopthologi Mediterrne 50, S Evidente A., L. Sprpno, A. Andolfi nd G. Bruno, Two nphthlenone pentketides from liquid ultures of Pheoremonium leophilum, fungus ssoited with es of grpevine. Phytopthologi Mediterrne 39, Feliino A.J., A. Esklen, nd W.D. Guler, Differentil suseptiility of three grpevine ultivrs to Pheomoniell hlmydospor in Cliforni. Phytopthologi Mediterrne 43, Fisher M., A new wood-deying sidiomyete speies ssoited with es of grpevine: Fomitipori mediterrne (Hymenohetles). Myologil Progress 1(3), Grmje D., L. Mostert, J.Z. Groenewld nd P.W. Crous, Pheoremonium: from es disese to pheohyphomyosis. Fungl iology, 119(9), Heth M.C., Hypersensitive response-relted deth. Plnt Moleulr Biology 44, Jendet P., A.C. Douillet-Breuil, R. Bressis, S. Deord, M. Spghi, nd M. Adrin, Phytolexins from the Vitee: Biosynthesis, phytolexin gene expression in trnsgeni plnts, ntifungl tivity, nd metolism. Journl of Agriulturl nd Food Chemistry 50, Kuć J., Phytolexins, stress metolism, nd disese resistne in plnts. Annul Review of Phytopthology 33, Lmert C., I.L.K. Khiook, S. Lus, N. Télef-Mioleu, J.M. Mérillon nd S. Cluzet, A fster nd stronger defense response: one of the key elements in grpevine explining its lower suseptiility to es? Phytopthology, 103(10), Leon G., G. Wojnrowiez, B. Holzpfel, F. Fontine, N. Villnt-Gveu, nd C. Clément, Sugrs nd flowering in the grpevine (Vitis vinifer L.). Journl of Experimentl Botny, 59(10), Lorenz D.H., K.W. Eihhorn, H. Bleiholder, R. Close, U. Meier nd E. Weer, Phenologil growth stges of the grpevine (Vitis vinifer L. ssp. vinifer). Enoding nd desription of the phenologil stges of the grpevine ording to the extended BBCH sheme. Austrlin Journl of Grpe nd Wine Reserh 1(2), Mgnin-Roert M., P. Letousey, A. Spgnolo, F. Renoelin, L. Jquens, L. Merier, C. Clément nd F. Fontine, Lef stripe form of es indues ltertion of photosynthesis nd defene retions in presymptomti leves. Funtionl Plnt Biology 38, Mrhi G., I.G. Psoe nd J. Edwrds, Young es in Austrli. Phytopthologi Mediterrne 40 (3), Mrhi G., F. Peduto, L. Mugni, S. Di Mro, F. Clzrno nd G. Surio, Some oservtions on the reltionship on mnifest nd hidden es to rinfll. Phytopthologi Mediterrne 45, Mrtos S., A. Andolfi, J. Luque, L. Mugni, G. Surio nd A. Evidente, Prodution of phytotoxi metolites y five speies of Botryospheriee using deline on grpevines, with speil interest in the speies Neofusioum luteum nd N. prvum. Europen Journl of Plnt Pthology 121, Mugni L., A. Grniti nd G. Surio, Es (lk mesles) nd rown wood streking: two old nd elusive diseses of grpevines. Plnt Disese 83, 5, Osti F. nd S. Di Mro, Iron-dependent, non-enzymti proesses promoted y Pheomoniell hlmydospor nd Pheoremonium leophilum, gents of es in grpevine. Physiologil nd Moleulr Plnt Pthology 74, Smith C.J., Tnsley review No. 86. Aumultion of phytolexins: defene mehnism nd stimulus response system. New Phytologist 132, Sprpno L., G. Bruno nd A. Grniti, Esopolisridi fitotossii sono prodotti in oltur d due speie di Pheoremonium ssoite l omplesso del ml dell es dell vite. Petri 8, Sprpno L., G. Bruno, C. Cirone nd A. Grniti, Infetion of grpevines y some fungi ssoited with es. II. Intertion mong Pheoremonium hlmydosporum, P. leophilum nd Fomitipori puntt. Phytopthologi Mediterrne 39, Sprpno L., G. Bruno nd A. Grniti, Three-yer oservtion of grpevines ross-inoulted with es-ssoited fungi. Phytopthologi Mediterrne 40, Surio G., Towrds redefinition of the diseses within the es omplex. Phytopthologi Mediterrne 48, Surio G., L. Mugni nd G. Mrhi, Older nd more reent oservtions on es, ritil overview. Phytopthologi Mediterrne 45, S68 S86. Thi R., A. Fkyert, C. Polirt nd G. Duin, Phytotoxins from fungi of es of grpevine. Phytopthologi Mediterrne 39, Vltud C., F. Thiult, P. Lrignon, C. Bersth, P. Fleurt-Lessrd nd A. Bourouloux, Systemi dmge in lef metolism used y es infetion in grpevines. Austrlin Journl of Grpe nd Wine Reserh, 17(1), ,. Aepted for pulition: Deemer 15, 2017 Vol. 56, No. 3, Deemer,