Levels of trans-resveratrol in leaves of vines affected with esca Francesco Calzarano 1, Vincenzo D Agostino 1, Fabio Osti 2, Stefano Di Marco 2 1 Università degli Studi di Teramo, Dipartimento di Scienze degli Alimenti, Via C.R. Lerici, 1, 64023 Mosciano S.A. (TE); 2 CNR, IBIMET, Via Gobetti 101, 40129 Bologna Abstract: A survey was carried out on esca symptomatic vines, diseased asymptomatic vines and healthy vines in vineyards previously inspected for more than 15 years. The levels of trans-resveratrol were determined on leaves with different symptoms severity and on leaves of asymptomatic vines, and compared with what assessed on leaves from healthy plants, at three different phenological growth stages. Moreover, in the laboratory, leaves collected from healthy vines were immersed in cultural filtrates of Phaeomoniella chlamydospora (Pch) or in sterile liquid growth medium and then in a suspension of trans-resveratrol or distilled water to assess the possible effects. The levels of transresveratrol in symptomatic leaves were particularly high at pre-bunch closure but also at harvest, increasing with the severity of foliar symptoms. At veraison, a dramatic decrease in trans-resveratrol content was observed and no differences were noticed among the different leaf groups or subgroups. In the laboratory test trans-resveratrol did not show any kind of effect on cut leaves previously immersed in Pch filtrates compared to what was observed for distilled water. The response of the plant to foliar symptoms as trans-resveratrol production varied in different phenological stages probably in relation to specific physiological conditions occurring at these stages. The correlation between transresveratrol levels and severity of symptoms on the leaf surface noticed at pre-bunch closure and at harvest but not at veraison as well as the lack of effects on cut leaves allowed us to hypothesize that trans-resveratrol is not involved in the control of foliar symptoms. However, taking into account the current hypothesis on the origin of foliar symptoms, trans-resveratrol may be considered as an important factor to better understand the plant response to symptoms expression. Key words: Trans-resveratrol, esca, phenological growth stages, foliar symptoms Introduction Many-year studies on esca led to an increase of knowledge about the nature of the disease hypothesizing a complex of different syndromes (Graniti et al., 2000; Surico et al., 2006). The esca proper is characterized by both a tracheomycosis by Phaeomoniella chlamydospora (Pch) and Phaeoacremonium aleophilum (Pal) also called young esca, and a white rot by Fomitiporia mediterranea. Although the pathogenicity of the three fungi also when inoculated singly on the trunk was demonstrated (Sparapano et al., 2001), further studies showed that white rot is not necessary for foliar symptoms occurrence, which appeared to be associated with Pch and Pal (Calzarano e Di Marco, 2007; 2008). Recently, the term grapevine leaf stripe disease was proposed instead of young esca to identify vines showing the typical foliar symptoms (Surico, 2009). Despite all these advances and new acquisitions, the origin of the foliar symptoms is not cleared yet. A commonly agreed hypothesis is based on substances produced by esca fungi during the wood colonization and translocated to the leaves by sap flow (Mugnai et al., 1999; Surico, 2009). These substances could be produced by the degraded wood or secreted by the fungi (Sparapano et al., 1998; Evidente et al., 2000; Tabacchi et al., 2000). Most of the tests carried out on detached leaves with cultural filtrates or toxins failed to yield any positive result. In some cases some toxic effects were obtained (Sparapano et al., 1998, 2000; Abou-
Mansour et al., 2004) and only Sparapano et al. (2001) and Feliciano et al. (2004) have reproduced symptoms rather similar to those observed in the field, but not clearly attributable to the typical leaf symptom. The erratic nature of symptoms in the vineyard in terms of disease incidence and symptom severity has led the involvement of physiological and environmental factors, such as the rainfall (Marchi et al., 2006; Calzarano and Di Marco, 2007; 2008). Moreover, the variations of the plant physiology during the vegetative growth season may interact with the biological activity of fungi inside the woody tissues. In the center Italy, vineyards affected with esca proper show foliar symptoms at the end of June (after the end of blooming) with a very low incidence up to end of July (pre-bunch closure). In August there is a consistent increasing in the incidence of symptomatic plants with a peak of incidence at harvest. The aim of the present study was to investigate the variations of the levels of trans-resveratrol occurring during the growing season in esca affected plant. Trans-resveratrol is considered the most representative grapevine phytoalexin, the real skeleton of the other stilbenes (Jeandet et al., 2002). The level of trans-resveratrol was analyzed in leaves with different type and extent of symptoms, at different plant phenological growth stages. Moreover, laboratory assays were conducted on leaves collected from healthy plants by immerging petioles in cultural filtrates of Pch and then in suspensions of trans-resveratrol, in order to assess the possible effects of trans-resveratrol on the cut leaves. Material and methods Vineyard trials The determination of the levels of trans-resveratrol were carried out on leaves collected from two vineyards in Central Italy, Abruzzo region, 34 years old, cv. Trebbiano d'abruzzo, affected with esca proper and monitored for foliar symptoms for more than 15 years. This long-period of investigation has made more consistent the classification of the plants into 3 groups: symptomatic vines, diseased asymptomatic vines, healthy vines (plants that did ever show foliar symptoms for all the period of investigations). Symptomatic vines were divided in four further subgroups according to the severity of leaf symptoms: T1) 5% chlorosis; T2) 20% chlorosis; T3) 40% chlorosis + necrosis T4) 65% chlorosis + necrosis. Leaves were collected at 3 phenological stages, according to BBCH (Lorenz et al., 2005): i) berries beginning to touch (pre-bunch closure); ii) berries developing colour (veraison); iii) berries ripe for harvest (harvest). In 2009 growing season, 6 plants were identified in each of the two vineyards and for each group or subgroup of plants. Eight leaves were collected from the central part of shoots and opposite to the clusters, of vines belonging to each group or subgroup. In each group or subgroup the collection of leaves at the 3 phenological stages was carried out on the same vines. For each of either group or subgroup, 6 replicates were set up each consisting of 8 leaves collected from a single plant. For each sample (as single repetition consisting of 8 leaves), the extraction and detection of trans-resveratrol were performed using the method developed by Calzarano et al. (2008). For each phenological growth stage, the average level of trans-resveratrol detected on each of the four subgroups of the symptomatic vines and on the asymptomatic vine group were statistically compared with the level of average level of trans-resveratrol assessed in leaves collected from healthy vines, by means of Student s t-test (P=0.05). For each investigated type of leaf, data represent the mean of the data obtained from the two vineyards. Laboratory assay
The experiment was conducted in two phases by taking 6 leaves of healthy plants whose petiole was immersed for 3 hours in cultural filtrates of Phaeomoniella chlamydospora or sterile growth liquid medium (1st phase) and then immersed for 15, 58, or 106 hours in suspensions of trans-resveratrol or in distilled water (2nd phase). The concentration of transresveratrol (80 ml l -1 ) was chosen in relation to the level previously found in the leaves taken from the vineyards. The following treatments were evaluated for the leaf turgor or the occurring of necrosis on the leaf surface: sterile medium + water; Pch medium filtrate + water; Pch medium filtrate + trans-resveratrol. For each time of immersion treatments were compared and data were statistically analyzed using Duncan s multiple range test, at P=0.05. Results and discussion Vineyard trials The levels of trans-resveratrol at pre-bunch closure were particularly high in the leaves of symptomatic vines, 39.72 and 21.05 µg g -1 d wt in the leaves T4 and T3, compared with asymptomatic vine leaves (9.08 µg g -1 d wt) or healthy vine leaves (6.12 µg g -1 d wt) according to Calzarano et al. (2010). Moreover, at pre-bunch closure, the content of transresveratrol in the leaves increased with the increasing of foliar symptom severity (Fig. 1). A similar trend was observed also at harvest, but with generally lower values of transresveratrol. At both pre-bunch closure and harvest, all symptomatic and asymptomatic leaves were statistically different for the content of trans-resveratrol compared to healthy leaves, with an increasing in statistical significance with the severity of the leaf symptoms (P=0.05). On the contrary, at veraison, a drastic decrease in the content of trans-resveratrol was observed; moreover, no statistically difference (P>0.05) among the different types of leaf was ever noticed. The high levels of trans-resveratrol recorded at both pre-bunch closure and harvest, and low at veraison, showed that the plant can react to foliar symptom expression through the production of trans-resveratrol in a diversified way, probably depending on plant physiology occurring at the different phenological growth stages. The direct correlation observed between levels of trans-resveratrol and extent of lesions in symptomatic leaves at the first foliar symptoms appearance in the vineyard, as well as the absence of this correlation at veraison when the symptoms are widely spread in the vineyard, demonstrated that trans-resveratrol is not active in limiting the development of symptoms in the leaf. Trans-resveratrol is formed as a plant response to injury caused by several unspecific biotic or abiotic stress factors that can damage the leaf tissue (Kuć, 1995; Smith, 1996; Bavaresco e Fregoni, 2001); the direct correlation noticed in this study between trans-resveratrol levels and severity of symptoms appear to be consistent with this statement: probably, the effects on leaf by fungal toxins or toxic metabolites induce an increasing in trans-resveratrol production ascribable to that of the above mentioned stress factors. The gene expression of phenylalanine ammonia lyase and stilbene synthase, enzymes involved in the synthesis pathway of trans-resveratrol, was repressed in the green area of symptomatic leaves, but no alteration was observed in the chlorotic area (Magnin-Robert et al., 2011). This assumption might be a confirmation of the increasing of production of trans-resveratrol only after the damage of the leaf by the disease observed in our study at pre-bunch closure and at harvest. The determination of the contents of trans-resveratrol in the leaves of diseased vines, seems to confirm the complex nature of the plant reaction to the foliar symptom development; moreover, the intensity of this reaction depends on the different stages of growth. As previously mentioned, the response of the plant as trans-resveratrol formation to symptoms development was rapid and correlated with the extent of the lesion on the leaf surface, and it
would indicate the highest response of the diseased plant at pre-bunch closure. The content of trans-resveratrol and δ-viniferine increased in leaves of healthy vines from the pre to the postflowering period in cultivars Merlot and Cabernet Sauvignon (Timperio et al., 2011). Similarly, vineyards inspected in the present study showed increased levels of transresveratrol in the leaves of healthy vines, until the pre-bunch closure (Calzarano, unpublished data). These observations strengthen the hypothesis of an increasing of the plant response capacity during the vegetative growth up to pre-bunch closure. The absence of the spread of foliar symptoms in this period cannot be attributable to the trans-resveratrol production, but rather to other factors maybe related to the plant physiology. However, the rapid synthesis of other stilbenes that are formed from trans-resveratrol as a result of different inductive stimuli (Keller et al., 2000; Commun et al., 2003; Slaughter et al., 2008) may suggest that the transresveratrol can be considered as an important factor for the study of the plant response to foliar symptoms expression, probably more important than pterostilbene or viniferines, that are active on pathogens (Pezet et al., 2004), because of the absence of pathogens in symptomatic leaves. 45 40 35 30 ppm d wt 25 20 15 10 5 0 Pre-bunch closure Veraison Harvest T4 T2 asymptomatic vine leaves T3 T1 healthy vine leaves Figure 1. Trans-resveratrol levels in leaves of esca affected vines at different symptom degree and in leaves of asymptomatic and healthy vines. Laboratory assays
Laboratory assays results showed statistically differences only comparing 3 hours Pch filtrates treatment with 3 hours sterile growth medium treatment (P=0.05). Finally, the results of the in vitro assay, where the trans-resveratrol did not show better effects in leaf turgor of leaves previously immersed in the culture filtrates of Pch, compared to distilled water, confirm a lack of effect of the stilbene. On the other hand, these results seemed to exclude the possible involvement of trans-resveratrol in the formation of foliar symptoms as reported by Bruno et al. (2005). Sterile growth medium + distilled water 3 hours sterile growth medium 15 hours water 58 hours water 106 hours water Pch filtrates + trans-resveratrol Pch filtrates + distilled water 3 hours Pch filtrates 15 hours water 58 hours water 106 hours water 3 hours Pch filtrates 15 hours t-resveratrol 58 hours t-resveratrol 106 hours t-resveratrol 0 1 2 3 4 5 Figure 2. Effect of Pch filtrates or sterile growth medium followed by trans-resveratrol or distilled water on cut leaves. A statistically difference (P=0.05) was assessed only for 3 hours Pch filtrates compared to 3 hours sterile growth medium, according to Duncan s MRT. Table 1. Arbitrary scale adopted for the evaluation of the status of the cut leaves immersed in filtrates and trans-resveratrol or water. Arbitrary scale values Status of the cut leaf 5 fresh, green 4 fresh, green with 5-10% marginal necrosis 3 withering + 2 withering ++ 1 withering +++
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