EFFECT OF SAR INDUCERS ON GRAPE SECONDARY METABOLITES

Fungicide application is now the most efficacious method for controlling plant diseases caused by oomycetes and fungi. As legislation is limiting and reducing their use, it is strongly stimulating studies for the identification of additional and environmentally friendly approaches in the control of their associated diseases. Among these, systemic acquired resistance (SAR) offers the prospect of long-lasting, broad-spectrum disease control through activation of the resistance defence machinery of the plant itself. Plant activators are products employed in crop protection able to elicit SAR. Therefore, they may trigger the plant own defence response against pathogen attacks, mainly stimulating mechanisms such as the biosynthesis of phytoalexins, plant secondary metabolites with a broad spectrum biological activity. In this study, two plant defence inducers, benzothiadiazole (BTH) and chitosan (CHT), deserving particular attention because of their efficacy and low toxicity, have been used. CHT is a natural and low-cost polymer (from the waste products of the crustacean carapace), obtained by chitin deacetylation. Its effectiveness is higher when molecular weight is between 10 and 100 kD and the deacetylation degree range is from 80 to 90 percent. BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester] is a synthetic compound and a functional analogue of salicylic acid, a plant hormone-like compound deeply involved in resistance against pathogens. Two different phytoiatric campaigns were planned, in 2009 and 2010, on two red grapevine (Vitis vinifera L.) varieties cultivated in experimental vineyards located at distinct sites: Groppello, an autochthonous cultivar of Lombardia, at Raffa di Puegnago (Brescia), and Merlot at Conegliano Veneto (Treviso). Open field treatments carried out on Groppello grapevines were: i) 0.03% (w/v) CHT (76 kDa molecular weight and 85% deacetylation degree), ii) 0.03% CHT in combination with 150 g hL-1 copper hydroxide (CHT/Cu) and iii) 0.3 mM BTH; whereas, on Merlot, besides CHT and CHT/Cu, BTH were replaced by 300 g hL-1 potassium phosphites. Untreated vines were used as negative control, while plants treated with conventional fungicides (penconazole and methyldinocap) were the positive control. In both field surveys, the trial was set up as a complete randomized block design in 4 replications, with 10 vines (a parcel) per treatment in each block. Plants were sprayed approximately every 10 days, according to the meteorological conditions, from the beginning of grape susceptibility to fungal diseases until the complete véraison. The phytosanitary status of vineyards was assessed weekly on leaves and bunches, by visual inspections, though both sites were not particularly predisposed to severe fungal or other epidemics. The epidemiological evaluations were performed on bunches alone and infection indexes were calculated. All the treatments were effective in controlling fungal infections (downy mildew, Plasmopara viticola and powdery mildew, Erysiphe necator), in terms of disease incidence (I %), disease severity (S %) and infection degree (ID %), though these indexes were low in untreated control grapevines, particularly in 2010. Sampling was scheduled at two phenological phases: pre-véraison and 100% véraison; bunches were randomly collected from plants during the morning and stored at -20 °C until analyses. Berry tissues were separated into skin, flesh and seeds, powdered and extracted. Then, extracts were tested to evaluate their melatonin levels [by ultra performance liquid chromatography (UPLC)-MS/MS], total polyphenols (TP, by Folin-Ciocalteau assay) and antiradical activity [by DPPH, 2,2-diphenyl-1-pycryl hydrazyl and ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay]. Groppello and Merlot experimental wines were produced, by standard microvinification techniques, in the Centro Vitivinicolo Provinciale of Brescia and Centro di Ricerca per la Viticoltura of Conegliano (TV), respectively, and stored at 4 °C in the dark until analyses. Microvinificates were produced from grapes treated with both elicitors and conventional fungicides (controls), and the following analyses, besides those also conducted for all berry tissues, were carried out: tryptophan, serotonin and melatonin detection (by UPLC-MS/MS); content of cis and trans resveratrol (by UPLC-MS/MS); levels of mycotoxins (manly ochratoxin A, OTA, by UPLC-MS/MS). In general, berry tissues treated with elicitors and the corresponding experimental wines showed higher levels of melatonin and polyphenols, as well as a higher antiradical activity than samples treated with conventional fungicides. High concentrations of tryptophan were detected in all samples, contrary to serotonin, which was not detected. The most effective elictors were CHT/Cu and CHT. In all wines, the level of OTA was below the allowable threshold of 2 ng/mL. To the best of our knowledge, these results represent the first data on the effects of agrochemicals on the melatonin content of red wine, and the presence of melatonin was reported, for the first time, in berry seeds and flash, after its previously detection in skin. Furthermore, the level of the indolamine in berry tissues varies according to the phenological stage, resulting more abundant in seed at pre-véraison and in skin at véraison. The good agreement between the data obtained in 2009 and 2010 for Groppello cultivar, and, in 2009, between Groppello and Merlot varieties cultivated in different geographical areas, suggests that, in general, plant activators may improve some qualitative/healthy treats ascribed to red wine, though their efficacy in controlling grapevine fungal diseases should be better ascertained. Finally, the role of melatonin, a powerful antioxidant, in grapevine physiology is still somewhat obscure. It is possible that, at pre-véraison, when skin anthocyanic pigmentation is still lacking, this compound may defend berry from damage due to photooxidation or UV radiation, whereas, at véraison, the indolamine may protect the germ tissues, particularly rich in storage lipids and membranes and vulnerable to oxidative damage. In conclusion, the possibility of enhancing the pharmaconutritional potential of grape/wine with phytosanitary treatments should be further tajen into account.