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.