EFFICIENCY OF CRIMSON SEEDLESS GRAPEVINES
4.2 Materials and Methods
The experiment layout, scenarios, climatic conditions, soil conditions and soil water status measurement methodology are described in Chapter III. Therefore, only relevant additional details are given here.
4.2.1 Infrared gas analyser measurements
Photosynthetic capacity and leaf gas exchange were measured during the 2013/14 and 2014/15 seasons. Net carbon assimilation rates (A), stomatal conductance (gs) and transpiration rates (E) were measured with an infrared gas analyser (IRGA) chamber (LI-6400, Li-Cor, Lincolin, Nedbraska, USA). The flow rate to the sample cell, reference CO2 and quantum flux on the IRGA was controlled
to the following values, respectively: 500 µmol.s-1, 380 µmol CO
2.mol-1 and 1500 µmol.m-2.s-1 for all
measurements. During the 2013/14 season, two vines that were randomly selected based on the stem diameter in all blocks (A, B, C & D) were measured during four different phenological stages (pea size berries, véraison, harvest & post-harvest). Due to logistical constraints regarding available equipment, it was decided to only conduct diurnal cycles at two of the four blocks for the next season. Consequently, only one vine in Blocks B and D were measured to determine diurnal cycles at the above-mentioned phenological stages for the 2014/15 season. The diurnal cycle measurements were done on the same leaf from 08:00 to 16:00 at two-hour intervals. Five leaves per grapevine were selected for the IRGA measurements in both seasons. These leaves were selected in categories as follows: one small shaded leaf, three fully expanded leaves, and a hardy sun exposed leaf for the 2013/14 season. Since there were no apparent differences between the small shaded leaves in all blocks in the 2013/14 season, it was decided to replace the small shaded leaf with a lateral fully expanded leaf for the 2014/15 season. In addition to the IRGA measurements, stomatal conductance was measured with a leaf porometer (Delta-T AP4, Cambridge, England and Decagon Devices, Inc., Pullman, WA). Leaf temperature (TLeaf) and vapour pressure deficit (VPD) measurements were also measured using the IRGA.
4.2.2 Light measurements
Canopy light measurements were conducted with an AccuPAR LP-80 ceptometer (Decagon Devices, Inc., Pullman, WA) in both seasons. Measurements were taken at the end of leaf gas exchange measurements per block and are expressed as a ratio of ambient radiation measured in units of μE.m-2.s-1. Four above canopy measurements were taken to calibrate the instrument where
after measurements below the canopy (30 cm below the trellising wire) were done at three positions. Selected positions were as follows: 1st wire (low), 4th wire (middle) and 6th wire (high). During the
2014/15 season, ceptometer readings were taken with the midday measurements (12:00-14:00) for all four blocks. In the 2014/15 season, the ratio of red: far red (660:730 nm) radiation was also measured around the leaf where gas exchange was measured using an R:FR sensor (Skye instruments, Powys, UK). These measurements were taken in conjunction with the IRGA measurements.
4.2.3 Reproductive measurements
4.2.3.1 Yield and its components
At harvest, yield (kg/vine) and export mass (kg/vine) of the marked data vines in all experimental units were determined. Grapes were sorted and classified according to the industry quality standards
81 into the following classes: Class 1 (Export), Class 2 (Local) and Class 3 (Cull) (Department of Agriculture, 2016).
4.2.3.2 Berry sampling and analysis
A random sample of 50 berries for each of the ten experimental units at each block was collected weekly from véraison to harvest for monitoring progression of berry ripening and quality evaluation in both seasons. However, for the 2014/15 season, berry sampling was done few weeks before harvesting (two to four weeks) in order to capture the final ripening stage before harvesting. After sampling, grapes were transported to the laboratory. Berry fresh mass (g) was determined with a digital scale, while the diameters (mm) were measured in the centre of the berry with a digital calliper. Grape samples for each of the ten experimental units were homogenised with a blender and juice was sieved into a clean measuring glass, from which 50 mL juice sample was extracted with a pipet for analysis. Total soluble solid concentration (TSS) was determined using a digital pocket refractometer (Atago PAL-1, Tokyo, Japan). Total titratable acidity (TTA) and pH was measured with an automatic titration device (Metrohm 785 DMP Titrino, Herisau, Switzerland). During the 2014/15 season, berry volume (mL) was also determined by water displacement.
4.2.3.3 Total anthocyanin analysis
Random samples of 50 berries per five experimental units were sampled as already described in Section 4.2.3.2. Samples collected throughout ripening were frozen at -20°C until further processing. An extraction solvent ethanol/water 50/50 (v/v) adjusted to pH 2 with 37% HCl was prepared. Anthocyanin extraction from berry skins (Iland/AWRI method – adapted) was used to determine the total anthocyanins (Iland et al., 2000). Berries were defrosted to facilitate the peeling process and 25 of the 50 berries were randomly selected. Berry skins were removed from the pulp using blades. Thereafter, berry skins were weighed. After extraction solvent (5 mL) was added to the berry skins, samples were homogenised with a homogeniser (IKA T18 basic, Germany) until smooth. Samples of 2 g homogenate were transferred to 50 mL Falcon tubes. The extraction solvent was then added to the Falcon tube at a ratio of 10 mL of solvent for 1 g of berry skin. Tubes were capped and the contents were mixed periodically by inverting the tube every 10 minutes over a period of 1 hour. After an hour, the tubes and contents were put in a centrifuge (Hermle Labortechnik GmbH, Germany) and centrifuged at 10 000 rpm for 5 min. During the 2013/14 season, the extract was scanned in an Analytic Jena Specord 50 UV−vis spectrophotometer (Jena, Germany), using a 10 mm path length quartz cuvette. A cuvette with extraction solvent was used as the reference blank. Unfortunately, during the 2014/15 season, the Specord spectrophotometer was not working so a Thermo Scientific Multiskan GO (Thermo Fisher Scientific Oy, Vantaa, Finland) spectrophotometer was used instead. As in the previous season, a 10 mm path length quartz cuvette, with extraction solvent as the reference blank, was used. In both seasons, absorption of the extract was read at 520 nm wavelength.
4.2.4 Yield water use efficiency and irrigation water use efficiency
Evapotranspiration was only determined from the second week of December until August during the 2014/15 season and did not account for the whole season. Therefore, the seasonal irrigation volumes for both seasons were determined in order to calculate irrigation WUE. The WUEy is defined
as total harvested yield per unit of water use (ET) (kg/m3 or mm), whereas the irrigation water use
82 Estimated ET and seasonal irrigation volumes (m3) were divided by the yield (kg/ha) per block to
determine WUEy and WUEirr. The following equations were used to calculate WUEy and WUEirr:
WUEy = Y/ ET (kg/m3) (Eq. 4.1)
WUEirr = Y/ I (kg/m3) (Eq. 4.2)
where Y is yield, ET is estimated ET from the water balance calculations (December–April) and I is seasonal irrigation volumes (September–April), respectively. Four different WUEy calculations were
done based on the type of ET correction used (Refer to Chapter 3). Thus, the following abbreviations were used for the uncorrected water balance method (WUEy(ET)), ET corrected with published crop
factors from Lategan (1996) (WUEy(ETL)) and (Myburgh, 2003b) (WUEy(ETM)) and ET corrected with
FruitLook derived crop factor (WUEy(ETFL)) as already discussed in Chapter 3.
4.2.5 Statistical analyses
Statistical analyses and graphs were done using Statistica 10 ® software (Statsoft, Tulsa, UK).