PROCEEDINGS OF THE INTERNATIONAL SYMPOSIUM

ON OLIVE IRRIGATION

AND OlL QUALITY

Co-Conveners S. Lavee

U. Yermiyahu

Nazareth, Israel

December 6-10, 2009

ISHS Section Nuts and Mediterranean Climate Fruits ISHS Commission Irrigation and Plant Water Relations

Acta Horticulturae 888 February 2011

ISSN 0567-7572 ISBN 978 90 6605 703 6, Acta Horticulturae nD

• 888 Price for non-members ofISHS: € 87,-Published by ISHS, February 2011

Executive Director ofISHS: J. Van Assche Technical Processing: J. Falleyn

ISHS Secretariat, PO Box 500, 3001 Leuven 1, Belgium

Printed by Drukkerij Station Drukwerk, PO Box 3099, 2220 CB Katwijk, The Netherlands

° 2011 by the International Society for Horticultural Science (ISHS). All rights reserved. No part ofthis book may be reproduced and/or published in any form or by any means, electronic or mechanical, including photocopying, microfilm and recording, or by any information storage and retrieval system, without written permission from the publishers.

Photographs on the front cover:

~2

3 4 5 6

1. Lysimeters for water and solute balance studies, Gilat, Israel. (Courtesy of Alon Ben-Gal) 2. Gas exchange measurement. (Courtesy of Alon Ben-Gal) 3. Desert olive orchard. Negev Highlands, Israel. (Courtesy of Amon Dag) 4. Olives. (Courtesy of Amon Dag) 5. Oil processing at Beit Shaan mill. (Courtesy ofNurit Agam) 6. Ancient olive tree in the Galilee. (Courtesy of Amon Dag)

2

The Leaf Patch Clamp Pressure Probe: a New Tool for Irrigation Scheduling and Deeper Insight into Olive Drought Stress Physiology

S. Rüger, W. Ehrenberger and U. Zimmermann Lehrstuhl für Biotechnologie, Biozentrum Universität Würzburg Am Hubland, D-97074 Würzburg Germany

D. Kool Irrigation and Water Engineering Wageningen University The Netherlands

A. Ben-Gal and N. Agam Environmental Physics and Irrigation Gilat Research Center Agricultural Research Organization Israel

Keywords: turgor pressure, water stress, monitoring, Olea europaea L.

Abstract Leaf turgor press ure provides a very sensitive indicator of plant water status.

Diurnal changes in turgor press ure of olives were measured over several months with a novel leaf patch elamp pressure (LPCP) probe. The LPCP probe is user-friendly, non-invasive, online-monitoring, robust and versatile, and is characterised by high precision, low-cost and automation suitability. Data are transferred wireless to an internet server via a mobile phone network for real-time evaluation and for remote regulation of irrigation or stress management. The probe measures the attenuated output pressure response (Pp) of a leaf patch upon application of an externally applied, constantly kept pressure, generated by two small magnets. Pp is sensed by a miniaturized press ure sensor embedded in silicon and integrated into one of the magnets. Concomitant measurements of balancing pressure values, Pb (i.e. stem water potential) using the pressure bomb technique revealed a very elose correlation between Pb and Pp' Both parameters depended inverselyon cell turgor pressure as evidenced by direct measurements of leaf turgor pressure using the cell turgor pressure probe. Measurements on olive trees plan ted in weighing lysimeters that allowed continuous monitoring of water balance of individual trees subjected to varying conditions of water status demonstrated the potential of the LPCP probe for irrigation scheduling and for elucidation of drought stress physiology.

INTRODUCTION Development of innovative sensors to provide sensitive and continuous plant

water status will aid preventing los ses by soil infiltration and evaporation. Water status monitoring of olives is of particular interest as stress conditions appear to be desirable for optirnization of oil yield and quality (Ben-Gal et al., 2009; Dag et al., 2008). Information on plant water status can be obtained by leafwater potential (Ferreira et al., 1997), stem water potential (Naor et al. , 1999), stomatal conductance (Tan and Layne, 1991), leaf temperature (Jones, 2004), variations in leafthickness (Mc Burney, 1992) and plant organ diameter (Goldhamer et al., 1999), xylem sap flow (Massai et al., 2000) as well as cell turgor and xylem pressure probe measurements (Zimmermann et al. , 2004; Tomos, 2000). Sensors monitoring soil water content and soil water potential in the root area have also been used to estirnate crop water status (Smith and Mullins, 2000; Dane and Topp, 2002; Iones, 2004).

Even though the plant-based sensors currently employed have been useful tools for basic research in plant physiology, none of them have found wide-spread application in agriculture and horticulture. Many sensor systems are highly sophisticated and expensive, susceptible to wind and rain and/or too insensitive to monitor water status. Some instruments used for monitoring water stress require removal of leaves or other organs. Furthermore, most sensor systems do not allow online monitoring over an entire

Proc. 1S on Olive Irrigation and Oil Quality Eds.: U. Yermiyahu et al. Acta Hort. 888, 1SHS 2011

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vegetation period; automation and remote control of irrigation are very often difficult or even impossible.

A novel, non-invasive and inexpensive probe for online monitoring of cell turgor pressure changes with high precision has recently been described (Zimmermann et al. , 2008, 2010; Westhoff et al., 2009). The so-called leaf patch clamp pressure (LPCP) probe (commercial name ZIM probe) consists of a miniaturised pressure sensor silicon­embedded and integrated into a magnetic clamp that is clipped to a patch of an intact plant leaf. The probe measures the pressure transfer function of the leaf patch, i.e. the attenuated output pressure, Pp, in response to the clamp pressure, Pclamp . The magnitude of the leaf pressure transfer function, and thus the attenuation of the constantly kept external pressure, is dictated by a plant-specific, turgor pressure-independent term (related with the compression of the silicone, the cuticle, cell walls and other structural elements) and a turgor pressure-dependent term. Thus, relative changes in turgor pressure are measured with high temporal resolution provided that the turgor pressure-independent term is kept constant. This was previously documented for leaves of grapevine and banana plants as weIl as for liana (Zimmermann et al. , 2008, 2010; Westhoff et al. , 2009). In this communication we demonstrate the potential for monitoring diurnal changes in leaf turgor pressure of olive trees using the LPCP probe.

MATERIALS AND METHODS

Plant Material Olives, Olea europaea 'Barnea', were planted in 15 2.5-m3 volume lysimeters

filled with a loamy sand soil at the Gilat Research Center in Israel. LPCP probes were attached to leaves of 3 year old non-bearing trees. The trees were normally irrigated daily with 120% of their measured evapotranspirative consumption. During the summer of 2009, the irrigation to 5 replicate trees was ceased for aperiod of one week (July 23 to July 28, inclusive) as available soil water was allowed to reach negligible levels while the other 10 trees continued with full irrigation.

Leaf Patch Clamp Pressure Probe: Principles and Theoretical Considerations The basic idea underlying the LPCP probe (Fig. 1 A) is that a small leaf patch is

used as a sensing element of turgor pressure changes in the surrounding uncovered tissue. A pre-condition is that the stomata of the sensing elements must be closed in order to avoid water loss (see the analogy to stern water potential measurements). This is achieved by clamping a leaf between two planar circular pads made of (non-transparent) aluminium as shown schematically in Figure 1 B. The lower pad contains an integrated pressure sensor chip. The clamp pressure is generated magnetically; the pad containing the sensor chip is fixed on a small toric magnet (see inset ofFig. IB), and the counter pad on a magnet that can be moved along a threaded rod. The distance between the two magnets dictates the clamp pressure. Variation of the distance allows adjustrnent of the magnetic force to the rigidity and thickness of the leaves.

Theoretical considerations have shown that the output patch pressure Pp sensed by the sensor chip upon application of an external clamp pressure, Pclamp, is apower function ofthe turgor pressure, Pe (Zimmermann et al., 2008; Westhoff et al., 2009):

I

Pp = (a/+b r ·Fa ·F::lamp (Eq.l)

where Fa is the attenuation factor and takes into account turgor pressure-independent losses due to the compressibility of the silicone of the sensor chip and leaf-specific structural elements such as cuticle, cell walls and intercellular air spaces. a and bare constants and equal or larger than unity. Eq. 1 does not hold for turgorless leaf tissue and was derived by assurning that the volumetrie elastic moduli of the cell walls depend linearlyon turgor pressure and are not temperature-dependent. The inverse relationship

224

between Pp and Pe beeomes particularly obvious ifthe constant ais equal to unity and bis mueh smaller than Pe. In this case, Eq. 1 becomes Pp = klPe where k = b . Fa . Pclamp, i.e. Pp and Pe show opposite changes. Fa is usually in the order of 0.2 to 0.3 and can be assumed constant over a large range of temperature and turgor pressures. However, upon approaching the plasmolytic point, Fa can vary considerably during day and night because of the dramatic water loss and the associated large midday temporary accumulation of air in the leaves. Under these conditions Fa assurnes very small values, i.e. the leaves are more compressible than during the night, and Eq. 1 loses validity.

Data Acquisition The LPCP probe values are continuously remotely recorded. The pressure sensor

chip is connected via a cable to a battery-powered wireless telemetrie transmitter (teleBITcom, Teltow, Germany). This transmitter sends the pressure signals together with the transmitter ID-code via an ISM band to a radio control unit located up to 500 m away (NTTB, Zeuthen, Germany). If needed, the distance between the transmitter and the radio control unit can be further increased using repeaters to amplify the transmitted signals. The radio control unit transfers the data with a time stamp to a GPRS modem linked to an internet server where the data are stored and subsequently analysed in a control centre. If the evaluation shows that the plants are subjected to drought, growers can be informed (e.g. by SMS or e-mail) or irrigation pumps/valves can be activate.

LPCP Calibration Calibration of the LPCP probe was performed using a cell turgor pressure probe.

The prineiple of this probe is described in detail elsewhere (Zimmermann et al. , 2004). Briefly, a microcapillary, sealed to a small Plexiglas chamber containing apressure transducer, is inserted into a leaf eell. Microcapillary and chamber are filled with ineompressible oil that transfers the turgor pressure to the pressure transducer. Beeause of their rigidity and due to logistic reasons, calibration was not done on olive leaves but was performed on leaves of a range of different perennial trees including grapevine, banana, liana and eucalyptus.

Pressure Charnber The Scholander pressure bomb (MRC, Israel) method was applied as described by

Shackel et al. (1997). For determination of balaneing pressure (stern water potential) values, leaflets were enclosed in darkened bags for at least two hours and allowed to equilibrate with the xylem water potential before measurements.

RESULTS As observed for leaves of other species (Zimmermann et al., 2008, 2010; Westhoff

et al. , 2009) homogenous contact between leaf and probe was aprerequisite for turgor pressure monitoring on olive leaves. Probe clamping partlyon the thick midrib of leaves led to erroneous results, because under these conditions, changes in the thickness of the rnidrib were measured. As expeeted, the initial Pclamp and, in turn, the Pp values and ranges had no effect on the diurnal Pp profiles of olive leaves. This was also proved by multiple probe readings on adjacent leaves exposed to the same microclimate (data not shown). Removal ofthe probes after 3 to 4 months showed no impressions or lesions. The adaxial and abaxial areas beneath the pads maintained the same colour as the surrounding tissue (Fig. 2). After longer clamping tirnes, the patch area can become somewhat brighter indicating some slight decrease in chlorophyll eontent. This had no apparent effect on the hydraulic connection ofthe cells in the patch with the surrounding uncovered cells.

Probe versus CeU Turgor Pressure and Stern Water Potential Measurernents According to Eq. 1 the LPCP probe measures relative ehanges in leaf turgor

pressure. This was proved by concomitant LPCP and cell turgor pressure probe measurements on leaves ofvarious plant species (Fig. 3). It is evident that the relationship

225

between Pp and Pe could be described quite weIl by Eq. 1 for all plant species independent of the Pp range determined by the selected clamp pressure P clamp.

Figure 4A shows a typical diurnal profile of Pp measured on an olive leaf of a weIl-watered tree together with balancing pressure values Pb (corresponding to negative values of stern water potential) on a sunny day (air temperature and relative humidity at noon: Ta = 35°C, RH = 26%). It is evident that the Pb values measured on several cut groups of leaflets show a similar diurnal trend as the Pp values measured on a single leaf. The small delay in response of Pb after sunrise and subsequent onset of transpiration resulted most likely from the fact that the leaves for the Pb measurements were taken from the north side of the tree. Leaves at this side became sun-exposed much later than the leaves located on the east side.

Despite the good agreement between the changes in the Pb and Pp values, incidents of variable weather and times when irrigation was ceased showed that the resolution of the LPCP probe was finer than that of the stern water potential measurements. An example is given in Figure 4B for another weIl watered tree. Under hot and dry conditions andlor water stress, oscillations of stomatal aperture occurred when turgor pressure dropped. These oscillations were reflected in the Pp values. Olives, similar to other plants (Farquhar and Cowan, 1974; Zimmermann et al., 2010), apparently keep transpiration water loss to a minimum under water shortage at the expense of photosynthetic productivity.

Drought Effects on LPCP Probe Readings Probe readings from periods in which olive trees were exposed to short-term

extreme drought conditions caused by cessation of irrigation are depicted in Figure 5. Irrigation was stopped on 23 July and reinstated on 29 July. Full tree transpiration decreased incrementally daily from the first day without irrigation and was less than 20% of that of fully irrigated trees on 28 July. From the first day of suboptimal conditions of soil water (24 July), an increase in the peak Pp value was recorded at noon, indicating lower turgor pressures compared to trees with optimal soil moisture (full irrigation). The time of turgor pressure recovery during the afternoon increased as the period of drought continued and the stress conditions increased. After 2 days (July 25) tree-scale transpiration was 50% of weIl watered tress and the Pp value established during the night due to water uptake via the roots also increased significantly, indicating that night-time high turgor pressure was not restored. Interestingly, when severe stress was reached after 4 days of drought (30% of transpiration relative to well watered trees), areversal in the diurnal Pp profiles was observed, i.e. Pp reached minimum values at noon and maximum values at night (see inset of Fig. 5). All the phenomena related to changes in turgor pressure due to drought stress were reversible and, following a single day ofre-irrigation, diurnal Pp profiles of the previously stressed trees were identical to those measured before the drought period.

DISCUSSION The data presented here demonstrate that the patch clamp pressure probe can

provide sensitive online monitoring of effects of transpiration andlor water stress on olive tree leaf water status. Experimental evidence is given that the LPCP output pressure Pp is inversely correlated with turgor pressure and is correlated linearly with balancing pressure values Pb (stern water potential). This suggests that both the pressure bomb and the LPCP probe measure relative changes in turgor pressure. We further suggest that the probe allows the definition of reliable threshold values for triggering irrigation or for maintaining desired conditions of water stress. Possible indicators of water deficits in olive trees (and other plants) are the increase ofthe peak Pp values at no on and ofthe base Pp values at night as weIl as the increase of the time of turgor pressure recovery during the aftemoon. The occurrence of Pp oscillations at severe turgor pressure losses is another important criterion for water deficits and particularly for fruit yield, because periodic closure of the stomata and, in turn, of CO2 assimilation, will certainly affect yield.

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Reversal of the diurnal changes of Pp as observed here for severely stressed olive leaves can be taken as evidence that the leaf cells were nearly turgorless and contained a large amount of air. Under such conditions the LPCP probe no longer sensed turgor pressure but rather changes in thickness were recorded (i.e. the attenuation factor Fa in Eq. 1 was extremely low during noon and higher at night). Interestingly, this dramatic change in the ratio of air to water in the leaf was not reflected in the Pb values (data not shown).

The determination of sub-optimum leaf water supply in real time opens up possibilities for water management. For irrigation monitoring calibration of the LPCP probes against the cell turgor pressure probe is not stringent. On the basis of the data depicted in Figure 5 it is sufficient to know the peak Pp values at no on and the minimum Pp values at night under appropriate irrigation conditions.

ACKNOWLEDGEMENTS This work was supported by a grant from the AIF (no. KF 0054703WM8) to U. Z ..

We would like to thank P. Geßner, G Zimmermann, A Yafe and E. Presnov for their support of the field studies and E. Stepien-Bötsch for her great help in evaluation of the LPCP datasets.

Literature Cited Ben-Gal, A, Agam, N., Alchanatis, v., Cohen, Y., Yermiyahu, U., Zipori, 1., Presnov, E.,

Sprints in, M. and Dag, A 2009. Evaluating water stress in irrigated olives: correlation of soil water status, tree water status, and thermal imagery. Irr. Sei. 27:367-376.

Dag, A. , Ben-Gal, A, Yermiyahu, U., Basheer, L., Nir, Y. and Kerem, Z. 2008. The effect of irrigation level and harvest mechanization on virgin olive oil quality in a traditional rain-fed 'Souri ' olive orchard converted to irrigation. J. Sci. Food and Agri. 88:1542-1528.

Dane, J.H. and Topp, GC. (eds). 2002. Methods of Soil Analysis. Part 4: Physical Methods. Soil Society of America Book Series: 5. Soil Science Society of America, Madison, WL

Farquhar, GD. and Cowan, LR. 1974. Oscillations in stomatal conductance: the influence in environmental gain. Plant Physiol. 54:769-772.

Ferreira, M.L, Pacheco, C.A, Valancogne, C., Michaelsen, J., Ameglio, T and Daudet, F.A 1997. Evapotranspiration, water stress indicators and soil water balance in a Prunus persica orchard, in central Portugal. Acta Hort. 449:379-385.

Goldhamer, D.A, Fereres, E., Mata, M., Girona, J. and Cohen, M. 1999. Sensitivity of continuous and discrete plant and soil water status monitoring in peach trees subjected to deficit irrigation. J. Amer. Soc. Hort. Sci. 124:437-444.

Jones, H.G 2004. Irrigation scheduling: advantages and pitfalls ofplant-based methods. J. Exper. Botany 55:2427-2436.

Massai, R., Ferreira, M.L, Par;o, TA and Remorini, D. 2000. Sap flow in peach trees during water stress and recovery in two environmental conditions. Acta Hort. 537:351-358.

McBurney, T 1992. The relationship between leaf thickness and plant water potential. J. ofExper. Botany 43:327-335.

Naor, A , Klein, 1., Hupert, H., Grinblat, Y., Peres, M. and Kaufman, A 1999. Water stress and crop level interactions in relation to nectarine yield, fruit size distribution, and water potentials. J. Amer. Soc. Hort. Sci. 124:189-193.

Shackel, K.A, Ahmadi, H., Biasis, w., Buchner, R., Goldhamer, D. , Gurusinghe, S., Hasey, J. , Kester, D., Krueger, B., Lampininen, B., McGourty, G, Micke, W., Mitcham, E., Olson, B., Pelletrua, K., Philips, H., Ramos, D., Schwankl, L. , Sibbett, S., Snyder, R. , Southwick, S. , Stevens on, M., Thorpe, M., Weinbaum, S. and Yeager, J. 1997. Plant water status as an index of irrigation need in deciduous fruit trees. Hort. Technol. 7:23-29.

Smith, K.A and Mullins, C.E. (eds). 2000. Soil and Environmental Analysis: Physical Methods, Second Edition. Marcel Dekker, New York.

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Tan, C.S. and Layne, R.E.C. 1991. Soil water content and stomatal conductance in a mature peach orchard as influenced by various irrigation regimes. Can. J. Soil Sei. 71 :253-258.

Tomos, A.D. 2000. The plant cell pressure probe. Biotechnology Letters 22:437-442. Westhoff, M. , Zimmermann, D., Schneider, H., Wegner, L.H., Geßner, P., Jakob, P.,

Bamberg, E. , Shirley, St. , Bentrup, F.-w. and Zimmermann, U. 2008. Evidence for discontinuous water colurnns in the xylem conduit of taU birch trees. Plant Biol. 11 :307 -327.

Westhoff, M., Reuss, R., Zimmermann, D., Netzer, Y. , Gessner, A. , Geßner, P., Zimmerniann, G. , Wegner, L.H., Bamberg, E. , Schwartz, A. and Zimmermann, U. 2009. A non-invasive probe for online-monitoring of turgor pressure changes under field conditions. Plant Biol. 11 :701-712.

Zimmermann, D., Reuss, R., Westhoff, M ., Geßner, P., Bauer, W., Bamberg, E., Bentrup, F.-W. and Zimmermann, U. 2008. A novel, non-invasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status. J. Exper. Botany 59:3157-3167.

Zimmermann, U., Rüger, S. , Shapira, 0 ., Westhoff, M., Wegner, L.-H.-, Reuss, R., Geßner, P. , Zimmermann, G. , Israeli, Y., Zhou, A. , Schwartz, A., Bamberg, E. and Zimmermann, D. 2010. Effects of environmental parameters and irrigation on the turgor pressure of banana plants measured using the non-invasive, online monitoring leafpatch clamp pressure probe. Plant Biol. 12:424-436.

Zimmermann, u., Schneider, H. , Wegner, L.H. and Haase, A. 2004. Water ascent in taU trees: does evolution of land plants rely on a highly metastable state? New Phytol. (Tansley Review) 162:575-615.

Figures

Fig. 1. The magnetic leaf patch clamp pressure (LPCP) probe. (A) Photograph of a probe clamped on an olive leaf in the field; (B) schematic diagram of the measuring principle of the probe (Pc/amp = applied clamp pressure; Pe = turgor pressure) and inset a schematic view of the lower part of the probe with the embedded sensor chip and silicone membrane.

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Fig. 2. Appearance ofthe adaxial (A) and ab axial (B) patches (marked by circles) of olive leaves upon removal of the probes after 3 months.

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Fig. 3. Calibration ofthe output pressure, Pp, values measured by the LPCP probe against the cell turgor pressure, Pe, using the cell turgor pressure probe. Measurements were performed on banana plants (Musa acuminate: open squares), oak (Qercus robur: filIed circles) eucalyptus trees (Eucalyptus gomphocephala: open triangles) and on grapevine (Vitis vinifera: filled triangles). Lines were firted to data points by Eq. 1.

229

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Fig. 4. (A) Concomitant measurements of output patch pressures, Pp (solid line) , and balancing pressure, Pb, values (using a Scholander pressure bomb) on a weH watered olive (28 July, 2009). The Pb values (circles) are averages, error bars are standard deviations, n=5. Note that Pb values reflect negative values of stern water potential. (B) Typical Pp oscillations of an olive leaf of a weH watered tree measured by the LPCP probe (8 November, 2009).

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Fig. 5. Part of a 4-month Pp recording on an olive tree under field conditions at the Gilat Research Center, Israel. The tree shown was subjected to drought by ceasing irrigation on 23 July and was re-irrigated on 29 July, 2009. Note the reversal in the diurnal Pp profiles after 3-days of non-irrigation: at this time Pp assumes maximum values during the night and minimum values at noon (see the enlargement in the inset).

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