Fruit attribute measurements 31

Fallen berries and berry shrivel

Fallen berries were counted using a 400 mm x 600 mm rectangular quadrat. Before assessment the vine trunk was shaken three times with similar force used each time. The quadrat was placed on the ground under the vines, four times per measurement panel. The under vine row was divided into four sections, two per measurement vine, and the quadrat placed at random in each section. All fallen berries inside the quadrat were then counted.

The nitrogen addition by exposure trial was used to further examine the shrivelled berries. During processing of the fruit, berries were divided into two groups, one for shrivelled berries and another for non-shrivelled, or full, berries. All berries in each group were weighed together, and then subsets counted to get an average berry weight. This allowed a calculation of total berries per group.

Rachis measurements

Stems from the nitrogen addition by exposure trial were measured for total length from the point of the first lateral rachis branching (or scar if it was not present) to the base of the pedicels at the tip of the rachis (Figure 2.5-1).

Figure  2.5-­‐1  Measurement  of  rachis  length  

counted on the 3rd_{, 6}th _{and 9}th _{rachis branching (Figure 2.5-2).}

Averages were calculated for all these values. Branchlets per mm and pedicels per mm were also calculated.

Flowering date

Flowering progression was assessed in the 2006-07 season by a survey when the vines were judged to be around the middle of capfall.

The number of bunches on each monitor shoot were counted, and then the degree of capfall was assessed as a visual estimation of the percentage of flowers that had released their caps. To minimise any variability the same researcher carried out all assessment. Frequent “calibrations” were done, where the percentage of capfall was calculated. Statistical analysis involved ANOVA of arcsine conversions of the percentages, to provide a normal distribution of data.

Flowering progression in the following season was estimated by grading shoots on the modified E-L scale developed in (Coombe, 1995). Modified E-L values between 20 and 26 are defined by the percentage of capfall.

Fruit set

In the 2007-08 season, fruit set was calculated by capturing and counting all flower caps. Fruit set assessments were carried out on vines that had formed the nitrogen by

exposure trial the previous season, and received either no nitrogen or 100 g N/vine pre- bloom. Four bunches were chosen per panel at random, and marked with a plastic bread tag. Caps were captured in paper bags secured on the inflorescence by a plastic cable tie. At the end of flowering, bags were removed. The caps were then cleaned to remove all anthers and other material. Any caps that were inadvertently retained in the non-cap material were counted to add to the total cap count later.

Cap counts were carried out by taking a random selection of caps, and two subsets of exactly 50 caps were separated from the main group. These were weighed, allowing calculation of an average cap weight, and then returned to the main group. The total weight of all the cleaned caps was then taken, allowing total caps to be calculated. This was added to the caps lost during cleaning.

The day before harvest of the main group of grapes, tagged bunches were removed. They had all berries removed, and rachis length, pedicels and branchlets counted. Fruit set was calculated by dividing pedicels by cap counts.

Fruit chemistry

Juice samples were taken from the grape must following crushing and destemming. Juice total soluble solids were assessed by a manual refractometer. In 2006 titratable acids (TA) were determined using the method of Iland et al. (2004), which involved a manual titration with NaOH to a pH of 8.2. A standard pH meter was used to find juice pH. In 2007 and 2008, a Metrohm 785 DMP Titrino autotitrator (Metrohm Ltd, Herisau, Switzerland) was used to determine both pH and TA. TA was calculated to an end-point of pH 8.2, against 0.333M Na OH, in accordance with the autotitrator instructions.

Malate

manufactured by Vintessential Laboratories (Dromana, Australia). Spectroscopy was carried out on a Metertech SP8001 spectrophotometer (Taipei, Taiwan).

Yeast Assimilable Nitrogen

Yeast source nitrogen from the metabolisation of ammonium and free alpha amino acids. The nitrogen available from these two components are calculated separately then totalled to give the final yeast assimilable nitrogen (YAN). While some low molecular weight grape proteins may also be used as sources for nitrogen these only contribute a small amount of the total YAN, and therefore are not measured (Bell and Henschke, 2005).

Primary alpha amino acids were assessed using a kit purchased from Vintessential (Dromana, Australia), which follows the method of Dukes and Butzke (1998).

Ammonium was also determined enzymatically, using a Vintessential kit. Both analyses were performed as detailed in the manufacturer’s directions, using a Metertech SP8001 spectrophotometer.

Yield and yield components

Within each trial, all fruit was harvested on the same day. Fruit from the central two vines was harvested and weighed together. Bunches were counted during harvesting, and 100- berry samples were randomly selected and manually removed from a significant

proportion of the harvested bunches and weighed to allow calculation of berry weight.

Berry extract analysis

Berry extraction and anthocyanin analysis followed the methods of the Australian Wine Research Institute recommended industry standard method, published by the

Cooperative Research Centre for Viticulture. Analysis was performed on the 100-berry samples following weighing.

Frozen berry samples were homogenized in a Waring blender for 60 seconds on high speed. Approximately one gram of homogenate was then removed and transferred to a tarred 10mL plastic centrifuge tube. The total mass of homogenate was recorded to three decimal places.

10mL of 50% aqueous ethanol was added to the centrifuge tube, which was capped and mixed by inversion. Mixing was carried out by further intermittent inversion for at least one hour.

This tube was then centrifuged at 3500 rpm (RCN = 2333G) for 10 minutes in a

Centurion EB series centrifuge (Centurion Scientific, West Sussex). The supernatant was then stored in the freezer until required.

The second stage of the analysis involved the transfer of 200 µL of extract to a plastic

vessel containing 10 mL of 1.0 M HCl. This was incubated for 3 hours.

A Metertech SP8001 model spectrophotometer (Metertech Inc., Taipei) was zeroed using 1mL HCl in a quartz cuvette as a blank. 2 mL was transferred from the sample to a quartz cuvette with a 10mm path length. Absorbance was then read for each sample following transfer to at 250 nm, 280 nm, 300 nm and 520 nm.

Anthocyanins (mg/g homogenate) = (10.5 x A520 x DF x 10.5 x 1000)/(500 x 100 x HW)

Where:

A520 is the absorbance at 520 nm

DF is the dilution factor from adding the extract to the HCl (11 in this case)

10.5 is the total extract volume estimate (based on a homogenate weight between 0.95 and 1.05 g)

HW is the homogenate weight in grams

Phenolics were calculated using a model developed by R. Dambergs of the Australian Wine Research Institute (Unpublished). The model had been developed comparing wine spectral signatures against methyl cellulose precipitatable tannins (Sarneckis et al., 2006). The spectral signatures were analysed to determine which wavebands were best able to indicate the different tannin levels. The model used in these trials was based on

absorbances at 250, 280 and 300 nm. These were assessed using quartz cuvettes with a 10 mm pathlength,