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WW IS 10-03

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Winetech

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______________________________________ FINAL REPORT 2013

Programme & Project Leader Information Research Organisation

Programme leader Project leader

Title, initials, surname Professor Alain Deloire & Dr Philip Myburgh

Mr. Ignacio Serra Stepke

Present position Professor (AD) & Specialist researcher (ARC Infruitec-Nietvoorbij) (PM)

Post graduate student: Dept Viticulture and Oenology; Lecturer, University of Concepción, Chile

Address NWGIC, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW, 2678, Australia. Stellenbosch University Private Bag X1 7602 MATIELAND

Facultad de Agronomía, Universidad de Concepción, Casilla 537, Chillán, Chile Stellenbosch University Private Bag X1 7602 MATIELAND

Tel. / Cell no. 0793628456 (56) (42) 2208817

Fax 021 808 4781 (56) (42) 2275309

E-mail [email protected] [email protected]

[email protected]

Project Information

Research Organisation Project number

WW IS 10-03

Project title Study of rootstocks water uptake on Pinotage (Vitis vinifera L.) leaf transpiration efficiency and on grapevine adaptation to drought

Fruit kind(s) Grapevines

Start date (mm/yyyy) 01/2010 End date (mm/yyyy) 12/2012

Project keywords climate change; drought tolerance; rootstocks; water deficit; water use

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Approved by Research Organisation Programme leader (tick box)

THIS REPORT MUST INCLUDE INFORMATION FROM THE ENTIRE PROJECT

Executive Summary Give an executive summary of the total project.

What to remember:

- Differences in stomatal conductance and plant water status among the different rootstocks were

detected.

- Stomatal conductance is the earlier response of the cultivar to soil water deficit and root to shoot

signalisation.

- The results showed that leaves growing in an environment with a lower light intensity (lower R/FR

ratio) had a lower stomatal density but bigger pore diameter.

- Differences in leave stomatal density and size were observed for Pinotage grafted on different

rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger

pore diameter than of 110 Richter and 1103 Paulsen.

- The results confirm that the rootstock is genetically controlling the cultivars’ stomatal functioning,

number and size. This can be used to understand the real rootstock – cultivar interaction and

refine rootstock classification.

- Thus the rootstocks are controlling the cultivar’s transpiration and adaptation to drought (soil

moisture availability). This fact should be considered for rootstock choice for a site.

- Rootstocks however have no effect on the leaf gas exchange under moderate water constraints

conditions. This fact should also be considered for rootstock choice for a site.

The main objective of this project was to study the role of rootstocks in the adaptation of the grapevine to

drought. Possible water scarcity in the near future (IPCC, 2008) increases the interest in drought

tolerance conferred by rootstocks. It has been postulated that the use of drought tolerant rootstocks can

help to minimize the effect of water constraints, by improving the water uptake and transport

(Carbonneau, 1985; Soar et al., 2006) and by controlling plant transpiration through chemical (Loveys

and Kriedemann, 1974; Stoll et al., 2000; Soar et al. 2006) and hydraulic signalling (Vandeleur et al.,

2009). Vines of cv. Pinotage (Vitis vinifera L.) grafted onto 5 rootstocks with different drought tolerances

were used in field and greenhouse experiments (Table 1). Field experiments were carried out in the

Welgevallen research farm (Stellenbosch University). Adult Pinotage grapevine plants, grafted onto 140

Ruggeri and 1103 Paulsen rootstocks, were sampled. Two levels of soil water content were studied:

irrigated grapevines without water constraint and rain-fed grapevines subjected to moderate water

constraint. Exposed and shaded adult leaves were sampled at harvest and analysed using scanning

electron microscopy (SEM) and the size and density of stomata were determined from the images

obtained. Two experiments were carried out in two different greenhouses. The first experiment was

located in a greenhouse provided with automatic temperature control (ATC) and the second in a

greenhouse without automatic temperature control (NoATC). One year old grafted vines, growing in 21 L

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pots filled with sandy soil, were used. Half of the vines were subjected to water constraint by withholding

irrigation. The other half was well watered throughout the whole experimentation. Leaf gas exchange and

plant water status were monitored and leaves were sampled to determine stomatal size and density by

SEM. As expected, water constraints induced a reduction in photosynthesis and stomatal conductance

and a reduction of stomatal size. Differences in stomatal conductance and plant water status in response

to drought among the different rootstocks were detected. The results showed that leaves growing in an

environment with a lower light intensity (lower R/FR ratio) had a lower stomatal density but bigger pore

diameter. Differences in stomatal density and size were observed on Pinotage leaves grafted onto

different rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger

pore diameter than 110 Richter and 1103 Paulsen.

Problem identification and objectives State the problem being addressed and the ultimate aim of the project.

Drought can reduce stomatal conductance and photosynthesis (Iacono et al., 1998; Koundouras et al.,

2008); decrease leaves expansion and internodes extension (Schultz and Mathews, 1988; Cramer et al.,

2007; Lovisolo et al., 2010), induce senescence of older leaves (Jackson, 1997) and reduce yield per vine

(dos Santos et al., 2003). The use of rootstocks to enhance drought tolerance in grapevine might be a

feasible solution to face drought and high temperature events (heat waves) link to a possible evolution of

the climate. The mechanisms of rootstocks tolerance to drought are not yet fully understood. The main

objective of this project was to study the role of rootstocks in the adaptation of the grapevine to drought.

This could allow giving recommendations on irrigation, canopy architecture, and rootstock selection for

specific soils and climate conditions.

Specific goals on Pinotage (V. Vinifera L.) were:

• To determine the effect of the rootstock on the vine water status (Pinotage) under increasing

water deficit conditions

• To evaluate the effect of the rootstock on Pinotage leaf gas exchange and immediate water use

efficiency under increasing drought

• To investigate the effect of vine water status on stomatal density and length

• To investigate the effect of light intensity on stomatal density and length

• To investigate the effect of light intensity on stomatal density and length in interaction with the

rootstocks

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Workplan (materials and methods) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages and parameters.

Plant material

Vines of cv. Pinotage (Vitis vinifera L.), grafted onto 5 rootstocks having different drought tolerance, were

used in a field and greenhouse experiments (Table 1).

Field grown grapevines: growth conditions and treatments

The field trial was carried out at the Welgevallen research farm Pinotage experimental vineyard

(Stellenbosch University; 33°56'S, 18°52'E, altitude: 157 m) during the 2010/2011 season. Adult Pinotage

grapevines (Vitis vinifera L.) clone 48A grafted onto 110 Richter (clone RQ28B), clone 50A grafted onto

140 Ruggeri (clone RU354B) and clone 48A grafted onto 1103 Paulsen (clone PS28A) rootstocks were

used. Grapevine ages were 19, 16 and 14 years old for grapevines grafted onto 110 Richter, 140 Ruggeri

and 1103 Paulsen, respectively. Grapevines were trained on a vertical shoot positioning system with a

single cordon de Royat, and pruned to two buds per spur. Replicates were randomized within a block

layout. Drip irrigation was installed for the treatments as prior to the onset of the project the experimental

vineyard was managed as a dryland vineyard. Two irrigation strategies were studied: irrigated grapevines

to avoid any water constraint and rain-fed grapevines subjected to moderate water constraint. Irrigation

timing were scheduled according to the soil water content and stem water potential measurements.

Based on these parameters, five irrigations were applied from véraison to harvest for the irrigated

grapevines treatment. Irrigations were scheduled in order to avoid stem water potential (ψstem) levels

lower than -0.6 MPa, whereas the water constraint grapevines received no irrigation. Irrigation was

applied by means of 2.3 L/h drippers spaced 600 mm apart in the grapevine row, whereas grapevine

spacing was 1.4 m. Leaf area was assessed, after the treatments were imposed, using equations

established within the study for the correlation between ‘Pinotage’ leaf area and leaf main vein length.

There were no significant differences found in the total leaf area among rootstocks (2.5, 2.2 and 2.0

m2/grapevine for 110 Richter, 140 Ruggeri and 1103 Paulsen respectively) or among irrigation treatments

(2.2 m2/grapevine for both no constraint and moderate water constraint treatments). In order to evaluate

the influence of light, full sun exposed and shaded adult leaves (growing inside the canopy) were

sampled at harvest.

Potted grapevines: growth conditions and treatments

Two experiments were carried out in two different greenhouses. The first experiment was located in a

greenhouse provided with automatic temperature control (ATC) and the second in a greenhouse without

automatic temperature control (NoATC). One year old grapevines, grafted the previous season, planted in

21 L pots filled with sandy soil, were used for both experiments in order to facilitate each water constraint

experiment. In 2011, twenty eight grapevines cv. Pinotage (clone PI48A) grafted onto 99 Richter (clone

RY25IM) and cv. Pinotage (clone PI48C) grafted onto 110 Richter (clone RQ28C) were used for the first

experiment. Replicates were randomized. Once all the grapevines selected for the study reached at least

15 leaves per shoot, half of the vines were subjected to water constraint by stopping the irrigation to the

pots for 12 days (5th to 17th October 2011), re-watering and thereafter exposed to a second water

Final report 5


constraint period for 5 days (2nd to 7th November 2011) in order to evaluate the recovery of the vines after

re-watering and drought. The other half of the vines were well watered throughout the whole

experimentation. Grapevines were irrigated at field capacity on alternate days. A second experiment was

performed in another greenhouse in order to evaluate the aforementioned experimentation under a semi

controlled environment. In 2012, twenty four Pinotage grapevines, consisting of clone PI48 grafted onto

99 Richter (clone RY25IM), clone PI48C grafted onto 110 Richter (RQ28C), clone PI48I grafted onto 140

Ruggeri (clone RU354E), clone PI48C grafted onto 1103 Paulsen (clone PS281) and clone PI48I grafted

onto Ramsey (clone 5C18AB), were selected for the study. Replicates were randomized. Once all the

grapevines of rootstocks 110 Richter, 140 Ruggeri and 1103 Paulsen reached at least 15 leaves per

shoot, half of the vines were subjected to water stress by stopping the irrigation of the pots. The other half

were maintained well watered throughout the whole experimentation which took 10 days. Grapevines

were irrigated at field capacity on alternate days. For the rootstocks 99 Richter and Ramsey, grapevines

were subjected to water stress only due to the limited number of grapevines with at least 15 leaves per

shoot. All the vines in the pots were single primary shoot vines without lateral shoots and inflorescence.

The grapevine characteristics used in the experiments and the duration of the water constraints are

described in Table 2.

Leaf water potential

Grapevine stem water potential (ψstem) at around midday was determined using a Scholander pressure

chamber (Choné et al., 2001). Each leaf was wrapped in a bag prior to excision using a razor blade and

the measurements were done on three to five leaves per treatment and per date. Each leaf was selected

from a single grapevine. Leaf gas exchange measurements were done prior to leaf water potential.

Soil water content

For the field trial, soil water content was determined by means of the neutron scattering technique at 0-

300 mm, 300-600 mm and 600-900 mm in three replications of each treatment. The measurements were

done around every 4 days from 5th November 2010 to 9th March 2011. The probe count ratios were

calibrated against gravimetric soil water content. For the greenhouse experiment, soil water content was

determined by means of gravimetric soil water content, where soil samples from three pots per treatment

were weighed (Tin + wet), dried at 105ºC for 16 hours. Soil water content was calculated as Soil water

content (%) = (((Tin + wet) - (Tin + dry)) / ((Tin + dry) – (Tin mass))) X 100. In the ATC greenhouse, the

measurements were carried out approximately every 4 days during the experimentation while in the

noATC greenhouse the measurements were done only at the beginning and at the end of the

experimentation.

Gas exchange measurements

Net photosynthesis (AN) and stomatal conductance (gs) was measured using three to five mature and

fully-exposed leaves per treatment and per date, using an open gas exchange analyser (Li-6400; Li-Cor,

Inc., Lincoln, NE). Each leaf was selected from a single grapevine. The measurements were carried out

approximately once a week, every four days and every three days for the field experiment, the ATC

greenhouse and the noATC greenhouse respectively. All measurements were performed at a quantum

flux of 1600 µmol m-2 s-1, which was determined to be above the light saturation level, with a CO2

Final report 6


concentration in the cuvette of 400 µmol CO2 mol-1 air and a flow rate of 350 µmol s-1. Block temperature

was maintained at 25ºC.

Air temperature and relative humidity

Air temperature and relative humidity were recorded using a data logger (Gemini Tiny Tag TGP-4500,

Gemini Dataloggers SA (PTY) Ltd) placed in a Gill screen above the canopy. The data were recorded

from September to March for the field experiment, while in the greenhouses the data were recorded for

approximate two months during the treatments’ duration. VPD data was obtained from the data loggers

for the time (at around midday) and days that the experimentation took place.

Light conditions

In order to characterise the leaf growing conditions for the field experiment in terms of light intensity of the

leaves under full sun exposure and for leaves growing inside the canopy in permanent shade, light

readings were carried out in the field to determine the quantum ratio of red light (660 nm) to far-red light

(730 nm) (R:FR) using a point sensor (Skye instruments, Powys, UK). In the ATC greenhouse, the solar

radiation at noon (clear sky) was measured at approximately 750 (W/m2). The measures were taken with

a light sensor (Davis Vantage Pro solar radiation sensor, Davis Instruments, Hayward, California, USA),

connected to a logger (DataTaker DT82E data logger, Thermo Fisher Scientific Australia Pty Ltd,

Scoresby, Victoria, Australia).

Leaf area

Leaf area was determined by measuring the lengths of the main leaf veins of the primary and secondary

shoots. Equations were established for the correlation between ‘Pinotage’ leaf area and leaf main vein

length, viz. y = -34.2857+13.163*x (r2 = 0.94) for primary shoots and y = -36.4218+12.8845*x (r2 = 0.84)

for secondary shoots, where “y” is leaf area (cm2) and “x” is the leaf main vein’s length (cm). Total leaf

area per grapevine was calculated by multiplying the total primary and secondary shoot leaf area by the

number of primary and secondary shoots per grapevine. This method was used for the field and potted

grapevines. In the case of field grapevines, 15 shoots per treatment were selected to determine leaf area

whereas in potted grapevines all the plants were assessed for leaf area.

Leaf stomatal density and size

Adult leaves were sampled from primary shoots according to the type of experiment (Table 3).

A leaf material sample of approximate 100 mm2 between the main leaf vein and the first right lateral vein

was taken from each fresh leaf. Prior to imaging, the samples were mounted on a stub with double sided

carbon tape. Specimens were coated with a thin gold layer and examined using a scanning electron

microscope (SEM) (LEO® 1430VP, LEO Co. LTD.). Beam conditions during surface analysis were 7 KV

and approximately 1.5 nA, with a working distance of 13 mm and a spot size of 150. All

photomicrographs were taken under the same conditions of magnification. The stomata counting and size

were analyzed using ImageJ software. The stomatal pore was measured considering the length in

micrometres between the junctions of the guard cells at each end of the stoma. To determine the

stomatal size, six stomata were randomly selected for each sample for the field experiment (2010/2011)

Final report 7


and 10 stomata for the rest of the experiments (Table 3). Due to the fact that in the greenhouse with

controlled environment the leaves were sampled during the whole period of physiological measurements,

and not only at the end of the treatments (like for the rest of the experiments), the leaves from the last two

dates were selected to determine the stomatal size.

Statistical analysis

Data were analysed using analysis of variance (ANOVA) and means were separated by Fisher’s least

significant difference (LSD) test.

Results and discussion State results obtained and list any industry benefits. If applicable, include a short discussion covering ALL accumulated results from the start of the project. Limit it to essential information only.

The project allowed getting the following results:

• As expected, water constraints induced a reduction in photosynthesis and stomatal conductance.

• Differences in plant water status and stomatal conductance were observed on Pinotage grafted

onto different rootstocks. This is important in terms of rootstock selection for cultivar drought

tolerance.

• Water constraint induced a reduction of the stomatal size. This is important in terms of the

understanding of grapevine adaptation to drought in interaction with the rootstock.

• The results showed that leaves growing in an environment with a lower light intensity (lower R/FR

ratio) had a lower stomatal density but bigger pore diameter. This is important in terms of the

understanding of grapevine adaptation to light intensity (canopy microclimate versus canopy

manipulation).

• Differences in stomatal density and size were observed on Pinotage leaves grafted onto different

rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger

pore diameter than 110 Richter and 1103 Paulsen. This helps to improve the interaction between

rootstocks and cultivars (Vitis vinifera L.) to enhance drought tolerance in grapevine.

• To demonstrate a moderate effect of the rootstock on the vine water status under no constraint

and moderate water constraint.

• To show the importance of the scion on the regulation of the leaf gas exchange, irrespective of

the rootstocks. The rootstocks have no effect on the leaf gas exchange under moderate water

constraints conditions. This fact should be considered for rootstock selection.

• To validate the stomatal conductance as an early response to soil water deficit and root to shoot

signalisation.

• To develop a method to study the stomata characteristics. This is relevant in terms of the

understanding of the capacity of the cv. Pinotage (and all cultivars) to adapt a new environment

conditions that include higher evapotranspiration conditions.

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Complete the following table

Milestone Target Date Extension Date

Date Completed Achievement

1. To characterize some climatic conditions under field and greenhouse conditions

2010 2011 2012

- 2012

Under greenhouse conditions, the maximum temperature during the day in general did not exceed 30º C, in contrast under field conditions the maximum temperatures were above 30º C. As expected the ATC greenhouse presented the lowest fluctuation of temperature and humidity in comparison with the noATC greenhouse and field conditions (Figure 1 and Table 4).

2. To apply water status treatments by creating extreme conditions in terms of vine water status

2010 2011 2012

- 2012

The water constraint treatments carried out in both greenhouses (Figures 2 and 3) produced clear differences in terms of plant water status in comparison to the well watered treatments (Figure 5A and B). Nevertheless, under field conditions (Figure 4), the water constraint treatments were not able to achieve a severe water constraint and only moderate water constraints were obtained, in comparison with well watered plants (Figure 5C and D). In the ATC greenhouse, it took approximately 10 days after withholding water to the pots to reach a severe water constraint resulting in visible effects such as senescence of older leaves and leaves wilting (Figure 6).

3. Determine the effect of the rootstock on the vine water status

2010 2011 2012

- 2012

Plant water status was affected by rootstocks under water deficit conditions in the noATC greenhouse and under field conditions. Plants grafted onto 99 Richter and 110 Richter presented a less negative ψstem under water constraint in comparison with Ramsey. The differences were found at the beginning and during the most part of the water withholding, nevertheless similar values of plant water status occurred at the end of the water constraint period (Figure 7).

4. To characterize light intensity for leaves under full sun exposure and shade conditions

2011 2012

- 2012

Under shaded conditions, the leaves, for the water constraint treatment of the vines grafted onto 1103 Paulsen, were exposed to a higher light

Final report 9


intensity in comparison with the rest of leaves probably due to less dense canopies (Figure 8).

5. Evaluate the effect of drought on leaf gas exchange and the influence of rootstock on the stomatal conductance and photosynthesis

2011 2012

- 2012

As expected, water constraints induced a reduction in photosynthesis and stomatal conductance (Figures 9 and 10). Differences in stomatal conductance, in response to drought among the different rootstocks, were detected (Figure 10).

6. Investigate the effect of vine water status on stomatal density and length

2011 2012

- 2012

In general stomatal density was not affected by soil water deficit, except for the field experiment where leaves under water constraint treatment presented a higher stomatal density in comparison to leaves under well watered treatment (Figures 11 and 12). In contrast, the pore diameter was affected by water constraint treatments in most of the experiments that reached severe water constraint (Figures 13A, 13B and 14) inducing a reduction in the pore diameter size. Nevertheless under moderate water constraint conditions, the stomata size was mostly not affected (Figures 13C and 13D).

7. Evaluate the effect of light intensity on stomatal density and length

2011 2012

- 2012

Leaves growing in an environment with a lower light intensity (lower R/FR ratio) had a lower stomatal density but bigger pore diameter (Figure 15).

8. To investigate the effect of light intensity on stomatal density and length induced by rootstocks

2011 2012

- 2012

Differences in stomatal density and size were observed on Pinotage leaves grafted onto different rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger pore diameter than 110 Richter and 1103 Paulsen (Figure 15). Differences in stomatal density and size were observed on Pinotage leaves grafted onto different rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger pore diameter than 110 Richter and 1103 Paulsen (Figure 16).

9. Article accepted in

Australian Journal of

Grape and Wine

Research

2012 - 2013

Serra I., Strever A., Myburgh P., and Deloire A. 2013. A review of the interaction between rootstocks and cultivars (Vitis vinifera L.) to enhance drought tolerance in grapevine. Australian Journal of Grape and Wine Research (Accepted).

10. Article submitted to Acta Horticulturae (Proceedings)

2012 2014 -

Serra I., Strever A., Myburgh P., Schmeisser M. and Deloire A. 2013. Grapevine (Vitis vinifera L., cv. Pinotage) leaf stomatal size and density as modulated by different rootstocks and scion water status. Proceedings Acta Horticulturae (Submitted).

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11. Article in preparation 2012 2014 -

Article in preparation “Stomatal development of grapevine leaves (Vitis vinifera L., cv. Pinotage) in response to the combined effect of light, plant water status and rootstocks”.

References Carbonneau A. 1985. The early selection of grapevine rootstocks for resistance to drought conditions.

American Journal of Enology and Viticulture 36: 195–198.

Chaves MM and Oliveira MM. 2004. Mechanisms underlying plant resilience to water deficits: prospects

for water-saving agriculture. Journal of Experimental Botany 55: 2365-2384.

Choné, X., van Leeuwen C., Dubourdieu D., Gaudellère J.P., 2001. Stem water potential is a sensitive

indicator of grapevine water status. Annals of Botany 87: 477-483.

Cramer GR, Ergül A, Grimplet J, Tillet RL, Tattersall EAR, Bohlman MC, Vincent D, Sonderegger J,

Evans J, Osborne C, Quilici D, Sclauch KA, Schooley DA and Cushman JC. 2007. Water and salinity

stress in grapevines: early and late changes in transcript and metabolite profiles. Funct Integr Genomics

7: 111-134.

dos Santos Tiago P., Carlos M. Lopes, M. Lucília Rodrigues, Claudia R. de Souza, João P. Maroco, João

S. Pereira, Jorge R. Silva and Chaves MM. 2003. Partial rootzone drying: effects on growth and fruit

quality of field-grown grapevines (Vitis vinifera). Functional Plant Biology 30: 663 – 671.

Iacono F, Buccella A and Peterlunger E. 1998. Water stress and rootstock influence on leaf gas

exchange of grafted and ungrafted grapevines. Scientia Horticulturae 75: 27-39.

IPCC, 2008. Climate change and water. In: Bates BC, Kundzewicz ZW, Wu S, Palutikof JP, eds. IPCC

Tech. Paper VI, IPCC Secretariat, Geneva.

Koundouras S, Tsialtas IT, Zioziou E and Nikolaou N. 2008. Rootstocks effects on the adaptive strategies

of grapevine (Vitis vinifera L. cv. Cabernet-Sauvignon) under contrasting water status: Leaf physiological

and structural responses. Agriculture, Ecosystems and Environment 128: 86-96.

Jackson M. 1997. Hormones from roots as signals for the shoots of stressed plants. Trends in Plant

Science 2: 22-28.

Loveys, BR, Kriedemann, PE. 1974. Internal control of stomatal physiology and photosynthesis, I.

Stomatal regulation and associated changes in endogenous levels of abscisic and phaseic acids. Aust. J.

Plant Physiol. 1: 407-415.

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Lovisolo C, Perrone I, Carra A, Ferrandino A, Flexas J, Medrano H, Scuhert A. 2010. Drought-induced

changes in development and function of grapevine (Vitis spp.) organs and in their hydraulic and non-

hydraulic interactions at the whole-plant level: a physiological and molecular update. Functional Plant

Biology 37: 98-116.

Schultz HR and Mathews MA. 1988. Vegetative growth distribution during water deficits in Vitis vinifera L.

Aust. J. Plant Physiol. 15: 641-656.

Soar CJ, Dry PR, Loveys BR. 2006. Scion photosynthesis and leaf gas exchange in Vitis vinifera L. vv.

Shiraz: mediation of rootstock effects via xylem sap ABA. Aust J. Grape Wine Res. 12: 82-96.

Stoll M, Loveys B, Dry P. 2000. Hormonal changes induced by partial rootzone drying of irrigated

grapevine. Journal of Experimental Botany 51: 1627-1634.

Vandeleur RK, Mayo G, Shelden MC, Gilliham M, Kaiser BN, Tyerman SD. 2009. The role of plasma

membrane intrinsic protein aquaporins in water transport through roots: diurnal and drought stress

responses reveal different strategies between isohydric and anisohydric cultivars of grapevine. Plant

Physiology 149: 445–460.

Accumulated outputs List ALL the outputs from the start of the project. The year of each output must also be indicated.

• Demonstrate an effect of light intensity on stomatal pore size (2011/2012).

• Demonstrate an effect of drought on stomatal pore size (2011/2012).

• Demonstrate a different response of plant water status to drought induced by rootstocks

(2011/2012).

• Demonstrate a different response of stomatal conductance to drought induced by rootstocks

(2011/2012).

• Show a possible role on stomatal development induced by rootstocks (2011/2012).

• To demonstrate a moderate effect of the rootstocks on the vine water status under no constraint

and moderate water constraint (2010/2011).

• Validation of the control by the scion on leaf gas exchange regulation. The rootstocks have no

effect on the leaf gas exchange under moderate water constraint conditions. This fact should be

considered for rootstock selection (2010/2011).

• Validation of the stomatal conductance as an early response to soil water deficit which addresses

the question of root to shoot signalisation (2010/2011).

• Method developed to study the stomata. This is relevant in terms of the understanding of the

capacity of the cv. Pinotage (and other cultivars) to adapt to new environmental conditions that

include higher evapotranspiration conditions (2010/2011).

Final report 12


Conclusions Rootstocks have a definitive influence in plant water status and leaf gas exchange on the scion which

implicates an influence in water uptake and transport and tight regulation of the stomatal conductance.

Rootstock influence in scion response to drought occurs from firsts stages of water constraint, up to a

point where the plant water status is the main driver of photosynthesis and the stomatal conductance.

This regulation occurred up to a point where the plant water status, linked to the level of drought, seemed

to become the main driver of stomatal conductance and consequently photosynthesis (Lovisolo et al.,

2010).

We have demonstrated that stomatal development is affected by light, drought and possibly by

rootstocks. Further research is still needed to elucidate the ways in which rootstocks can affect the

stomatal size and conductance and therefore potentially the photosynthetic capacity in response to

drought. The results confirmed that the rootstock is regulating the cultivar's stomatal size (anatomical

changes during leaf growth) and functioning (stomatal regulation) through a complex signalisation

process. The transpiration rate of leaves was more related to stomatal size than density. Thus one

possible mechanism of Pinotage leaf adaptation to water constraints was structural during leaf growth,

with a reduction in pore size to reduce plant water loss. The effect of light is interesting in the context of

canopy microclimate and canopy manipulation (choice of the vine architecture in the context of climate

change versus increase in drought and water scarcity). The interaction/signalling of the combination

rootstock-cultivar is important for the understanding of grapevine adaptation to sites (soil x climate) where

drought and water scarcity will be the main concerns.

Technology development, products and patents Indicate the commercial potential of this project, eg. Intellectual property rights or commercial product(s) Fabrication of six root chambers (2009) Method to describe the stomata at the leaf level (2011). This can be used to understand the real interaction rootstock – cultivar and refine the rootstock classification. No patents

Suggestions for technology transfer List any suggestions you may have for technology transfer Popular article will be written for Wineland magazine. In a dry land or in irrigated conditions with a limited amount of available water, it is proposed that grafting on water tolerant rootstocks is still the best option for the cultivar to adapt to drought. For wet lands or soils with a high water holding capacity and for deep soils, or in irrigated conditions with no water restriction, the choice of a rootstock will depend on the desired canopy vigour, yield and wine style.

Human resources development/training Indicate the number and level (eg. MSc, PhD, post doc) of students/support personnel that were trained as well as their cost to industry through this project. Add in more lines if necessary.

Student level (BSc, MSc, PhD, Post doc) Cost to Project

Final report 13


PhD M Ignacio Serra (Chile), thesis defence in January or March 2014

Publications (popular, press releases, semi-scientific, scientific) 2013 Article in preparation “Stomatal development of grapevine leaves (Vitis vinifera L., cv. Pinotage) in

response to the combined effect of light, plant water status and rootstocks”.

Serra I., Strever A., Myburgh P., and Deloire A. 2013. A review of the interaction between rootstocks and

cultivars (Vitis vinifera L.) to enhance drought tolerance in grapevine. Australian Journal of Grape and

Wine Research (Accepted).

Serra I., Strever A., Myburgh P., Schmeisser M. and Deloire A. 2013. Grapevine (Vitis vinifera L., cv.

Pinotage) leaf stomatal size and density as modulated by different rootstocks and scion water status.

Proceedings Acta Horticulturae (Submitted).

2012 Serra-Stepke, I. 2012. Living with almost no water: when surviving is just a matter of root to shoot

communication. New voices in Science 2013, 10-11.

2011 Deloire, A.J., Z.A. Coetzee, M. Muller, J. Brand, M. van der Rijst & I. Serra-Stepke. 2011. Madurez óptima

de la baya. La importancia del color (Optimum berry ripeness. The importance of colour). Vitis Magazine

43: 44-48.

Presentations/papers delivered 2013 Serra I., Strever A., Myburgh P., Schmeisser M. and Deloire A. 2013. Grapevine (Vitis vinifera L., cv.

Pinotage) leaf stomatal size and density as modulated by different rootstocks, scion water status and

irradiance. Presentado en: Ninth International Symposium on Grapevine Physiology & Biotechnology, La

Serena, Chile, 21-26 Abril. (Poster).

2012 Serra I., Strever A., Myburgh P., Schmeisser M. and Deloire A. 2012. Stomatal density of grapevine

leaves (Vitis vinifera L., cv. Pinotage) responds to light and vine water status. Presentado en: SASEV

34th Congress, Allée Bleue, Groot Drakenstein, South Africa. Novembre 14-16. (Poster).

2011 Serra I., A. Deloire, P. Myburgh and M. Schmeisser. 2011. Preliminary results on the interaction between

rootstocks and vine water status on Vitis vinífera L. cv ‘Pinotage’ physiology. XIII Latin-American

Congress of Viticulture and Enology, Santiago Chile (21-23 November) (Oral presentation).

2010

Final report 14


Serra Stepke, I., A.J. Deloire & P. Myburgh. 2010. Quantification of root growth using an image analysis

tool. First Wine Sciences Research Day. DVO-IWBT, Stellenbosch University. (26November) (Oral

presentation).

A visit of the layout have been organised (2010) for a group of viticulturists from the RSA wine industry on

the theme of root and grapevine. The possibility for them to visualize the root system was appreciated

and relevant exchanges around this theme were made.

Tables and figures

TABLE 1

An indication of the rootstocks used for the different experiments.

Year Type of

experiment

Rootstocks Treatments

2010/2011 Field

experiment

1103 Paulsen Well watered

Water deficit

110 Richter Well watered

Water deficit

140 Ruggeri Well watered

Water deficit

2011 Greenhouse

experiment

(ATC)


Water deficit


Water deficit

2012 Greenhouse

experiment

(NoATC)

1103 Paulsen Well watered

Water deficit


Water deficit

140 Ruggeri Well watered

Water deficit

99 Richter Water deficit

Ramsey Water deficit

2011/2012 Field

experiment

1103 Paulsen Well watered leaves full sun exposure

Leaves in the shade

Water deficit leaves full sun exposure

leaves in the shade

140 Ruggeri Well watered leaves full sun exposure

leaves in the shade

Water deficit leaves full sun exposure

Final report 15


TABLE 2

Characteristics of the plants used in the greenhouse at the onset of the experiments and the duration of

the water constraint treatments.

Greenhouse Experiments

Shoot lenght (cm) Total leaf area (m2)

Duration of the water constraint (days)

Automatic temperature control 99 Richter 92.3±3.8 0.16±0.01 12 (first water constraint)

5 (second water constraint) 110 Richter 91.7±3.8 0.17±0.01 12 (first water constraint)

5 (second water constraint) Without automatic temperature control

99 Richter 130.2±7.8 0.22±0.02 10 110 Richter 159.0±7.8 0.27±0.02 10 140 Ruggeri 157 .2±7.8 0.30±0.02 10

1103 Paulsen 116.2±7.8 0.23±0.02 10 Ramsey 179.3±10.0 0.27±0.02 10

TABLE 3

Leaf and stomata sampling.

Type of experiment Number of leaves per

treatment and per

date

Timing of the

sampling

Number of stomata

randomly selected for

stomatal size

Field (2010/2011) 8 harvest 96

Field (2011/2012) 5 harvest 400

Greenhouse

experiment (ATC)

4 From the beginning till

the end of the water

constraint

(four different dates)

320

Greenhouse

experiment (NoATC)

3 After 10 days of water

constraint

150

leaves in the shade

Final report 16


FIGURE 1

Temperature and humidity. A: Greenhouse experiment (ATC); B: Greenhouse experiment (NoATC); C:

Field experiment 2010/2011; D: Field experiment 2011/2012. Under greenhouse conditions, the

maximum temperature during the day in general did not exceed 30º C, in contrast under field conditions

the maximum temperatures were above 30º C. As expected the greenhouse (ATC) presented the lowest

fluctuation of temperature and humidity in comparison with greenhouse (NoATC) and field conditions.

TABLE 4

Average temperatures and VPD during experimentation

Experiments Mean temp max Mean temp min VPD

Greenhouse experiment (ATC) 28,1 °C 20,9 °C 1,58

Greenhouse experiment (NoATC) 30,0 °C 19,7 °C 2.39

Field experiment 32,1 °C 17,0 °C ND*

*No data.

Final report 17


FIGURE 2

Soil water content for pots in greenhouse (ATC). Under greenhouse (ATC), during the physiological

measurements, the soil water content was 14.7% ± 0.57 on a dry-mass basis for the well watered

treatments. At the end of the water deficit treatments, the soil water content dropped to 1.3% ± 1.61 on a

dry-mass basis for the water deficit treatments.

Final report 18


FIGURE 3

Soil water content in greenhouse (NoATC). The first and second dates correspond to the measurements

prior to respectively the onset and the end of irrigation treatments. At greenhouse (NoATC) the well

watered treatments had a soil water content of 14.5% ± 1.72 while the water deficit treatment dropped to

1.5% ± 2.44 on a dry-mass basis.

Final report 19


FIGURE 4

Soil water content during field experiments of cv. Pinotage grafted onto 110 Richter, 140 Ruggeri and

1103 Paulsen subjected to (A) water constraint and (B) without water constraint. Under field conditions,

irrigation commenced late December and ended late February. The volume of irrigation applied was

about 163, 181 and 213 mm for 110 Richter, 140 Ruggeri and 1103 Paulsen respectively divided among

five irrigations.

100

120

140

160

180

200

220

240

260

0 30 60 90 120 150 180 210

mm

wate

r in

top 0

.9

m s

oil

depth

Days

A

R110

Rug

Paulsen

100

120

140

160

180

200

220

240

260

0 30 60 90 120 150 180 210

mm

wate

r in

top 0

.9 m

soil

depth

Days

B

R110RugPaulsen

Final report 20


FIGURE 5

Plant water status for stomatal density and size experiments. A: Greenhouse experiment (ATC); B:

Greenhouse experiment (NoATC); C: Field experiment 2010/2011; D: Field experiment 2011/2012. The

water constraint treatments carried out in the greenhouses produced clear differences in terms of plant

water status in comparison to well watered treatments. Nevertheless under field conditions, the water

constraint treatments were not able to reach a severe water constraint obtaining only a moderate water

constraint in comparison with well watered plants.

Final report 21


FIGURE 6

Senescence of older leaves (left) and leaves wilting (right) in vines under water deficit treatments. In the

greenhouse with controlled ambient, it took approximately 10 days after withholding water to the pots to

reach a severe water constraint resulting in visible effects such as senescence of older leaves and leaves

wilting.

Final report 22


FIGURE 7

Plant water status. Greenhouse experiment (NoATC) under increasing water deficit. Plant water status

was affected by rootstocks under water deficit conditions in greenhouse (NoATC) and field conditions.

Plants grafted onto 99 Richter and 110 Richter presented a less negative ψstem under water constraint in

comparison with Ramsey. The differences were found at the beginning and during most part of the water

withholding, nevertheless similar values of plant water status occurred at the end of the water constraint

period.

Final report 23


FIGURE 8

Red : Far red ratio. Field experiment 2011/2012. As expected, leaves under full sun exposure were

exposed to a higher light intensity (higher R/FR ratio) in comparison to leaves growing inside the canopy

under permanent shade. Under shade conditions, the leaves from the water constraint treatment of the

vines grafted onto 1103 Paulsen were exposed to a higher light intensity in comparison with the rest of

leaves probably due to a less canopy density.

Final report 24


FIGURE 9

Photosynthesis. A: Greenhouse experiment (ATC); B: Greenhouse experiment (NoATC) considering

grapevines grafted onto rootstocks 110 Richter, 140 Ruggeri and 1103 Paulsen under well watered and

water deficit conditions; C: Field experiment 2010/2011; D: Greenhouse experiment (NoATC) considering

grapevines grafted onto rootstocks 110 Richter, Ramsey, 99 Richter, 140 Ruggeri and 1103 Paulsen

under only water deficit conditions. As expected, water constraints induced a reduction in photosynthesis.

Final report 25


FIGURE 10

Stomatal conductance. A: Greenhouse experiment (ATC); B: Greenhouse experiment (NoATC)

considering grapevines grafted onto rootstocks 110 Richter, 140 Ruggeri and 1103 Paulsen under well

watered and water deficit conditions; C: Field experiment 2010/2011; D: Greenhouse experiment

(NoATC) considering grapevines grafted onto rootstocks 110 Richter, Ramsey, 99 Richter, 140 Ruggeri

and 1103 Paulsen under only water deficit conditions. As expected, water constraints induced a reduction

in stomatal conductance. Differences in stomatal conductance in response to drought among the different

rootstocks were detected.

Final report 26


FIGURE 11

Stomatal density. A: Greenhouse experiment (ATC); B: Greenhouse experiment (NoATC) C: Field

experiment 2010/2011; D: Field experiment 2011/2012. In general stomatal density was not affected by

soil water deficit, except for the field experiment where leaves under water constraint treatment presented

a higher stomatal density in comparison to leaves under well watered treatment.

FIGURE 12

Stomatal density. Greenhouse experiment (NoATC) under increasing water deficit.

Final report 27


FIGURE 13

Stomatal size. A: Greenhouse experiment (ATC); B: Greenhouse experiment (NoATC) C: Field

experiment 2010/2011; D: Field experiment 2011/2012. In contrast, the pore diameter was affected by

water constraint treatments in most of the experiments that reached severe water constraint (Figure 13A

and 13B) inducing a reduction in the pore diameter size. Nevertheless under moderate water constraint

conditions the stomatal size was mostly not affected (Figure 13C and 13D).

Final report 28


FIGURE 14

Stomata on the abaxial surface of cv. Pinotage grafted onto 1103 Paulsen. A: water constraint. B: no

water constraint. The figure clearly shows how the soil water deficit induces a response in the stomatal

development that results in a reduction of the pore size.

Final report 29


FIGURE 15

Stomata on the abaxial surface of cv. Pinotage grafted onto 1103 Paulsen and 140 Ruggeri. A:

1103 Paulsen water constraint and full exposure. B: 1103 Paulsen water constraint and shade. C: 140

Ruggeri water constraint and full exposure. D: 140 Ruggeri water constraint and shade. The figures

clearly show that leaves growing in an environment with a lower light intensity (lower R/FR ratio) had a

lower stomatal density but bigger pore diameter. Differences in stomatal density and size were observed

on Pinotage leaves grafted onto different rootstocks, where plants grafted onto 140 Ruggeri presented

lower stomatal density but bigger pore diameter than 110 Richter and 1103 Paulsen.

Final report 30


FIGURE 16

Stomatal size. Greenhouse experiment (NoATC) under increasing water deficit. Differences in stomatal

density and size were observed on Pinotage leaves grafted onto different rootstocks, where plants grafted

onto 140 Ruggeri presented lower stomatal density but bigger pore diameter than 110 Richter and 1103

Paulsen.

Final report 31


Total cost summary of the project

TOTAL COST IN

REAL TERMS

COST

CFPA DFTS Deciduous SATI Winetech THRIP OTHER TOTAL

YEAR 1

YEAR 2

YEAR 3

YEAR 4

YEAR 5

TOTAL

sati winetech cfpa - sawis library

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