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Phytopathologia Mediterranea (2017) 56, 3, 494−501 DOI: 10.14601/Phytopathol_Mediterr-22055

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

1 2 1 1 1 Francesco CALZARANO , Fabio OSTI , Vincenzo D’AGOSTINO , Alessia PEPE , Flavio DELLA PELLE , Mirko DE 3 3 2 ROSSO , Riccardo FLAMINI and Stefano DI MARCO

1 Università degli Studi di , Facoltà di BioScienze e Tecnologie Agro-Alimentari ed Ambientali, Via Renato Balzarini, 1, 64100 Teramo, 2 CNR, IBIMET, Via Gobetti 101, 40129 Bologna, Italy 3 CREA-VIT, Viale XXVIII Aprile 26, 31015 Conegliano (TV), Italy

Summary. Grapevine leaf stripe disease (GLSD) is one of the most common diseases in the esca complex. Losses in grape yields and quality caused by GLSD are correlated with the severity of the leaf symptoms. The time course of phytoalexin levels was examined in vine leaves of two vineyards, in leaves of healthy, asymptomatic/diseased and symptomatic vines. Symptomatic leaves were further divided into four categories according to symptom severity: 1, 5% chlorosis; 2, 20% chlorosis; 3, 40% leaf surface covered with tiger stripes; or 4, 65% leaf surface covered with tiger stripes. Leaves were sampled at three growth stages: ‘berries beginning to touch’; ‘berries developing colour’; and ‘berries ripe for harvesting’. The phytoalexins trans-resveratrol, trans-ε-viniferin; trans-δ-viniferin; and trans- pterostilbene were detected and assayed. Patterns of each of the phytoalexins were similar at each growth stage to those found earlier for trans-resveratrol, with an increased phytoalexin levels with increasing leaf symptom sever- ity on the leaf blade, especially at the stages ‘berries beginning to touch’ and ‘berries ripe for harvest’. A laboratory test was also carried out. Petioles of healthy grapevine leaves were immersed in culture filtrates of Phaeomoniella chlamydospora and then dipped in solutions of trans-resveratrol or trans-pterostilbene to assess the effects of these substances on the leaf blades. Adding these phytoalexins did not influence the effects of P. chlamydospora. These results indicated that phytoalexins increased in the leaves after GLSD symptoms appeared, and not before host response (phytoalexin synthesis) to control symptom development.

Key words: phytoalexins, GLSD, leaf symptoms.

Introduction ‘esca proper’, but experimental findings have made it clear that the three fungi mainly involved in esca Grapevine leaf stripe disease (GLSD) is a vine proper, Phaeomoniella chlamydospora (Pch) (Crous and wood disease, and one of the Esca complex of dis- Gams, 2000), Phaeoacremonium minimum (syn. P. aleo- eases. GLSD is widespread in all vine-growing ar- philum, Gramaje et al., 2015) and F. mediterranea, can eas, but despite considerable experimental advances each colonise vines without any of the others, and made in recent years (Di Marco et al., 2011a; 2011b), it in no particular chronological order (Sparapano et al. is still difficult to control. 2000). Esca proper has now been confirmed to de- Initially, GLSD was associated with white rot rive from at least two distinct diseases; a trachaeo- caused by the basidiomycete Fomitiporia mediterra- mycosis, caused by P. chlamydospora, P. minimum and nea (Fischer, 2002; Surico et al., 2006) and was named probably some other species of Phaeoacremonium; and a white wood rot, caused by F. mediterranea. It was subsequently found that white rot caused by Corresponding author: F. Calzarano F. mediterranea was not necessary for the expression E-mail: [email protected] of leaf symptoms attributed to P. chlamydospora and

494 ISSN (print): 0031-9465 www.fupress.com/pm ISSN (online): 1593-2095 © Firenze University Press

© 2017 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Phytoalexins in vine leaves with GLSD symptoms

P. minimum (Calzarano and Di Marco, 2007; 2008). al., 2013). The availability of a new analytical tech- Consequently, the term GLSD was proposed to des- nique for the extraction and identification of trans- ignate specifically the disease that causes the typical resveratrol and other derived phytoalexins, namely leaf symptoms on infected grapevines (Surico, 2009). viniferins and trans-pterostilbene, has made it possi- The fungi mainly associated with GLSD are P. ble to better assess the levels of trans-resveratrol, and chlamydospora and P. minimum. These fungi colo- to measure the amounts of the other stilbene phyto- nise the grapevine wood and produce brown necro- alexins in healthy and diseased leaves (Calzarano et sis and dark streaking of the vessels (Marchi et al., al. 2016). 2001; Calzarano and Di Marco, 2007; Surico, 2009). The aim of the present study was to advance un- The toxic metabolites produced by these fungi in derstanding of the roles of these phytoalexins in the colonised portions of vines are translocated to the formation of leaf symptoms in the crowns of grape- crowns by the sap stream, and the GLSD symptoms vines infected with GLSD. In particular, we deter- develop on leaves (Sparapano et al., 1998; Mugnai et mined the levels of various phytoalexins in leaves al., 1999; Evidente et al., 2000; Tabacchi et al., 2000; with different GLSD symptom severity at different Surico, 2009). These symptoms are: leaf chlorosis vine growth stages. The laboratory test with the Pch and necrosis (tiger stripes), purple spots, and more culture filtrates was repeated measuring the effects or less extensive withering of the grape bunches that of trans-resveratrol and trans-pterostilbene. result in losses in yield and quality proportional to the severity of the leaf symptoms (Calzarano et al., 2001; 2004; Bertsch et al., 2013). Materials and methods Despite the advances of knowledge on these dis- Foliar symptom survey, sampling of leaves and eases, the idea that the leaf symptoms of GLSD are phytoalexin extraction due to translocated toxic metabolites is still only a hypothesis. Tests undertaken with culture filtrates The study was carried out in 2014 in two 36-year- or with the constitutent fungal metabolites involved old vineyards, planted with the cv. have produced uncertain results (Sparapano et al., d’ on 420A rootstock, managed with IPM 1998, 2000; Abou-Mansour et al., 2004). Only Spara- strategies, and located at Controguerra (Geneva pano et al. (2001) and Feliciano et al. (2004) were able Double Curtain system) and (Tendone to induce leaf symptoms, but these were different system) in the , Abruzzo, Italy. from typical GLSD symptoms. The well-known phe- Both vineyards were on clay-calcareous soils. The cv. nomenon of the partial remission or total disappear- Trebbiano d’Abruzzo is usually severely affected by ance of the foliar symptoms (but not of the disease GLSD. itself) on certain plants in certain years suggests that The sanitary status of each vine (healthy or GLSD- other physiological or environmental factors may affected) in both vineyards was known because the also be involved in symptom expression. These may vines had been monitored for GLSD symptoms since include rainfall early in the growing season (Marchi 1994. In the 2014 test year, the vines could be posi- et al., 2006; Calzarano and Di Marco, 2007; 2008). tively identified as either healthy, diseased/asymp- More recently, variations in the levels of the phy- tomatic or diseased/symptomatic. toalexin trans-resveratrol, the main phytoalexin in Incidence and severity of GLSD foliar symptoms grapevine (Jeandet et al., 2002), have been examined were evaluated in 2014 on 20 September in Contro- in leaves of vines infected by GLSD in some vine- guerra and 22 September in Giulianova, during the yards at different growth stages (Calzarano et al., maximum of symptom expression, recorded and 2008). In a following study, trans-resveratrol levels analyzed using the methods described in Calzarano were measured in leaves differing for GLSD symp- et al. (2014). toms severity (Calzarano et al., 2013). Furthermore, Leaf samples were collected from each of the laboratory tests with healthy vine leaves dipped by three groups of vines, designated: H (healthy), AS petiole, first in P. chlamydospora culture filtrate and (diseased/asymptomatic) or T (diseased/sympto- then in a trans-resveratrol suspension, showed that matic). Leaves from T vines were arbitrarily divided trans-resveratrol did not influence the effects of Pch into four subgroups: T1, exhibiting 5% chlorosis; T2, filtrates on the withering of the leaves (Calzarano et 20% chlorosis; T3, 40% leaf surface covered with ti-

Vol. 56, No. 3, December, 2017 495 F. Calzarano et al. ger-stripes (chlorosis + necrosis); or T4, 65% leaf sur- stage, from positions opposite to grape bunches to face covered with tiger-stripes (chlorosis + necrosis). minimise natural variations along the shoots. Peti- This gave a total of six categories of leaves. Leaves oles of leaves from healthy vines were individually were sampled at three growth stages, according to immersed in 50 mL of Pch culture filtrate (for a total BBCH-scale for grapevine (Lorenz et al., 1995): i, ‘ber- of 18 leaves) or in sterile liquid growth medium as ries beginning to touch’ (BBCH 77); ii, ‘berries de- controls (additional 18 leaves) for 3 h, and were then veloping colour’ (BBCH 83); or iii, ‘berries ripe for evaluated using an arbitrary leaf withering severity harvest’ (BBCH 89). From each of the three groups scale; where 1 was maximum withering and 5 was of vines, six vines were selected, and from each of H absence of withering (fresh green leaf). Leaves from and AS vines eight leaves, and from each of T vines Pch culture filtrate treatment were then randomly 32 leaves (eight leaves for each symptom subgroup), subdivided into three groups of six leaves each. at each growth stage, were sampled from the central Leaves in these groups were individually immersed portion of a vine shoot, located in a position oppo- in 50 mL of distilled water or in distilled water con- site a grape bunch, to minimise natural variations taining 80 mg L-1 of trans-resveratrol, or 15 mg L-1 of between leaves due to leaf position along each shoot. trans-pterostilbene, corresponding to the maximum The leaves sampled at the three growth stages were concentrations found previously in GLSD-affected always taken from the same vines in each groups. Trebbiano d’Abruzzo grapevine leaves in both vine- Each sample then consisted of eight leaves. The 8 yards (Calzarano et al., 2001; 2004; 2008; 2013; 2014). leaves of each sample were combined and treated The control was a group of six leaves immersed in as one replicate, from which phytoalexins were ex- sterile growth medium and in distilled water. All tracted and identified according to the procedure de- leaves were evaluated for leaf withering at 15 h, 58 h, scribed in Calzarano et al. (2016). or 106 h of immersion.

Laboratory assay Statistical analyses

A laboratory assay was carried out to determine Data of the concentrations of each phytoalexin in the effect of the interactions among phytoalexins - the different GLSD categories of leaves were- com grapevine leaf - Pch filtrate. To obtain Pch filtrates, pared separately at each growth stage using Tukey’s an adequate volume of Czapeck–Dox medium, honest significant difference (HSD) test atP = 0.05. In modified by adding 0.5% yeast and 0.5% maltex- the laboratory test for each time of immersion, treat- tract (pH 6.8), was autoclaved, and 150 mL was ments were compared and data were statistically added to each of 1 L capacity Roux flasks. Half of analyzed using Tukey’s honest significant difference the prepared flasks were inoculated with of agar/ (HSD). Statistical analyses were carried out using mycelium obtained from the edges of 2-week-old SAS version 9.3 (SAS Institute Inc.). Pch colonies grown on potato dextrose agar. The re- sulting liquid cultures were incubated in stationary phase at 25°C in darkness. After 28 d, mycelium was Results removed by filtration through Whatman No. 4 filter Phytoalexins determinations paper (Martos et al., 2008; Osti and Di Marco, 2010). Filtrates form the liquid cultures were pooled, mixed In 2014, the mean amounts of trans-resveratrol and observed with a light microscope to ensure that levels in T4 leaves, recorded at the BBCH 77 vine fungal structures had been excluded by the filtration. growth stage, were 34.67 mg kg-1 d wt in Contro- The remaining half of the flasks were subjected to the guerra and 47.99 mg kg-1 d wt in Giulianova. These same procedure except that they were not inoculat- levels were among the greatest amounts of trans- ed, to provide sterile liquid growth medium. resveratrol recorded in those vineyards (Calzarano The petioles of healthy grapevine leaves were et al., 2016). These high levels were consistent with immersed in Pch culture filtrate, and then in solu- the high incidence (30.4%) and severity (11.8%) of tions of trans-resveratrol or trans-pterostilbene. The GLSD leaf symptoms recorded in the Controguerra leaves were taken from the middle portions of the vineyard, and 9.2% incidence and 5.8 % severity re- primary grapevine shoots at the BBCH 77 growth corded in the Giulianova vineyard.

496 Phytopathologia Mediterranea Phytoalexins in vine leaves with GLSD symptoms

A trans-resveratrol B trans-resveratrol Controguerra Vineyard Giulianova Vineyard 45,00 50,00 a 40,00 45,00 a 35,00 40,00 35,00 30,00 b d wt d wt

1 30,00 1 - - 25,00 bc 25,00 20,00 b c 20,00 mg kg mg kg a c 15,00 d 15,00 ab ab 10,00 a 10,00 cd abc c bc 5,00 5,00 d 0,00 0,00 25/07/2014 25/08/2014 23/09/2014 26/07/2014 26/08/2014 24/09/2014 berries beginning to touch berries developing colours berries ripe for harvest berries beginning to touch berries developing colours berries ripe for harvest BBCH 77 BBCH 83 BBCH 89 BBCH 77 BBCH 83 BBCH 89

healthy vine leaves asymptomatic vine leaves healthy vine leaves asymptomatic vine leaves symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe

C trans-ε viniferin D trans-ε viniferin Controguerra Vineyard Giulianova Vineyard 4,50 4,50 a 4,00 a a 4,00 3,50 3,50 3,00 3,00 d wt d wt 1 1 - - 2,50 2,50 2,00 b 2,00 b mg kg mg kg 1,50 1,50 1,00 1,00 bc 0,50 bc 0,50 c c 0,00 0,00 25/07/2014 25/08/2014 23/09/2014 26/07/2014 26/08/2014 24/09/2014 berries beginning to touch berries developing colours berries ripe for harvest berries beginning to touch berries developing colours berries ripe for harvest BBCH 77 BBCH 83 BBCH 89 BBCH 77 BBCH 83 BBCH 89 healthy vine leaves asymptomatic vine leaves healthy vine leaves asymptomatic vine leaves symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe symptomatic vine leaves with 40 % tiger -stripe symptomatic vine leaves with 65 % tiger -stripe

Figure 1. Levels of trans-resveratrol (A, B) and trans-ε-viniferin (C, D) in the leaves of vines infected with GLSD with varying degrees of chlorosis or tiger-stripes and in Figurethe leaves 1. Levelsof diseased/asymptomatic of trans-resveratroland healthy (A,vines B) atandvarious transgrowth-ε-viniferinstages in the (C,2014 D)growing in theseason leavesin the of Controguerravines infectedand Giulianova with GLSDvineyards with. Statistical vary- inganalysis degreeswas performed of chlorosisaccording or totiger-stripesTukey’s honest andsignificant in thedifferences leaves(HSD) of diseased/asymptomatictest. Each of the six grapevine category and healthy(4 subgroups vinesof diseased/symptomatic, at various growth1 stagesgroup ofindiseased/asymptomatic the 2014 growingand season1 group ofinhealthy) the Controguerrahad 6 replications andfor each Giulianovacategory of leavesvineyards.in each growth Statisticalstage and analysisin each vineyard was performed. Different letters ac- represent significant differences at P = 0.05. cording to Tukey’s honest significant differences (HSD) test. Each of the six grapevine category (4 subgroups of diseased/ symptomatic, 1 group of diseased/asymptomatic and 1 group of healthy) had 6 replications for each category of leaves in each growth stage and in each vineyard. Different letters represent significant differences at P=0.05.

In both vineyards, trans-resveratrol levels were and δ-viniferin, increased in proportion to the chlo- particularly high in symptomatic leaves at the BBCH rosis and necrosis severity on symptomatic leaves 77 and BCCH 89 growth stages, compared with (Figures 1A–1D and 2A–2B), as observed for trans- healthy and asymptomatic/diseased leaves. At the resveratrol at BBCH 77 and BCCH 89 stages, and for BBCH 77 stage, trans-resveratrol levels declined ε-viniferin and δ-viniferin at all of the growth stages showing similar values for the three groups of vines (Figures 1 and 2). These findings agree with those for (Figure 1A–1B). Levels of ε-viniferin and δ-viniferin trans-resveratrol reported by Calzarano et al. (2013). in symptomatic leaves exhibited similar patterns over Trans-pterostilbene levels were always very low and the growth stages, as compared with healthy and dis- similar in all types of leaves and at all growth stages eased/asymptomatic levels, except that they were (Figure 2C–2D). higher than in healthy and diseased/asymptomatic leaves, also at the ‘berries developing colour’ stage. Laboratory assay This was despite the generally lower amounts of the phytoalexins at this stage (Figures 1C–1D and 2A–2B). Trans-resveratrol or trans-pterostilbene failed to Concentrations of trans-resveratrol, ε-viniferin improve the turgor of the healthy grapevine leaves

Vol. 56, No. 3, December, 2017 497 F. Calzarano et al.

A trans-δ viniferin B trans-δ viniferin Controguerra Vineyard Giulianova Vineyard 1,40 0,80 a 0,70 a 1,20 0,60 ab 1,00 d wt 0,50 d wt 1 1 ab - - 0,80 0,40 bc 0,60 mg kg 0,30 mg kg bc 0,20 cd 0,40 0,10 0,20 d d b c b 0,00 0,00 25/07/2014 25/08/2014 23/09/2014 26/07/2014 26/08/2014 24/09/2014 berries beginning to touch berries developing colours berries ripe for harvest berries beginning to touch berries developing colours berries ripe for harvest BBCH 77 BBCH 83 BBCH 89 BBCH 77 BBCH 83 BBCH 89 healthy vine leaves asymptomatic vine leaves healthy vine leaves asymptomatic vine leaves symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe

C trans-pterostilbene D trans-pterostilbene Controguerra Vineyard Giulianova Vineyard 0,50 0,50

0,40 0,40 a a a 0,30 d wt 0,30 d wt

1 a - 1 - 0,20 0,20 mg kg mg kg 0,10 0,10

0,00 0,00 25/07/2014 25/08/2014 23/09/2014 26/07/2014 26/08/2014 24/09/2014 berries beginning to touch berries developing colours berries ripe for harvest berries beginning to touch berries developing colours berries ripe for harvest BBCH 77 BBCH 83 BBCH 89 BBCH 77 BBCH 83 BBCH 89 healthy vine leaves asymptomatic vine leaves healthy vine leaves asymptomatic vine leaves symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 5 % chlorosis symptomatic vine leaves with 20 % chlorosis symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe symptomatic vine leaves with 40 % tiger-stripe symptomatic vine leaves with 65 % tiger-stripe

Figure 2. Levels of trans-δ-viniferin (A, B) and trans-pterostilbene (C, D) in the leaves of vines infected with GLSD with varying degrees of chlorosis or tiger-stripes and in Figurethe leaves 2. Levelsof diseased/asymptomatic of trans-δ-viniferinand healthy (A, B)vines andat various trans-pterostilbenegrowth stages in the (C,2014 D)growing in theseason leavesin the of Controguerravines infectedand Giulianova with GLSDvineyards with. Statistical var- yinganalysis degreeswas performed of chlorosisaccording orto tiger-stripesTukey’s honest significant and in differencesthe leaves(HSD) of testdiseased/asymptomatic. Each of the six grapevine category and(4 healthysubgroups ofvinesdiseased/symptomatic, at various growth1 group of diseased/asymptomatic and 1 group of healthy) had 6 replications for each category of leaves in each growth stage and in each vineyard. Different letters represent stagessignificant in thedifferences 2014 atgrowingP = 0.05 season in the Controguerra and Giulianova vineyards. Statistical analysis was performed ac- cording to Tukey’s honest significant differences (HSD) test. Each of the six grapevine category (4 subgroups of diseased/ symptomatic, 1 group of diseased/asymptomatic and 1 group of healthy) had 6 replications for each category of leaves in each growth stage and in each vineyard. Different letters represent significant differences at P=0.05.

collected at BBCH 77 growth stage and immersed of withering after 15 or 58 h, but these were signifi- in Pch culture filtrate and then in phytoalexin sus- cantly different from the leaves immersed in distilled pensions (Figure 3). In the first part of the laboratory water after immersion in sterile growth medium as assay, results showed statistically significant differ- controls (Q =44.6825, d.f: = 3, P<0.0001 after 15 h; ences comparing the 3 hours Pch filtrate treatment and Q = 40.8333 d.f: = 3, P<0.0001, after 58 h) (Figure (mean leaf withering score = 1.39) with the 3 h ster- 3). At these times, control leaves showed no wither- ile growth medium treatment (mean leaf withering ing. At 106 h, all leaves dried up and no significant score = 5) (Q = 229.8043, d.f. = 1, P<0.0001). differences in withering scores could be detected The three groups of six leaves each treated with among treatments, according to Tukey’s HSD test (Q Pch culture filtrate did not differ significantly for leaf = 0.1724, d.f. = 3; P=0.9138) (Figure 3). turgor, with mean leaf withering scores ranging from 1.3 to 1.5 (Q = 0.2, d.f: = 2, P=0.8209). These leaves Discussion immersed for 15 or 58 h in suspensions of trans-res- veratrol, trans-pterostilbene or distilled water after Results of this study highlighted the differences immersion in Pch filtrates, showed similar levels in phytoalexin levels in GLSD-symptomatic grape-

498 Phytopathologia Mediterranea Phytoalexins in vine leaves with GLSD symptoms

15 h a ethylation of trans-resveratrol was much less; conse-

58 h a quently, amounts of viniferin, and especially trans- . pterostilbene, were much less in 2014 than in previ- 106 h a Sterile growth distilled water then dipped in ous growing seasons (Calzarano et al., 2016). medium (3 hours) Amounts of phytoalexins in leaves at the 2014 15 h b growth stages exhibited the same fluctuations as 58 h b those seen in previous years (Calzarano et al., 2008;

distilled water 106 h a 2013; 2016). These findings suggest that the time- course of phytoalexin amounts may also depend on 15 h b variations in vine physiology during the growing resveratrol - 58 h b season. The renewal of carbohydrate reserves in the

trans 106 h a vine wood at the ‘berries developing colour’ stage (Lebon et al., 2008), and the need to translocate en-

15 h b ergy for berry ripening, could explain the reduced Pch filtrates (3 hours) then dipped in synthesis of the phytoalexins at that stage.

pterostilbene 58 h b - Leaves showing the same extension of tiger-stripe

trans 106 h a symptoms synthesised different amounts of phyto- 0 1 2 3 4 5 alexins in different years. The amounts of the phyto- Figure 3. Effect on leaves of the immersion of petioles in Pch filtrates or sterile growth medium then dipped in solutionsalexinsof trans in- leaves with the greatest severity of tiger- resveratrol or trans-pterostilbeneFigurein 3.distilled Effectwater, oronin leavesdistilled water of thealone immersion. Results were expressed of petiolesusing an arbitraryin 1-5 scale of leaf withering severity where Pch1 is the filtratesmaximum withering or sterileand 5 is the absencegrowthof withering medium(fresh green thenleaf) . dippedThe statistical in stripeanalysis symptomswas varied considerably between grow- carried out at each time of immersion using Tukey’s honest significant difference (HSD). suspensions of trans-resveratrol or trans-pterostilbene in ing seasons (Calzarano et al., 2016). For example, distilled water, or in distilled water alone. Results were ex- amounts of trans-pterostilbene in 2014 (0.25–0.35 mg -1 pressed using an arbitrary 1-5 scale of leaf withering sever- kg d wt) were considerably less than those recorded ity where 1 is the maximum withering and 5 is the absence in previous years (2.0–5.5 mg kg-1 d wt; Calzarano et of withering (fresh green leaf). The statistical analysis was al., 2016). Similarly, trans-resveratrol amounts fluctu- carried out at each time of immersion using Tukey’s honest ated from 20 to 50 mg kg-1 d wt in the years examined significant difference (HSD). (Calzarano et al., 2016). These fluctuations probably indicate that environmental factors also affect the amounts of phytoalexins produced in symptomatic vine leaves, depending on different symptom severi- leaves, i.e., the rainfall amount during the growing ties, at different plant growth stages. This study has season. These factors could increase the incidence of also identified in vitro activity of trans-resveratrol GLSD symptoms (Marchi et al., 2006), and could also and trans-pterostilbene towards the phytotoxic ef- determine the amounts of phytoalexins produced in fects of Pch culture filtrates on grapevine leaves. grapevine leaves. In the Controguerra vineyard, the high trans-res- The fact that levels of trans-resveratrol, ε-viniferin veratrol levels in leaves corresponding to the maxi- and δ-viniferin increased with increasing severity mum symptom severity were consistent with the of GLSD symptoms on the leaf, confirmed that all high incidence and severity of GLSD leaf symptoms these substances are largely synthesised as a reaction recorded in 2014. In Giulianova, the incidence and to lesions suffered by leaves, similarly to any biotic severity of leaf symptoms were less than in Contro- or abiotic events that damage the leaves (Kuć, 1995; guerra, but not so low as to lead to decreases in phy- Smith, 1996; Bavaresco and Fregoni, 2001). Toxic toalexin levels, even though lower severity of leaf metabolites produced by fungi in wood (Andolfi et symptoms are always associated with lower levels al., 2011) and translocated to the leaves may trigger of phytoalexins (Calzarano et al., 2016). A substan- plant responses that include the formation of ne- tial difference between the levels oftrans- resveratrol, crotic lesions on leaves, as a hypersensitive reaction. trans-ε-viniferin, trans-δ-viniferin and trans-pter- These reactions are usually followed by formation ostilbene recorded in the present study and those of antimicrobial compounds such as stilbene deriva- previously noted in the same vineyards (Calzarano tives (Heath, 2000). In the green portions of sympto- et al., 2016) was observed. Thus, in the grapevines matic leaves, gene expression of phenylalanine am- analysed in this study, the dimerisation and dem- monia lyase and stilbene synthase, involved in the

Vol. 56, No. 3, December, 2017 499 F. Calzarano et al. trans-resveratrol synthesis pathway, did not occur. in determining the amount of phytoalexins occur- These enzymes are found only in the chlorotic ar- ring in leaves. The study also revealed that these eas of leaves where symptoms have already formed phytoalexins are not involved, under natural condi- (Magnin-Robert et al., 2011). Moreover, in asympto- tions, in the mechanisms that inhibit development of matic grapevine leaves before the ‘berries beginning GLSD leaf symptoms. to touch’ growth stage (when any GLSD symptoms that may appear later are not yet visible), levels of trans-resveratrol and ε-viniferin were low and levels Literature cited of δ-viniferin and trans-pterostilbene were undetect- Abou-Mansour E., E. Couchè and R. Tabacchi, 2004: Do fun- able (Calzarano et al., 2016). The amounts of all these gal naphthalenones have a role in the development of esca substances in asymptomatic/diseased leaves then symptoms? Phytopathologia Mediterranea 43, 75–82. increased at the ‘berries beginning to touch’ stage, Andolfi A., L. Mugnai, J. Luque, G. Surico, A. Cimmino and A. Evidente, 2011. Phytotoxins produced by fungi associated when GLSD symptoms begin to appear, as reported with grapevine trunk diseases. Toxins, 3 (12), 1569–1605. in other studies (Magnin-Robert et al., 2011; Valtaud Bavaresco L. and C. Fregoni, 2001: Physiological role and mo- et al., 2011; Lambert et al., 2013), and levels of phyto- lecular aspects of grapevine stilbenic compounds. In: Mo- alexins were especially high in symptomatic leaves. lecular Biology and Biotecnology of the grapevine (K.A. Roube- All these results indicate that the phytoalexins do lakis-Angelakis, ed.), Kluwer Acad. Publ., Dordrecht, The not prevent leaf symptoms to form until the ‘ber- Netherlands, 153–182. Bertsch C., M. Ramírez-Suero, M. Magnin-Robert, P. Larignon, ries beginning to touch’ growth stage (Calzarano et J. Chong, E. Abou-Mansour, A. Spagnolo, C. Clèment and al., 2016). From that stage onward, the differences in F. Fontaine, 2013. Grapevine trunk diseases: complex and the levels of the phytoalexins between healthy and still poorly understood. Plant Pathology 62(2), 243-265. asymptomatic/diseased leaves were so small that Calzarano F., A. Cichelli and M. Odoardi, 2001. Preliminary it is unlikely that these phytoalexins have any roles evaluation of variations in composition induced by esca on cv. Trebbiano D’Abruzzo grapes and wines. Phytopatho- in the mechanisms that inhibit symptom formation. logia Mediterranea 40, Supplement, S443–S448. An alternative hypothesis could be that symptoms Calzarano F., L. Seghetti, M. Del Carlo and A. Cichelli, 2004. sometimes do not appear in diseased vines during Effect of esca on the quality of berries, musts and wines. an entire growing season is due to environmental Phytopathologia Mediterranea 43, 125–135. factors, or to the particular conditions of individual Calzarano F. and S. Di Marco, 2007. Wood discoloration and vines, or to interactions between environmental fac- decay in grapevines with esca proper and their relation- ship with foliar symptoms. Phytopathologia Mediterranea, tors and the conditions of vines. That phytoalexins 46, 96–101. have no role in prevention of leaf symptom develop- Calzarano F. and S. Di Marco, 2008. Approfondimenti sulla ment seems more likely since both trans-resveratrol dinamica di colonizzazione e sulla manifestazione dei sin- and trans-pterostilbene failed to improve the turgor tomi fogliari nell’esca propria. Micologia Italiana, 2, 29–41. of the leaves, when these leaves had previously been Calzarano F., V. D’Agostino and M. Del Carlo, 2008. Trans-res- immersed in Pch culture filtrates, any more than did veratrol extraction from grapevine: application to berries and leaves from vines affected by esca proper. Analytical immersion in distilled water. Nevertheless, it is yet Letters 41, 1–13. to be determined if increased amounts of the phy- Calzarano F., V. D’Agostino, F. Osti and S. Di Marco, 2013. toalexins induced in asymptomatic/diseased vine Levels of trans-resveratrol in leaves of vines affected leaves before leaf symptom appearance (i.e. prior to with “esca”. In: Protection and Production in Viticulture, the ‘berries beginning to touch’ growth stage) could IOBC/WPRS Bulletin Vol. 85, 129–136. contribute vines remaining asymptomatic during Calzarano F., S. Di Marco, V. D’Agostino, S. Schiff and L. Mug- nai, 2014. Grapevine leaf stripe disease (esca complex) are particular growing seasons. Repeated applications reduced by a nutrients and seaweed mixture. Phytopatho- of a leaf fertilising mix containing calcium, magne- logia Mediterranea 53(3), 543–558. sium and extracts of algae during vegetative growth Calzarano F., V. D’Agostino, A. Pepe, F. Osti, F. Della Pelle, strongly reduced the incidence of GLSD symptoms, M. De Rosso, R. Flamini and S. Di Marco, 2016. Patterns and caused high phytoalexins levels at the ‘fruit-set’ of phytoalexins in the grapevine leaf stripe disease (esca and the ‘pea-sized berries’ stages (Calzarano et al., complex)/grapevine pathosystem. Phytopathologia Medi- terranea 55(3), 410–426. 2014; 2017). Calzarano F., F. Osti, V. D’Agostino, A. Pepe, and S. Di Marco, In conclusion, this study has confirmed that the 2017. Mixture of calcium, magnesium and seaweed affects growth stage of grapevines plays an important role leaf phytoalexin contents and grape ripening on vines

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