View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Firenze University Press: E-Journals 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 Teramo, Facoltà di BioScienze e Tecnologie Agro-Alimentari ed Ambientali, Via Renato Balzarini, 1, 64100 Teramo, Italy 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. Trebbiano have produced uncertain results (Sparapano et al., d’Abruzzo 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 Giulianova (Tendone to induce leaf symptoms, but these were different system) in the province of Teramo, 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.