Effects of regulated deficit irrigation under subsurface drip irrigation ...

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Five treatments were applied: T1 (surface drip), which was irrigated at 100%. ETc (crop evapotranspiration) ... E-mail: [email protected] with dry conditions ...
Plant and Soil 260: 155–168, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

155

Effects of regulated deficit irrigation under subsurface drip irrigation conditions on water relations of mature almond trees Pascual Romero1 , Pablo Botia1,2 & Francisco Garcia1 1 Instituto

Murciano de Investigaci´on y Desarrollo Agrario y Alimentario (IMIDA), Estaci´on Seric´ıcola 30150, La Alberca, (Murcia, Spain). 2 Corresponding author∗

Received 6 August 2003. Accepted in revised form 15 October 2003

Key words: Gas exchange response, Prunus dulcis, regulated deficit irrigation, subsurface drip irrigation, soil-plant relationships.

Abstract The influence of several regulated deficit irrigation (RDI) strategies under subsurface drip irrigation (SDI) conditions on water relations and gas exchange activity was analysed during a four year period for mature trees of a local variety of almond (Prunus dulcis (Mill.) D.A. Webb, cv. Cartagenera), in a commercial plantation in the district of Aljorra, Murcia (Southern Spain). Five treatments were applied: T1 (surface drip), which was irrigated at 100% ETc (crop evapotranspiration) for the full season, applying 603 mm per year; T2 (surface drip), irrigated at 100% ETc, except during the kernel-filling stage (from early June to early August), when 20% ETc was applied; T3 (SDI), equal to T2, but in subsurface drip irrigation; T4 (SDI), irrigated at 100% ETc, except during kernel- filling (20% ETc) and post-harvest (75% ETc); T5 (SDI), equal to T4, but a greater reduction of post-harvest irrigation (50% ETc). The severity of water stress was characterised by measurements of soil water content (θv ), predawn leaf water potential (pd ), osmotic potential (π ), relative water content (RWC), and gas exchange rates. Under stress (during the kernel-filling stage) and non-stress conditions (during active growth and post-harvest periods) in treatments T2, T3 and T4 there were not significant differences in the soil water content, nor in the plant water status and gas exchange parameters measured. Only T5 (SDI) showed a significant reduction in gas exchange activity at the end of the kernel-filling stage (point of maximum stress). This response was closely correlated with the severity of the stress reached. Minimum values of pd reached in this period were −2.37 MPa in T2, −2.26 MPa in T3, −1.95 MPa in T4 and −2.52 MPa in T5. Maximum reductions in photosynthesis rate (A), with regard to the control, were 64%, 61%, 58% and 75%, respectively. Reductions in A, E and stomatal conductance (gs ) in response to severe water stress were reversible. Although soil and plant water status recovered rapidly when trees were irrigated post-harvest, gas exchange activity (gs , E and A) recovered more slowly in all treatments. T5 showed a recovery of soil water status that was slower and incomplete compared to the other treatments, postharvest (early August-early September), although gas exchange activity was not affected in this period. These results indicate that these RDI strategies, with a severe irrigation deprivation during kernel-filling (20% ETc), and a recovery post-harvest at 75% ETc or up to 50% ETc under SDI, can be adequate in this orchard under semiarid conditions, due to a higher water application efficiency of this irrigation system, saving between 220–273 mm year−1 irrigation water.

Introduction In semiarid regions of Spain, almond (Prunus dulcis (Mill.) D.A. Webb) has been associated traditionally ∗ FAX No.: +34 968 366792. E-mail: [email protected]

with dry conditions, resulting in a low productivity. Only about 10% of this crop is irrigated (Girona, 1987). However, nowadays the greater profitability of modern orchards and the higher yields obtained, are causing a transition from traditional orchards, generally of low profitability to modern orchards, under ir-

156 rigation conditions. Although almond shows a positive response to irrigation, (León et al., 1985; Torrecillas et al., 1989; Hutmacher et al., 1994), in these semiarid regions, water scarcity is the main factor limiting yield, and it is difficult to apply full tree water requirements, to sustain maximal growth and yield. So there is interest in determining how to maintain optimum crop yields in water deficit conditions. Recently, this has stimulated research in new technologies, systems and irrigation strategies to improve water use efficiency. One of the most promising methods applied in almonds to improve irrigation efficiency has been the application of regulated deficit irrigation (RDI) strategies, reducing applied water in the low water stress sensitivity periods to obtain a beneficial horticultural response, such as a reduction in vegetative growth, while maintaining or increasing fruit growth (Lampinen et al., 1995). A second method involves the use of subsurface drip irrigation (SDI) systems, which improve the water application efficiency to the plant, delivering water and nutrients directly to the root zone and minimising soil surface evaporation, runoff and deep percolation (Hoffman and Martin, 1993; Phene et al., 1993; Phene, 1999). Moreover, SDI typically results in a larger wetted volume of soil and surface area available for root proliferation than surface drip irrigation, and a deeper root development associated with the depth of the water source (Phene, 1999). Up to now, the results show that, almond orchards can be suitable for application of RDI by reducing irrigation water during the kernel-filling stage, in which the main event is dry weight accumulation in the seed (kernel) and applying the full amount of water during most the stress-sensitive times, bloom, the active vegetative development phase and post-harvest (Girona and Marsal, 1995; Goldhamer and Shackel, 1989, 1990; Goldhamer, 1996). These studies have shown satisfactory results, with no significant reductions of yield and substantial water savings and increased irrigation efficiency (Andriani et al., 1989; Girona, 1992, 1994; Goldhamer and Viveros, 1991, 2000; Goldhamer and Smith, 1995; Goldhamer, 1996, 1997a, 1997b). Although the application of RDI in almond has been studied intensely, few studies report the physiological behaviour of almond under subsurface drip irrigation (Botía et al. 1998, 2000; Del Amor and Del Amor, 1999). These studies indicate that the subsurface irrigation system maintains better soil and plant water status than surface irrigation systems (Botía et al., 2000; Romero, 2002). So far, both techniques (RDI and SDI) have been applied separ-

ately; this study combines both technologies in order to improve water use efficiency in the almond orchard, saving water and maintaining high productivity. The aim of this first work was to determine the effects of several RDI strategies under subsurface drip irrigation conditions on soil and leaf water relations parameters. Specifically, we examined soil and tree water status, leaf gas exchange response, osmotic adjustment and the interrelationships between them. Moreover, we quantified the degree of water stress reached and the following recovery in order to evaluate, from a physiological point of view, the long-term response of field-grown almond trees under these irrigation conditions. We identified the pre-dawn leaf water potential threshold as being related to adequate soil water content and leaf function.

Material and methods Plant material, experimental conditions and treatments The study was carried out from 1997 to 2000, on a commercial plantation of 13-year old-almond trees, cv. Cartagenera, grafted on almond rootstock, with tree-spacing at 7 × 5 m (285 trees ha−1 ) on drip-line irrigation. The plantation is situated in the district of Aljorra, Murcia (Southern Spain). The orchard comprised rows of Prunus dulcis cv. Cartagenera (70%) planted alternately with pollenizer rows of cv. Ramillete (30%). All measurements were made on cv. Cartagenera trees. The soil is of a fine-loam texture to a depth of 100 cm and clay-loam below this. The irrigation water during the experimental period had an average electrical conductivity (EC) of 1.3 dS m−1 . The weather is Mediterranean semiarid with scarce annual rainfall at the experimental site (280 mm) and total annual reference Evapotranspiration (ETo), calculated via Pan evaporation Class-A method, U.S. Weather Bureau, between 1100 and 1200 mm. The experiment was started with 9-year-old (in 1997) trees, with the canopy fully developed. No weeds were allowed to develop within the orchard, resulting in a clean orchard floor for the duration of the experiment. Pest control practices and pruning were those commonly used by growers. The trial involved five irrigation treatments that were applied in four consecutive years, using two irrigation systems, surface and subsurface drip and three different regulated deficit irrigation strategies

157 Table 1. Irrigation treatments

Treatment

Irrigation system

January–early June Bloom, active vegetative and fruit growth

early June–early August Kernel-filling stage

early August–end of cycle Post-harvest

T1 T2 T3 T4 T5

Surface Surface Subsurface Subsurface Subsurface

100% ETc 100% ETc 100% ETc 100% ETc 100% ETc

100% ETc 20% ETc 20% ETc 20% ETc 20% ETc

100% ETc 100% ETc 100% ETc 75% ETc 50% ETc

Table 2. Annual applied water for each treatment. Annual rainfall and ETo (reference evapotranspiration) at the experimental site Water applied (mm)

Rainfall ETo

Year

T1

T2

T3

T4

T5

mm

mm

1997 1998 1999 2000 Mean % reduction

571 602 595 644 603 0

387 442 441 475 436 28

385 442 441 475 436 28

346 382 384 416 382 37

312 322 328 358 330 45

295 200 243 389 277

1103 1136 1227 1099 1120

(Table 1). The lay-out of the experiment took the form of four complete randomised-selected plots. Four repetitions (one per plot) were performed for each treatment, with seven trees per repetition. Border rows of trees were avoided, to eliminate potential border effects (water extraction by roots from an adjacent row). T1 (control) was irrigated at 100% crop evapotranspiration ETc (ETo calculated via Pan evaporation method-Class A, U.S. Weather Bureau) over the entire crop season (Table 2). Crop coefficients applied were 0.22 in January, 0.33 in February, 0.42 in March, 0.52 in May, 0.61 in June, July, August and September, 0.54 in October, 0.38 in November and 0.23 in December. High frequency irrigation (in short pulses) was applied daily and was controlled and adjusted weekly according to the soil matric potential (measured by tensiometers located at 15, 30 and 60 cm depth) and daily climatic data at a weather station in the vicinity (1 km) of the experiment. In both irrigation systems, a drip-line was utilised with four self-compensating drippers (type RAM, 3.5 l h−1 ) per tree, 1 m apart. In the subsurface drip irrigation system, the drip-line was buried at 35 cm depth. A root growth-inhibiting chemical, trifluralin (dinitro- N, N-

dipropyl-4 trifluoro methylanidine), was used in the filtration system to avoid root intrusion in the buried drips. All treatments received the same annual amount of fertilisers: 130 kg N, 58 kg P2 O5 and 78 kg K2 O per ha, which were supplied through the irrigation system. Nitrogenous solution (32% N) was applied in January and September and from 16th–31st October, potassium nitrate in February, March (1st–15th), May, July and October (1st–15th), ammonium nitrate in March (16th– 31st), April, June, August and phosphoric acid in November and December. Irrigation was controlled automatically by a head-unit programmer and electrohydraulic valves. The amounts of water applied for each irrigation treatment (Table 2) were measured with flowmeters. Water relations and gas exchange measurements Volumetric soil water content (θv ) was measured over the course of the experiment using a neutron probe (Troxler, model 3332; North Carolina, USA). Readings were taken close to trees, 25 cm from the drip head and perpendicularly oriented to the drip-lines, at depths of 20 cm intervals to a maximum depth of 160 cm, for four repetitions per treatment. Each year from April to October, leaf water potential at dawn (pd ) was measured periodically before actual sunrise. Two expanded leaves from the middle third of the tree, and in several orientations were taken from twelve trees per treatment. Leaf water potential at dawn was measured by using a pressure chamber (model 3000; Soil Moisture Equipment. Corp., Santa Barbara, California, USA), according to the Scholander et al. (1965) and Turner (1988) technique. In the same way, stem water potential (x ) was measured at noon in six leaves per treatment per plot. The leaves were enclosed within foil-covered plastic and aluminium envelopes at least 1 h before the midday

158 measurement (midday stem water potential) (Garnier and Berger, 1985; McCutchan and Shackel, 1992). In 1998 and 1999, relative water content at dawn (RWC) was obtained under the same conditions as pd . Leaves were sampled in the same way as for pd . Relative water content was calculated using the equation: RWC (%) = [(wf-wd)/(wt-wd)] ×100 (Slavik 1974), in which wf, wd and wt refer to fresh weight, dry weight and turgid weight respectively. Rehydration was carried out by submerging leaf petioles in distilled water overnight (Yoon and Richter, 1990). In 1998 and 1999, estimates of leaf osmotic potential at full turgor (π100), leaf water potential at the turgor loss point (π0 ), relative water content at the turgor loss point (RWC0 ) and bulk modulus of elasticity (ε) were obtained in May (before stress), July (point of greatest stress) and October (recovery period), via pressure volume (PV) analysis (Tyree and Hammel, 1972; Turner, 1988) of eight fully-expanded leaves per treatment. Pressure volume curves were generated using the free transpiration dehydration method (Hinckley et al., 1980) and the analysis was realised using a type IIa transformation (Tyree and Richter, 1981), drawing graphically the relationship between 1/ and RWC. Average cell wall elasticity was estimated as the slope of the relationship between p and relative water content, by the equation ε = RWC∗0 tan a (Wilson et al., 1979), tan a, being the slope of the regression line for p vs. relative water content (Warren-Wilson, 1967). p was calculated from the difference between π and . Gas exchange measurements were taken between 9:00 and 11:00 a.m., daylight hours, weekly from June to September in 1998 and 1999. These were taken from eight entirely unfolded, healthy leaves per treatment, exposed and perpendicularly-oriented to the sun, from young branches in the middle third of the tree, using a portable photosynthesis analyser IRGA (model LCA-4, ADC Bioscientific Ltd., Hoddesdon, U.K.) and a leaf chamber PLC-4N (11.35 cm2 ), configured to an open system. Atmospheric CO2 concentrations varied between 370 and 385 µmol mol−1 . All measurements were taken with a PAR solar radiation above 1500 µmol m−2 s−1 and an internal chamber temperature between 30–35 ◦ C. The rate of molar air flow (U ) inside the chamber was 155 µmol s−1 . Simultaneously, the net rate of photosynthesis (A), stomatal conductance (gs ), and transpiration rate (E) were measured. Calculations of these parameters were performed using Von Caemmerer and Farquhar (1981) equations and adjusted according to the leaf area .

Regressions and statistical analysis Relationships between parameters were fitted to linear and non-linear regressions. Analysis of variance (ANOVA) was used in order to discern the main treatment effects. Results and discussion Soil water content In the deficit treatments a severe irrigation cut-off during kernel-filling (early June – early August), produced a decrease of soil water content (θv ) in the zone of highest root density. After three weeks, minimum values of θv were around 16–20% (Table 3). This strong decrease in θv was aggravated by a high evaporative demand and rainfall absence in this period. In the surface treatment, T2, θv in the root zone (0–40 cm soil profile) was significantly lower than for T1 in the irrigation-deprivation period (Table 3). The mean reductions were about 24% and 35%, respectively, compared to T1. In the post-harvest period, after the re-irrigation, the recovery of soil water content was very rapid, reaching values similar to T1 or even higher in some years (Table 3). This could be due to a high water storage and water retention capacity of the soil at the test site, given its loamy nature. In the subsurface treatments, water content at the soil surface (0–20 cm) was lower, (θv = 15%) compared to the surface system, reducing soil surface evaporation (data not shown). In all years, T3 showed values of θv significantly higher than T1 and T2 in the 100% ETc period (March–May) and post-harvest (in 1998 and 1999) (Table 3). During these periods, values were maintained at about 28–32%. During kernelfilling (in 1999 and 2000), the maximal reduction of θv in the subsurface treatments was around 25% in T3 and 30% in T4 and T5 compared to T1. Post-harvest, the recovery of water content in the soil profile was related to applied water amounts in this period and to the irrigation system. The recovery of T3, irrigated at 100% ETc, was very rapid, showing in 1998 and 1999 a significantly higher θv than the surface treatment T2 and higher even than T1 in the post-harvest period (Table 3). The recovery of irrigation at 75% ETc (T4) in subsurface drip conditions was enough to recover soil water content in the root zone, to similar levels as T1 (Table 3). However, in T5, irrigation to 50% ETc post-harvest was not enough for recovery of soil water content to the level of the other

159

Figure 1. Seasonal patterns of pre-dawn leaf water potential (pd ) and midday stem water potential (x ) in 1998 (A and B). Seasonal patterns of pre-dawn leaf water potential (pd ) and relative water content (RWC) in 1999 (C and D). Vertical bars indicate the standard error of the mean. Each point is the average of twenty four measurements per treatment.

deficit treatments, being significantly lower than T2, T3 and T4. During the months immediately after the harvest (August and September), T5 had significantly lower soil water content in the root zone compared to T1, about 18%, 12% and 4% lower depending on the year (Table 3). Plant water status The decrease of θv produced a rapid reduction of pd , x and RWC (Figure 1), such that by seven days after irrigation decrease, significant differences between T1 and the other treatments had occurred (Figure 1). The differences between the deficit treatments were accentuated during water stress development, the largest

differences being at the end of kernel-filling and at the beginning of post-harvest (Figure 1). During this 30 or 40 day period (July 23rd to August 24th), mean pd values of about −1.12 MPa in T4 and −1.48 MPa in T5 were reached, the latter value being significantly lower than the other treatments. The water stress level and the water stress development rate (indicated by decrease of pd ) depended on the year. In 1999, a higher level of water stress was reached than in the other years due to environmental conditions (higher evaporative demand). In this year mean pd values at the end of kernel-filling were −0.76 MPa in T1, −2.37 MPa in T2, −2.26 MPa in T3, −1.95 MPa in T4 and −2.52 MPa in T5 (Figure 1C). More severe levels of water stress in this phase have been recorded

160

Figure 2. Relationship between soil water content (θv ) and pre-dawn leaf water potential (pd ) for surface (A) and subsurface (B) drip irrigation systems. Each point is the average of four repetitions for the soil water content, taken 25 cm from the drip head in the root zone (0–60 cm in surface and 40–80 cm in subsurface drip irrigation), and for pre-dawn leaf water potential measured in the same tree. Long dashed lines indicate the threshold value of soil water content in the root zone (see text). Surface Irrigation System (pd = −1440.93θv−2.46 , r = 0.72, P < 0.001). Subsurface Irrigation System (pd = −395.01θv−1.97 , r = 0.69, P < 0.001). The threshold values were placed (long-dashed lines) when the percentage of change in the slope of the curve (pd /θv ) was above 5%. In this point the slope started to increase faster, coinciding with a pre-dawn leaf water potential of around −1 MPa.

in almonds under RDI by Goldhamer (1996). RWC was also significantly different between treatments, confirming the results obtained for pd (Figure 1D). For T5, mean values of RWC in 1999, were around 80% at the point of greatest water stress. There was a

high linear correlation between these two parameters (RWC = 0.1352pd −13.42, r 2 = 0.87). Two weeks after the renewal of irrigation, in the post-harvest period, the recovery of plant water status was complete, not only in the treatments in which 100% ETc was applied, but also in the subsurface

161

Figure 3. (A), Relationship between osmotic potential at full turgor and pre-dawn leaf water potential (pd ) (y= 0.71 + 0.17x, r = 0.69, P < 0.001). (B), Relationship between osmotic potential at full turgor and relative water content (RWC) (y= −3.17 + 0.02x, r = 0.61, P < 0.001). (C) and (D), Relationship between turgor potential and leaf water potential for each treatment.

Table 3. Mean values of volumetric soil water content (θv ) in the zone of highest root density for each treatment at three important periods in three years of the experimental period. The zone of highest root density for surface treatments T1 and T2 was 0–40 cm depth and for subsurface treatments T3, T4, and T5 was 40–80 cm depth (data not shown) Volumetric soil water content (%) Treatment T1 T2 T3 T4 T5 ANOVA

March-May 27.7 a 27.4 a 31.0 b 30.4 b 27.9 a ***

1998 June-July 24.9 a 16.0 c 23.1 a 20.6 b 18.6 b ***

Aug-Sept 24.7 b 26.3 b 30.2 a 22.9 c 20.2 d ***

March-May 24.0 a 24.9 a 28.1 b 27.6 b 27.0 b ***

1999 June-July 24.8 c 18.0 ab 18.6 b 17.5 a 17.7 ab ***

Aug-Sept 26.9 ab 28.8 c 30.8 d 27.6 b 25.8 a ***

March-May 26.0 a 27.3 ab 29.2 c 26.9 ab 28.0 bc **

2000 June-July 26.4 a 20.0 b 20.0 b 18.2 c 19.9 b ***

∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. Separation by Duncan’s multiple range test at the 95% confidence level.

Aug-Sept 30.8 bc 31.1 bc 32.5 c 29.3 ab 26.9 a ***

162

Figure 4. (A), Seasonal patterns of transpiration rate, E; (B), stomatal conductance, gs , and (C), net photosynthesis rate, A, during the kernel-filling stage and post-harvest, in 1999, for the treatments. Vertical bars indicate the standard error of the mean. Each point is the average of eight measurements.

treatment T4, irrigated in this phase at 75% ETc. This recovery was associated with a rapid recovery of the soil water content. Treatments T2, T3 and T4 reached values of pd similar to T1, between 12 and 15 days after the renewal of irrigation, depending on the year. However, the subsurface treatment T5, watered postharvest at 50% ETc, had pd and RWC values, that were slightly lower than the other treatments, even two months after re-irrigation (Figure 1). This was

associated with a lower soil water content of this treatment. The relationship between the volumetric soil water content in the root zone and pre-dawn leaf water potential, observed in this study (Figure 2), was similar to others observed for different fruit trees under RDI, for example peaches (Girona et al., 1993; Natali et al., 1985), apricots (Perez, 2001) and citrus (Gonzalez, 1998). This soil-plant relationship was similar in both irrigation systems and suggests a threshold value of soil moisture, below which pd decreases greatly as a result of small changes in soil water content, inducing tree water stress. These values were slightly lower in the surface system (19%) than in the subsurface (21%) (Figure 2). These threshold values of soil water content coincide with pd values of about −1 MPa, and reflect an adequate water status in the tree. These results may indicate optimum levels of soil moisture which should be maintained in the root zone during critical stages to avoid deleterious effects on tree growth and productivity. Moreover they can be used as a useful tool for irrigation scheduling as a function of plant water status, Similar threshold levels of pd (between −1 MPa and −1.5 MPa) have been suggested for almond grown under different edaphoclimatic conditions by Girona and Marsal (1995) and Prichard (1996) in order to minimize the deleterious effects of a reduced water supply. The osmotic potential at full turgor (π100) during water stress decreased in the deficit treatments, reaching values significantly lower than the control at the greatest point of stress (31st July). We observed a high correlation between π100, RWC and pd (Figure 3A and B). Based on P-V curves, the deficit treatments T2 and T3 had π100 values of −1.13 and −1.15 MPa, respectively, as opposed to −0.90 MPa in the control (Table 4). The osmotic adjustment (active accumulation of solutes in the cells due to metabolic activity in response to stress) observed was −0.23 MPa in T2 and −0.25 MPa in T3. Similar values of osmotic adjustment (−0.3 MPa) have been recorded in almond under water stress (Castel and Fereres, 1982). These results show the limited osmotic regulation capacity of almond, with respect to other fruit trees, as pointed out by Goode and Higgs (1973). The observed decrease in π100 from May to October (post-harvest) (Table 4), could be due to ontogenetic processes in the leaf (Yoon and Richter, 1990). Post-harvest, there were no significant differences between treatments for π100 or bulk modulus of elasticity (ε) (Table 4). These results indicate the reversibility of the osmotic adjustment when

163 Table 4. Pressure-volume curve parameters for each treatment: leaf osmotic potential at full turgor (π100 ), leaf water potential at turgor loss point (π0 ), relative water content at the turgor loss point (RWC0 ) and bulk modulus of elasticity (ε), in the irrigation treatments in three points of the experiment: May 31st (no stress), July 31st (greatest stress point) and October 19th (post-harvest) sampling dates (1999) 31st May (no water stress) Treat. π0 RWCo ε (MPa) (MPa) (%) (MPa) T1 −0.8 −1.2 92.9 10.3 T2 −0.7 −1.0 94.3 10.9 T3 −0.8 −1.2 94.0 11.3 T4 −0.8 −1.1 93.3 10.2 T5 −0.8 −1.2 93.1 10.3 ANOVA n.s. n.s. n.s. n.s. π100

31st July (Severe water stress) π0 RWCo ε (MPa) (MPa) (%) (MPa) −0.90 c −1.4 c 92.2 c 10.4 b −1.13 a −1.8 a 89.6 a 9.3 ab −1.15 a −1.8 a 88.7 a 8.9 a −0.98 bc −1.6 bc 91.1 b 10.3 b −1.05 ab −1.7 ab 89.8 a 8.8 a *** *** *** ** π100

Post-harvest (Recovery period) π100 π0 RWCo ε (MPa) (MPa) (%) (MPa) −1.0 −1.7 ab 89.5 a 8.3 −1.2 −1.9 a 88.6 a 9.1 −1.0 −1.6 ab 89.7 ab 8.2 −0.9 −1.4 b 91.4 b 8.9 −1.2 −1.9 a 89.3 a 9.8 n.s. * * n.s.

n.s. not significant; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. Separation by Duncan’s multiple range test at the 95% confidence level.

Figure 5. (A), Relationship between photosynthesis, A and pre-dawn leaf water potential, pd (y= 10.18 + 2.61x, r = 0.82∗∗∗ ). (B), Relationship between stomatal conductance and pre-dawn leaf water potential (y= 0.15 + 0.045x,r = 0.85∗∗∗ ). (C), Relationship between instantaneous water use efficiency, A/E and pre-dawn leaf water potential, pd (y = ln 30.08 + 9.01x, r = 0.56∗∗∗ ). (D), Relationship between intrinsic water use efficiency, A/gs , and pre-dawn leaf water potential, pd y(= 88.33e(−0.5∗(x+1.77)/1.79)2 , r = 0.76∗∗∗ ). Each point is the average of eight measurements. Measurements were taken from June to September 1999. ∗∗∗ P < 0.001.

164

Figure 6. (A), Relationship between photosynthesis, A, and stomatal conductance, gs .(B), Relationship between transpiration rate, E, and stomatal conductance, gs . Each point is the average of two measurements. Measurements were taken from June to September 1999.

the severe water stress was eliminated. The trees of the treatments T2, T3 and T5 had lower relative water content at the turgor loss point (88–89%), compared to T1, (92.2%) (Table 4). This suggests a higher capacity to maintain cell turgor to a lower RWC and  (Figure 3C and D). During water stress, ε decreased significantly in the subsurface treatments T3 and T5 (Table 4), indicating almond trees are able to adjust elastically under severe water stress, as pointed out by Ruiz-Sanchez et al. (1993).

Gas exchange response During 100% ETc application in the active period of growth (March – May), there were no significant differences between treatments in the gas exchange parameters (Table 5). During the stress, a gradual decrease in E, gs and A was observed in all deficit treatments (Figure 4A, B and C, respectively), reaching values significantly lower than T1 a week after the commencement of irrigation deprivation. In 1999, after 64 days of stress (at the end of the kernel-filling stage), reductions of A were maximal; 64% in T2, 61% in T3, 58% in T4 and 75% in T5 (Table 5). In this period there were no significant

a RWC gs A E A/E A/gs

For each row values followed by distinct letters are significantly different as determined by Duncan’s multiple range test at the 95% confidence level. Units: pd (MPa), RWC (%), A (µmol m−2 s−1 ), gs (mol m−2 s−1 ), E (mmol m−2 s−1 ), A/gs (µmol mol−1 ), A/E (µmol mmol−1 ).

30 days after stress (10th September) T1 T2 T3 T4 T5 −0.4a −0.3b −0.3b −0.3b −0.5a 92.8 93.2 93.4 92.8 93.3 0.13 0.17 0.13 0.11 0.12 8.9 9.6 8.8 9.3 9.1 2.2 2.4 2.4 2.3 2.3 4.01 4.01 3.83 4.19 4.12 69.0 57.6 73.9 85.8 82.7 15 days after stress (24th August) T1 T2 T3 T4 T5 −0.6b −0.5b −0.5 b −0.5b −0.7a 93.3b 93.2b 93.1b 93.5b 90.7a 0.17c 0.13ab 0.16bc 0.13a 0.12a 11.3b 9.0 a 9.6a 8.9a 8.7a 2.9 c 2.6 ab 2.9 bc 2.5a 2.7abc 3.92 3.54 3.34 3.70 3.31 69.6 70.9 65.0 73.1 72.6 7th August T1 T2 T3 T4 T5 −0.8 c −2.4 a −2.2ab −1.9b −2.5a 93.0 c 82.4a 83.2ab 85.1b 81.7a 0.10 b 0.04 a 0.05a 0.05a 0.04a 9.1 c 3.3 b 3.5b 3.8b 2.3a 3.6 b 1.6 a 1.8a 1.9a 1.7a 2.64 a 1.68bc 1.81b 1.85b 1.27c 74.1 71.4 75.7 77.9 67.0 31st May T1 T2 T3 T4 T5 −0.6 −0.5 −0.4 −0.4 −0.5 96.0 95.5 97.7 96.8 95.8 0.14 0.13 0.15 0.14 0.15 9.9 9.7 10.9 10.4 11.0 3.1 3.3 3.6 3.3 3.4 3.2 2.9 3.0 3.2 3.3 70.7 74.6 72.6 74.3 73.3 Param.

Post-harvest (Recovery period) Vegetative growth phase (No water stress) End of kernel-filling stage (Severe water stress)

Table 5. Mean values of pd , RWC, A, gs , E, A/E and A/gs at four important points of the experimental period during a single year of the experiment (1999)

165 differences in A between treatments T2, T3 and T4. Transpiration rate also decreased significantly in the deficit treatments compared to T1, with reductions of 56% in T2, 50% in T3, 47% in T4 and 54% in T5 (Table 5). Only T5 showed values of A significantly lower than the other deficit treatments after one month of stress (during the maximum stress period, 9 July – 7 August), indicating a greater effect of water stress on photosynthetic activity in this treatment. However, this response depended on the year. So, in 2000 there were no significant differences in gas exchange parameters between treatments (data not shown), possibly due to lower water stress levels being reached during the kernel-filling stage (minimum values of pd > −2 MPa in 2000, as opposed to 1999, pd < −2 MPa, see Table 5). Post-harvest, there was a delay in the recovery of gas exchange compared to plant water status (Table 5). The delay in recovery of gs with regard to plant water status has been observed in other varieties of almond under water stress (Torrecillas et al., 1996). Such a response indicates that stomatal aperture and gas exchange could be related not only to mesophyll water status, but also to other factors, such as the transmission of chemical signals originating in the roots, such as abscisic acid, which could regulate the stomatal response in plants under water stress (Davies and Zhang, 1991), as shown for young almond plants by Wartinger (1990). There were no significant differences in E and gs between T2 and T3, 15 days after the renewal of irrigation at 100% ETc (Table 5). Also, T4 and T5, irrigated in this phase at 75% and 50% ETc, respectively, had stomatal conductances similar to T2, although significantly lower than T1. The recovery of A was similar in all deficit treatments, with no significant differences between them after 15 days, but there were differences from the control. Despite the significant reduction in A at the point of maximum stress, the subsequent recovery of A was relatively rapid, reaching, at day 15, values of 79% in T2 and T4, 85% in T3 and 77% in T5. One month later values of A, E and gs were similar or even higher than the control (Table 5). This also suggests that as far as the photosynthesis is concerned under severe stress, damage is reversible. These results also indicate that the application of less water post-harvest (T4 and T5) under SDI was sufficient to recover gas exchange activity after severe water stress, maintaining rates of A and E similar to T2 and T3. Having a photosynthetic

166 apparatus capable of achieving recovery after stress is also typical of drought tolerant plants (Ni and Pallardi, 1992) and can be advantageous in the application of RDI strategies. However, other studies in almonds have shown that the recovery of A after severe water stress was slow and incomplete (Klein et al., 2001). After three years, subsurface treatments T4 and T5 did not show a cumulative effect of water stress on gas exchange response post-harvest. A possible explanation could be that, in all years, the levels of θv and pd maintained in this period were above threshold values estimated (θv > 21% and, pd > −1 MPa, see Figure 2, Tables 3 and 5), indicating the presence of a slight stress that did not produce effects on gas exchange. The decreases of A and gs were closely correlated with the severity of stress, showing a high linear correlation with pd (Figure 5A and B) and RWC (data not shown). Previous work in almonds under water stress has shown different patterns in the relationship between gs and pd (with threshold levels of pd ) (Torrecillas et al., 1988). Instantaneous water use efficiency (A/E) decreased gradually during the water stress development (Figure 5C). When the water stress was severe (at the end of the kernel-filling stage), A/E was significantly lower in the deficit treatments compared to T1 (Table 5). The treatment T5 was the most affected, showing significant reductions in A/E compared to the other deficit treatments (T2, T3 and T4). On the other hand, the intrinsic water use efficiency (A/gs ) increased linearly with increasing water stress over a wide range of potentials (pd > −2.0 MPa) (Figure 5D). Below these values (pd < −2.0 MPa), the efficiency (instantaneous and intrinsic) dropped, suggesting a threshold level of pd in order to maintain an adequate leaf function under moderate water stress. A close correlation was also observed between A and gs and E and gs (Figure 6A and B). Photosynthesis values increased gradually with gs , but tended to stabilise for high values of gs in all treatments. Similar correlations have been observed in other varieties of almond (Rieger and Duemmel, 1995; Marsal et al., 1997). At high conductances (gs > 0.1 mol m−2 s−1 ), for the same value of gs , the deficit treatments had lower transpiration rates than for T1 (Figure 6B). From these results we conclude that deficit irrigation applied under SDI did not affect clearly the studied tree water status parameters and their relationships, and produced a higher water application efficiency post-harvest. Only subsurface drip irrigation treatment

T5, with an irrigation deprivation (80% ETc) in the kernel-filling stage and post-harvest recovery at 50% ETc, showed a slower recovery of soil water content post-harvest (from early August to early September), although plant water status and gas exchange activity in this period was not affected. Maintenance of pd above (less negative) threshold levels in the kernelfilling stage (−2 MPa) and post-harvest (−1 MPa) was correlated positively with adequate soil water content and leaf function, and can be a useful tool for application of RDI to almond. In semi-arid zones with strictly limited water, these results could be relevant for applying long-term RDI under subsurface drip systems, saving a substantial amount of irrigation water in periods of high evaporative demand, in the kernel-filling stage (up to 80%) and post-harvest (25–50%).

Acknowledgements The authors would like to thank Dr David Walker for correction of the English. This work has been supported partially by a grant of The Institute Euromediterráneo de Hidrotecnia Foundation, awarded to Pascual Romero Azorín.

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