2.3 Leaves sampling and characterization
Leaves (10 per vine) were randomly picked (as replications) from the 15 selected vines at stretched-out leaves (SL), fruit setting (FS), cluster closing (CC) and bunch ripening (BR) phenological phases (Baggiolini, 1979). At CC and BR collections, symptomless and symptomatic leaves were taken from diseased vines.
Leaves were transported to the laboratory in an icebox, deprived of the petiole, photographed and subjected to morphological and physiological characterization.
Each leaf was weight with a Sartorius BP 210S analytical balance (Data Weighing Systems, Inc., Wood Dale, IL, USA) to assess fresh weight (Lfw).
Leaf size was measured with ImageJ (National Institutes of Health, MD, USA) an open-source image-processing program.
Leaf dry weight (Ldw) was estimated by drying 100 mg of leaves for 20 min at 105 °C with the infrared Mettler LP 16-M desiccator (Mettler-Toledo SpA., Milan, I). Leaf moisture (Lm) was calculated on a wet-weight basis.
Chlorophyll was determinate as described by Harborne (1973) using about 2 g of leaf samples and 80% acetone (16 ml) in ice bath. Total chlorophyll concentration (mg g-1 Lfw) was calculated as: [((20.2 × A645) + (8.02 × A663)) / (100 × W)] × V, where A645 and A663 = absorbance at 645 and 663 nm respectively, V = volume (ml), W = Lfw (g).
Hydrogen peroxide concentration was determined as previously described (Lee & Lee 2000) using 1 g of leaf lamina ground with 4 ml of sodium phosphate buffer (0.1 M; pH 6.5).
Lipid peroxidation was estimated as malondialdehyde (MDA) on 200 mg of the lamina (Heath & Packer, 1968). The results were expressed as nmol MDA per gram of Lfw.
The levels of ascorbic acid (AsA), dehydroascorbic acid (DHA), reduced (GSH) and oxidized (GSSG) glutathione were quantified on 2 g of leaf lamina ground, at 4°C, in 5% metaphosphoric acid (6 ml). After centrifugation (20,000×g , 15 min, 4°C) the supernatant was used as proposed by Zhang & Kirkham (1996).
The ascorbate (A-RS) and glutathione (G-RS) redox state were calculated as A-RS = [AsA / (AsA + DHA)] and G-RS = [GSH / (GSH + GSSG)].
For enzymes involved in ascorbate regeneration, leaf lamina (2 g) was homogenised in 6 ml of extraction buffer (50 mM Tris-HCl pH 7.8, 0.3 mM mannitol, 10 mM MgCl2, 1 mM EDTA, 0.05% cysteine) at 4°C. The homogenate was centrifuged (25,000×g , 15 min, 4°C). The supernatant was dialysed against 50 mM Tris-HCl (pH 7.8) and used for ascorbate peroxidase (EC 1.11.1.11, APX), dehydroascorbate reductase (EC 1.8.5.1, DHA-R), glutathione reductase (EC 1.6.4.2, G-R) and ascorbate free radical reductase (≡ monodehydroascorbate reductase, EC 1.6.5.4, AFR-R) activities determination according to Paciolla et al. (2008).
2.4 Statistical analysis
Data were subjected to general linear analysis of variance models using the SAS/STAT version 9.0 (SAS Institute Inc., Cary, NC, USA). Normal distribution and homoscedasticity were tested using the Shapiro-Wilk and Bartlett’s tests, respectively. Pair-wise comparisons of means were performed according to the Tukey test at P ≤0.05. Data of leaves morphological and physiological features were analysed for vine typology (BWSV, BWSWRV and HV), phenological phases (SL, FS, CC and BR), symptoms (presence, absence) and their interactions.