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Chapter 8 Effects of sodium molybdate foliar sprays on Merlot fruit composition

8.1 Introduction

In the previous chapters (Chapter 4, Chapter 5 and Chapter 7) the effects of sodium

molybdate foliar sprays on vine nutrient status, vegetative growth and yield of Merlot were

discussed in detail. Given the significant uptake of molybdenum when Mo-deficient vines

were treated with sodium molybdate (Chapter 4), significant increases in yield (Chapter 6)

and the consequent change in vine balance (Chapter 5), it follows that fruit composition may

potentially be affected by Mo-treatment. Gridley (2003) reported that the application of

sodium molybdate to Mo-deficient Merlot significantly improved yield. In that study Mo-

treatment had no demonstrable effects on total soluble solids, pH, titratable acidity, total

phenolics or colour in berries. There is currently no information on the effects of sodium

molybdenum treatment on the concentration of molybdenum in the resultant fruit.

In the current study, control vines in the Hills had an adequate level of molybdenum at

flowering time in 2003-04 and in 2004-05. The application of sodium molybdate sprays at

modified E-L stage (MELS) 12 (shoots 10 cm) significantly increased the concentration of

molybdenum in Mo-treated vines; however, it had no effect on yield or vine balance.

At McLaren Vale, control vines were deficient in molybdenum in 2003-04 and in 2004-05.

The application of sodium molybdate spays at MELS 12 significantly increased the

concentration of molybdenum in the vines at flowering time. Mo-treatment resulted in a

significant improvement in yield compared to Mo-deficient controls. The increase in yield

altered vine balance such that the yield to pruning weight ratio (Y/P) was significantly higher

for Mo-treated vines (8.6 and 9.7 for rates 1 and 2 respectively) than the controls (Y/P = 3.5).

A similar difference in vine balance between the Mo-treated and untreated vines was also

observed in 2004-05.

These changes in vine balance may have potentially affected the vine’s ability to ripen the

fruit and ultimately fruit composition. Furthermore, if molybdenum is transported to the fruit

on Mo-treated vines, its presence in the fruit at harvest may have implications for human

health.

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8.1.1 Aims

The aim of this experiment was to compare the composition of berries from Mo-treated vines

and untreated controls.

8.2 Materials and methods

8.2.1 Experimental design

Fruit for analysis was collected from the molybdenum experiments in the Hills and at

McLaren Vale. Details of the treatments and experimental design are provided in Chapter 3.

8.2.2 Berry sampling method

Close to commercial harvest a 100-berry sample was collected from each replicate. The

sample was collected to represent all the berries on the vines. This was achieved by randomly

selecting berries from the top, bottom, interior, exterior and middle of bunches, and from

bunches in the interior and exterior of the canopy. Fruit was stored in insulated containers and

transported to the laboratory for analysis on the day of collection.

8.2.3 Sample processing

From the 100-berry sample, 50 berries were selected at random for processing while the

remaining 50 berries were placed into labelled bags and frozen at -23°C. The berries for

processing were crushed using a ‘Simplex 2’ citrus juicer and the juice was clarified by

centrifuging at 5000 rpm for 5 minutes. Total soluble solids (TSS) was measured using an

ERMA BRX-242 bench-top refractometer and a PHM62 standard pH meter was used for the

measurements of pH and titratable acidity (TA), following the procedures described by Iland

et al. (2000). Surplus juice was frozen at -23°C.

8.2.4 Nutritional analysis

Nutritional analysis was performed on residual clarified juice samples collected from the Hills

and McLaren Vale in 2003-04 (section 8.2.3). Three replicates were selected from each

treatment based on the uniformity of TSS concentration. Hills samples were selected as close

as possible to 22˚Brix. Samples from McLaren Vale were all approximately 24˚Brix.

125

Waite Analytical Services performed the analyses of Mo, Fe, Mn, Cu, B, Zn, Ca, Mg, K, P,

and Na using Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES) in the

first instance and thereafter using Inductively Coupled Plasma Mass Spectrometry (ICPMS)

to measure molybdenum concentration below 0.06 mg/L.

8.3 Results

In the Hills in 2003-04 there were no significant differences in berry composition between

treatments. In 2004-05 there was no significant difference in TSS or pH between treatments

however, the TA of juice from rate 2-treated vines was significantly higher than the control

(Table 8.1 and Table 8.2).

At McLaren Vale in 2003-04 the concentration of juice TSS tended to be higher for fruit from

Mo-treated vines. Juice TSS was significantly higher for rate 2 than the control. Juice pH was

significantly lower from the Mo-treated vines than from the controls. There was no significant

difference in TA between treatments (Table 8.1).

Table 8.1. Total soluble solids (TSS), pH and titratable acidity (TA) of berries from the Adelaide Hills and McLaren Vale in 2003-04. Vines were treated with rate 0 (control), rate 1 and rate 2 molybdenum sprays at modified E-L stage 12 in 2003-04.

2003-04 TSS (˚Brix) pH TA Adelaide Hills

Rate 0 22.6 a 3.26 a 6.3 a Rate 1 21.3 a 3.19 a 6.5 a Rate 2 21.5 a 3.22 a 6.4 a

McLaren Vale

Rate 0 23.6 a 3.53 a 5.6 a Rate 1 24.1 ab 3.44 b 5.5 a Rate 2 24.5 b 3.45 b 6.0 a

In the new block in 2004-05 there were no significant differences in juice TSS or TA between

treatments; however, juice pH from rate 2-treated vines was significantly lower than for the

control and rate 1. In the old block there were no significant differences in juice composition

between treatments (Table 8.2).

126

Table 8.2. Total soluble solids (TSS), pH and titratable acidity (TA) of berries from the Adelaide Hills and McLaren Vale in 2003-04. In the Hills vines were treated with rate 0 (control), rate 1 and rate 2 molybdenum sprays at modified E-L stage (MELS) 12 in 2003-04 and were left untreated in 2004-05. At McLaren Vale vines in the new block were treated at MELS 12 in 2004-05 only. Vines in the old block were treated at MELS 12 in 2003-04 and again at MELS 12 in 2004-05. Letters after the values represent significant differences between treatments within in an experiment (p<0.05).

2004-05 TSS (˚Brix) pH TA Adelaide Hills

Rate 0 24.1 a 3.40 a 5.46 a Rate 1 23.7 a 3.40 a 6.00 ab Rate 2 23.8 a 3.43 a 6.43 b

McLaren Vale New block

Rate 0 24.7 a 3.37 a 3.6 a Rate 1 24.0 a 3.32 a 3.6 a Rate 2 24.2 a 3.21 b 3.6 a

Old block Rate 0 24.6 a 3.34 a 3.2 a Rate 1 23.9 a 3.28 a 3.6 a Rate 2 23.8 a 3.38 a 3.8 a

In the Hills in 2003-04, juice from rate 1-treated vines had a significantly higher

concentration of molybdenum than the control. The concentration of molybdenum in juice

from rate 2-treated vines was significantly higher than the control and rate 1. At McLaren

Vale in 2003-04 there was no significant difference in juice molybdenum concentration

between treatments (Figure 8.1).

a

b

c

A A

A

0

5

10

15

20

25

Rate 0 Rate 1 Rate 2

Mo

(ug/

L)

Figure 8.1. Concentration of molybdenum (µg/L) in Merlot juice from the Adelaide Hills (■) and McLaren Vale (■) at modified E-L stage (MELS) 38 (berries harvest ripe) in 2003-04. Vines were treated with rate 0 (control), rate 1 and rate 2 molybdenum sprays at MELS 12 (shoots 10 cm). Different letters above the columns indicate significant differences between treatments at a site (Hills = lower case, McLaren Vale = upper case) (p<0.05).

127

In the Hills, juice from Mo-treated vines tended to have a lower concentration of potassium

than the control. There were no other significant differences in juice nutrient analyses from

the Hills or McLaren Vale (Table 8.3 and Table 8.4).

Table 8.3. Nutritional analysis of juice at modified E-L stage (MELS) 38 (berries harvest ripe) from vines treated with rate 0 (control), rate 1 and rate 2 molybdenum sprays at MELS 12 (shoots 10 cm) in the Adelaide Hills in 2003-04. Different letters after values indicate significant differences between treatments (p<0.05).

Molybdenum treatment Rate 0 Rate 1 Rate 2

Fe (mg/L) 0.37 a 0.30 a 0.32 a Cu (mg/L) 0.75 a 0.61 a 0.62 a Mn (mg/L) 0.56 a 0.43 a 0.56 a Mo (µg/L) 4.57 a 11.59 b 22.22 c B (mg/L) 3.50 a 3.04 a 3.31 a Zn (mg/L) 0.31 a 0.94 a 0.74 a Ca (mg/L) 46.76 a 53.76 a 48.08 a K (mg/L) 1677 a 1550 ab 1260 b Mg (mg/L) 89 a 90 a 85 a P (mg/L) 72 a 64 a 60 a Na (mg/L) 27 a 21 a 19 a Al (mg/L) 0.31 a 0.26 a 0.22 a S (mg/L) 38 a 31 a 34 a

Table 8.4. Nutritional analysis of juice at modified E-L stage (MELS) 38 (berries harvest ripe) from vines treated with rate 0 (control), rate 1 and rate 2 molybdenum sprays at MELS 12 (shoots 10 cm) at McLaren Vale in 2003-04. There were no significant differences between treatments (p<0.05).

Molybdenum treatment Rate 0 Rate 1 Rate 2

Fe (mg/L) 0.38 0.40 0.44 Cu (mg/L) 0.46 0.43 0.48 Mn (mg/L) 0.59 0.50 0.54 Mo (µg/L) 11.12 10.60 12.92 B (mg/L) 13.74 13.42 12.25 Zn (mg/L) 0.38 0.32 0.31 Ca (mg/L) 90 64 69 K (mg/L) 1497 1673 1470 Mg (mg/L) 89 87 79 P (mg/L) 71 66 67 Na (mg/L) 32 29 31 Al (mg/L) 1.09 0.83 0.82 S (mg/L) 42 38 38

128

8.4 Discussion

8.4.1 Berry total soluble solids, pH and titratable acidity

In the Hills the absence of significant differences for berry TSS, pH and TA may have been

due to the absence of any effect of Mo-treatment on the vegetative growth or balance of those

vines (Chapter 5). At McLaren Vale in 2003-04, fruit on Mo-treated vines tended to ripen

earlier. This may have been a result of improved balance on those vines given that fruit on

well-balanced vines tends to ripen earlier than on unbalanced vines (Dry et al. 2005).

However, the same differences in composition were not observed in 2004-05, therefore no

conclusions can be drawn from those data. Furthermore, in both 2003-04 and 2004-05, Merlot

was harvested earlier than average (pers. comm., R. Birchmore 2005); therefore the small

differences observed in 2003-04 probably had no practical significance.

8.4.2 Nutritional composition

At McLaren Vale, the application of sodium molybdate to vines at MELS 12 did not affect

the concentration of molybdenum in the juice at harvest. However, in the Hills, rate 1-treated

vines produced fruit with a significantly higher concentration of molybdenum than the

controls. This is in agreement with the findings of Tilbrook (2006 unpublished data) who

measured a significantly higher concentration of molybdenum in wine made from Shiraz fruit

from vines that had been sprayed with sodium molybdate at flowering time. In the current

study, juice from rate 2-treated vines had approximately double the concentration of

molybdenum than the standard Mo-treatment; however, the concentration of molybdenum in

the juice did not reach a level that is likely to exceed the recommended daily intake for

humans (45 µg/day) (National Health & Medical Research Council 2005).

The differences between McLaren Vale and the Hills may be a consequence of different

levels of molybdenum in the vines at flowering time. In the Hills, the application of sodium

molybdate to vines which already had an adequate level of molybdenum probably resulted in

an abundance of molybdenum, which may have allowed it to be more readily transported to

the fruit. At McLaren Vale however, the application of sodium molybdate to Mo-deficient

vines was sufficient to improve fruitset, but may have been inadequate for its translocation to

the fruit.

If molybdenum is translocated to the fruit when it is in abundant supply, there may be

implications for growers who routinely apply sodium molybdate to vines as a means of

129

insurance against poor fruitset. If the vines already have an adequate supply of molybdenum

when sodium molybdate is applied, molybdenum may be transported to the fruit and end up in

the wine at potentially unsafe levels for human consumption. Further experimentation should

be carried out to measure the concentration of molybdenum in fruit and wine from vines

treated with varying levels of sodium molybdate.

8.5 Conclusions

• In most cases Mo-treatment did not affect TSS, pH or TA of berries despite its effects on

yield and vine balance.

• At McLaren Vale foliar application of sodium molybdate in spring did not affect the

concentration of molybdenum in the juice at harvest.

• In the Hills, the concentration of molybdenum in berry juice was higher in Mo-treated

vines than in controls. This may have implications for growers who spray vines with

sodium molybdate as a precautionary measure where the vines may not be Mo-deficient.

130

Chapter 9 Effects of mode of pollination on yield of Merlot and the interacting effects of sodium molybdate sprays

9.1 Introduction

The impacts of mode of pollination on yield in other crop species are well documented.

However, there is disagreement in the literature about pollination of grapevines. Numerous

factors point toward the existence of self-pollination or ‘autogamy’: complete pollination and

fertilisation; short life of the stigma; and ‘cleistogamy’, the ability for fertilisation to take

place under the intact calyptra (Lavee & Nir 1985). Many grape varieties self-pollinate

successfully and, in a large genetic study, it was found that only 1-2% of seeds are formed

through fertilisation with pollen from other sources (Antcliff 1980). However, self-pollination

is not obligatory (Suessengrath 1953 and Winkler 1962, cited in Pratt 1971).

Many researchers report that fruitset and yield of some grape varieties can be improved when

inflorescences are artificially pollinated with a mixture of pollen or are cross-pollinated

(Einset 1930, Olmo 1943 and Shoemaker 1955, cited in Free 1993; Hale & Jones 1956; Uppal

et al. 1975; Bordeu & Gil 1984; Ebadi 1996). In fact, in the 1950s, table grapegrowers in the

Swan Valley, Western Australia were advised to either interplant or artificially pollinate

Ohanez grapes in order to achieve commercially viable yields (Hale & Jones 1956). There are

few comprehensive reports or statistical data describing the effects of mode of pollination on

yield of commercial winegrape varieties under Australian conditions.

Pollination experiments were conducted on field-grown own-rooted Merlot (clone D3V14)

vines in commercial vineyards in the Adelaide Hills and at McLaren Vale in 2003-04 and

2004-05. Given the improvements in yield gained by spring foliar applications of sodium

molybdate to Mo-deficient Merlot vines (Chapter 6), in 2004-05 an additional treatment was

incorporated to observe the effects of pollination with Mo-treated pollen. In 2005-06 a

reciprocal experiment was conducted to separate the effects of Mo-treatment and mode of

pollination on the male and female flower parts. This involved application of supplementary

pollination treatments to both Mo-treated and Mo-deficient vines.

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9.1.1 Aims

The aims of these experiments were to:

• Examine the effects of different modes of pollination on components of bunch yield of

own-rooted Merlot (clone D3V14).

• Determine whether the male or female reproductive organs are more important in

determining the success of fruitset of Mo-deficient Merlot.

• Determine which remedial measure, Mo-treatment or pollination, is more effective in

overcoming poor fruitset.

132

9.2 Materials and methods

9.2.1 Vine and inflorescence selection

All experiments were conducted on own-rooted Merlot (clone D3V14) vines in commercial

vineyards in the Adelaide Hills and at McLaren vale (details provided in Chapter 3).

Experimental vines were selected for uniformity based on visual observation of trunk

circumference and canopy size. Each pollination treatment was applied to one inflorescence

per vine selected on the basis of uniformity of flower development.

Each inflorescence was enclosed inside a bag primarily to collect and count the caps shed

from the flowers. In 2003-04 inflorescences were enclosed inside paper bags; however,

problems with insect and weather damage led to a change to mesh fabric bags in subsequent

seasons (Figure 9.1).

Figure 9.1. A Merlot inflorescence enclosed inside a mesh bag at McLaren Vale. Note the shed caps in the bottom of the bag.

133

9.2.2 Experimental design

Adelaide Hills

In 2003-04 the pollination experiments were conducted on eight vines within the same row.

Three treatments were applied to each vine and an additional fourth treatment, supplementary

self- Mo-treated pollination, was added in 2004-05 (Table 9.1). Mo-treated pollen for the self-

Mo-treated pollination treatment was sourced from Mo-treated vines in the molybdenum

experiment (details provided in Chapters 3).

Table 9.1. Pollination treatments applied to Merlot (clone D3V14) in the Adelaide Hills in 2003-04 and 2004-05. All pollen was sourced from the Adelaide Hills.

Treatment Pollen variety

Open-pollination (Open) None Supplementary self-pollination (Self -Mo) Merlot Supplementary cross-pollination (Cross)

2003-04 2004-05

Zinfandel Semillon

Supplementary self Mo-treated-pollination (Self +Mo) Merlot (Mo-treated)

McLaren Vale

At McLaren Vale supplementary pollination treatments were applied to eight vines within two

neighbouring rows of vines, four replicates in each. Three treatments were applied to each

vine and in 2004-05 an additional treatment, supplementary self-Mo-treated pollination, was

added (Table 9.2). Mo-treated pollen for the self-Mo-treated pollination treatment was

sourced from vines that were treated with rate 1 sodium molybdate spray at modified E-L

stage (MELS) 12 (shoots 10 cm) (details provided in Chapter 3). The pollen source vines

were situated two rows to the south of the pollination experiment.

Table 9.2. Pollination treatments applied to Merlot (clone D3V14) at McLaren Vale in 2003-2005.

Pollination treatment Pollen variety Pollen source

Open-pollination (Open) None Supplementary self-pollination (Self-Mo) Merlot McLaren ValeSupplementary cross-pollination (Cross) Semillon Urrbrae Supplementary self Mo-treated-pollination (Self +Mo) Merlot (Mo-treated) McLaren Vale

134

In 2005-06 the pollination experiment was performed on vines which had not previously

received any molybdenum treatment. The experimental vines were within two rows of vines,

three replicates in each. Eight spray and pollination treatments (Table 9.3) were applied to the

vines in a completely randomised design. One vine on each side of the experimental vines

was sprayed to provide a buffer to the other vines in the row and a buffer row was left

unsprayed between the two rows of vines (details provided in Appendix B).

Vines were treated with either rate 0 (control) or rate 1 sodium molybdate sprays at MELS 12

applied using the same procedure as for the molybdenum experiments (details provided in

Chapter 3). Semillon pollen collected from the Coombe Vineyard at Urrbrae was used as the

pollen source for the cross-pollination treatment. A Merlot pollen collection area was

designated two rows to the south of the experimental vines at McLaren Vale. Vines in the

pollen collection area were sprayed with rate 0 and rate 1 sodium molybdate sprays (details

provided in Chapter 3).

Table 9.3. Spray and pollination treatments applied to Merlot at McLaren Vale 2005-06.

Molybdenum treatment Rate 0 Rate 1 Pollination treatment Cross Cross Open Open Self (-Mo) Self (-Mo) Self (+Mo) Self (+Mo)

9.2.3 Pollen collection and storage

Pollen was obtained by collection of whole inflorescences with 50-80% of their flowers open

in the afternoon of the day prior to pollination. To ensure adequate pollen supply, two

inflorescences were collected for each inflorescence to be pollinated. The inflorescences were

processed as follows:

1. Place the harvested inflorescences into a labelled paper bag and transport back to the lab in an insulated container.

2. Open the bags containing the inflorescences and lay them flat in a sealed vessel containing dry silica gel.

3. When the inflorescences are dry (1-2 days) remove from the bags and place into labelled, sealed plastic jars.

For short storage periods (1-2 days) the dried inflorescences were kept below 5°C or for

longer periods below 0°C.

135

9.2.4 Pollination

Pollination started once the first flowers on an inflorescence started to open and was repeated

approximately every second day until flowering was complete. Pollen was applied to the

inflorescences systematically, pollinating with one pollen type at a time, and care was taken to

avoid disturbing the flowers as much as possible. Pollination was performed using the

following procedure:

1. Enclose all inflorescences in mesh bags prior to flowering. 2. Wash hands with 70% ethanol and allow to air dry. Shake the jar containing the dried

inflorescences to release the pollen. Pollen from sufficiently dried inflorescences should stick to the walls and inside the lid of the jar.

3. Using a soft, clean artists brush, brush the inside walls and/or cap of the jar to ‘pick up’ the pollen in the bristles.

4. On the first vine, open the mesh bag taking care not to spill any shed flower caps. 5. Apply the pollen to the newly opened flowers by gently flicking the bristles of the

brush directing the pollen towards the flowers. Take care not to touch and disturb the flowers. Repeat this step 2-3 times ensuring that each newly opened flower has received pollen.

6. Replace and secure the mesh bag on the inflorescence. 7. Move to the next vine and repeat steps 2-6 until each vine has received that treatment. 8. Repeat steps 2-6 for each treatment. 9. Collect fresh pollen for pollinating in 2 days time using the pollen collection and

storage procedure described above

9.2.5 Fruitset and berry weight

The methods used for calculating fruitset and berry weight are described in Chapter 3.

In 2003-04 flower caps from inflorescences of the self-pollination treatment at McLaren Vale

were not collected. Therefore, fruitset was calculated using the average number of flower caps

from the other two pollination treatments. Fruitset for that treatment was not included in the

statistical analysis. In the Hills the flower caps were lost in 2003-04 because of insect and

weather damage to the bags. The average number of berries per bunch is presented as an

estimate of fruitset.

9.2.6 Nutritional analysis

In 2005-06 petiole analysis was performed on the pollinated vines and the vines in the pollen

collection area using the procedures described in Chapter 4. For the pollinated vines, petiole

samples were collected from each replicate; however, in the pollen collection area a single

sample was collected from each spray treatment.

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9.3 Results

9.3.1 Hills

Mode of pollination did not affect yield of Merlot in the Adelaide Hills in the two seasons of

experimentation. In 2003-04 there was a trend toward more berries per bunch for the cross-

and self-pollination treatments compared to the open treatment; however, this was not

statistically significant (Figure 9.2). Berry weight was also unaffected by pollination

treatment in 2003-04 (Figure 9.3). In 2004-05, both fruitset and berry weight were unaffected

by mode of pollination (Figure 9.2 and Figure 9.3).

0

40

80

120

160

200

Cross Self Open

Berr

ies

/ bun

ch

0

10

20

30

40

Cross Self Self+Mo Open

% F

ruits

et

Figure 9.2. Mean berry number per bunch 2003-04 (■) and percent fruitset 2004-05 (■) on bunches from pollination treatments: supplementary cross- (cross), supplementary self- (self), supplementary self- Mo-treated pollination (Self +Mo) & open-pollination (open) on Merlot in the Adelaide Hills. There were no significant differences between treatments (p<0.05).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cross Self Open

Ber

ry w

eigh

t (g)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cross Self Self+Mo Open

Figure 9.3. Average berry weights from bunches of the pollination treatments: supplementary cross- (cross), supplementary self- (self), supplementary self- Mo-treated (self+Mo) & open-pollination (open) on Merlot in the Adelaide Hills in 2003-04 (■) and 2004-05 (■). No significant differences were found between treatments in the same season (p<0.05).

137

9.3.2 McLaren Vale 2003-04 and 2004-05

In 2003-04 cross-pollinated bunches set significantly better than those from the open-

pollination treatment at McLaren Vale. Self-pollinated bunches also tended to have improved

fruitset compared to open-pollinated bunches. Mode of pollination did not affect berry weight.

In 2004-05 mode of pollination did not affect fruitset or berry weight of Merlot at McLaren

Vale (Figure 9.4).

b

a

AA

AA

0

10

20

30

40

50

Cross Self Self+Mo Open

% F

ruits

et

a aa

AA

AA

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Cross Self Self+Mo Open

Berr

y w

eigh

t (g)

Figure 9.4. Percent fruitset (left) and mean berry weight (g) (right) for pollination treatments: supplementary cross- (cross), supplementary self- (self), supplementary self- Mo-treated pollination (Self +Mo) & open-pollination (open) on Merlot at McLaren Vale in 2003-04 (■) and 2004-05 (■). Different letters above the columns represent significant differences between treatments within the same season (p<0.05). Because percent fruitset was estimated on the self-pollination treatment, this was not included in the statistical analysis.

9.3.3 McLaren Vale 2005-06

Nutritional analysis

At MELS 25 (80% flowering) in 2005-06 the petiolar molybdenum concentration of control

vines in the pollination experiment was within the range suggested to result in deficiency

(0.05-0.09 mg/kg). The petiolar molybdenum concentration on rate 1-treated vines was

significantly higher than the controls. In the pollen collection area the petiolar molybdenum

concentration of the rate 0-treated vines marginally exceeded the suggested deficiency range;

however, that value was from a single petiole sample and may have been subjected to

analytical error (Table 9.4). Given that all other samples taken from rate 0-treated vines in the

same vineyard in 2005-06 fell within the deficiency range (Table 9.4 and Chapter 4) it was

assumed that the vines in the pollen collection area were also Mo-deficient.

138

Table 9.4. Concentration of molybdenum (mg/kg) in Merlot petioles at modified E-L stage (MELS) 25 (80% flowering) at McLaren Vale in 2005-06. Vines were treated with rate 0 (control) and rate 1 molybdenum sprays at MELS 12 (shoots 10 cm). Letters after the values represent significant differences between treatments (p<0.05). The absence of letters after the values indicates that samples were not replicated and statistical analysis was not performed.

Molybdenum treatment Rate 0 Rate 1 Pollination vines 0.088 a 6.444 b Pollen source vines 0.095 7.449

Yield

Bunches from the Mo-treated vines had a significantly higher percent fruitset than those from

Mo-deficient vines, irrespective of pollination treatment. Within the Mo-deficient vines,

supplementary cross-pollination significantly improved fruitset compared to the open- and

self- (-Mo) pollination treatments. The self- (+Mo) pollination treatment also improved

fruitset, however it was not significantly different to the open- and self- (-Mo) pollination

treatments (Table 9.5). There were no significant differences in berry weight between

treatments (Table 9.6).

Table 9.5. Percent fruitset of bunches from the pollination experiment at McLaren Vale in 2005-06. Vines were treated with rate 0 (control) or rate 1 molybdenum sprays at modified E-L stage 12 (shoots 10 cm) and inflorescences were subjected to pollination treatments: supplementary cross- (Cross), supplementary self- Mo-treated pollen (Self +Mo), supplementary self- Mo-deficient pollen (Self –Mo) or open-pollination treatments at flowering time. Different letters within the same row represent significant differences (p< 0.05) (LSD=7.57).

* Significant difference between values within the same column (p<0.05) (LSD=8.01).

Pollination treatment Cross Open Self +Mo Self -Mo

Molybdenum treatment Rate 0 32 x 21 y 27 xy 22 y Rate 1 42 ab 43 a 34 b 39 ab * * * *

Table 9.6. Mean berry weight (g) from the pollination experiment at McLaren Vale in 2005-06. Vines were treated with rate 0 (control) or rate 1 molybdenum sprays at modified E-L stage 12 (shoots 10 cm) and inflorescences were subjected to pollination treatments: supplementary cross- (Cross), supplementary self- Mo-treated pollen (Self +Mo), supplementary self- Mo-deficient pollen (Self –Mo) or open-pollination treatments at flowering time. There were no significant differences between treatments (p< 0.05).

Pollination treatment Cross Open Self +Mo Self -Mo

Molybdenum treatment

Rate 0 1.12 1.09 1.18 1.11 Rate 1 1.20 1.21 1.11 1.27

139

9.4 Discussion

Mode of pollination did not affect either fruitset or berry weight of Merlot over the two

seasons of experimentation in the Adelaide Hills. As discussed in previous chapters (Chapter

4, Chapter 5 and Chapter 6) vines at the Hills experimental site had an adequate level of

molybdenum at flowering time which may have been sufficient for the inflorescences to

achieve optimal fruitset.

At McLaren Vale none of the pollination treatments affected berry weight however, cross-

pollination improved fruitset in two out of three seasons. In 2005-06 when the supplementary

pollination treatments were applied to inflorescences on both Mo-treated and Mo-deficient

vines, cross-pollination improved fruitset on bunches only on Mo-deficient vines. This

suggests that the female gametophyte may play a greater role in the determination of fruitset

of Merlot than the male flower parts.

9.4.1 Effects of mode of pollination on berry size

In the current study berry weight was not affected by mode of pollination suggesting that the

number of fertilised ovules per ovary and subsequent seed numbers were unaffected. In grape

flowers, ovule development is complete at the time of flowering; therefore neither type nor

amount of pollination can affect the proportion of functional ovules available for fertilisation

(Stout 1936). This suggests that the full complement of ovules in an ovary may not be

available for fertilisation, and therefore the number of fertilised ovules could not be improved

by other pollination treatments. This supports the finding reported in Chapter 7 that Mo-

deficient Merlot vines produce a higher proportion of faulty ovules which in turn affects their

ability to attract pollen tubes.

9.4.2 Effects of mode of pollination on fruitset

The improvement in fruitset in bunches on Mo-deficient vines supplied with supplementary

Merlot pollen at McLaren Vale in 2003-04 hints that Merlot might not be able to produce a

sufficient supply of pollen for adequate fruitset. However, failure for that treatment to

improve fruitset in subsequent seasons does not support that theory.

At McLaren Vale, cross-pollination of Merlot inflorescences with Semillon pollen

significantly improved fruitset on bunches on Mo-deficient vines in two out of three seasons.

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This suggests some level of self-incompatibility. However, spraying vines with sodium

molybdate in springtime had a greater effect on fruitset than supplying inflorescences on Mo-

deficient vines with supplementary pollination treatments.

It is not possible to discriminate between self-incompatibility at the pre- or post-zygotic

stages. Inhibition of pollen tube growth in ovaries of Mo-deficient vines (Chapter 7) suggests

that Mo-deficiency may contribute to self-incompatibility at the pre-zygotic stage (Sedgley &

Griffin 1989). The improvement in fruitset with cross-pollination suggests that those pollen

tubes are more successful at reaching the ovules and fertilisation taking place. In other species

post-zygotic self-incompatibility, acting at the early stages of embryo development, has been

associated with small fruit, which may also drop prematurely as a result of genetic selection

(Degani et al. 1986, cited in Sedgley & Griffin 1989). These characteristics are typical of

ovary abortion or poor fruitset in Mo-deficient Merlot.

Potential mechanism for molybdenum to overcome self-incompatibility

In some species ovule development occurs asynchronously, thereby reducing the availability

of functional ovules at flowering, which in turn reduces the fertilisation potential. However,

when those species are cross-pollinated, the number of functional ovules increases, a

phenomenon thought to occur via stimulus from the polleniser (Pimienta and Polito 1983;

Sage et al. 1999). If Mo-deficiency delays ovule development in Merlot such that there are

fewer functional ovules available for fertilisation at flowering (as discussed in Chapter 7) then

perhaps cross-pollination provides the stimulus required for ovule development.

9.4.3 Supplementary pollination or Mo-treatment?

When Merlot vines are Mo-deficient, fruitset may be improved by cross-pollination. However

cross-pollination did not improve fruitset to the same level as that achieved by rectification of

the effects of Mo-deficiency. This finding is similar to that of Ebadi (1996) who found that

supplementary pollination could only partially reverse the effects of low temperature on

fruitset.

As previously discussed, Mo-treatment can be applied in consecutive years without any

detrimental effect on yield (Chapter 6). However, the long-term effects of sodium molybdate

sprays on vines and soil are unknown. Artificial pollination, while not producing equal

improvement in fruitset as Mo-treatment, may be considered as a more ‘environmentally

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friendly’ option to improve yield. On the other hand, as seen in the current study, artificial

pollination may not always produce consistent results because of the interacting effects with

the environment. Therefore the use of artificial pollination should not be relied upon to

improve yield.

9.5 Conclusions

• Supplementary cross-pollination significantly improved fruitset on bunches from Mo-

deficient Merlot vines; however, it had no effect on berry weight. The improvement in

fruitset achieved with supplementary cross-pollination was not as great as that

achieved by treatment of vines with sodium molybdate sprays.

• When Merlot vines had adequate levels of molybdenum, cross-pollination did not

affect either fruitset or berry weight.

• Mo-deficiency appears to primarily affect the female part of the flower. This effect

can be moderately improved by the addition of Mo-treated pollen.

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Chapter 10 The occurrence of star flowers in Australia

10.1 Introduction

‘Star’ or ‘avalidouire’ flowers were described in the French literature as early as 1866

(Planchon 1866), and much later star flowers feature in descriptions of flower aberrations by

several authors (for example Pratt 1971). Star flowers differ from normal Vitis flowers in that

the petals open freely from the top in star formation similar to those of the genus Cissus. This

is in contrast to the release of the petals of normal Vitis flowers, which abscise from the base

of the calyptra and subsequently shed as a ‘cap’.

The structure of star flowers is also reported to be anomalous, resulting in male sterility and in

some cases, an inability to set fruit (Mares 1865 cited in Planchon 1866, Portele 1883,

Despeissis 1921, Pratt 1971). In fact, the star flower condition has been reported as an

hereditary ‘disease’ (causing infertility), that is transmissible by vegetative propagation and

cured only by grafting or vine removal (Planchon 1866, Despeissis 1921).

During the flowering period of spring-early summer in 2003, aberrant flower types were

observed on Vitis vinifera x Vitis labrusca cv. Canada Muscat and Vitis vinifera cvs. Gamay

and Pinot Meunier vines in the Coombe Vineyard on the University of Adelaide’s Waite

Campus. Prior to this there had been no previous reports of star flowers in Australia.

Dissemination of information about Canada Muscat star flowers resulted in reports of other

variants of star flowers.

This chapter describes flowering and the structure of Canada Muscat, Chardonnay and Shiraz

star flowers. Observations of other star flower variants are also presented.

10.1.1 Aims

• To describe the morphology and development of star flowers and the resultant berries.

• To observe the functionality of the male and female organs of Canada Muscat star flowers

by cross-pollination with another variety.

• To determine whether the star flower phenotype is a ‘disease’.

• To compare and contrast other star flower variants.

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10.2 Materials and methods

10.2.1 Nomenclature

In the text, flowers, berries, bunches and vines may be referred to as either ‘star’ or ‘normal’.

Preceding ‘berry’ those prefixes refer to the flower type from which the berry arose. The

prefixes refer to the predominant flower type produced on the inflorescence or vine.

10.2.2 Microscopy

Pollen tubes and ovule structure

Samples for observation of pollen tubes and ovules were prepared using the procedures

described in Chapter 3.

Scanning electron microscopy

Fresh samples were placed in electron microscope (EM) fixative (1.25% EM grade

glutaraldehyde, 4% sucrose, 4% paraformaldehyde in phosphate buffered saline (PBS) buffer

[0.70g di-sodium hydrogen orthophosphate, 0.16g potassium dihydrogen orthophosphate,

8.50g NaCl in 1L distilled water, pH 7.2]) and stored at room temperature until further

processing following the schedule below.

1. Fix in EM fixative for at least 30 minutes 2. Wash in PBS + 4% sucrose – two changes of 5 minutes each. 3. Post-fix in 1% OsO4 in H2O for 1 hour. 4. Dehydrate in a series of 70%, 90%, 95% – two changes of 10 minutes each, followed

by 100% ethanol - two changes of 10 minutes plus one change of 30 minutes. 5. Dry samples in Balzers critical point drier 6. Mount samples on stubs and coat with 100 angstrom spectroscopically pure carbon

and 200 angstrom gold palladium (pers. comm., Adelaide Microscopy 2004)

All samples were observed and photographed using a Philips XL20 scanning electron

microscope.

10.2.3 Berry classification

Berries were categorized as ‘hens’, ‘chickens’ or ‘live green ovaries’ (LGOs) according to the

classification outlined in Chapter 3.

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10.2.4 Flower and berry development on Canada Muscat

Observations were made during the flowering period of spring-early summer in 2003-2005 on

three Canada Muscat vines growing in the variety collection of the Coombe Vineyard at The

University of Adelaide’s Waite Campus. All vines were on their own roots and were

approximately 10 years old in the first year of observation.

In 2004 observations were made to elucidate whether it was indeed possible for star flowers

to set normal seeded berries. At the beginning of flowering seven inflorescences were tagged

with coloured flagging tape and the pedicels of each star flower were marked with coloured

acrylic ink applied with a Rotring technical isograph pen (Gray 2002) (Figure 10.1). Over the

next three weeks newly opened star flowers were marked in a similar manner and their

development was monitored.

Figure 10.1. A Canada Muscat inflorescences with the pedicels of star flowers marked with red acrylic ink (indicated by arrows).

10.2.5 Cross pollination of Canada Muscat

On 29 October 2003, four Canada Muscat inflorescences were selected for uniformity by

visually assessing the proportion of opened flowers, and were enclosed inside paper bags. At

this stage all flowers were opening in star formation. Two of these inflorescences were not

supplied with any additional pollen (OP) and two were supplied with pollen from the variety

‘Baco Noir’ (CP). Baco Noir was chosen due to its early flowering. Inflorescences intended as

a pollen source for cross-pollination of Canada Muscat were harvested in the afternoon prior

to pollen application. On each harvest date, two Baco Noir inflorescences, with approximately

50-80% of the flowers open, were selected to ensure an adequate amount of pollen. These

Pollen tube growth

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inflorescences were collected in labelled paper bags and returned to the laboratory where they

were allowed to air-dry overnight at room temperature.

The following day the Baco Noir inflorescences were placed inside a labelled plastic vial.

Using a soft artist’s brush, the pollen was brushed from the sides of the vial and gently flicked

onto the freshly opened flowers of Canada Muscat. This was repeated every two to three days

during the early flowering period. Application of supplementary pollen to these inflorescences

ceased after approximately 14 days when the flowers began to open normally. The four

bunches were harvested approximately three weeks after pollination ceased. The berries were

removed from the rachis, counted and weighed.

10.2.6 Double pruning of Canada Muscat

In 2003, one of the Canada Muscat vines was double-pruned after fruitset so that the vine

would produce a second generation of flowers in the same season (Dry 1987). This pruning

intervention was intended to test the assertion by Planchon (1866), and Despeissis (1921) that

star flowers on a given grapevine will occur in each and every generation of flowers. On 26

November 2003, approximately three weeks after spring flowering had finished, the Canada

Muscat vine was pruned to a total of eight, six-node shoots (all bunches and lateral shoots

were removed). That action promoted burst of latent buds (which were in a state of

paradormancy), and flowering on this double-pruned vine commenced on 27 December 2003.

10.2.7 Observations of Chardonnay and Shiraz star flowers

Following dissemination of the description of Canada Muscat star flowers in Longbottom et

al. (2004) other possible star flower variants on Chardonnay and Shiraz were reported. The

Chardonnay star flowers were collected from three vines growing in the University of

Adelaide’s Alverstoke orchard, Waite Campus. While a thorough examination or description

of the flowers had not previously been undertaken, the three vines were known to produce

bunches of seedless berries. Henceforth these vines will be known as ‘star vines’.

In 2005-06, general observations of the Chardonnay vines were made. At around the time of

commercial harvest a random selection of bunches was collected from both star and normal

Chardonnay vines. A photographic comparison of bunches was made to illustrate the

difference in size of the bunches from the two vines. Bunches were individually weighed and

the berries removed from the rachis for closer observation. The berries from the star vines

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were separated into four categories (Figure 10.2) whilst the berries of the normal vines were

separated into either hens or chickens. The seeds were removed from the berries of each

bunch type, cleaned, weighed and arranged in size categories to illustrate the proportions of

each.

The Shiraz star flowers were collected from vines grown in the Orlando Wyndham,

Richmond Grove vineyard at Padthaway, South Australia. The vines had been noted to

produce abnormal flowers; however, identification of the aberration and observations past

flowering had not been previously performed. General observations were made in the 2005-

06 season.

10.2.8 Other star flower variants

Further star flower variants were observed in commercial vineyards at Padthaway,

Coonawarra and in the Adelaide Hills, South Australia.

A

B

C

D

Figure 10.2. Photographic characterisation of berry and seed development of star Chardonnay. Photographs of the categories are arranged in pairs to represent the initial grading made looking through the uncut berry (left) and an actual representation of the exposed seeds / seed traces (berries dissected longitudinally through the centre). A: no visible seed trace, B: visible seed trace, C: solid, short seed trace, D: normal seed.

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10.3 Results and discussion

10.3.1 Structure and development of Canada Muscat star flowers

The first recorded observations of Canada Muscat were made on 8 October 2003, shortly after

commencement of flowering. Canada Muscat was the first variety in the collection with

flower and flowering lasted for a particularly long period (approximately 6 weeks) compared

to other varieties. At the outset the Canada Muscat flowers appeared immature. Flower buds

were bright green and more compact than normal Vitis flowers. The earliest flowers began

opening from the top of the calyptra (Figure 10.3) in contrast to normal flower opening which

starts when petals abscise from their base and lift up to shed the cap (Figure 10.4).

Figure 10.3. Star flowers on a Canada Muscat inflorescence from the variety collection in the Coombe Vineyard at the Waite Campus at The University of Adelaide on 12 October 2003.

Figure 10.4. The progression of flowering of normal Vitis vinifera flowers. A: The petals separate from the base of the calyptra, B: the petals curl upward and C: the cap lifts off releasing the anthers and exposing the stigma.

A BC

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The early Canada Muscat star flowers were very slow to open and the anthers were short and

tended to stick inside the petals (Figure 10.5). The anthers were sometimes irregularly shaped

and remained intact well after flower opening. In some, but not all cases, the anthers were

pale in appearance indicating pollen sterility (May 2004) compared to the anthers of normal

flowers which were bright yellow. At this stage most star flowers did not have any nectaries.

In some, but not all cases, the star flowers had a necrotic patch at the tip of the flower bud and

on opening it became evident that the necrosis extended to the anthers (Figure 10.6). This

description of star flowers is in agreement with that of Mares (1865 cited in Planchon 1866).

When whole inflorescences were brought into the laboratory and held for 24 hours, the

flowers did not open readily nor did the anthers dehisce.

Figure 10.5. Canada Muscat star flowers showing A: The petals start to separate from the top of the compact flower bud and remain attached at the base. Anthers remain intact inside the petals and B: the relative size of the filaments and anthers (petals have been removed). C: A normal Shiraz flower after capfall showing the relative size and position of the stamens (NB. The anthers have dehisced).

Figure 10.6. Canada Muscat star flowers at different stages of opening showing necrotic patches on the tips of the petals extending to the anthers.

B C

A

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150

Approximately one month after the first observations of the star flowers on Canada Muscat in

2003 the flowers appeared to be developing more normally. Those flowers elongated and

changed colour from bright green to pale green/yellow before opening in star formation. The

stamens were standing erect and the anthers were released from inside the petals (Figure

10.7). The anthers of some flowers dehisced normally (Figure 10.8 and Figure 10.9);

however, it was more commonly observed that the anthers remained intact and eventually

necrosed. When the flowers were newly-opened the stigma appeared shiny and receptive, and

pollen was clearly visible (Figure 10.10 and Figure 10.11) on the stigma.

Figure 10.7. A Canada Muscat star flower with the stamens standing erect and the anthers separated from the petals. Anthers are pale in appearance, have necrotic patches developing and have not dehisced.

151

Figure 10.8. A scanning electron micrograph of a Canada Muscat star flower. The petals separate from the tip of the calyptra to expose the stigma. Note that the anthers have begun to dehisce (indicated by arrow).

Figure 10.9. A side view of a Canada Muscat star flower showing the stigmatic surface and a dehisced anther.

152

Figure 10.10. Pollen grains (indicated by arrows) being released from the anther of a Canada Muscat star flower.

Figure 10.11. Pollen grains (indicated by arrows) on the surface of the anthers of a Canada Muscat star flower.

153

Approximately five weeks after the beginning of flowering, newly-opened flowers on the

Canada Muscat vines opened normally, that is, the petals detached from the base of the flower

(Figure 10.12). A high proportion of the normal flowers were cleistogamous and self-

pollinating prior to capfall. The ovaries of these flowers enlarged beneath the lifted but

persistent cap (Figure 10.13). In some cases, both star flowers and normal flowers were found

on the same branches of an inflorescence (Figure 10.14).

Figure 10.12. A scanning electron micrograph of a normal Canada Muscat flower showing relative sizes of the male and female flower parts. Anthers are dehisced, nectaries (n) present.

Figure 10.13. Swelling of an ovary beneath the lifted but persistent calyptra (cleistogamy) of a Canada Muscat flower

A

n

A

B

n

Figure 10.14. A branch of Canada Muscat with A: a normal flower shedding its cap. Note also the presence of nectaries (n), and B: a star flower with persistent petals and stamens.

10.3.2 Pollen tube growth in Canada Muscat star flowers

On 13 October 2003, pollen grains could be seen on the stigmas of about two-thirds of the star

flowers observed and, in one-third of the flowers, pollen tubes were observed in the style

(Figure 10.15). The presence of a pollen tube in the style suggests that the stigma and style of

the star flowers were sufficiently developed to support pollen tube growth, and, potentially,

fertilisation.

Figure 10.15. Photographs of the stigma and style of two Canada Muscat star flowers taken using fluorescence microscopy showing A: pollen grains on the stigma and B: pollen tubes in the style.

In 2005 it was hoped that with further observations of pollen tube growth in Canada Muscat

star flowers, changes in stigma receptivity from early to later flowering star flowers could be

A

B

154

155

determined. However, in spring of 2005, the period of time from the beginning of flowers

opening in star formation until the flowers started opening normally (two weeks) was much

shorter than in the previous seasons.

Observations on Canada Muscat flowers revealed that normal flowers were more likely to

have pollen tubes growing on the stigma than star flowers. The frequency of stigmas with

pollen tubes increased later in the flowering period. Normal flowers also tended to have more

pollen tubes in the style and ovary than star flowers. None of the star flower pistils observed

had ovules that had been penetrated by a pollen tube (Table 10.1).

Table 10.1. Observations on star and normal Canada Muscat flowers collected from the Coombe Vineyard at the University of Adelaide’s Waite campus in 2005. Different letters after the values represent significant differences (p<0.05).

These data suggest that normal flowers may be more conducive to pollen tube growth than

star flowers. As discussed in Chapter 7, the success of pollen tube growth may be affected by

differences in the structure or chemical composition in the transmitting tissue, or pollen tube

signalling along the pathway from stigma to ovule. It may be that the stigma and transmitting

tissue of star flowers is immature at the time of opening and therefore incapable of supporting

pollen tube growth. Later in the flowering period as the newly opening flowers are more

mature, pollen tube growth may be sustained.

10.3.3 Fruitset of Canada Muscat star flowers

In 2003, swelling of the ovaries of the star flowers was first noted on 27 October, three weeks

after the beginning of flowering. All of the berries produced from star flowers observed in

2003 were seedless chicken berries. The petals and stamens of the star flowers persisted at the

base of the flower (Figure 10.16) as described by Mares (1865 cited in Planchon 1866) and in

Portele (1883).

Flower type Date collected

# pistils examined

Incidence of pollen tube presence on

stigma (%)

Average # pollen tubes on stigma /

upper style

Average # pollen tubes in lower

ovary

Average # penetrated

ovules / ovary

# ovules per ovary

Star 14.10.05 13 8 0.5 ab 0.1 ab 0 a 4 Star 18.10.05 19 21 0.4 a 0 a 0 a 4 Normal 18.10.05 10 50 4.7 b 0.7 b 0 a 4 Normal 31.10.05 8 75 4.9 b 1.1 b 0 a 4

Figure 10.16. A swollen ovary of a Canada Muscat star flower. Note the persistent stamens and petals at the base of the berry.

In 2004, the first Canada Muscat flowers were noted as opening in star formation earlier than

in the previous season (22 September) and had similar features to those observed in 2003. The

period of star flower opening lasted approximately three weeks (until 12 October) when

flowers began to open normally. Over the following few days the star flowers began to set.

While many of the ovaries initially began to swell, they subsequently became necrotic and

abscised from the rachis, a phenomenon described by Portele (1883) as associated with

flowers whose anthers are shorter than the ovaries. The necrotic ovaries were still present on

the bunches on 17 December (Figure 10.17); however, a small number of the star flowers

remained attached and continued to develop.

Approximately one month after ovary abscission began, full sized berries with petals stuck to

their base were observed indicating that they had developed from star flowers. Those berries

were removed from the bunch and photographed alongside berries derived from normal

flowers on the same bunch for size comparison (Figure 10.18). Each berry derived from a star

flower contained at least one seed demonstrating that star flowers can set normal seeded

berries (Figure 10.19).

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Figure 10.17. Canada Muscat inflorescences showing setting berries and necrotic ovaries abscising from the rachis (indicated by arrows) A: 28 October 2004 and B: 17 December 2004

Figure 10.18. Typical Canada Muscat berries from A: normal flowers and B: star flowers, 19 November 2004. Note the persistent petals and stamens (indicated by arrow).

A B

A

B

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A

B

158

Figure 10.19. Seed development in Canada Muscat berries from A: normal and B: star flowers, 19 November 2004.

10.3.4 Ovule structure of Canada Muscat star flowers

Preliminary observations of Canada Muscat ovules collected in 2005 revealed that they were

typical for Vitis and there were no differences between normal and star flowers (Figure

10.20). This indicates that the ovules were mature and fertilisation could occur.

However, due to the particularly short period of star flower opening in 2005, the pistils

collected for structural analysis were collected relatively late in the star flower opening

period. As previously discussed, as flowering progressed, the newly opening star flowers

changed in appearance from immature to mature. This is supported by observations of more

frequent pollen tube growth on the stigma later in the flowering period (section 10.3.2). It is

likely that earlier in the flowering period ovule development is also incomplete, therefore

fertilisation cannot occur, and those flowers abscise from the rachis. Star flowers that open

later are functionally mature and are capable of setting normal seeded fruit.

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Figure 10.20. Typical ovules from Canada Muscat A: normal and B: star flowers. The inner (ii) and outer (oi) integuments are complete and embryo sac (es) is present

10.3.5 Cross-pollination of Canada Muscat star flowers

Bunches that were supplied with supplementary pollen from another variety (CP) tended to

have larger berries than those that had other pollen sources excluded (OP) (Table 10.2)

indicating more seeds per berry. From these preliminary results, pollination of Canada Muscat

flowers with Baco Noir pollen resulted in more successful fertilisation.

It is assumed that the earliest opening star flowers were not affected by pollination treatment

given that they were probably functionally immature. The improvement in berry size with

cross-pollination may have been from both star and normal flowers, a consequence of either

an inadequate pollen supply or else poor pollen viability brought about by the presence of star

flowers. Self-incompatibility may be ruled out on the grounds of observed cleistogamy. By

implication, pollination had occurred within the flowers prior to capfall.

B

es

oi ii

A

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Table 10.2. Results of supplementary cross-pollination (CP) and open-pollination (OP) of Canada Muscat in 2003. Statistical analysis not performed.

Treatment Berries / bunch Berry weight (g) CP # 1 28 0.63 CP # 2 64 0.73 OP # 1 41 0.39 OP # 2 37 0.29

10.3.6 Second generation flowering of Canada Muscat

The second generation flowers produced on the double-pruned vines all opened normally

(from their bases) and shed their calyptra. In the case of Canada Muscat, and in contrast to

suggestions by Planchon (1866) and Despeissis (1921), where aberrant star flowers occur on a

given grapevine they are not necessarily a feature of each and every generation of flowers.

This result suggests that star flowers were produced on Canada Muscat in response to

environmental conditions during flower development.

Canada Muscat was observed to be the earliest variety in the Waite variety collection to go

through budburst and flowering in three seasons. As a result the developing flowers were

exposed to the low temperatures prevalent in early spring. This suggests that Canada Muscat

has a genetic predisposition to produce star flowers when they are exposed to low

temperature. As flowering progressed and the temperature increased, flowers tended to form

normally.

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10.3.7 Observations of star flowers on Chardonnay and Shiraz

Vegetative growth of star Chardonnay and Shiraz vines

The vegetative growth of the star and normal Chardonnay vines was dissimilar. Normal

Chardonnay leaves are only slightly lobed, almost hairless, and the petiolar sinus is U-shaped

with a naked base (Kerridge & Antcliff 1999). Leaves from the vines that produced star

flowers were shiny, hairless and had a wide open V-shaped petiolar sinus with a naked base

(Figure 10.21). Inflorescences from the star and normal Chardonnay vines were similar in size

(Figure 10.22). There were no differences in the vegetative growth of Shiraz vines with

normal or star flowers. Inflorescences with normal flowers were similar in size and

appearance to those with star flowers (Figure 10.23).

Figure 10.21. Typical leaves from Chardonnay vines with A: normal flowers and B: star flowers

A B

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Reproductive development of star Chardonnay and Shiraz vines

The inflorescences of star and normal vines were similar in length and structure, however

shorter pedicels on star inflorescences gave them a more compact appearance (Figure 10.22

and Figure 10.23).

Figure 10.22. Inflorescences from Chardonnay vines that produce A: normal flowers and B: star flowers.

1 cm

B

5 cm

5 cm

1 cm

A

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Figure 10.23. Shiraz inflorescences with A: star and B: normal flowers 28th November 2005.

Prior to opening, flowers on star Chardonnay vines were similar in appearance to immature

normal flowers. Star flowers were smaller and more compact than normal flowers. The pistils

of star flowers were round compared to the elongated pistils of normal flowers and contained

small, round ovules. Star flower anthers were pale in appearance and filaments were shorter

than in normal flowers (Figure 10.24). In many cases the stigma appeared undeveloped and

nectaries were absent. This is in agreement with the description by Portele (1883) of flowers

with an undeveloped stigma.

Chardonnay and Shiraz star flowers began opening precociously. They remained in compact

groups (Figure 10.25) and resembled immature normal flowers (Figure 10.26). Both

Chardonnay and Shiraz star flowers had bright pink/red pigmentation at the tips of their

petals. This pigmentation tended to fade as the flowers matured. The inner organs of the star

flowers continued to develop over time. As the pedicel lengthened the stigma took on a more

normal appearance and the ovary began to swell. Late in their development the star flower

petals tended to reflex (Figure 10.27 and Figure 10.28).

For both Chardonnay and Shiraz, normal and star flowers were observed on the same

inflorescences towards the end of the flowering period (Figure 10.29).

A B

Figure 10.24. Chardonnay flowers dissected longitudinally just prior to opening on 19 October 2005, A: normal, B: star flower. Note the differences in shape of the flower buds and the relatively small size of the ovules and the pale coloured anthers of the star flower. The anther in the foreground of B has been positioned deliberately to more clearly expose the inner organs of the flower.

Figure 10.25. The onset of flower opening of star Chardonnay 29 September 2006. Flowers remain in compact groups.

Figure 10.26. Chardonnay flowers A: a normal immature flower with petals removed to show the relative sizes of the developing pistil and anthers 19 October 2004, and B: a star flower at the onset of flower opening 18 October 2004.

A B

A B

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165

Figure 10.27. Typical Chardonnay star flowers. A: early in development the ovary is small and B: later in development when the ovary has enlarged.

Figure 10.28. Stages of opening on Shiraz star flowers, 15 November 2005. A: Early separation of the petals at the tip of the flower, B: Petals fully opened and anthers released, C: Petals reflexed and anthers erect.

C

B A

A

B

166

Figure 10.29. A: Star and B: normal flowers on the same Shiraz inflorescence 30th November 2005.

10.3.8 Pollen tube growth in Chardonnay and Shiraz star flowers

No pollen was observed on the stigmas of the Chardonnay star flowers and no pollen tubes

were observed in the style. Many of the star Chardonnay pistils had hollow styles and the

ovules in the dissected pistils appeared abnormal (Figure 10.30).

No pollen tubes were observed in Shiraz star flowers. This was in contrast to proliferous

pollen tube growth and ovule penetration observed in normal Shiraz pistils collected on the

same day. The ovules of star Shiraz also appeared abnormal compared to those from normal

flowers (Figure 10.31). This is discussed in more detail in section 10.3.9.

A

B

A B

Figure 10.30. A star Chardonnay pistil collected 18 October 2005 showing A: no pollen grains on the stigma and no pollen tubes in the style and B: a hollow section in the style (indicated by arrow) and aberrant-shaped ovules.

Figure 10.31. A: Pollen tube growth and ovule penetration in a normal Shiraz ovary and B: aberrant ovules of a Shiraz star flower.

A

B

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10.3.9 Ovule development in Chardonnay and Shiraz star flowers

Mature grapevine ovules consist of a massive nucellus, two integuments and a funiculus

(Reiser and Fischer 1993). Ovules develop anatropically, that is, the main body is bent down

against its stalk so that the micropyle is directed towards the placenta (Pearson 1933; Fritsch

& Salisbury 1965). The integuments continue to enlarge such that they overgrow the nucellus

(Reiser & Fischer 1993) (Figure 10.32).

Ovules from star Chardonnay and Shiraz were similar in appearance; however, they did not

resemble ovules from normal flowers. Ovules from star flowers had incomplete inner and

outer integuments which formed a cap-like structure over the nucellus. Given that ovules from

normal flowers develop anatropically, the incomplete integuments of the star flower ovules

suggest that the ovules were arrested in development (Figure 10.33 and Figure 10.34).

Evidence of an embryo sac was observed in some, but not all, star flower ovules. Existence of

the egg cell, two synergids and the two polar nuclei or a polar fusion nucleus could not be

confirmed.

Figure 10.32. Anatropic ovule development.

A. Ovule shortly after initiation, showing a single megasporocyte (ms). nu, nucellus.

B. Ovule after both integuments have been initiated. At this time, the megasporocyte has undergone first meiotic division. The axis of the nucellus is transiently perpendicular to the axis of the funiculus (fu). ii, inner integument; oi, outer integument.

C. Ovule after meiosis. The functional megaspore (fm) at the chalazal end has expanded, and the non-functional megaspores are degenerated. The axis of the nucellus is now parallel to the funiculus due to unequal growth, primarily of the integuments. Dm, degenerate megaspores.

D. Ovule after megagametogenesis. The mature embryo sac contains seven cells and eight nuclei.

Reproduced from Reiser & Fischer (1993)

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Figure 10.33. Typical ovules from A: normal and B: star Chardonnay flowers.

Figure 10.34. Typical ovules from Shiraz star flowers

A similar ovule defect to that observed in Chardonnay and Shiraz star flowers was reported

for the varieties ‘Red’ and ‘White Corinth’. Those ovules were misshapen and did not become

anatropous; however, in those varieties the inner integument was absent and the nucellar

B

Bunch development

A

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tissue overgrew the outer integument (Pearson 1933). In Red and White Corinth, berry

development is parthenocarpic.

10.3.10 Star bunch, berry and seed development on Chardonnay and Shiraz

Chardonnay

In 2005 an inspection of the star Chardonnay vines revealed that a large proportion of

bunches were shedding the ovaries at the time (Figure 10.35). This was similar to the

observation made on Canada Muscat (Figure 10.17).

Figure 10.35. An inflorescence of star Chardonnay shedding its ovaries, 22 November 2005.

At maturity the remaining bunches on those vines had mostly small berries, compared to

normal sized berries on bunches from normal vines (Figure 10.36). Berries from star bunches

were mostly true chicken berries given that they contained only small seed traces or small

abnormally formed seeds. This is in contrast to the normal berries which contained mostly

normal seeds (Figure 10.37). The lack of pollen or pollen tubes on star Chardonnay suggests

that berry development on star Chardonnay (10.3.8) may be parthenocarpic.

A B

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Figure 10.36. Typical bunches from A: star and B: normal Chardonnay at maturity

Figure 10.37. Seeds extracted from the berries of a star bunch (above the horizontal line) and a normal bunch (below the horizontal line) showing the relative proportions of seeds from each bunch in each of the four categories; 1: enlarged, solid seed traces, 2: small seeds with abnormal appearance, 3: small seeds with normal appearance, 4: seeds of normal size and appearance. Scale bar represents 1cm.

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Shiraz

In 2005-06, Shiraz star bunches were arrested in development after flowering. All bunches on

star vines necrosed but were still present on the vines at veraison (Figure 10.38). Failure of

the ovaries to develop into berries suggests that the mutation of star Shiraz may be different to

that of the star Chardonnay.

Figure 10.38. Bunches from A: star and B: normal Shiraz, 28 January 2006

A B

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10.4 Other star flower variants

The star flower aberration, while previously unreported in Australia, seems to have been

present; however, it may have been overlooked or regarded as unimportant. In 2006,

Sreekantan et al. reported different gene expression patterns in two grapevine mutants that

produced aberrant flowers. Emphasis was placed on abnormalities of the filaments and

anthers and functionality of the flowers. However, the photographs provided (Figure 10.39)

clearly show that the flowers of both mutants also opened in star formation. No reference to

this peculiarity was made by those authors.

Figure 10.39. Dissected flowers of wild type grapevine and the Mourvèdre and Bouchalès mutants before flowering (A) V. vinifera wild type, (B) Mourvèdre mutant showing carpelloid structures (sic) and pseudo-ovules (sic) and (C) Bouchalès mutant with double row of petals and pseudo-anthers (sic) prematurely opened. a, Anther: a*, pseudo-anther; c, carpelloid structure; o, ovule; o*, pseudo-ovule; ov, ovary; p, petal; s, stigma; sep, sepal. Scale bars = 1 mm (reproduced from Sreekantan et al. 2006).

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Fungal infection

In 2005 Cabernet Sauvignon (clone G9V3) flowers opening in star formation were observed

in the Fosters Wine Estates’, Cluny Vineyard at Coonawarra, South Australia. The vines in

that vineyard displayed symptoms of infection with Phomopsis on the shoots (Figure 10.40)

and leaves. All star flowers had a necrotic area at the tip of the calyptra (Figure 10.41).

Phomopsis was isolated from the necrotic regions of the flowers (pers. comm., B. Hall 2005).

Figure 10.40. Phomopsis infection on a Cabernet Sauvignon shoot at Coonawarra, 15 November 2005.

Figure 10.41. Phomopsis infection on Cabernet Sauvignon flowers from Coonawarra causing them to open in star formation, 15 November 2005.

Flowers affected by glyphosate

During a routine inspection of Merlot (clone D3V14) vines at Padthaway, South Australia

several vines showing symptoms of exposure to the herbicide ‘glyphosate’ were noted. All

flowers on those vines were opening in star formation. The star flowers were opening

precociously, were smaller than normal and appeared to be immature, and had pink

pigmentation at the tip of the calyptra (Figure 10.42) similar to that observed on Chardonnay

and Shiraz star flowers (Figure 10.27 and Figure 10.28).

Figure 10.42. Star flowers from a Merlot vine affected by glyphosate herbicide at Padthaway, 15 November 2005.

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Insect feeding damage to flowers

Star flowers were also observed on Merlot vines (clone D3V14) in the Adelaide Hills, South

Australia. Inflorescences with star flowers were localised on a single vine. Opening of the

flowers in star formation was associated with necrotic patches which appeared consistent with

insect feeding damage similar to that caused by thrips (Figure 10.43).

Figure 10.43. Star flowers (indicated by arrows) on Merlot vines in the Adelaide Hills probably caused by insect feeding damage.

Other star flower observations

Star flowers were identified during routine inspections of Merlot vines at the experimental

sites at McLaren Vale and in the Hills. In those cases, the star flowers were sporadic and

occurred singly on inflorescences. Star flowers resembling those of Canada Muscat were also

identified on Pinot Noir vines in California in spring 2006 (pers. comm., P. Dry 2006).

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10.5 Future directions for star flower research

There are currently several researchers working on the characterisation of genes involved in

grapevine flowering (Boss et al. 2003), floral organ differentiation (Sreekantan et al. 2006),

the regulation of flowering (Boss et al. 2001) and fruit development (Fernandez et al. 2006).

The results of that work may contribute to our current knowledge about the mechanisms

involved and the impacts from external influences.

Canada Muscat may provide valuable material for future molecular research. The ability of

that variety to regulate the production of different flower types dependant upon external

environmental conditions suggests regulated expression of a controlling factor. There is also

scope to compare the star flowers of Chardonnay and Shiraz at the molecular genetic level.

Both varieties display similar flower phenotypes, yet they differ in their ability to set fruit.

10.6 Conclusions

• There are several star flower variants. Canada Muscat may have genetic predisposition to

produce star flowers under specific environmental conditions; however, the Chardonnay

and Shiraz vines observed in this study always produced star flowers.

• Pollen tube growth in Canada Muscat star flowers tended to improve later in the flowering

period. This supports the theory that newly-opened Canada Muscat star flowers tend to be

more mature later in the flowering period.

• Ovules from Canada Muscat star flowers were similar in appearance to those from normal

Canada Muscat flowers when observed late in the flowering period. This suggests that, at

this stage, the ovules are functional and capable of fertilisation.

• Canada Muscat star flowers can produce either seedless or seeded berries.

• Cross-pollination of Canada Muscat star flowers may improve fruitset and berry size.

• No pollen or pollen tubes were observed on Chardonnay and Shiraz star flowers

• Star Chardonnay vines produced bunches with mostly seedless berries suggesting that star

Chardonnay berries develop parthenocarpically.

• Star Shiraz vines did not produce any bunches.

• Chardonnay and Shiraz star flowers both contained aberrant ovules.

• Ovules of star Shiraz are similar in structure to those of star Chardonney; however, the

inability of the star Shiraz flowers to develop into berries suggests that there may be two

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different mutations affecting these varieties or the difference may lie in the parthenocarpie

tendencies of the two.

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Chapter 11 General discussion

11.1 Addressing the confusion about fruitset and fruitset disorders

One of the critical observations arising from this research is the confusion amongst

researchers, academics, viticulturists and vineyard personnel regarding fruitset, its

significance and relative contribution to yield, and the mis-identification of fruitset disorders.

The challenge arising from this has been to redefine the disorders and mechanisms and to

disseminate this information so that it reaches an appropriate audience to allow its adoption

and correct use.

‘Fruitset’ is a term that is used commonly, both in the general and scientific literature.

However, the contribution of fruitset to yield is commonly misunderstood and misquoted

because it is seldom measured. This, in turn, results in the dissemination of inaccurate

information which causes further confusion.

Qualitatively, fruitset is the development of an ovary into a berry, whilst quantitatively,

fruitset defines the proportion of flowers that become berries (May 2004). In the research and

academic environment the use of berries per bunch alone is not an adequate measure of

fruitset, nor is the calculation of fruitset from estimates of flower numbers (Chapter 6). The

inclusion of ‘live green ovaries’ (LGOs) or ‘shot’ berries in the calculation of fruitset is also

unacceptable given that they do not constitute the definition of berries and they do not

contribute to yield (Chapter 1).

The mis-identification of ‘poor fruitset’ (or ‘coulure’), ‘millerandage’ and ‘hen and chicken’

has been problematic to the current research, in terms of comparing the current research

results with those of other authors. The following definitions and use of the following

common terms are recommended:

‘Coulure’ is characterised by infertility of grapevine flowers and their subsequent shedding

from the inflorescence resulting in poor fruitset. The term is derived from the French ‘elles

coulent’ or ‘they run or flow’ (Chancrin 1908).

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‘Filage’ refers to the condition of excessive flower shedding, such that the inflorescence

becomes tendril-like. It is derived from the French ‘becoming a thread’ (May 2004).

‘Hen and chicken:’ by implication, refers to the presence of a high proportion, or at least

presence of, seedless chicken berries on a bunch (as previously discussed, chicken berries

must be confirmed as such by the absence of seeds). Bunches displaying hen and chicken are

affected by millerandage (see below); however, bunches affected by millerandage do not

necessarily display hen and chicken (see Chapter 1 for a more detailed discussion).

‘Live green ovary/s’ (LGOs) may resemble an ovary, that is, with the style intact, or they may

be round in shape (May 2004) and remain on the bunch at harvest. LGOs do not develop

colour, flavour, sugar or seeds. LGOs should not be included when quantifying fruitset. The

term live green ovary (or LGOs) should replace the term ‘shot’ berry/s (pers. comm., P. Dry

2003) (see Chapter 1).

‘Millerand’ may be used as a prefix to describe undeveloped berries or bunches with

undeveloped berries (Chancrin 1908).

‘Millerandage’ is a condition defined by the presence of berries arrested at different stages of

development and, by implication, of different sizes, on the same bunch. This may include, but

is not limited to, LGOs, chicken berries or a combination of both.

It is critical that the above terminology is utilised correctly at all levels from research to the

general viticultural environment. This will avoid further confusion, perpetuation of misuse,

and ensure that future research is conducted efficiently.

11.2 Understanding the contribution of different mechanisms of berry development

As discussed in Chapter 1, and demonstrated in Chapter 6, not all flowers on an inflorescence

develop into berries. At harvest, bunches may contain a combination of berry types, formed

via different mechanisms.

Berries are commonly classified according to the presence or absence of seeds. However,

there is a tendency to assume berry seededness based on berry size. In the current study, it

was not uncommon to find seeds in small berries that may have been classified as ‘chickens’,

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had the classification had been based on size alone. This highlights the shortcomings of

previous studies in which berries were classified according to size alone without confirming

seededness, and thereby adds confusion to the interpretation of those results. In the research

environment, the correct classification of berry types is essential.

This being said, the classification of small berries as ‘chickens’ according to their seededness,

may not be as imperative from an agronomic perspective. When berry size is small, the

impact on grapegrowers is on yield, regardless of whether the small berries are seeded or not.

Grape varieties are commonly classified according to the mechanism by which the fruit is

formed (i.e. seeded, parthenocarpic or stenospermocarpic). However, this method of

classification fails to acknowledge that for many varieties, berries within a bunch may be a

combination of types depending on the ability of the ovules to function in fertilisation and

develop into seeds (Chapter 7).

Ebadi (1996) demonstrated that Chardonnay and Shiraz flowers exposed to low temperatures

just prior to opening produced a high proportion of faulty ovules. Those two varieties differed

in their susceptibility to low temperature and therefore produced different proportions of

faulty ovules. Cold-treated flowers had poor fruitset and some of the resultant berries suffered

from arrested development. In the current research, Mo-deficiency of Merlot was also found

to be detrimental to ovule development and it is hypothesised that different levels of Mo-

deficiency may result in different proportions of faulty ovules (Chapter 7). In Ebadi’s study

(1996) and in the current study, berry development was arrested and resulted in the formation

of seedless LGOs or chicken berries. By implication, berry development was not via

fertilisation and the production of seeds.

These examples demonstrate that although Chardonnay, Shiraz and Merlot are classed as

seeded varieties, seededness is facultative, depending on the availability of healthy ovules for

fertilisation. The varying degrees that the different mechanisms contribute to fruit

development should be considered in future research.

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11.3 Advances in understanding the relationship between Merlot and molybdenum

The current research has highlighted the fact that molybdenum may have two distinct effects

on Merlot: a) the vegetative disorder of young Merlot vines (described in detail in Chapter 5);

and b) the effect on reproductive growth and yield (Chapter 6 and Chapter 7).

In the current study, Mo-deficient Merlot vines did not display any of the vegetative

symptoms described by Robinson & Burne (2000), nor did tissue analysis reveal any

significant differences in nitrogen levels between controls and Mo-treated vines. This

suggests that the Mo-deficiency that affected the reproductive growth of Merlot in the current

study was a) not a result of excessive nitrogen and b) that the concentration of molybdenum in

the vines was sufficient to maintain Mo-dependent enzymatic activity. The higher

molybdenum concentration in the stamens and pistils (Chapter 7) relative to that in the

vegetative tissues (Chapter 4) suggests that the requirement for molybdenum by the

reproductive organs is greater than the vegetative parts of the vine, thereby making them more

sensitive to Mo-deficiency.

The effects of molybdenum deficiency on the vegetative growth of Merlot, or the ‘Merlot

problem’, may be more of an issue in young vines because of their limited root system. The

treatment of young vines suffering from poor vegetative growth with molybdenum sprays

may improve vegetative growth which in turn promotes root growth. Once the vines produce

fruit, the demand for molybdenum is higher, in some vines necessitating further remedial

treatment.

11.3.1 Effects of molybdenum on yield of Merlot

In the current study the application of sodium molybdate to Mo-deficient Merlot vines at

modified E-L stage (MELS) 12 (shoots 10 cm) consistently improved yield on those vines.

However, contrary to previous reports, the component of yield that was most affected by Mo-

deficiency was fruitset, not berry size (Chapter 6). The differences between the current study

and previous reports may be a result of one or a combination of the following factors:

• Mis-diagnosis of the fruitset disorder in previous studies (see section 11.1) • Failure to measure fruitset at all in previous studies (see section 11.1) • Incorrect conclusions drawn in previous studies • Differences in clonal sensitivity to Mo-deficiency • Different levels of molybdenum affecting the proportions of healthy ovules and

consequent different mechanisms of berry development (Chapter 7)

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11.3.2 Mechanism by which molybdenum affects yield of Merlot

At the commencement of the current study, Williams & Bartlett (2002) and Gridley (2003)

had shown that foliar sprays of sodium molybdate applied to Mo-deficient Merlot vines

improved yield. These authors suggested that the mechanism was likely to be related to

pollination or fertilisation. However there was a lack of evidence to support either contention.

In this study, the disparity of molybdenum concentration between the stamens and pistils

suggests that molybdenum may be preferentially partitioned to the stamens at the expense of

the pistils during flower development under Mo-deficient conditions. This was perhaps the

first indicator that Mo-deficiency has a greater effect on the female organs than on the male

organs. The absence of any effect of Mo-treatment on pollen vitality further supports this

theory (Chapter 7).

While the application of sodium molybdate to Mo-deficient vines did not affect pollen

vitality, it significantly improved pollen tube growth and penetration of the ovules. Mo-

treatment increased the number of penetrated ovules per ovary and the frequency of ovaries

with at least one penetrated ovule. The strong relationship between the proportion of ovaries

with at least one penetrated ovule and percent fruitset confirms that the improvement in

fruitset gained by Mo-application was the result of more successful fertilisation.

The inhibition of pollen tube growth in ovaries of Mo-deficient vines may be a consequence

of numerous factors such as: changes to pollen morphology or enzymatic activity; hollow or

diverted stylar tissue; or unfavourable conditions in the style (further details provided in

Chapter 7). These factors were not examined in detail, therefore their involvement is

speculative. However, there is evidence to suggest that Mo-deficient vines produce a higher

proportion of ovules without an embryo sac. It was also shown that the incidence of ovaries

with at least one ovule without an embryo sac was significantly higher under Mo-deficient

conditions (Chapter 7). Given the pivotal role of the embryo sac in pollen tube signalling, it is

likely that this is the mechanism affected by Mo-deficiency which causes poor fruitset in

Merlot vines.

11.3.3 Predisposing conditions for molybdenum deficiency

In previous reports and anecdotally, Mo-deficiency of Merlot has been suggested to occur

when vines are grown in low pH soils and when springtime weather conditions are cool and

wet (Williams & Bartlett 2002; pers. comm., C. Williams 2003; Williams et al. 2004).

However, to date no data has been provided to support those theories. It is possible that the

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assumptions made about the role of weather conditions determining vine molybdenum status

relate more to the direct effect of those weather conditions on the success of flowering and

fruitset, rather than their effect on molybdenum status.

There are several factors in this study which suggest that there is an underlying cause of Mo-

deficiency which has not previously been expounded. In this study, no link was found

between Mo-deficiency and cool, wet conditions, nor did soil pH determine vine molybdenum

status. Based on the information available at the time of commencement of this study, vines in

the Hills were expected to be Mo-deficient; however, they were not. By comparison, at

McLaren Vale, where long-term climate averages are higher than in the Hills and soils are

neutral, vines were deficient in molybdenum in the three seasons of experiments. The

difference in vine molybdenum status between the Hills and McLaren Vale experimental sites

remains unclear.

At both sites, the vines were the same age and clone (Chapter 3) and had a similar history of

fertiliser use (data not presented). The soil analyses from both sites were as expected (Chapter

4) and did not contribute to our understanding of molybdenum availability or uptake.

Preliminary analysis of the irrigation water from both sites is currently being conducted;

however, at this time there is no indication that irrigation water contributes to vine Mo-status.

Springtime weather conditions were similar in the Hills and at McLaren Vale, as were those

during the flowering period. Despite the differences in climate between the two sites, the

similarity in weather conditions at flowering time was not unexpected, given that flowering

occurred later in the Hills. However, this does contradict the common belief amongst

grapegrowers that flowering time temperatures are lower in cool climate regions.

The underlying cause of Mo-deficiency in Merlot warrants further investigation to enable the

problem of Mo-deficiency to be more effectively and efficiently addressed.

11.3.4 Adoption by the industry and practical implications of the current molybdenum research

At the commencement of the current study, springtime treatment of Merlot with molybdenum

foliar sprays had already been readily adopted by grapegrowers in South Australia as a

remedial measure against low and inconsistent yield. The espousal of Mo-treatment was

based on a report of interim results (Williams & Bartlett 2002) and information disseminated

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to the general viticultural community by growers involved in on-farm trials (for example,

Miller & Karetai 2002), whose experiments showed that Merlot yield could be improved by

Mo-treatment.

The current recommendation regarding the use of sodium molybdate sprays every season on

entire vineyards as ‘insurance’ against poor fruitset (SARDI 2006) requires further

investigation. The effects of routine molybdenum applications to vineyards may potentially be

detrimental to the health of the vines, the environment and the consumers of the resultant

wine (see section 11.3.5 for further details).

In 2006, at least one fertiliser company began advertising molybdenum preparations and

prescriptions for their application to improve Merlot fruitset and enhance fruit quality (see

Appendix C). To the best of my knowledge, these recommendations have no scientific basis.

The results of this study, and previous studies, have shown that the application of

molybdenum to Mo-deficient Merlot vines can improve yield. However, there is currently no

information about the sustainability of this practice in the long-term.

11.3.5 Long-term and environmental implications of Mo-treatment

Previous research into the effects of molybdenum on Merlot did not address the potential

long-term effects of repeated application of molybdenum preparations to vines. While a

disclaimer stated that further investigation was being made into the potential impacts of

molybdenum sprays on fruit composition and quality (Williams & Bartlett 2002), no allusion

was made to the potential long-term effects of Mo-treatment on vine health or the

environment. In 2005, claims were made that the standard rate of molybdenum application

(300g/ha Na2MoO4) was sustainable in terms of yield (Williams et al. 2005); however, the

inconsistent yield responses presented in 2004 (Williams et al. 2004) and preliminary data

collected over two years in this study (Chapter 6) do not support that opinion.

What is the fate of molybdenum in the vines?

In the current study foliar application of sodium molybdate to vines in two consecutive

seasons resulted in lower petiolar molybdenum concentration at flowering time compared

with vines treated in one season only (Chapter 4). The reason for the decline in molybdenum

concentration after successive seasons of Mo-treatment is unknown. However, it was

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proposed that once the initial starvation for molybdenum is satisfied (in the first season of

treatment), additional molybdenum application is superfluous, resulting in its transport away

from those parts of the vine that are annually renewed (pers. Comm., B. Kaiser 2006).

Delayed budburst in the season following Mo-treatment (Chapter 5) may be evidence of this.

The fate of molybdenum in the vines when the same vines receive Mo-treatment each season

is unknown. Do the vines continually divert molybdenum away from the annual growth

resulting in a gradual decline in yield improvement or does the level of molybdenum in the

vine eventually achieve equilibrium? These questions can be answered only with further field

experimentation and analysis of molybdenum in different parts of the vines over a number of

seasons.

What is the fate and consequence of molybdenum build-up in the soil?

In the current study, the concentration of molybdenum in the soil was higher beneath Mo-

treated vines suggesting that molybdenum accumulates in the soil when molybdenum sprays

are applied on a seasonal basis (Chapter 4). Accumulation of molybdenum in the soil may

affect soil micro-organisms either directly or via its effect on soil chemistry. Little is currently

known about the influence of high levels of molybdenum in soil and the consequent

interaction with soil micro-organisms. This is an area for further investigation.

At the current time there is little available data about the effects of Mo-treatment on varieties

other than Merlot. However, it has been hypothesised that Merlot is affected by Mo-

deficiency because of its inability to extract molybdenum from the soil (pers. comm., C.

Williams 2003). In the case of routine application of molybdenum to varieties other than

Merlot as insurance against poor yield, this practice may have potentially detrimental effects.

If those other varieties are effective at extracting molybdenum from the soil and it is

constantly being added to the soil via runoff from foliar application, then molybdenum may

accumulate to toxic levels in the vines. Clearly, more information about soil extraction ability,

sensitivity to molybdenum and responses to Mo-application across different varieties is

required.

Potential health issues

Nutrient analysis of grape juice from the Hills and McLaren Vale suggests that the treatment

of vines with molybdenum sprays, when molybdenum is already present in the vines at

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adequate levels, may result in significantly higher levels of molybdenum in the fruit (Chapter

8). Despite the levels of molybdenum being relatively low compared to the Australian and

New Zealand recommended daily intake (45 µg/day) (National Health and Medical Research

Council 2005), the effect of application of molybdenum sprays to vines with already high

levels of molybdenum is unknown. Again this may have implications for growers who apply

molybdenum sprays to vines as insurance against poor fruitset, especially in varieties other

than Merlot. This also highlights the importance for growers to monitor molybdenum levels in

vines before remedial Mo-treatments are applied.

11.4 Advances in grapevine tissue analysis

In the current molybdenum experiments, higher levels of molybdenum were measured in the

petioles at MELS 25 (80% flowering) from Mo-treated vines than the controls. This was in

agreement with previous studies of Gridley (2003), Phillips (2004) and Williams et al. (2004).

However, the usefulness of nutrient analyses from petioles that are potentially contaminated

by foliar sprays is limited.

To overcome the potential contamination problem, shoot tip analysis was conducted on fresh

growing tips from Mo-treated vines in conjunction with petiole analysis so that the

relationship between the two tissue types could be examined. The benefits of doing this were

two-fold. Not only was there a strong relationship between the concentration of molybdenum

in the shoot tips and the petioles, shoot tip analysis also confirmed that the sodium molybdate

applied to the vines in spring time penetrated the tissue and was mobile within the vines at

flowering time. It also confirmed the vine’s ability to absorb and maintain higher levels of

molybdenum in the shoots when sodium molybdate was applied at double the standard rate of

application (Chapter 4). In situations where molybdenum sprays are applied to vines at MELS

12, shoot tip analysis should be used in preference to petiole analysis to avoid possible

contamination.

11.4.1 What is the critical level of molybdenum in Merlot?

In 2004, Williams et al. reported that the critical level of molybdenum at which yield of

Merlot was compromised was between 0.05-0.09 mg/kg measured in the petioles at 80%

flowering.

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In the current study, when the concentration of molybdenum in the shoot tips was greater than

0.1 mg/kg, there was a significant increase in yield — primarily a function of improved

fruitset on bunches (Chapter 6). Mo-treatment reduced the variability in percent fruitset and

increased the likelihood of obtaining fruitset greater than 30%. Given the strong relationship

between petiole and shoot tip molybdenum concentrations (Chapter 4), it is suggested that the

critical level of molybdenum for optimal fruitset on own-rooted Merlot is 0.1 mg/kg in the

shoot tips at MELS 25 (80% flowering) (Chapter 6).

11.4.2 Nutrient analysis of anthers and pistils

Vine nutrient analysis was taken a step further in the current study with the aim of identifying

any incongruities between the male and female flower parts. As discussed earlier (section

11.3.2) the consistently higher concentration of molybdenum in the anthers than in the pistils

from control vines was perhaps the earliest indication of the significant effect of Mo-

deficiency on the female flower parts.

11.5 The potential importance of the star flower discovery to Australian viticulture

It is likely that the Canada Muscat vines observed in the current study had been producing star

flowers for years before their discovery and identification in 2003 (Chapter 10). As with

many novel discoveries, after the initial identification, the observer becomes attuned to that

specific peculiarity. The ability to recognise star flowers after the initial observations on

Canada Muscat allowed for the identification and description of several previously

undescribed star flower variants, and sporadic observations of other flower mutations (data

not presented). However, this was only possible via intense scrutiny of hundreds of thousands

of flowers at several different sites over the course of four seasons, and via dissemination of

the discovery to the broader viticultural community.

Canada Muscat appears to have the ability to regulate the type of flowers produced in

response to different environmental conditions. The earliest flowers produced were immature

and probably formed seedless berries. However, as flowering progressed, newly-opened

Canada Muscat star flowers appeared mature and probably produced normal seeded berries.

In contrast to this, the Chardonnay and Shiraz star flowers always opened precociously when

the floral organs were immature. For star flowers of Chardonnay, berry development probably

occurred parthenocarpically, resulting in small seedless fruit; however, star flowers of Shiraz

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did not proceed past flowering and no fruit was produced. These three star flower variants

provide the basis for a molecular study of flower development, flowering and fruitset which

may be extremely valuable to our understanding of those processes, and indeed normal flower

development. Additional star flower variants, such as those present and overlooked by other

authors (for example, Sreekantan et al. 2006) and those described in Chapter 10, may provide

further material for investigation.

Other flower abnormalities that inhibit fruitset or produce seedless berries have been

described for various grape varieties and are summarised by May (2004). One such

aberration, the formation of an ‘internal ovary’, was reported to be common in Pinot Noir

vines grown in Burgundy. Of the 24% of ovaries containing an internal ovary, only 3% of

those ovaries transformed into berries, suggesting a detrimental effect on fruitset (May 1987).

The identification of star flowers on numerous varieties, in several different regions and their

production in response to different environmental conditions, suggests that the occurrence of

star flowers may be more widespread than previously acknowledged. The association of star

flowers with the production of seedless berries and poor fruitset suggest that star flowers may

also play a significant role in the problem of poor fruitset, especially under certain

environmental conditions. This warrants further investigation.

11.6 Remaining questions and areas for further investigation

• What is the fate of molybdenum in the vines after multiple seasons of Mo-treatment?

• What are the long-term effects of Mo-treatment on vine and soil health?

• What is the fate of molybdenum when vines with adequate levels of molybdenum are

treated with sodium molybdate sprays?

• What is the mechanism by which molybdenum affects embryo sac development?

• What is the underlying cause of Mo-deficiency in Merlot?

• What is the genetic pathway that governs flower development and flowering?

11.7 Conclusions

• Clarification of commonly used terms such as millerandage and fruitset is required in

order that future research is conducted more effectively and efficiently.

• Mo-deficiency may have two distinct effects on Merlot: a) the vegetative disorder of

young vines; and b) the effect on reproductive growth and yield. These effects may

operate at different levels of molybdenum in the vines.

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• The cause of Mo-deficiency of Merlot remains unclear and warrants further

investigation.

• The current study has made significant progress toward the elucidation of the

mechanism by which Mo-deficiency affects yield of Merlot. Flowers on Mo-deficient

vines produce a higher proportion of faulty ovules which is detrimental to pollen tube

signalling and consequently fertilisation.

• Long-term effects on vine, soil and human health and sustainability of Mo-treatment

should be addressed, especially the routine use of molybdenum sprays as ‘insurance’

on varieties other than Merlot.

• Growers wishing to apply Mo-treatments to vines should perform flowering time

tissue nutrient analysis to assess molybdenum levels in the season prior to spraying.

This will avoid unnecessary treatment and potential accumulation of molybdenum in

grape juice.

• Significant advances have been made in the area of tissue nutrient analysis. Shoot tip

analysis has enabled the proposal of a critical level of molybdenum for adequate

fruitset of Merlot. Shoot tip analysis should be used in preference to petiole analysis

when vines are sprayed in springtime to avoid potential contamination. The analysis of

pistils and anthers has highlighted that there is specific partitioning of molybdenum to

the male and female parts of the flowers during development.

• The discovery of star flowers in Australia may have significant implications for future

research and the understanding of grapevine flowering and fruitset.