Ben-Gurion University of the Negev Jacob Blaustein Institutes for Desert Research Albert Katz International School for Desert Studies
Characterization of new olive ( Olea europea L) varieties response to irrigation with saline water in the Ramat Negev area
Thesis submitted in partial fulfilment of the requirements for the degree of "Master of Science in Deserts Studies"
By Sebastian Weissbein
October, 2006 II
Ben-Gurion University of the Negev Jacob Blaustein Institutes for Desert Research Albert Katz International School for Desert Studies
Characterization of new olive ( Olea europea L) varieties response to irrigation with saline water in the Ramat Negev area
Thesis submitted in partial fulfilment of the requirements for the degree of "Master of Science"
By Sebastian Weissbein
Under the Supervision of: Prof. Zeev Wiesman 1 Prof. Moshe Silberbush 2 Dr. Jhonathan Ephrath 2
1. Department of Biotechnology Engineering The Institutes for Applied Research Ben-Gurion University of the Negev 2. The Jacob Blaustein Institutes for Desert Research Ben Gurion University of the Negev
Author's Signature …………….……………………… Date …………….
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Approved by the Director of the School …………… Date ……………. III
Characterization of olive ( Olea europea L) varieties response to irrigation with saline water in the Ramat Negev area
Sebastian Weissbein Ben- Gurion University of the Negev 2006
Thesis submitted in partial fulfilment of the requirements for the degree of "Master of Science in Deserts Studies"
In many countries of the Mediterranean basin, especially those in the arid zone with high rates of population growth, urbanization and industrialization, water is becoming a scarce resource. High-density olive orchards are increasing around the world, many of which may be potentially affected by salinity. In this study, the vegetative, reproductive and oil quality parameters of eleven olives varieties were measured in a saline water drip irrigated existing olive orchard situated in the central area of the Negev desert of Israel. Root development of olive cv Barnea was studied measuring total root number, root length and surface area during two growing seasons. Saline water significantly affected trunk circumference at the early stage of trunk growth. During the 2002 growing season fruit weight was affected by salinity but did not affected fruit and oil yield after four years. Basic oil quality parameters (acidity and peroxides value) were not affected by salinity. No significant differences were found in the oleic acid percentage, while saline water irrigation improves in some varieties olive oil quality increasing antioxidant concentrations. Minirhizotron observations demonstrated that the total root number in the saline water irrigated trees were considerably smaller in comparison to the control treatment. A “compensation pattern”, in the total root surface area, between diameter, length and root number in response to saline water irrigation was observed. Saline levels at the rhizosphere zone varied between 7.05 and 3. 92 dsm -1 after soil leaching, which is the upper limit where olive can grow and produce with no significant differences to olive trees irrigated with fresh water. Root distribution with depth and distance from the trunk varied between the two water quality treatments. The data obtained in the present study clearly show that with adequate saline water irrigation management olive can grow and produce under arid conditions.
IV Acknowledgments
My deepest thanks to my supervisors Prof. Zeev Wiesman, Dr Jhonathan Ephrath and
Prof. Moshe Silberbush. I am especially grateful to Prof. Zeev Wiesman, who not only introduced and guided me in the academic world, he also advised me in my new Israeli life.
Thanks to the Albert Katz International School for Desert Studies and to the MASHAV-
Center for International Cooperation, Ministry of Foreign Affairs of the State of Israel, who supported in part this work through a scholarship.
Thanks to all the members of the laboratory and institute. Special thanks to Bishnu
Chapagain for his significant help on my work and this writings, but most important I want to thank him for giving me his friendship. Thanks to Arik Yashiv, Yariv Yehoshua and Shosh Avni.
My heartfelt thanks to my girlfriend Ariela, for her love and patience, without her this work wouldn’t have been possible. Thanks to my mother and father in Argentina for their love and moral support. V
Table of Content Page
Title page II Abstract III Acknowledgments IV Table of content V List of figures VII List of tables IX Abbreviations and terms XII 1. General Introduction 1.1 Olive 1 1.1.1 Background 1 1.1.2 Flowering and fruiting 3 1.1.3 Varieties 3 1.1.4 Olive cultivation practices 4 1.2 Salinity effect on fruit trees 7 1.2.1 Root stock effect 7 1.2.2 Seed germination 8 1.2.3 Chloride and Sodium accumulation 8 1.3 Irrigation with saline water 9 1.4 Hypothesis and objectives 11 2. Response of vegetative and reproductive growth of olive to saline water irrigation 2.1 Introduction 12 2.2 Materials and Methods 15 2.2.1 Experimental 15 2.2.2 Field measurements 18 2.2.3 Leaf mineral analyses 18 2.2.3 Oil percentage determination 18 2.2.4 Soil mineral analysis 19 2.2.5 Statistical analysis 19 2.3 Results and discussion 20 2.3.1 Trunk development 20 2.3.2 Fruit development 25 2.3.3 Leaf (Na +, Cl -) and soil (EC) analysis 29 2.3.4 Fruit Yield 34 2.3 .5 Oil Percentage and oil yield 3. Effect of saline water irrigation on Olive oil quality 3.1 Introduction 43 3.2 Materials and methods 46 3.2.1 Acidity determination (free fatty acid) 46 3.2.2 Peroxides determination 46 3.2.3 Fatty acid profile 47 3.2.3.1 Gas chromatography conditions 47 3.2.4 Total polyphenols determination 48 3.2.5 Vitamin E determination 48 3.2.5.1 Tocopherol standard solutions 49 3.2.5.2 Mixed tocopherol standards working solution 49 3.2.5.3 Working conditions 49
VI Page
3.2.5.4 Test sample 49 3.2.6 Sterol determinations 50 3.2.6.1 Preparation of the unsaponifiables 50 3.2.6.2 Separation of the sterol fraction 51 3.2.6.3 Preparation of the trimethylsilyl ethers 52 3.2.6.4 Gas chromatography analysis 52 3.2.6.5 Peak identification 52 3.2.6.6 Expression of the results 53 3.2.7 LC-MS Phenols determination 53 3.2.7.1 Phenol extraction 53 3.2.7.2 Working conditions 54 3.2.7.3 Optimization procedures 54 3.2.8 Chemical reagents 55 3.2.9 Standards 55 3.2.10 Instruments 56 3.2.11 Statistical analysis 56 3.3 Results and Discussion 57 3.3.1 Free fatty acid (Acidity) 57 3.3 2 Peroxides value 60 3.4.3 Fatty acid composition 63 3.4.4 Polyphenols 68 3.4.5 Phenol composition 70 3.3.7 Vitamin E 77 3.4.8 Sterol concentrations 4. Olive c.v Barnea root development under saline water drip irrigation 4.1 Introduction 86 4.2 Material and Methods 89 4.2.1 Field Experiment 91 4.2.2 Soil salt concentration 93 4.3 Results and Discussion 93 5. Conclusions 104 6. References 106 VII
List of Figures Page
Figure 1.1 A mature olive tree (cv Barnea). 2
Figure 1.2 Flower and fruit of olive a) flower, b) young fruit and c) mature Fruits. 3
Figure 2.1 Map of the studied varieties and their distribution in the two sub- plots; the schematic trees indicate the number of trees of each analyzed olive variety. 17
Figure 2.2 IR Horiba 350 used for olive oil percentage determination; A: Oil content analyzer B: cell holder for oil determinations. 19
Figure 3.1 The olive oil mill (a); real photographs of the used mill to oil extraction (b)and sequence of the extraction process (c). 62
Figure 3.2: Olive oil GC fatty acid profile of six different varieties irrigated with fresh water (a-f) and saline water (A-F).Barnea (a/A), Picual (b/B), Leccino (c/C), Koroneiki (d/D), Picholine (e/E) and P. Morocco (f/F) 16:0: Palmitic acid; 18:0 Stearic acid; 18:1 Oleicacid;18:2 Linoleic acid. 67
Figure 3.3 LC-MS Phenol Chromatogram mix standards. 71
Figure 3.4 GC Chromatogram of sterol mix standards. a Cholesterol, b Brassicasterol, c Campesterol, d Campestanol, e Stigmasterol, f 7 Campesterol, g 5.23 Sigmastadienol, h Cholesterol, i Sitostanol, j b-sitosterol, k Avenasterol, l 5,24 stigmastadienol, m 7- stigmastenol, n 7- avestanol. 80
Figure 4.1 Picture showing the tube disposition in the Minirhizotron olive experiment. 90
Figure 4.2 “Straight lines” used method for tip number, length and diameter root determinations in the Minirhizotron olive experiment. 91
Figure 4.3 Olive cv Barnea root development under saline water irrigation at 50 (a, b and c) and 150 (d, e and f) cm distance from the trunk; Total root number (a and d), Total root length (b and e) and Total root surface area (c and f). 96
Figure 4.4 Total root diameter (mm) and total root length (cm) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk. 97
Figure 4.5 Total root number and total root length (cm) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk. 98
VIII Page Figure 4.6 Total root diameter (mm) and total surface area (cm 2) development under fresh water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk. 98
Figure 4.7 Total root number and total surface area (cm 2) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk. 98
Figure 4.8 Olive cv Barnea root development under fresh water irrigation at 50 (a, b and c) and 150 (d, e and f) cm distance from the trunk; Total root number (a and d), Total root length (b and e) and Total root surface area (c and f). 100
Figure 4.9 Schematic diagrams showing the position of minirhizotron acrylic tubes in olive irrigated with fresh water (1.2 dS m-1) and soil salt concentration salt different depths (cm). 101
Figure 4.10 Schematic diagrams showing the position of minirhizotron acrylic tubes in olive irrigated with saline water (4.2 dS m-1) and soil salt concentrations at different depths (cm). 102
IX List of tables Page
Table 2.1. Variety, number of plants and origin of the olive used in the study. 15
Table 2.2. Annual distribution of the irrigation water supplied to the tested olive plots. 16
Table 2.3 A. Annual trunk circumference of different olive varieties irrigated with saline and tap water. 24
B. Tukey Kramer analysis of variance of trunk perimeter. 25
C. Grouping of olive varieties based on trunk circumference at October 2005 according to the response of the variety to saline water irrigation. 25
Table 2.4 A. Effect of saline water irrigation on fruit weight in different olive varieties. 26
B. Analysis of variance of ten fruit weight by Tukey Kramer 27
C. Grouping of olive varieties based on fruit average yield according to the response of the variety to saline water irrigation 27
Table 2.5 A. Comparison of leaf sodium and chloride content in trees of all tested olive varieties irrigated with saline and tap water 30
B. Fisher analysis of sodium and Chloride content in leaf of olive trees 30
Table 2.6 A. Soil fractions EC in tap water subplot (1.2 dS m-1) and moderate saline water subplot (4.2 dSm -1). 31
B. Soil Fractions EC Tukey Kramer analysis of variance. 32
Table 2.7 A. Comparison of fruit yield per tree in all tested olives varieties irrigated with saline and tap water 35
B. Fisher analysis of variance of fruit yield 35
C. Grouping of olive varieties based on fruit average yield according to the response of the variety to saline water irrigation 36
X Page
Table 2.8 A. Comparison of olive oil percent in all tested olive varieties irrigated whit saline and tap water 39
B. Fisher analysis of variance of oil percentage 39
C. Grouping of olive varieties based on average fruit oil percentage according to the response of the variety to saline water irrigation 39
Table 2.9 A. Comparison of olive oil yield in all tested olives varieties irrigated with saline and tap water. 41
B. Fisher analysis of variance of olive oil yield. 41
C. Grouping of olive varieties based on average olive oil yield according to the response of the variety to saline water irrigation. 41
Table 3.1 A. Comparison of free fatty acid (% of oleic) of oils obtained from all tested olive varieties irrigated whit saline and tap water. 58
B. Tukey Kramer analysis of variance of olive oil acidity. 58
C. Grouping of olive varieties based on percentage of free fatty acid. 59
Table 3.2 A. Comparison of peroxide concentrations (milleq. peroxide / Kg oil) of oils obtained from all tested olive varieties irrigated whit saline and tap water. 60
B Tukey Kramer analysis of variance of olive oil peroxides Concentration. 60
Table 3.3 A. Comparison of fatty acid composition of oils obtained from all tested olive varieties irrigated whit saline and tap water. 65
B. Tukey Kramer analysis of olive oil fatty acid composition. 66
C. Grouping of olive varieties based on percentage of Oleic Acid (18:1) according to the response of the variety to saline water irrigation. 66
Table 3.4 A. Comparison of total polyphenols concentrations (ppm of caffeic acid) of oils obtained from all tested olive varieties irrigated whit saline and tap water. 69
B. Fisher analysis of variance of olive oil polyphenols content. 69
C. Grouping of olive varieties based on polyphenols concentrations according to the response of the variety to saline water irrigation. 69
XI Page
Table 3.5 A. Effect of saline irrigation water on Phenol composition (ppm) in subjected to different varieties. 75
B. Tukey Kramer analysis according to each of the analyzed phenol concentration subjected to different varieties. 76
Table 3.6 A. Comparison of Vitamin E concentration (µg/g) of oils obtained from all tested olive varieties irrigated whit saline and tap water. 78
B. Fisher analysis of variance of olive oil Vitamin E concentration. 78
C. Grouping of olive varieties based on Alpha Tocopherol concentration according to the response of the variety to saline water irrigation. 79
Table 3.7 Effect of moderate saline water irrigation on sterol olive oil profile concentration (µg/Kg) in different varieties olive varieties. 83
Table 3.8 Effect of moderate saline water irrigation on Barnea olive oil sterols concentration (µg/Kg) at three different saline levels. 84
Table 4.1 Total root number at different depths in each of the three analyzed distances from the trunk (50 cm, 100 cm and 150 cm) of the two treatments: fresh water 1.2 dS m -1 (f) and saline water 4.2 dS m -1 (s) irrigation. 94 XII
Abbreviations terms
IOOC International Olive Oil Council EU Europe Union UNCTD United Nations Conference Trade Development USDA Unite State Department of Agriculture FAO Food and Agriculture Organization ISO International Organization for Standardization IUPAC International Union of Pure and Applied Chemistry g gram kg kilogram meq milliequivalent mg milligram µg microgram ppm parts per million ml millielitre µl microliter l litre M molar N normal v/v volume/volume v/v/v volume/volume/volume cm centimetre s second nm nanometre dw dried weight ha hectare UV ultra violet IR infra red HPLC high performance liquid chromatography GC gas chromatography LC/MS liquid chromatography/mass spectra GC/MS gas chromatography/mass spectra MR mini rhizothron
1 1. General Introduction
1.1 Olive
1.1.1 Background
The cultivated olive ( Olea europaea L, Oleaceae) is a long-lived, evergreen tree native to the Mediterranean basin (Figure 1.1). It is appreciated for its fruit and oil.
Mediterranean countries account for around 95% of the world's olive cultivation
(8,702,000 ha). In the Mediterranean 90% of the olive trees are grown for the oil
(IOOC, 1994). World olive oil production is currently increasing. The commercial production during 1990-1994 was 1,796,900 tons (IOOC, 1994), with the European
Union (EU) producing around 1,337,000 tons (Spain 40%, Italy 33%, and Greece 22%),
Tunisia 194,000 tons and Turkey 61,500 tons of oil. World table olive production in
1990-1994 was 954,500 tons (IOOC, 1994). Spain is the world's largest producer (23%) and exporter of table olives, followed by Turkey (12%), U.S. (11.4%), Morocco (8.5
%), Syria (7.5%), and Greece (7.3%). In Argentina, Australia, Chile, China, Mexico,
New Zealand, and South Africa, olives are considered a new crop. In the land of Israel, historically, most of the olive orchards were established and cultivated by traditional methods on the mountains of Samaria, since the beginning of the Jewish settlement, early in the 20 th century (Rymon et al., 1997).
The olive oil and table olive industries play an important role in the agricultural and processing sectors of the major olive producing countries. Most of the olive oil is consumed within the Mediterranean countries and only 18% of production enters in the world trade. On average from 1988 through 1991, the world olive oil marketed represented 6% of the quantity and 23% of the value of the world trade in fluid edible oils (UNCTD, 1993). To a large extent olive oil does not compete with other vegetable oils but occupies a specialty niche market. 2 The olive tree has a wide range of adaptability. It requires a mild climate with warm summers and cold winters. The tree requires substantial chilling for good fruiting
(Martin et al., 1994), but is injured when temperatures fall below -10º C. Olive is considered as drought-tolerant species because it thrives in areas where drought is frequent like Mediterranean climates. It has been postulated that the minimum water requirement for olive is 2,000 m 3/ha per year mainly during flowering and fruit setting in late spring and again in the summer as the fruit increases in size (Tous and Romero,
1990; Bongi and Palliotti, 1994). Olive trees will grow on poor soils and rocky hillsides but deep soils produce the best quality fruit. They tolerate saline or alkaline soils and those with high lime content. Their root system is relatively shallow and not tolerant to waterlogged soils.
Figure 1.1 A mature olive tree (cv Barnea).
1.1.2 Flowering and fruiting
The small, cream-colored olive flowers are largely hidden by the evergreen leaves and grow on a long stem arising from the leaf axils (Figure 1.2A and 1.2B). Olive produces two kinds of flowers: a perfect flower containing both male and female organs, and a staminate flower with stamens only. The flowers are largely wind pollinated with most 3 olive varieties being also self-pollinating, although fruit set is usually improved by cross pollination with other varieties. There are self-incompatible varieties that do not set fruits without other varieties nearby, and there are varieties that are incompatible with some others (Bianchini, 1976). Incompatibility can also occur for environmental reasons such as high temperatures (Griggs et al., 1975). Mature olive trees produce huge numbers of flowers (Figure 1.2A) but the fruit set is normally below 5% (Martin et al.
1994). The olive fruit (Picture. 1.2B 1.2C) is classified as a “drupe”, similar to other drupes of stoned fruits such as apricot and cherries with the same anatomy (Garrido
Fernandez et al., 1997). Most olive cultivars will set some fruits in a monocultivar culture. However, they benefit greatly from cross-pollination. Olives are picked late in autumn or winter as the oil content and fruit characteristics change with ripening.
A B C
Figure 1.2 Flower and fruit of olive A) flower, B) young fruit and C) mature fruits.
1.1.3 Varieties
Olive cultivars are often classified as "Oil Olives" and "Table Olives" where oil cultivars predominate. The most popular are: 'Picual', 'Arbequina', 'Cornicabra',
Hojiblanca', and 'Empeltre' in Spain; 'Frantoio', 'Moraiolo', 'Leccino', and 'Pendolino' in
Italy; 'Koroneiki' in Greece; 'Chemlali' in Tunisia; 'Ayvalik' in Turkey; 'Mission' in
California and Australia. The "table olive" cultivars include 'Manzanilla' and 'Gordal'
(syn. 'Sevillano' in California and 'Queen' in Australia) from Spain; 'Kalamata' from 4 Greece; 'Ascolano' from Italy; 'Barouni' from Tunisia. (Jacoboni and Fontanazza, 1981;
Barranco, 1984; Tous and Romero, 1993)
Tree management of olives has undergone many changes in the recent decades including increased planting densities (100 to 300 trees/ha). New methods of propagation, improved cultural practices and mechanical harvesting, are some examples.
1.1.4 Olive cultivation practices
Propagation
Olives are propagated primarily by cuttings, truncheons or by budding seedling rootstocks. The trees propagated by rooting semi-hardwood cuttings come into bearing within three to four years after planting (Barranco, 1984; Wiesman and Lavee, 1994).
Irrigation
Olive is traditionally cultivated in marginal areas with no irrigation, where rainfall does not meet potential evaporation demand (Fernandez and Moreno, 1999). To improve yield and maintain low water consumption, modern olive plantations with relatively high number of trees per hectare require modern techniques of irrigation, like drip irrigation, to support this tree production (Girona et al., 1993; Marsal et al., 2002).
Indeed, a sagacious irrigation approach is a major task in modern olive growing due to the limited water resources available in Mediterranean countries (Villalobos et al.,
2000).
5 Fertilization
Nitrogen (N), phosphorus (P), potassium (K) and boron (B) are essential nutrients in olive orchards. Application of N is the main aspect of the olive orchard fertilization requiring annual soil and leaf applications. Nitrogen fertilization is usually applied to the soil at 0.5–1.5 kg per olive tree at the end of winter, using urea, ammonium sulphate, or ammonium nitrate; and to the foliage in spring using urea solution at 4%
(Fernandez-Escobar, 1999). Nitrogen fertilization consistently increases olive yield but only when leaf N is below the sufficiency threshold (Hartmann, 1958).Deficiencies of any nutrients in trees can be correctly detected through a leaf analysis, being the best diagnosis method to determine the nutrient status and to plan fertilizer recommendations
(Fernández-Escobar, 1999). However, olive fertilization is not in practice by farmers based on recommendations from a leaf analysis, but rather from intuitive and visual observations.
Pruning
Proper pruning is one of the most critical activities for successful olive orchard. Pruning both regulates production and shapes the tree for easier harvest. The trees can withstand radical pruning, so it is relatively easy to keep them at a desired height. The problem of alternate bearing can also be avoided with careful pruning every year. It should be kept in mind that the olive tree never bears fruit in the same place twice, and usually bears on the previous year's growth. For a single trunk, prune suckers and any branches growing below the point where branching is desired. For the gnarled effect of several trunks, stake out basal suckers and lower branches at the desired angle. Prune flowering branches in early summer prevent olive fruits from forming. Olive trees can also be pruned to espaliers (Gucci and Cantini, 2000).
6 Diseases and pest
Olives are affected by some pests and diseases, even fewer than most fruit trees. Around the Mediterranean the major pests are med fly ( Bactrocera oleae ) ( Calvitti et al., 2002) and the olive fruit fly, Dacus oleae (Carey et al., 1986). Verticillium (Verticillium dahliae) is a serious fungal disease in olive trees; its incidence and severity could be higher in irrigated orchards and also in orchards previously cropped or actually intercropped whit cotton or vegetables (Blanco-Lopez et al., 1984). There is no effective treatment other than avoiding planting on infested soils and removing damaged trees and branches. Because the olive has fewer natural enemies than other crops, and because the oil in olives retains the odour of chemical treatments, the olive is one of the least sprayed crops (Sanchez Hernandez et al., 1998)
Yield and harvesting
One characteristic of olive is the “Alternate bearing” which is defined as a sequence of a high yield followed by a low yield in the subsequent year. These alternate bearings have a strongly relation with the variety of the olive (Gucci and Cantini, 2000). The type of harvest depends upon fruit use; oil olives are harvested by hand or by mechanically shaking the tree. Olive harvesting must be done during the final stages of maturation that is when the oil content is highest. (Wiesman et al., 2002)
Olive Oil
Olives are nutritious, the oil component includes unsaturated fatty acid (70% to 80% oleic acid and 7% to 12% linoleic acid), and small amounts of polyphenols, tocopherols, sterols, and many aromatic compounds (Mataix and Martinez, 1988; Tous and Romero,
1993). In addition to culinary uses, olive oil, an unsaturated fat, has recently become more valued for its health benefits. Mainly for this reason, through the last decade the 7 olive oil consumption has increased in several non-Mediterranean countries such as
USA, Australia, Japan, and others (IOOC, 1995)
Olive oil and table olive industries play an important role in the agricultural and processing sectors of the major olive producing countries of the Mediterranean basin.
World olive oil production and exports are projected to increase slightly in the next few years, while table olive production will remain stable. While Italian olive oil production is expected to stabilize or decrease, Spain, Tunisia, Morocco, and other countries
(Argentina, Australia) are expected to achieve substantial increases in this crop production (UNCTD, 1993).
1.2 Salinity effect on fruit trees
Salinization transforms fertile and productive land to barren land. Salinity limits vegetative and reproductive growth of plants by inducing physiological dysfunctions and causing widespread direct and indirect harmful effects, even at low salt concentrations (Epstein, 1980). Salt tolerance of plants is difficult to quantify because it varies appreciably with many environmental factors (e.g., soil fertility, soil physical conditions, salinity components (ions), distribution of salt in the soil profile, irrigation methods, and climate) and plant factors (e.g., stage of growth, variety, and rootstock).
Woody plants are relatively salt tolerant during seed germination, much more sensitive during the emergence and young seedling stages, and become progressively more tolerant with increasing age through the reproductive stage (Kozlowski, 1997).
1.2.1 Root stock effect
There are wide variations in tolerance of rootstocks to salinity; salt-sensitive species sometimes accumulate toxic amounts of salts over a long period of time, even from soils 8 considered to be non saline. Salinity adversely affects nonhalophytes by inducing injury, inhibiting growth, altering plant morphology and anatomy, often as a prelude to tree mortality (Yeo 1983); injury is more severe when salts absorbed from the soil are augmented by salts deposited on leaves. Injury is induced not only by the osmotic effects of salts but also by specific toxic effects resulting from the accumulation of Cl - and Na + (Munns, 1983).
1.2.2 Seed germination
Salinity reduces the total number of seeds germinating and postpones initiation of germination processes that influences seed germination primarily by lowering the osmotic potential of the soil solution sufficiently to retard water absorption by seeds; finally salinity adversely influences several aspects of reproductive growth, including flowering, pollination, fruit development, yield and quality, and seed production
(Munns, 1983).
1.2.3 Chloride and Sodium accumulation
The decrease in growth is related to the chloride content of the leaves. High salinity in irrigation waters reduced flowering intensity, fruit set, number of fruits, and fruit growth. Salinity often alters the morphology and anatomy of woody plants. Leaves of plants that grow on saline soils often are thicker and more succulent than those of trees growing on salt-free soil (Seaman, 2002). The epidermal cell walls and cuticles of leaves of salinized plants are also thicker. By increasing the internal surface area per unit of leaf surface, leaf succulence may increase CO 2 absorption per unit of leaf area.
Increase in leaf thickness in response to salinity has been attributed to an increase in number of mesophyll cell layers or cell size, or both. Salinity reduces shoot growth by 9 suppressing leaf initiation and expansion as well as internode growth and by accelerating leaf abscission (Epstein, 1980).
When irrigating with saline water the sodium ions displace other more useful ions such as calcium and magnesium. If irrigation with saline water is continued, over time the soil becomes concentrated with sodium. When fresh, less saline water is applied, clay bonds become weak. The weakening of the bonds results in the swelling of clay particles, which then disperse forming a soil with little or no structure. The clay particles move through the soil clogging pores and reducing the volume of water that can move into or through the soil profile (Bethune and Batey 2001).
Combined flooding and salinity typically decrease growth and survival more than does either stress alone. In contrast to the effect of flooding with fresh water, flooding with saline water typically inhibits shoot growth more than root growth. Olive is considered as a moderately salt tolerant plant (Rugini and Fedeli, 1990). Comparing the olive with others Mediterranean-grown tree crops, olive is more tolerant than citrus but less tolerant than date palm (Ayers and Westcot 1976). Tolerance of olive to salinity could be related to a mechanism of salt exclusion and low transport of Na + and Cl - to the aerial part of the tree (Tattini, 1994; Munns, 1983). This classification should be used as a first approximation t salinity tolerance/sensitivity, due to the limited and contradictories reports found in the literature especially as most of them were done under greenhouse conditions and with young plants cultivated in pots (Aragues, 2005).
1.3 Irrigation with saline water
Many problems associated with irrigated agriculture arise from the chemical composition of the water applied. The use of various qualities for irrigation, as well as 10 the advantage of predicting problems that might develop when different quality of irrigation water is being used (Frenkel, 1984).
With the use of saline waters for irrigation, there is a need to undertake appropriate practices to prevent the development of excessive soil salt concentration for crop production. Management not only need to control salinity at the lowest possible level, but also to keep it within limits commensurate with sustained productivity. Crop, soil and irrigation practices can be modified to help to achieve these limits (Feinerman et al.,
1984). Management practices for the control of salinity include: selection of crops or crop varieties that will produce satisfactory yields under the resulting conditions of salinity, use of land-preparation and planting methods that aid in the control of salinity, irrigation procedures that maintain a relatively high soil-moisture regime and that periodically leach accumulated salts from the soil, and maintenance of water conveyance and drainage systems. The crop type, the water quality and the soil properties determine, to a large degree, the management practices required to optimize production (Bressler and Hoffman, 1986).
Irrigation practices which are important in the management of saline water are: irrigation scheduling (amounts and interval); leaching scheduling (amount and timing); irrigation method and management of multi-source irrigation water of different qualities
(Shalhevet, 1984). Leaching is the key factor by which soil salinity can be maintained at acceptable levels without undue damage to crops. Thus appropriate natural or installed drainage and disposal systems are essential (Bernstein and François, 1973). 11 1.4 Hypothesis and objectives
Hypothesis
In recent years, the consumption of olive oil and table olives has steadily increased, even in countries that do not have such tradition. This trend is being fostered by the recognized nutritional value of the Mediterranean diet. To meet the increasing demand, olives are being cultivated and produced in areas that do not have a tradition of olive growing, while both semi arid conditions and saline water irrigation is the “new” environment for new olive orchards.
Taking into account the trend that new olive orchards are planted in semi arid areas and irrigated with poor quality of water, we hypothesized that saline water could be used for olive irrigation applying a conjugated agronomic techniques including an adequate management of the irrigation. The main hypothesis of the present work was that olive, which shows moderate tolerance to salinity, can grow and produce in semi arid areas irrigated with saline water.
Objectives
The main aim of this study was to investigate the effects of saline water on olive; investigating and comparing a large number of varieties from different origins, planted under desert conditions and simulating a commercial orchard. The specific objectives were as follow:
1. To measure and compare the vegetative and reproductive olive varieties
parameters response to saline water irrigation
2. To analyze and characterize the obtained oil from the studied olive varieties.
3. To compare root development of olive irrigated with saline and fresh water
irrigation. 12 2. Response of vegetative and reproductive growth of olive to saline water irrigation
2.1 Introduction
Due to the increasing demand for healthy sources of fat for human consumption and the fact that olives are considered to be well adapted to semiarid conditions, an intensive wave of olive planting had been taken place in the last decade in many places world- wide (FAO, 1989). Traditionally, olives have been cultivated in the Mediterranean region and many superior cultivars have been selected in most of the countries of this area. However, due to a continuously increasing shortage of fresh water, mainly in semiarid areas, the irrigation of most new olive plantations is based on available low quality sources of water that are all characterized by a relatively high salinity (Wiesman,
2004). The relationship between saline water and olive cultivation has been intensively studied for many years and significant progress has been made in the knowledge of this topic (Aragues et al., 2005; Wiesman et al., 2004; Gucci and Tattini, 1997; Munns,
1993; Mass and Hoffman, 1977; Bernstein, 1964).
It is well established that saline conditions limit olive vegetative and reproductive development; interfering in the osmotic balance between the soil and the root system, causing toxic accumulation of Cl - and Na + in the leaves (Hassan et al., 2000; Maas and
Grattan, 1999; Tattini, 1994; Benlloch et al., 1991; Bongi and Loreto, 1989) Salinity is known to be a common limiting factor in semiarid areas, even when fresh water is used, due to the high rate of water evaporation which can cause salt accumulation in the soil
(Shalhevet, 1994).
The FAO (1985) classifies olive trees as a 'moderately tolerant crop' to salinity, suggesting a threshold electrical conductivity of the soil saturation extract of between 3 13 and 6 dSm -1. Although the threshold of Cl - and Na + toxic concentrations varied, due to different experimental conditions and tested genotypes, most studies concluded that they are approximately 2 mg g -1 of Cl - and 4–5 mg g -1 Na + on a leaf dry weight basis, and it was suggested that injury is better correlated with Na + than with Cl - (Gucci and
Tattini, 1997; Klein et al., 1994; Al-Saket and Aesheh, 1987; Bernstein, 1975;).
Recently, Aragues (2005) reported on Arbequina olive tree trunk growth reduction in
Cl - levels higher than 2.3 mg g -1 and Na + levels higher than 1.5 mg g -1 leaf dry weight.
Most reports have focused on the physiological mechanisms involved in olive tree response to saline soil and water conditions. Due to the long time until olive trees reach maturity and their yielding phase, the majority of these studies had been carried out with solution cultures or greenhouse pot trials using young olive seedlings, and were focused on specific cultivars tested in various environmental conditions and cultivation practices. As a result these experiments cannot be extrapolated to field conditions. A limited number of field trials analyzing the response of some specific olive varieties cultivated using various cultivation practices and at different maturation stages have been reported in recent years (Aragues et al., 2005; Wiesman et al., 2004; Murillo et al.,
2000; Klein et al., 1994;). Even in these studies, relatively young trees were used and the sustainable effect of salinity on the olive trees was not studied for a long enough time. In addition, salinity tolerance parameters were not well established, especially in mature trees, and were not well enough analyzed in field conditions. Thus, the actual impact of salinity on the yield of olives is uncertain. It is difficult to reach general conclusions and to predict the response of most olive varieties to intensive cultivation under saline irrigated semiarid conditions.
14 In order to support the rapidly growing olive industry in the semiarid south part of Israel and similar areas in the world, we established in 1997 a new special saline irrigation controlled experimental plot. Twelve selected outstanding local olive varieties and from various Mediterranean countries were planted in this plot that was divided in to two subplots. One subplot was irrigated with fresh water (1.2 dS m-1) and the second subplot was irrigated with moderate saline water (4.2 dS m-1). In the present study we aimed to evaluate and compare the vegetative and reproductive multi-annual response of mature yielding trees of the selected olive varieties drip irrigated with fresh water and moderate saline water in a commercial orchard in a desert area. 15 2.2 Materials and Methods
2.2.1 Experimental
This study was conducted in the Ramat Negev Experimental Station situated in the central-south part of the Israeli semiarid Negev area, near Wadi Ha Besor, 30 km south of Beer-Sheva city; latitude 31° 05' 00", longitude 34° 41' 03", altitude 305 m above sea level. The soil verge common in the experimental station is light loess; 6–8% clay and pH 7.2 up to 8.0. The average annual rain fall in this area is about 50 mm. Twelve olive varieties from various olive growing countries in the Mediterranean basin were planted in 1997 in the saline water irrigated testing plot. Olive varieties selected from Israel,
Italy, Spain, Greece, France, and Morocco in ten or six replications were planted in spaces 4 m × 6 m, representing a density of 420 plants/ha (Table 2.1).
Table 2.1 Variety, number of plants and origin of the olive used in the study. Variety Number of trees Origin Barnea 10 Israel
Souri 10 Israel Maalot 6 Israel
Frantoio 10 Italy Leccino 10 Italy Arbequina 10 Spain Picual 10 Spain Picudo 10 Spain Kalamata 6 Greece Koroneiki 10 Greece Picholin 10 France Picholin di Morocco 6 Morocco
The experimental plot was divided to two subplots, each containing the same varieties in a mirror-like image. One subplot was irrigated with fresh water (EC 1.2 dS m -1) as a control and the second with moderate saline water (EC 4.2 dS m-1) (Figure 2.1).
The amount of water per month supplied to the olive trees by drip irrigation are exposed in Table 2.2. Values were calculated using a basic monthly irrigation formula that was 16 developed based on multi-year local pan evaporation data. This formula was continuously rechecked on a daily basis and corrected accordingly. The average amount of water applied annually was 6560 m 3 per hectare. Usually twice a year, in March and
November, a supplemental amount of 1000 m 3/ha was added to the monthly irrigation water to leach the soil and to remove the salt excess from the root zone. During the first three years after planting, the trees were irrigated following any rain fall to prevent re- salinization of the root zone area (Wiesman, 2004). The fresh water used was supplied by the Israeli National Water Carrier while the saline water was drawn from local wells.
The Electrical Conductivity of the water for the saline treatment was adjusted by mixing the two types of water or adding NaCl. The rate of NPK fertilization was determined based on the results of annual nutrient leaf analysis.
Table 2.2 Annual distribution of the irrigation water supplied to the tested olive plots.
Month Quantity (m3 ha-1 month -1 January 230 February 270 March 450+1000* April 700 May 780 June 810 July 900 August 1000 September 760 October 460 November 1000* December 200 *Additional 1000 m 3/ha were added in order to leach the soil
17
North
Saline irrigation water (4.2 dS m -1) Fresh irrigation water (1.2 dS m -1)
Kalamata Barnea Frantoio Picholin Barnea Frantoio Picholin Kalamata
Arbequina Leccino Souri Arbequina Leccino Souri Picholin di Morocco Picholin di Morocco
Maalot Maalot
Koroneiki Picudo Picual Koroneiki Picudo Picual
Figure 2.1 Map of the studied varieties and their position in the two sub- plots; the schematic trees indicate the number of trees of each analyzed olive variety . 18 2.2.2 Field measurements
Every year during the study, the trunk circumference of each tree was measured twice in the same marked location (30 cm above the ground). At harvesting, yield of individual trees was collected separately and the olive yield of each variety was assessed for every tree in each subplot (group of trees of the same variety and the same saline treatment).
Olive fruit samples of each variety were collected in different developmental stages, beginning from fruit stone hardening, till final harvesting. Five replicated batches of ten olives were randomly chosen and used for average weight and determination.
2.2.3 Leaf mineral analyses
The leaf analyses were carried out in three batches of thirty leaves collected from all the trees of each variety (and treatment). Leaf samples were dried at 70ºC, ground, and digested with sulphuric acid. Chloride (Cl -) was determined by titration with 0.5 N
+ AgNO 3, while Na and other cationic elements were determined using Flame Atomic
Absorption Spectrometry (Varian, Australia).
2.2.3 Oil percentage determination
The oil percentage was determined using IR Horiba 350 (Horiba Instruments, USA)
(Figure 2.2), an infrared system specific for determination of oil content in olive tissues
(Wiesman, 2004). Briefly, one gram of crushed fruits from each variety (and treatment) was taken and 10 ml of tetrachoroethylene was added. The sample was vortexed, and kept over night at room temperature to insure the total oil extraction. After centrifugation, one aliquot of 0.06 ml from the tetrachoroethylene donweer phase was taken and measured on the Horiba system. The results were expressed as a percentage of the total fresh weight.
19 A B
A
Figure 2.2 IR Horiba 350 used for olive oil percentage determination; A: Oil content analyzer B: cell holder for oil determinations.
2.2.4 Soil mineral analysis
Soil samples were taken from 0–30, 30–60, 60–90 and 90-120 cm soil depth column fractions. At least four randomized sub-samples of each subplot irrigated with 1.2 dS m-
1 and 4.2 dS m-1, respectively, were collected. The soil analyses were done at the soil laboratory of the Gilat Experimental Station. Electrical conductivity and chloride concentration were done in saturated soil extracts, as described by Sparks, (1996).
2.2.5 Statistical analysis
At least three replicates were (i.e., trees) used for each field test. The data were statistically analyzed with JMP software (SAS, 2000) using either the Tukey–Kramer
HSD or Fisher LSD test for determining significant differences among the treatments at
P≤ 0.01 or P≤ 0.05 .
20 2.3 Results and discussion
As a follow-up to in the present study, the response of twelve selected olive varieties from Mediterranean olive growing countries to saline irrigation water in the present study was analyzed in a separate saline irrigated experimental plot established at the
Ramat Negev Station in 1997. The tested plot was used as a simulation of a commercial olive plantation in a semiarid area. The data collected during five years of intensive drip irrigation cultivation under orchard conditions are presented in this study and the results are discussed.
2.3.1 Trunk development
Changes in the trunk circumference over five years of mature olive trees irrigated with moderate saline water (4.2 dS m-1) in comparison to fresh water irrigated trees of the same varieties are presented in Table 2.3A.
Previous studies showed that the response of olive trees to salinity changes during different developmental stages of the tree (Aragues et al., 2005; Wiesman et al., 2004;
Gucci and Tattini, 1997). In order to evaluate the vegetative response of mature olive trees to saline irrigation water, the changes in trunk circumference were recorded. In agreement with many reports concerning olive tree development in saline conditions
(Aragues et al., 2005; Chartzoulakis and Ragab, 2005; Wiesman et al., 2004;; Cresti et al., 1994; Klein et al., 1994; Klein et al., 1992; Bouaziz, 1990), a pattern of decrease in trunk circumference of saline treated trees compared to the control was observed in the first three years (2001–2003). However, no significant differences were found in the statistic analysis carried out in between pairs of among olive varieties treated with fresh water and moderate saline water. In the next two years (2004–2005), this pattern changed and the saline irrigated trunk circumference seemed to develop at a faster rate 21 than the control trees. But again, no significant differences were found among all olives varieties irrigated with fresh and saline water.
In the selected Israeli olive varieties, Barnea and Maalot, this changed pattern could be seen and in October 2005. The saline irrigated Barnea trunk circumference was equal to that of the control trees (57 cm for both). The increasing average trunk circumference size per year evident a higher value for the saline irrigated trees than for control trees.
This pattern of trunk growth is in agreement with previous reports dealing with Barnea trees irrigated with saline water (Wiesman et al., 2004; Klein et al., 1994). A stronger trend for trunk growth was observed for Maalot variety irrigated with saline water that reached 69 cm compared to 65 cm in the control trees. In this case, the difference between the average trunk growth rates of saline irrigated trees compared to the control trees was even higher (8.7 versus 6.2 cm/year for control trees). In agreement with most of the earlier reports (Gucci and Tattini, 1997; Klein et al., 1994; Bouaziz, 1990), Souri, the third selected Israeli olive variety, demonstrated a pattern of reduced trunk circumference growth in response to the saline irrigation water during the whole tested period of five years. Accordingly, the average trunk increase per year was higher in control trees than in saline irrigated trees.
Among the Italian varieties, Frantoio showed a similar behaviour to Barnea. In the first phase of trunk growth in relatively young trees, the saline irrigated tree tended to be reduced in comparison to the control trees but later phase, the difference was diminished saline irrigated trees respect to the control trees. Trunk circumference values in Leccino trees showed a minimal difference between the two irrigation treatments during the whole trial period. To the best of our knowledge, no previous reports of similar saline conditions concerning these Italian varieties are available. 22 Arbequina, a variety from the Spanish group, showed only minor differences between the saline treated trees and the control trees during the entire testing period. The average trunk growth per year suggests a slightly higher rate of growth of the control trunk in comparison to the saline trunk (5.9 versus 5.3 cm/year, respectively). Picual trees irrigated with saline water were somewhat smaller in all measured years compared to the control trees, but the rate of trunk growth was similar for both treatments.
Picudo saline irrigated trees showed a similar type of growth like Maalot which began with a higher trunk circumference in the control trees and ended with a larger trunk circumference in the saline irrigated trees. Indeed, the average rate of trunk growth of the saline irrigated trees was bigger that of the fresh irrigated trees, (6.7 versus 4.7 cm/year, respectively).
The two varieties from Greek origin, Kalamata and Koroneiki, and Picholin from
France, also showed somewhat smaller initial trunk values in saline irrigated trees than the control trees. In Picholin di Morocco, only in the initial date (November 2001), the trunk circumference was smaller in saline treatment compared to the control and since then, the trunk of the saline irrigated trees developed similarly and even at a faster rate than the control trees.
It seems to us that due to limited number of trees (5 replicates) used for each treatment in the present study, no significant differences could be obtained in between the paired treatment of all the olive varieties. However, a Tukey Kramer analysis of the trunk circumference response of the tested olives to saline water clearly showed that the significant differences observed in November 2001; September 2003; and October 2005 are given by the genetic differences between verities (Table 2.3B). A low significant 23 difference between saline and fresh water treatment was obtained only at the beginning of the study in November 2001. This difference was disappeared in the next two dates.
No interaction in between olive varieties and irrigation water treatments was obtained at all.
Generally, it seems that the response of most varieties of olive trees can be divided in two phases. In the first phase of relatively young mature trees that took part in the present study, during the forth, fifth, and sixth years of the tree growth, some reduction in vegetative growth of saline irrigated trees could be observed in terms of trunk growth, in good agreement with previous studies (Wiesman et al., 2004; Klein et al.,
1994) and with other reports dealing with the growth reducing effect of saline on young olive trees (Aragues et al., 2005 Gucci and Tattini, 1997; Bernstein, 1964). In the second phase, this pattern of reduction gradually changed, and the rate of growth of saline irrigated trees increased and was even higher than in the fresh water irrigated trees, again in good agreement with a previous report (Wiesman et al., 2004).
The analysis of the changes in trunk circumference during the course of this study, in the two types of water used for irrigation, could be divided to three main groups (Table
2.3C): Group A – including Leccino and Maalot – at the final date of October 2005 their trunk circumference ranged from 63–69 cm; group B – including Barnea, Souri,
Frantoio, Picual, Picholin, and Picholin di Morocco – their trunk circumference ranged from 55–62 cm; group C – including Arbequina, Picudo, Kalamata, and Koroneiki – ranged from 48–53 cm. These data may assist in the efficient selection of olive varieties, especially for the new olive industry trend of establishing high density intensive cultivated olive plantations in semiarid conditions (Aragues, 2005). 24
Table 2.3 A. Trunk circumference measurement (30 cm from the soil surface) of different olive varieties irrigated with saline and fresh water.
Irrigation Water Trunk Circumference (cm) Average trunk Olive Variety (dS m -1) Nov 2001 Oct 2002 Sep 2003 Dec 2004 May 2005 Oct. 2005 growth (cm/year) Israeli origin Barnea 1.2 37.0 a x y 42.4 a 48.5 a x y 50.8 a 52.8 a 57.4 a x y 5.1 Barnea 4.2 34.5 a x y 38.2 a 45.9 a x y 51.2 a 54.1 a 57.6 a x y 5.8 Souri 1.2 33.4 a x y 42.8 a 50.5 a x y 55.3 a 59.6 a 60.9 a x y 6.9 Souri 4.2 32.6 a x y 38.8 a 46.5 a x y 51.5 a 53.7 a 55.6 a x y 5.7 Maalot 1.2 37.8 a x 47.3 a 54.7 a x 59.7 a 62.3 a 65.0 a x 6.8 Maalot 4.2 34.2 a x y 43.6 a 53.0 a x 60.7 a 63.5 a 69.2 a x 8.7 Italian origin Frantoio 1.2 37.2 a x y 41.8 a 47.7 a x y 51.4 a 52.7 a 56.3 a x y 4.8 Frantoio 4.2 31.1 b x y 37.0 a 44.8 a x y 48.7 a 55.0 a 55.9 a x y 6.2 Leccino 1.2 38.7 a x 46.2 a 53.2 a x 56.6 a 58.5 a 63.0 a x 6.1 Leccino 4.2 37.7 a x 44.4 a 52.5 a x 57.5 a 59.5 a 63.7 a x 6.5 Spanish origin Arbequina 1.2 27.1 a y 35.2 a 41.0 a x y 44.1 a 45.9 a 50.8 a y 5.9 Arbequina 4.2 27.0 a y 33.2 a 40.3 a y 43.2 a 45.8 a 48.1 a y 5.3 Picual 1.2 37.8 a x 43.2 a 50.0 a x y 54.1 a 56.7 a 62.9 a x y 6.3 Picual 4.2 34.0 a x y 38.2 a 46.0 a x y 51.0 a 53.0 a 59.7 a x y 6.4 Picudo 1.2 31.5 a x y 35.1 a 43.8 a x y 46.3 a 47.6 a 50.2 a y 4.7 Picudo 4.2 26.4 a y 32.6 a 40.3 a y 46.9 a 49.5 a 53.3 a y 6.7 Greek origin Kalamata 1.2 25.9 a y 33.5 a 41.7 a x y 46.5 a 49.0 a 51.2 a y 6.3 Kalamata 4.2 22.2 a x y 30.7 a 39.7 a y 45.0 a 48.2 a 50.5 a y 7.0 Koroneiki 1.2 29.9 a x y 35.6 a 43.5 a x y 47.9 a 49.4 a 50.6 a x y 5.2 Koroneiki 4.2 26.4 a y 30.4 a 42.2 a y 43.8 a 44.1 a 48.1 a y 5.4 French origin Picholin 1.2 33.8 a x y 39.4 a 48.5 a x y 53.6 a 55.2 a 59.2 a x y 6.3 Picholin 4.2 35.3 a x y 37.8 b 48.1 a x y 52.5 a 54.4 a 60.0 a x y 6.2 Morocco origin P. Morocco 1.2 28.9 a x y 38.0 a 46.8 a x y 52.0 a 54.0 a 57.0 a x y 7.0 P. Morocco 4.2 23.5 a y 38.7 a 45.3 a x y 52.3 a 58.5 a 60.7 a x y 9.3
Tukey Kramer test (p=0.01) was carried out between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different 25 B. Tukey Kramer analysis of variance of trunk circumference.
Source DF Nov 2001 Sep 2003 Oct 2005 Variety 11 *** *** *** Treatment 1 * NS NS Variety*Treatment 11 NS NS NS DF: degree of freedom; *** high significant effect; * low significant effect; NS: non significant effect .
C. Grouping of olive varieties based on trunk circumference on October 2005 according to the response of the variety to saline water irrigation.
Group Range (cm) Olive Variety A 63-69 Maalot, Leccino B 55-62 Barnea, Souri, Frantoio, Picual, Picholin, P. Morocco C 48-53 Arbequina, Picudo, Koroneiki, Kalamata
2.3.2 Fruit development
Fruit weight was used for analyzing the effect of saline irrigation water on olive fruit development from the end of August until the harvest date of each variety in the 2002 season (Table 2.4A). Due to the high variation between olives of the same variety even on the same tree, the average weight of 10 fruits of each variety in both treatments was used.
Fruit weight of Barnea control trees was significantly higher in most of the tested dates in comparison to the saline irrigated olives. This result is in agreement with a previous report done under the same conditions showing an increased weight of fresh water irrigated Barnea olives in comparison to 4.2 dSm -1 saline irrigated olives (Wiesman et al., 2004). Weight of saline irrigated Souri olives was significantly higher on most of the tested dates in comparison to the control irrigated trees. 26
Table 2.4 A. Effect of saline water irrigation on fruit weight in different olive varieties.
Salinity Average of ten fruit Weight (g.) in the 2002 season Variety (dS m -1) 28 Aug. 19 Sep 2 Oct 9 Oct 13 Oct 16 Oct 30 Oct 6 Nov Israeli Origin Barnea 1.2 25.0 a 21.1 a x 28.4 a x 32.9 a x nd 33.2 a 30.1 a Barnea 4.2 18.2 b 19.0 a x 29.4 a x 28.5 b x nd 30.2 b 26.6 b Souri 1.2 23.3 b 31.5 a s 31.5 b x 35.5 a t nd 36.0 b Souri 4.2 26.4 a 33.4 a s 37.1 a t 37.2 a t nd 43.7 a Maalot 1.2 nd 26.7 b t 26.3 a x 27.0 a x 26.5 a Maalot 4.2 nd 27.5 a t 25.4 a x 26.7 a x 23.4 b Italian Origin Frantoio 1.2 12.5 a 13.6 b y 16.5 b y 16.4 b y nd 16.5 b 19.2 b 18.1 b Frantoio 4.2 13.9 a 16.3 a y 20.9 a x 20.5 a y nd 20.9 a 26.6 a 24.2 a Leccino 1.2 nd 24.2 a t 29.3 a x 29.0 a x Leccino 4.2 nd 22.5 b x 26.8 b x 28.5 a x Spanish Origin Arbequina 1.2 12.0 b 13.3 a y 17.7 a y 19.8 a y 16.4 a Arbequina 4.2 14.5 a 15.1 a y 18.1 a y 21.4 a y 18.7 a Picual 1.2 nd 26.1 a t 31.8 b x 33.0 a x nd nd 38.0 a 40.8 a Picual 4.2 nd 26.2 a t 36.9 a t 33.5 a t nd nd 31.6 a 30.6 a Picudo 1.2 nd 33.3 a s 37.7 b t 45.3 a s nd nd 41.4 b Picudo 4.2 nd 34.4 a s 45.0 a s 47.3 a s nd nd 48.5 a Greek Origin Kalamata 1.2 nd 33.4 a s 42.9 a s 46.3 a s 54.3 a Kalamata 4.2 nd 34.4 a s 37.4 b t 46.7 a s 43.9 b Koroneiki 1.2 nd 7.7 b z 8.6 b z 9.8 a z nd 9.7 a 10.1 a 12.7 a Koroneiki 4.2 nd 8.8 a z 10.0 a z 8.5 a z nd 9.0 b 10.7 a 11.3 b French Origin Picholin 1.2 14.9 b 22.2 b x 24.8 b x 26.0 b y nd 26.6 b 30.8 b nd Picholin 4.2 28.4 a 30.5 a t 37.5 a t 42.1 a t nd 40.4 a 46.3 a nd Morocco Origin P. Morocco 1.2 nd 27.2 a t 29.9 b x 34.8 a t P. Morocco 4.2 nd 30.3 a t 34.5 a t 37.9 a t Tukey Kramer test (p=0.01) was done between two salinity levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters s, t, x and z). Levels not connected by same letter are significantly different, nd= not available. 27 B. Analysis of variance of ten fruit weight by Tukey Kramer.
Source DF 19 Sep 2 Oct 9 Oct Variety 11 *** *** *** Treatment 1 *** *** *** Variety*Treatment 11 *** *** *** DF: degree of freedom; *** high significant effect at p=0.01
C. Grouping of olive varieties based on fruit average yield according to the response of the variety to saline water irrigation.
Group Range (g of ten fruits) Olive Varieties A 44-55 Picholine, Picudo, Kalamata B 30-43 Souri, Picual P. Morocco C 22-30 Barnea, Maalot, Frantoio, Leccino D 15-20 Arbequina E 5-15 Koroneiki
Analysing the olive weight of the third Israeli selected variety, Maalot, did not show a clear pattern of difference between olives of the two irrigation treatments. A higher weight was seeing at the beginning but gradually lowered to the harvesting month.
In the Italian group, olive fruit weight of saline irrigated Frantoio trees was significantly higher than the control plants for all tested dates except the first date (August 28). In
Leccino, although in two test dates weights were higher than in the control than the saline olives, at harvesting time no significant difference was noted between control and saline treated trees.
Again, apart from the initial testing date, no clear pattern of differences was noted in fruit weight between Arbequina fruits irrigated with saline and control water. Olive fruit weight in Picual showed a variation with no clear tendency between the treatments. In
Picudo, a pattern of greater weight was found in olives from saline irrigated trees in comparison to olives of control trees. 28 In selected olive varieties from Greek origin, the olive weight of Kalamata in control was higher on most of the tested dates up to harvesting time in comparison with the saline irrigated olives. In the case of Koroneiki small olives, no clear pattern could be observed. From the end of August until the beginning of October, the weight of saline irrigated olives was greater than the olive controls after and later on, random changes in weight could be observed in the two irrigation treatments.
Fruit weight of Picholin showed a tendency of higher values in saline irrigated treatment in comparison to the control till harvesting date. In the final results Picholin di
Morocco, showed a trend of increasing olive fruit weight in saline irrigated trees compared to the control, but only on October 2 was significantly different.
Tukey Kramer analysis of variance of olive varieties development response of the tested olive varieties to saline water clearly showed a significantly differences during the whole period of time that was tested (19 September; October 2; and October 9) (Table
2.4B). Significant differences were obtained also in between the two irrigation water salinity treatments and an interaction between treatments and olive varieties is clearly shown in relation to olives weight.
In summary, the data olive weights of the various varieties cultivated in Ramat Negev under arid conditions with saline water versus fresh water shows no clear effect and it vary between the olive varieties. These results are in partial agreement with previous reports suggesting no clear and significant pattern of olive growth in response to saline conditions (Aragues et al., 2005; Chartzoulakis and Ragab, 2005 Cresti and Tattini,
1994; Klein et al., 1994; Klein et al., 1992; Bouaziz, 1990). However, based on the data collected in the present study we could divide the fruit weight increase of the tested 29 varieties in response to saline water into five groups (Table 2.4C): Group A – including
Picudo, Kalamata; Group B – Souri, Picual, Picholin and Picholin Morocco; Group C –
Barnea, Maalot, Frantoio and Leccino; Group D – Arbequina; Group E – Koroneiki.
The systematic information concerning the olive size and weight may be important for future development of table olive processing in plantation cultivated in arid areas and irrigated with saline water, but further studies are needed before reaching final conclusions.
2.3.3 Leaf (Na +, Cl -) and soil (EC) analysis
In order to explain the further about the differences in horticultural response between the trees of various olive varieties irrigated with saline (4.2 dS m-1) and fresh water (1.2 dS m-1), specific soil EC in various fractions of the soil depth in the root zone, and the leaf sodium and chloride content were analyzed in the all twelve tested olive varieties cultivated. As expected and in agreement with previously reports (Aragues et al., 2005;
Gucci and Tattini, 1997; Klein et al., 1994; Benlloch et al., 1991; Al-Saket and Asheh,
1987; Bernstein, 1975; Bernstein, 1964), the level of Na + and Cl - was higher (but not significantly) in leaves of saline irrigated trees compared to the fresh water irrigated trees, with one exception in the case of Cl - in Arbequina leaves (Table 2.5A). Even in the case of Arbequina a slightly (but not significantly) increased chloride level in the leaves of fresh water irrigated trees in comparison to saline irrigated trees, this data well fit a previous report concerning the response of this specific olive variety to salinity
(Aragues et al., 2005). The highest level of Na + was found in saline irrigated leaves of
Picual, Arbequina, Koroneiki, and Picholin (1.455, 1.291, 1.268, and 1.125 mg g -1/dw, respectively). The highest level of Cl - was found in leaves of Picual, Picholin di
Morocco, Kalamata, and Souri (1.480, 1.466, 1.333, and 1.275 mg g -1, respectively).
30 Table 2.5 A. Comparison of leaf sodium and chloride content in trees of all tested olive varieties irrigated with saline and fresh water.
Salinity Variety Na + (mg g -1/d.w) Cl - (mg g -1/d.w) (dS m -1) Israeli Origin Barnea 1.2 0.338 a x 0.550 a y Barnea 4.2 0.540 a x 1.105 a y Souri 1.2 0.518 a x 0.857 a y Souri 4.2 0.600 a x y 1.275 a y Maalot 1.2 0.600 a x y 0.533 a y Maalot 4.2 0.725 a x y 0.866 a y Italian Origin Frantoio 1.2 0.600 a x y 0.700 a x y Frantoio 4.2 0.725 a x y 0.655 a x y Leccino 1.2 0.570 a x y 0.932 a y Leccino 4.2 0.575 a x y 0.115 a y Spanish Origin Arbequina 1.2 0.938 a x y 0.597 a x Arbequina 4.2 0.129 a y 0.430 a x Picual 1.2 1.213 a x y 0.840 a y Picual 4.2 1.455 a y 1.480 a y Picudo 1.2 0.405 a x 0.460 b y Picudo 4.2 0.612 a x y 1.100 a y Greek Origin Kalamata 1.2 0.400 a x 0.700 a y Kalamata 4.2 0.508 a x 1.333 a y Koroneiki 1.2 0.956 a y 0.782 a x y Koroneiki 4.2 1.268 a x y 0.697 a x y French Origin Picholin 1.2 0.493 a x 0.735 a x y Picholin 4.2 1.125 a x y 0.845 a x y Morocco Origin P. Morocco 1.2 0.650 a x y 0.566 b y P. Morocco 4.2 0.808 a x y 1.466 a y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not shared with the same letter are significantly different.
B. Fisher analysis of variance of Sodium and Chloride content in leaf of olive trees.
Source DF Na + CL - Variety 11 * NS Treatment 1 NS * Variety*Treatment 11 NS NS DF: degree of freedom; * low significant effect at p=0.05. NS: non significant effect.
In agreement with Aragues (2005) who reported on growth reduction in Arbequina olive trees only when leaf Cl - and Na + levels were higher than 2.3 and 1.5 mg g -1/dw, respectively leaf content of both elements, in the present study was higher than these levels were not recorded in the two salinity treatments. Interestingly, apart from the significantly higher Cl - level in Picudo and Picholin Di Morocco leaves from saline 31 treated trees, compared with control trees, no significant differences in Na + and Cl - level could be observed between trees treated with the two treatments in the other varieties.
Fisher analysis of variance of sodium content in the olive varieties leaves showed a low significant difference effect of olive varieties but no significances in relation to the varieties effect on chloride effect (Table 2.5B).The two irrigation water salinities showed a low significant effect (p=0.05) on chloride content in the leaves, whereas, no significant effect was obtained on sodium content in the leaves. No interaction between varieties and treatment was found.
Furthermore, electrical conductivity (EC) of three fractions (0–30, 30–60, and 60–90 cm) of the soil depth (Table 2.6A) showed a peak of 4.47 dSm -1 in the middle soil fraction of the fresh water irrigated trees that was significantly different from the lower depth fraction (2.80 dS m-1). The lower fraction was also significantly different from the upper soil fraction (2.03 dS m-1). A non-significant differ gradient, from 4.51 dS m-1 to 6.06 dS m -1, was found in the soil column of the saline irrigated trees. Tukey Kramer analysis of variance showed a low significant effect of the depth and a strong significant effect of the treatments (Table 2.6B). No effect of the interaction between the treatment and the depth was found.
Table 2.6 A. Soil fractions EC in fresh water subplot (1.2 dS m-1) and moderate saline water subplot (4.2 dS m-1).
Soil Column Soil EC (dS m -1) Fraction (cm) Fresh Water Saline Water 0–30 2.03+0.1 c x 4.51+0.6 a y 30–60 4.47+0.2 a x 5.34+0.7 a x 60–90 2.80+0.1 b x 6.06+0.4 a y Tukey Kramer test (p=0.01) was done in each of the treatment along the soil profile in three different depths (letters a and b), and between the two treatments (letters x and y). Levels not connected by same letter are significantly different. Values are the mean of three ±SE. 32
B Soil Fractions EC Tukey Kramer analysis of variance.
Source DF Significance Depth 2 * Treatment 1 *** Treatment*Depth 2 NS DF: degree of freedom; *** high significant effect at p=0.01; * low significant effect at p=0.05; NS: non significant effect.
The analysis of the difference in each of the three soil depth fractions between the two irrigation treatments showed that aside from the middle fraction, EC in the upper and lower fractions were significantly higher in the saline irrigated subplot than in the control sub-plot. The data obtained from EC soil analysis are in good agreement with previous reported data suggesting a range between 3 and 6 dS m-1 as enabling most olive trees to develop well with no significant horticultural parameter reduction
(Aragues et al., 2005 Maas and Hoffman, 1977; FAO, 1985; Bernstein, 1964).
Based on the data obtained in the present study, it seems that the key factor for cultivation of various olive varieties using moderate saline water for irrigation lies in the ability to maintain the EC of the soil in the root zone growth area at a level lower than 6 dSm -1, as suggested by the FAO (1985) recommendation of olive cultivation and supported by many other reports (Aragues et al., 2005; Maas and Hoffman, 1977;
Bernstein, 1964). In semiarid regions, where most of the crop water requirement is supplied through irrigation and water often contains large amounts of dissolved salts, salinity control is frequently a major objective of irrigation management (Shalhevet,
1994). Leaching is the process of applying more water to the crop than can be held by the root zone so that the excess water drains below the root system, carrying soluble salts with it (Grattan and Oster, 2002; Shalhevet, 1994). When saline water is supplied to crops, leaching become indispensable in order to exclude or reduce the salt excess 33 from the root zone (Beltran, 1999). Therefore, a proper leaching methodology during the entire year was developed specifically for olive cultivation in arid areas and had been used in previous studies carried out in the Ramat Negev area (Wiesman, 2004;
Wiesman, 2002). In the previous study carried out with Barnea olives irrigated with three levels of saline irrigation water (1.2, 4.2, and 7.5 dS m-1), the soil leaching enabled cultivation of the trees. A gradual and significant reduction of vegetative and reproductive development of the 7.5 dS m-1 in comparison to the other lower saline treatments suggested that in the long term, the high salinity concentration water is not suitable for sustainable olive cultivation (Bernstein, 1964; Maas, 1977; FAO, 1985;
Aragues, 2005). In the present study, the leaching methodology based on drip irrigation was further developed. It was found that application of an additional 1000 m 3/hectare of saline water (4.2 dS m-1) in March at the end of the winter season and in November at the beginning of the next winter (Table 2.2), enabled the reduction of the salt level in the developing root zone to a level lower than 6 dS m-1, essential for olive normal development. This leaching regimen scheme, together with accurate weekly drip irrigation, kept the soil conditions well set for most of the tested olive varieties to develop very similarly to the fresh water irrigated trees of the same variety.
The EC in these soil fractions is lower than the upper level of 6 dS m-1 which is well recognized as limiting olive varieties regular development (FAO, 1985). The data obtained in the present chapter clearly show the ability to cultivate all the tested olive varieties in an arid area with moderate saline water irrigation. A significant variation in terms of horticultural performance was found between the various tested olive varieties.
These differences may be attributed to the natural characteristics of each variety and/or to their rate of adaptation to the environmental conditions in the tested area, rather than related to the moderate saline drip irrigation in this study. 34 2.3.4. Fruit Yield
The effect of saline irrigation water on average fruit yield per tree from 2001 to 2004 is presented in Table 2.7A. General fluctuation in tree productivity was observed among all the olive varieties across the various years analyzed in this study. Indeed, fluctuation of olive tree yield is a common phenomenon due to alternate bearing (Barranco, 1998).
However, even after clearing the alternate bearing effect, a high level of variation is still observed, and the ratio between yields of the same olive variety irrigated with the two types of water changes during the four tested years.
Fresh water irrigated trees of Barnea (2003), Frantoio (2002), Picudo (2002), Picholin
(2002), and Picholin di Morocco (2002) yielded significantly more than trees irrigated with saline water. Usually, but not significantly, in the following year the yields of the saline irrigated trees tended to be greater than those of the control trees. Picholin trees irrigated with saline water did not follow this order and were found to produce a significantly higher yield than the control trees in years 2003 and 2004. Calculation of the average yield during the four analyzed years shows that no significant differences could be found between trees of all the varieties irrigated with the two irrigation treatments. Analysis of variance of the yielding clearly showed a significant effect to varieties (Table 2.7B). No significant effect was obtained in between the two salinity irrigation water tested and no interaction between olive varieties and treatments was found. 35 Table 2.7 A. Comparison of fruit yield per tree in all tested olives varieties irrigated with saline and fresh water.
-1 Salinity Olives yield (kg tree ) Variety -1 Average (dS m ) 2001 2002 2003 2004 Israeli Origin Barnea 1.2 38.3 a 55.1 a 41.3 a 76.6 a 54.8 a x Barnea 4.2 33.7 a 52.8 a 23.0 b 84.5 a 52.2 a x Souri 1.2 5.0 a 20.3 a 33.1 a 51.1 a 31.8 a y Souri 4.2 4.2 a 22.6 a 36.9 a 52.8 a 31.5 a y Maalot 1.2 nd 32.0 a 39.6 a 40.7 a 37.5 a y Maalot 4.2 nd 21.0 a 27.5 a 52.8 a 33.8 a y Italian Origin Frantoio 1.2 10.5 b 54.5 a 0.0 66.7 a 38.6 a y Frantoio 4.2 23.8 a 36.6 b 0.0 66.0 a 31.6 a y Leccino 1.2 9.2 a 36.0 a 38.4 a 71.2 a 46.1 a x y Leccino 4.2 12.2 a 37.2 a 27.7 a 78.7 a 45.7 a x y Spanish Origin Arbequina 1.2 12.3 a 38.2 a 43.7 a 51.6 a 42.5 a x y Arbequina 4.2 25.5 a 31.9 a 50.0 a 57.9 a 45.3 a x y Picual 1.2 30.5 a 41.4 a 11.0 a 77.4 a 42.5 a x y Picual 4.2 16.5 a 44.3 a 13.0 a 77.9 a 43.3 a x y Picudo 1.2 2.0 b 42.6 a 8.3 a 50.6 a 31.9 a y Picudo 4.2 5.5 a 25.0 b 17.0 a 55.0 a 30.7 a y Greek Origin Kalamata 1.2 nd 16.2 a 37.5 a 40.4 a 30.3 a y Kalamata 4.2 nd 15.1 a 41.1 a 51.9 a 38.5 a y Koroneiki 1.2 11.0 a 53.5 a 24.1 a 58.0 a 44.4 a x y Koroneiki 4.2 15.8 a 28.6 b 21.7 a 55.7 a 33.1 a y French Origin Picholin 1.2 19.1 a 67.6 a 11.5 b 88.4 b 53.6 a x Picholin 4.2 26.6 a 57.0 b 23.4 a 102.9 a 59.0 a x Morocco Origin P. Morocco 1.2 nd 34.0 a 21.7 a 54.0 a 36.6 a y P. Morocco 4.2 nd 7.33 b 28.6 a 69.1 a 35.0 a y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different. Nd= no available.
B. Fisher analysis of variance of fruit yield.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant effect at p=0.01, NS: non significant effect.
36 C. Grouping of olive varieties based on fruit average yield according to the response of the variety to saline water irrigation.
Group Range (kg of fruit) Olive Varieties A 52-59 Barnea, Picholin B 43-46 Leccino, Picual, Arbequina C 30-38 Souri, Maalot, Frantoio, Picudo, Kalamata, Koroneiki, P. Morocco
It is generally reported and accepted that a significant yield reduction occurs in olives
cultivated in saline conditions in comparison to control conditions (Gucci and Tattini,
1997). The olive yield data obtained in the present study seem to contradict this
common suggestion. However, a similar pattern of variation in olive yield as found in
the present study was previously reported by Bouaziz (1990) concerning saline brackish
water used for irrigation of olive trees cultivated in field conditions. Murillo (2000)
have reported that in olive trees irrigated with wastewater characterized by a salinity
level between 4.3 and 6.0 dS m-1, an estimated 30% reduction in fruit yield. Previous
studies carried out in the same location and based on similar intensive agricultural
practices (Wiesman et al., 2004; Klein et al., 1994), clearly showed that olive yield
reduction in response to irrigation with saline water is dependent on the level of salinity
in terms of EC and on proper methodology of soil leaching, as will be discussed later.
Based on these studies, water containing 4.2 dS m-1 was used for commercial olive
cultivation and, indeed, successful large scale olive plantations irrigated with this type
of water (4.2 dS m-1) already exist and are increasing significantly in the Ramat Negev
arid areas (Wiesman et al., 2002).
The reproductive response of olive varieties to the Ramat Negev area to the two types of
water used for irrigation, enable us to determine three main groups in term of their yield
in saline irrigation water (Table 2.7C): Group A – including Barnea and Picholin – their
average yield ranged from 52 - 59 kg/tree; group B – including Leccino, Picual and 37 Arbequina - their average yield ranged from 43 -46 kg/tree; Group C – Souri, Maalot,
Frantoio, Picudo, Kalamata, Koroneiki, and Picholin di Morocco – their average yield ranged from 30 – 38 kg/tree. This data may directly contribute to the information required by the olive industry for its promotion initiative of cultivation in semiarid areas.
2.3 .5. Oil Percentage and oil yield
The percentage of olive oil in saline and fresh water treated trees is presented in Table
2.8A. The average oil percentage in the four tested years (2001–2004) showed that in all olive varieties, no significant differences were obtained between olives produced on trees irrigated with the two types of water. However, in the year 2001, significantly higher oil levels were obtained in saline irrigated olives compared to the control olives in Barnea, Souri, Frantoio, Arbequina, and Koroneiki.
In 2002, except for Frantoio and Koroneiki, the oil percentage was similar in most of the varieties. In these two varieties saline treatment outline higher oil percentage.
In 2003, the oil percentage measured in most of the trees irrigated with saline water was significantly lower than the control trees. In 2004, the oil percentage of saline irrigated Barnea, Frantoio, Leccino, and Arbequina trees was significantly higher than in control trees. These data suggest that no clear pattern of oil accumulation could be observed in response to irrigation with saline water. These results are in good agreement with previous reports concerning the effect of salinity on olive oil accumulation
(Wiesman et al., 2004; Wiesman et al., 2002; Murrilo et al., 2000; Gucci and Tattini,
1997; Klein et al., 1994).
Fisher analysis of variance clearly showed a significant difference between olive varieties (Table 2.8B), as expected due to the literature available on potential oil 38 production of the various tested olive varieties (Barranco, 1998). No significant differences were obtained concerning to the effect of the two salinity irrigation treatments and concerning to the interaction between varieties and treatments.
Based on oil percentage of deferent olives varieties response to saline water used for irrigation can be divided in to three groups as showed in Table 2.8C: Group A – Barnea,
Souri, Maalot, Arbequina, and Koroneiki – their average oil percentage ranged from
18–22%; group B – Leccino, Frantoio, Picudo and Kalamata – their average oil percentage ranged from 15 - 17%; group C – Picual, Picholin and Picholin di Morocco
– ranged from 13 – 14%.
Oil yield is one of the most important parameters from the horticultural point of view in the olive oil industry, it is determined by multiplying fresh olive weight by the oil percentage; these data are presented in Table 2.9A.
As expected, due to a large fluctuation in olive production and oil accumulation of all the varieties, a large variation could be seen during the four tested years (2001–2004).
However, the average olive oil yield of all tested varieties was not significantly different between the trees irrigated with saline and fresh water. In agreement with previous reports (Wiesman et al., 2004; Wiesman et al., 2002; Murrilo et al., 2000; Barranco,
1998).
39 Table 2.8 A. Comparison of olive oil percent in all tested olive varieties irrigated whit saline and fresh water.
Salinity Oil Content (%) Variety Average (dS m -1) 2001 2002 2003 2004 Israeli Origin Barnea 1.2 21.8 b 22.9 a 21.7 a 16.0 b 20.4 a x Barnea 4.2 25.7 a 21.1 a 18.7 b 19.3 a 19.6 a x y Souri 1.2 20.3 b 16.8 a 23.0 a 21.0 a 20.3 a x y Souri 4.2 25.6 a 18.7 a 16.4 b 21.1 a 19.4 a x y Maalot 1.2 nd 16.5 a 29.8 a 27.4 a 24.6 a x Maalot 4.2 Nd 17.3 a 19.3 b 29.2 a 22.0 a x Italian Origin Frantoio 1.2 14.0 b 17.4 b 0.0 19.5 b 12.5 a y Frantoio 4.2 19.1 a 23.0 a 0.0 23.5 a 15.9 a x y Leccino 1.2 25.6 a 14.7 a 19.5 a 15.4 b 17.5 a x y Leccino 4.2 16.7 b 13.1 a 18.6 a 17.6 a 16.6 a x y Spanish Origin Arbequina 1.2 19.0 b 13.1 a 21.1 a 21.7 b 18.7 a x y Arbequina 4.2 26.7 a 12.9 a 17.1 b 25.5 a 19.3 a x y Picual 1.2 15.7 a 13.2 a 16.9 a 13.7 a 14.7 a y Picual 4.2 16.5 a 14.2 a 13.3 b 15.0 a 14.7 a y Picudo 1.2 14.4 a 12.5 a 21.9 a 15.8 a 16.5 a x y Picudo 4.2 13.5 a 11.9 a 20.0 a 15.2 a 15.5 a x y Greek Origin Kalamata 1.2 nd 8.9 a 22.5 a 18.2 a 16.6 a x y Kalamata 4.2 nd 7.3 a 18.9 b 19.7 a 15.4 a x y Koroneiki 1.2 15.0 b 18.4 b 21.9 a 19.9 a 19.6 a x y Koroneiki 4.2 22.1 a 23.1 a 20.4 b 18.5 a 20.9 a x French Origin Picholin 1.2 20.3 a 15.6 a nd 16.7 a 16.8 a x y Picholin 4.2 20.2 a 16.0 a nd 11.0 b 14.4 a y Morocco Origin P. Morocco 1.2 nd 12.6 a 11.9 a 15.8 a 13.5 a y P. Morocco 4.2 nd 14.0 a 11.7 a 16.2 a 13.8 a y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different. Nd: not available.
C. Fisher analysis of variance of oil percentage.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant differences at p=001 NS: non significant.
C Grouping of olive varieties based on average fruit oil percentage according to the response of the variety to saline water irrigation.
Group Range (% oil) Olive Varieties A 18-22 Barnea, Souri, Maalot, Arbequina, Koroneiki B 15-17 Frantoio, Leccino, Picudo, Kalamata C 13-14 Picual, Picholin, P. Morocco 40 Fisher statistic analysis showed significant differences between the olives varieties tested in the present study (Table 2.9B). No significant difference was obtained between the two irrigation water treatments and no interaction between olive varieties and the two treatments was found.
A summary of the average olive oil yield by the olive varieties in response to Ramat
Negev arid environmental conditions and saline water used for irrigation, suggest three main groups: Group A – including Barnea, Arbequina and Picholin – their average oil yield ranged from 9–10 kg/tree; group B – including Souri, Maalot, Frantoio, Leccino,
Picual, Kalamata and Koroneiki – their average oil yielded ranged from 6–8 kg/tree; group C – including Picudo and Picholin di Morocco – ranged from 4–5 kg/tree (Table
2.9C). These last data demonstrate the lack of significant response by the tested olive varieties in the present experimental set-up based on intensive drip application of saline
(4.2 dSm -1) and fresh water (1.2 dSm -1), suggesting that when selecting olive varieties for cultivation in semiarid areas, most attention should be directed to the genetic differences between varieties in terms of vegetative growth, productivity, oil yield, and quality, rather than to water quality.
The vegetative and reproductive responses of mature yielding trees of many olive varieties commonly cultivated in Mediterranean countries, irrigated with saline water has not yet been studied under intensive orchard management. Previous studies carried out with mature yielding trees of common local Israeli Barnea variety cultivated in an orchard irrigated with three levels of saline water (1.2, 4.2, and 7.5 dS m-1), clearly showed the advantage of using 4.2 dS m-1 water for optimization of the horticultural performance of Barnea trees in terms of growth and olive yield (Wiesman et al., 2004;
Wiesman et al., 2002; Klein et al., 1994). 41 Table 2.9 A. Comparison of olive oil yield in all tested olives varieties irrigated with saline and fresh water.
Salinity Oil Yield (kg oil/tree -1) Variety Average (dS m -1) 2001 2002 2003 2004 Israeli Origin Barnea 1.2 8.3 12.6 9.0 12.2 11.0 a x Barnea 4.2 8.6 11.1 4.3 16.3 10.5 a x Souri 1.2 1.0 3.4 7.6 10.7 6.6 a x y Souri 4.2 1.1 4.2 6.0 8.5 5.9 a y Maalot 1.2 nd 5.3 11.8 11.1 9.4 a x y Maalot 4.2 nd 3.6 5.2 15.4 8.1 a x y Italian Origin Frantoio 1.2 1.5 9.5 0.0 13.0 7.1 a x y Frantoio 4.2 4.5 8.4 0.0 15.5 7.3 a x y Leccino 1.2 2.3 5.3 7.5 11.0 7.6 a x y Leccino 4.2 2.0 4.9 5.1 13.8 7.6 a x y Spanish Origin Arbequina 1.2 2.3 5.0 9.2 11.2 8.1 a x y Arbequina 4.2 6.8 4.1 8.5 14.8 9.0 a x y Picual 1.2 4.8 5.5 1.8 10.6 7.8 a x y Picual 4.2 2.7 6.3 1.7 11.7 6.0 a y Picudo 1.2 0.3 5.3 1.8 8.0 4.8 a y Picudo 4.2 0.7 3.0 3.4 8.4 4.6 a y Greek Origin Kalamata 1.2 nd 1.4 8.4 7.3 5.7 a x y Kalamata 4.2 nd 1.1 7.8 10.2 6.5 a x y Koroneiki 1.2 1.6 9.8 5.3 11.5 8.4 a x y Koroneiki 4.2 3.5 6.6 4.4 10.3 6.9 a x y French Origin Picholin 1.2 3.9 10.5 - 14.8 11.9 a x Picholin 4.2 5.4 9.1 - 11.3 9.8 a x y Morocco Origin P. Morocco 1.2 nd 4.3 2.6 8.5 5.1 a y P. Morocco 4.2 nd 1.0 3.3 11.3 5.2 a y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different. Nd: not available.
B. Fisher analysis of variance of olive oil yield.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant effect at p=0.05, NS: non significant effect.
C. Grouping of olive varieties based on average olive oil yield according to the response of the variety to saline water irrigation.
Group Range (kg oil) Olive Varieties A 9-10 Barnea, Arbequina, Picholine B 6-8 Souri, Maalot, Frantoio, Leccino, Picual, Kalamata, Koroneiki, C 4-5 Picudo, P. Morocco
42 Vegetative and reproductive response to saline water irrigation of Olive ( Olea europea
L), as was well demonstrated in the present study, are strongly correlated to the genetics differences existing between the eleven studied olive cultivars and not to the fact that trees are irrigated with moderate saline water. Trunk circumference (Table 2.1A, B and
C) results show clearly that some of them were most tolerant at the initial phase of growth like, Leccino and Maalot, than others cultivars.
Salt tolerance in olive plants mainly depends on exclusion mechanisms that reduce the uptake and transport of sodium and chloride to the canopy (Gucci and Tattini, 1997).
The sodium and chloride leaf content (Table 2.5A, B and C) in the present study were found higher but no significant different in trees irrigated with saline water (except of
Arbequina).
An oscillation in the fruit yield (Table 2.7A, B and C) was observed along the four studied years; assuming the evidence that alternate bearing is a common phenomenon in olive, average of fruit yield of the studied cultivar was found no significant affected by salinity. Indeed, yields of olives irrigated with saline water was somewhat to be greater than those of the control.
Finally olive oil percentage (Table 2.8A, B and C) and oil yield (Table 2.9A, B and C), the most two important parameters in the olive oil industry, were not significantly affected by the presences of salt in the irrigation water in the average of the four studied years.
43 3. Effect of saline water irrigation on Olive oil quality
3.1 Introduction
Oil is one of the most important products obtained from olives and it represents in economic terms, around 15 % of the total vegetable oil produced around the world
(Luchetti, 2002) and almost two percent of the total fat consumed (USDA, 2004); 85% of this consumption are concentrated in countries of the Mediterranean basin (IOOC,
2004) who are also the principal producers with nearly 98 % of the total olive cultivated area in the world (Luchetti, 2002). Since the mid 1990s available data shows an increase in the olive oil production. This trend is likely to continue in the foreseeable future: predictions in amounts and quality of olive oil converge in projecting a future scenario with increase in world supply above those in potential demand (IOOC, 2001; Mili and
Rodriguez Zuñiga, 2001). Production is gradually rising in the traditional producers countries such as Spain, Italy, Greece, Turkey and Morocco, but also and more recently in non-traditional producing countries such as Argentina, Australia, New Zealand,
Chile, Mexico, South Africa, USA and China (IOOC, 2001-2004). These gains in oil productivity are the result of a substantial improvement in cultivation techniques, new varieties, localized drip irrigation and the implantation of new olive orchards in semiarid areas where poor quality sources of water, especially saline water, are the main water available to irrigation (FAO, 1985).
The detrimental effects of salts on plants are the consequent of water deficits that result from the relatively high solute concentrations in the soil as well as a stress specific to
Cl - and Na + resulting in a wide variety of physiological and biochemical changes.
High concentration of salt, can affect the CO 2 assimilation in the photosynthesis apparatus (Chartzoulakis et al., 2002) and reduce the amount of assimilates produced by the plant, affecting finally the growth and plant productivity (Munns, 2002). 44 In terms of olive oil quality, salinity cannot affect or induce to some variations in several oil parameters; Wiesman (2004) report that acidity which express the total free fatty acid present in the oil cannot be affected or show no significant variation by salinity in oils obtained from Barnea trees irrigated with three saline water levels; Royo
(2005) found that the peroxides values was not affected by salinity in a multi-annual
Arbequina olives saline water irrigated experiment.
There is a growing evidence that Mediterranean diet, in witch olive oil is the main source of fat, has a beneficial effect on diseases associated with oxidative damage such as coronary heart diseases (Renaud et al.,1995). One of the biological virtues of olive oil is due to its balanced fatty acid composition and antioxidant components. Cresti (1997) reported that salinity short the olive maturation time, increase the total concentration of aliphatic and terpenic alcohols, decreasing the oleic/linolenic acids ratio and modify the linoleic/linolenic acids ratio.
Phenolics compounds are antioxidant agents present in several foods of the
Mediterranean diet among which olive oil stands out. The biological benefits of olive consumption in preventing low-density lipoproteins (LDL) by oxidation could be linked to its antioxidants content, mainly phenolic compounds vitamin E and sterols.
The amount of phenols in olive oil depends on several factor including cultivars, degree of maturation and climate (Visioli and Galli, 2001); at the present, the most widely employed methods for phenols quantification are the Folin- Ciocalteu colorimetric and high performance liquid chromatography (HPLC) (Visioli et al., 1995). These two methods are economic and easy to perform, but are limited by the low specificity of the reagent toward phenolic compounds and it does not give qualitative information of 45 single phenolics present in the sample. Among the phenols present in olive oil with antioxidant capacity, the most important class is polar phenolic compounds, such as tyrosol derivates (Montedoro et al., 1992). Even today, no clear information are found in literature about the effect of salinity in the phenols presents in olive oil; Royo (2005) indicated that the polyphenols content in oil obtained from saline treatments was around
42 % higher compared to oils obtained from olives irrigated with fresh water, and
Wiesman (2004) reported that total polyphenols concentration was increased by salinity but non significant differences were found between polyphenols concentration in
Barnea oil extracted from olives irrigated with three saline water levels.
Tocopherols are natural antioxidants which play a key role in preserving oil from rancidity during storage thus prolonging its shelf-life; α tocopherol is traditionally considered as the major antioxidant in olive oil and it comprises about 90% of the total tocopherols, its natural concentration varies between few ppm up to 300 ppm (Blekas et al., 2002); tocopherols also effectively inhibit lipid oxidation in foods and in biological systems. Wiesman (2004) found a significant increase in vitamin E concentrations in
Barnea oil obtained from trees irrigated with saline water.
Sterol profiles are used to characterize olive oils and especially to detect the adulteration with hazelnut oil. Recently, it also has been proposed that this profile could be used to classify virgin oil according to their fruit variety (Aparicio et al., 1997). Phytosterols, like other antioxidants, apparently help to reduce the total plasma and LDL-cholesterol, and as a result these compounds are being considerer as functional foods, (Ostlund, et al., 2002), unfortunately till today no works were done about the effect of saline water irrigation in both total sterol concentration and sterol profile in olive oil.
46 3.2 Materials and methods
3.2.1 Acidity Determination (free fatty acid)
Acidity was calculated and expressed as percentage of oleic acid, according to the
International Olive Oil Council regulations (IOOC 1995; ISO 660).
In brief, 5 g of oil was weighed in Erlenmeyer flask and 10 ml of 95 % boiling ethanol and 0.5 ml of Phenolphthalein (10 g/l solution in ethanol 95% v/v) were added.
The titration was carried out drop by drop with a 0.5 M NaOH solution until the colour change.
Acidity was calculated as percentage by mass and is equal to:
Acidity (% oleic acid) = V.c.M/10.m
Where
V = volume in ml of the standard volumetric NaOH solution c = concentration, in moles per litter, of the standard volumetric NaOH
M = molar mass, in g per mole of oleic acid m = mass in g, of the test portion
3.2.2 Peroxides Determination
Peroxide value were calculated according to the International Olive Oil Council regulations (IOOC 1995; ISO 3960), and expressed in mill equivalents (me) of active oxygen per kilogram. Five g of oil was taken in an Erlenmeyer and 50 ml of mix solution of acetic acid/isooctane 60:40 v/v was added to it, and then 0.5 ml of saturated potassium iodide solution was added and mixed together with the initial solution and the titration was done with thiosulfate solution 0, 01 mol/l. After the solution is coming 47 light yellow about 0.5 ml of starch solution (5 g/l) was added. The end point was determined when the solution changed the brown colour to transparent.
The peroxide value was calculated by the following equation:
Peroxide Value (me. peroxide / kg oil) = 1000(V-V0).c/m
Where:
V = volume of sodium thiosulphate solution used, in ml
V0 = volume sodium sulphate used for the blank determination, in ml c = concentration of the sodium thiosulphate solution, in moles per litter m = mass of the test portion, in grams
3.2.3 Fatty Acid Profile
The methyl esters of the oil were prepared according to the procedure of the
International Olive Oil Council (IOOC) regulations. In a 5 ml tube 0.1 g oil was weighed and diluted with 2 ml heptane and 0.2 ml of 2 N methanolic potassium hydroxide solutions was added. The combined solution was shaken vigorously for 30 seconds and left to stratify until the upper solution become clear. This solution was collected and evaporated to dryness under N 2 gas flow; the methyl esters were re- suspended in 1000 µl of heptane and injected into the GC for determination.
3.2.3.1 Gas chromatography conditions
The operating conditions were as follows:
- Column temperature: 120 to 190 ± 5 °C,
- Injector temperature: 250°C,
- Detector temperature: 300 °C, 48 - Linear velocity of the carrier gas: Nitrogen 30 cm/s; hydrogen 20 to 30 cm/s and air
300 cm/s
- Amount of substance injected: 4 µl of the re-suspended methyl esters solution.
The percentage of each Acid was calculated according to the formula:
% X = (Area X x 100) / Total Area
3.2.4 Total polyphenols determination
The total polyphenols in olive oil was estimated by colorimetric analyses using the
Folin-Ciocalteu reagent Gutfinger (1981) . One g of olive oil was dissolved in 5 ml of hexane and 2 ml of aqueous methanol (60:40 v/v) were added, the sample was mixed vigorously for two minutes, the methanolic phase was pippeted off and put it in a beaker, this process was repeated twice. The combined methanolic solution was evaporated to dryness in a vacuum rotary evaporator at 40° C, and the residues were re- suspended in 1 ml of methanol. An aliquot of 0.05 ml of the concentrate phenol solution was transferred to 5 ml volumetric flask and 2.5 ml of distilled water, 0.125 ml of Folin
& Ciocalteu reagent 2N, and 0.5 ml of Na 2HCO 35 % solution was added and filled with distilled water up to the mark. After 1 hour the sample was measured at the UV
Spectrophotometer at 725 nm wavelength.
Reference curve was prepared using the same procedure and the results were expressed as ppm of caffeic acid.
3.2.5 Vitamin E determination
The tocopherol (Alpha, Beta and Gamma) concentrations were prepared and calculated according to the International Olive Oil Council regulations (IOOC 1995, IUPAC
1998). Vitamin E concentration was determined by High Performance Liquid
Chromatography (HPLC) 49 3.2.5.1 Tocopherol standard solutions
Ten g of the standard were weighed into a 100 ml volumetric flask and filled up to the volume with hexane. Ten ml of this solution were pippeted out and evaporated to dryness at 40°C and combined with 10 ml of methanol. The absorbance of each standard was measured at the UV Spectrophotometer and the final concentration for each of them was calculated by using the adequate divisor factor:
292 nm alpha tocopherol = 0.0076
298 nm gamma tocopherol = 0.0091
298 nm delta tocopherol = 0.0087
3.2.5.2 Mixed tocopherol standards working solution
Appropriate volumes of the tocopherol standard solutions were diluted with hexane in order to give a solution containing between 1 and 5 µm per ml of each tocopherol.
3.2.5.3 Working conditions
Isocratic acetate/hexane mobile phase (10/90 v/v) was pumped through the column at flow rate of 1 ml/min for 15 min.
3.2.5.4 Test Sample
One ml of oil were diluted with 10 ml of hexane and one aliquot of 20 µl of this solution was injected into the column and identified the tocopherol presence by reference to the chromatograph obtained from the standards.
The alpha tocopherol content in the sample was given by:
Alpha tocopherol concentration (µg/g oil) = C.a.D.25/A.m
50 Where:
C = concentration of the alpha tocopherol standard (µg/g oil)
A = mean of the peak area obtained for the alpha tocopherol standard a = mean of the peak obtained for the alpha tocopherol in the test sample m = mass of the test sample taken (g)
D = dilution factor
The gamma and delta tocopherol content were calculated in the same way using the data obtained from the chromatography of the corresponding tocopherol standard.
3.2.6 Sterol determinations
3.2.6.1 Preparation of the unsaponifiables
Five g of oil were put into 250 ml flask and 500 µl of 0.2 % α-cholestanol solution were added. The combined solution was evaporated to dryness with nitrogen and 5 g of this dry filtered were put in the same flask. Fifty ml of 2 N ethanolic potassium hydroxide solutions were added and the saponification was carrying out by boiling and stirring the sample. The sample was heating for 20 minutes, then 50 ml of distilled water were added and the sample was cooled to approximately 30 °C. The content was transferred to a separating funnel using rinses of distilled water, all about 50 ml, approximately 80 ml of ethyl ether were added, and the sample was shaken vigorously for 30 seconds and allowed to settle. The lower aqueous phase was separated and collected into a second separating funnel. Two more extractions were done from the water-alcohol phase using
65 ml of ethyl ether on each time.
The ether extracts were pooled into a single separating funnel and washed with distilled water (50 ml each time), until the wash gave a neutral reaction. Then the wash water has 51 been removed, the sample was dried with anhydrous sodium sulphate and filtered on anhydrous sodium sulphate into a previously weighed 250 ml flask and the funnel and filter were washed with small aliquots of ethyl ether.
The ether was evaporated to few ml, and then was dried with nitrogen; dryness was completed in a stove at 100°C for approximately 15 min and then weighed after cooling in a diseccator.
3.2.6.2 Separation of the sterol fraction
Approximately 5 % solution of the unsaponifiables in chloroform was prepared and a chromatographic plate was streaked with 0.3 ml of this solution. At the same time 2 to 3
µl of the sterol reference solution were streaked, as the sterol band was identified after developing.
The plate was placed into the developing chamber, and allowed to elute until the solvent reached 1 cm from the upper edge of the plate, the plate was left inside the hood in order to evaporate the solvent. The plate was sprayed with 2, 7-dichloroflouredciein solution, and the sterol band was identified under ultraviolet light. This band was scraped from the silica gel and the final comminuted material was placed into the filter funnel; 10 ml of hot chloroform were added and filtered under vacuum, collecting the filtrate in a conical flask attached to the filter funnel.
The collected residue was washed three times with ethyl ether (10 ml each time), and the filtrate was collected in the same flask attached to the funnel, this filtrate was evaporate to a volume of 4-5 ml and the residual solution was transferred into 10 ml test tube; this solution was dried by heating in a gentle flow of nitrogen, upped again with acetone (few drops) and evaporated to dryness again. The sample was placed in a stove 52 at 105°C for approximately 10 minutes and then allowed to cool in a diseccator and weighed. The residue contained in the tube consists of the sterol fraction.
3.2.6.3 Preparation of the trimethylsilyl ethers
Silylation reagent was added consisting of a 9:3:1 (V/V/V) mixture of pyridine/hexamethyl disilazane/ trimethyl chlorosilane in the ration of 50 µl for every milligram of sterol to the test tube containing the sterol fraction, the tube was shaken until the sterols were completely dissolved. The sample was leaved for at least 15 minutes at ambient temperature and then centrifuged for a few minutes. The obtained clear solution is ready for gas chromatography.
3.2.6.4 Gas chromatography analysis
The operating conditions were as follows:
- Column temperature: 260 ± 5 °C,
- Injector temperature: 280°C,
- Detector temperature: 290 °C,
- Linear velocity of the carrier gas: helium 20 to 35 cm/s; hydrogen 30 to 500 cm/s,
- Splitting ration: from 1:50 to 1:100,
- Instrument sensitivity: from 4 to 16 times the minimum attenuation,
- Recording sensitivity: 1 to 2 mV
- Paper speed: 30 to 60 cm/ hour,
- Amount of substance injected: 0.5 to 1 µl.
3.2.6.5 Peak identification
Individual peaks were identified on the basis of retention times and by comparison with mixture of sterol TMSE analyzed under the same conditions. 53 The sterols were eluted in the following order: cholesterol, brassicasterol, 24-methylene cholesterol, campesterol, campestanol, stigmasterol, ∆ 7-campesterol, ∆ 5, 23- stigmastadienol, clerosterol, β-sitosterol, sitostanol, ∆ 5-avenasterol, ∆ 5, 24 stigmastadienol, ∆ 7 –stigmastenol, ∆ 7 –avenasterol. The concentration of each individual sterol, expressed in mg/kg of fatty material, was calculated as follows:
Sterol concentration (mg/kg) = Ax.m s.1000 /A s. m
Where:
Ax: peak area for sterol x, in square millimetres;
As: area of the α-cholestanol peak, in square millimetres; ms: mass of α-cholestanol added, in milligrams; m: mass of the samples used for determination, in grams;
3.2.6.6 Expression of the results
Individual sterol concentration was recorded as mg/kg of fatty material and their summed as "total sterols"
3.2.7 LC-MS Phenols Determination
3.2.7.1 Phenol Extraction
The Phenols were extracted following the procedure described in the Total Polyphenols
Determinations. One g of olive oil was dissolved in to 5 ml of hexane and 2 ml of aqueous methanol (60:40 v/v) were added, the sample was mixed vigorously for two minutes, the methanolic phase was pippeted off and put it in a beaker, this process was repeated twice. The combined methanolic solution was evaporated to dryness in a vacuum rotary evaporator at 40° C, and the residues was re-suspended in 1 ml of 54 methanol. An aliquot of 20 µl of this solution was injected into the column and identified the phenols present by reference to their mass weight.
3.2.7.2 Working conditions
Five µl of the methanolic extract was injected onto a reversed-phase column and analyzed using an Agilent 1100 series HPLC (Palo Alto, CA) with a G1314A UV detector and Lichrospher RP-18 column (5 µm, 4 x 250 mm, code 1.50983—Merck,
Darmstadt, Germany). The mobile phase consisted of (A) methanol and (B) 0.1% aqueous acetic acid. The flow rate was 1 mL/min with a gradient profile consisting of B with the following proportions (v/v) of A: 0-30 min, 5-35%; 30-35 min, 35-65%; 35-50 min, 65-100%; 50-55min, 100%.
Mass spectra were obtained with a Bruker MS Esquire 3000 Plus (Billerica, MA) with an electro spray source and ion trap detector operated in negative mode. The system was run on Bruker Daltonics Data Analysis 3.0 software.
3.2.7.3 Optimization procedures
We experimented with different mobile phases and gradients in an HPLC system comprising a Varian ProStar 240 solvent delivery module and ProStar 330 PDA detector (Walnut Creek, CA) monitored at 280 nm. After achieving effective peak separation, we switched to LC-MS analysis. The molecular weights of the compounds giving the major HPLC peaks were determined by MS (by comparison with those of known phenolic compounds). Solutions prepared for each standard were injected into the LC-MS system, and MS-MS fragmentation spectra of the standards were recorded in the system library. MS analysis was optimized for each LC peak time and target compound so that the fragmentation spectra of peak compounds could be isolated and 55 compared with library records. Comparisons were scored with the Bruker Daltonics system software on a 1,000 point scale, with 1,000 being a perfect match. (Maranz et al., 2003)
3.2.8 Chemical reagents
Tetrachoroethylene (J.T Baker, NJ, USA), Ethanol (J.T Baker, NJ, USA), Na (OH)
(Frutarom, Haifa, Israel), Acetic acid (J.T Baker, NJ, USA), Iso-octane (Riedel-deHaen,
Germany), Potassium iodide (J.T Baker, NJ, USA), Potassium hydroxide (J.T Baker,
NJ, USA ), Thiosulfate (Riedel-deHaen, Germany), Starch (Sigma, Rehovot, Israel),
Hexane (J.T Baker, NJ, USA), Methanol (J.T Baker, NJ, USA), Folin & Ciocalteu 2N
(Sigma Aldrich, Rehovot Israel), Na 2HCO (J.T Baker, NJ, USA), Toluene (Frutarom,
Haifa, Israel), Heptane (Frutarom, Haifa, Israel), Acetone (Frutarom, Haifa, Israel),
Chloroform (J.T Baker, NJ, USA), Ethyl ether (Frutarom, Haifa, Israel), Anhydrous sodium sulphate (J.T Baker, NJ, USA), silylation reagents: Hexamethyl disilazane and trimethylchlorosile (Sigma, Rehovot, Israel), 2,7-dichlorofluorescein (Sigma, Rehovot,
Israel), Anhydrous pyridine (Sigma, Rehovot, Israel), were used for different chemical analyses.
3.2.9 Standards
Alpha, Gamma and Delta Tocopherols (Sigma Aldrich, Rehovot Israel), rapeseed oil standard (Sigma Aldrich, Rehovot Israel), cholesterol (Sigma Aldrich, Rehovot Israel),
α-cholestanol (Sigma Aldrich, Rehovot Israel) were used for different chemical analyses.
56 3.2.10 Instruments
UV Spectrophotometer (Jasco V-350, Tokyo, Japan), Rotor Evaporator ( Rotovapor R,
Buchi Germany), Centrifuge (Hermle Z 400, Germany), Gas chromatograph (Hewlett-
Packard 6890 series) , FID detector and column SP-2330 (30 m, 0.2 _m film thickness)
(Supelco, PA, USA), Pump ( Varian Prostar 240, Japan), UV Detector ( Varian Prostar
330, Japan), Auto sampler (Varian Prostar 410, Japan), ICP-AES 3000 (Germany) ,
HPLC Column 250 x 4mm, 5µm Spherisorb SS NH 2 (Regis Technologies, Morton
Grove, IL), Glass plates (PolyGram SIL, Macherey-Nagel, Germany), condenser reflux
( Isopad, USA) were used .
3.2.11 Statistical analysis
At least three replicates were used for each field test and at least three replicates for the laboratory tests. The data were statistically analyzed with JMP software (SAS, 2000) using the Tukey–Kramer HSD and Fisher LDS tests for determining significant differences among the treatments at P≤ 0.01 and P≤ 0.05 . 57 3.3 Results and Discussion
3.3.1 Free fatty acid (Acidity)
Only a few studies have been carried out on the effect of salinity on olive oil quality.
Acidity reflects the rate of triglycerides cleavage as percentage of oleic acid, it is well demonstrated that acidity is affected principally by the treatment gives to the olives after harvesting till oil extraction (Perez Arquillue et al., 2003); an increase in the acidity value is due to the enzymatic activity caused by tissue damages in the olives
(Boskow,1996).
No clear answers are available in the literature for the effect of saline water irrigation on olive oil acidity behaviours (Wiesman et al., 2004; Klein et al, 1994; Bouazi, 1990).
Acidity of oils from four different years are shown in Table 3.1A; the results are expressed as percentage of oleic acid.
Significant differences were found between varieties from Israel in years 2003 and
2004, in the average of the four analyzed years only in Maalot significant differences were found between the treatments (0.99 % and 0.88 %).
Among the varieties from Italy only in Leccino in years 2004 and 2005 significant differences were found between the treatments, in the rest of varieties no significant differences were observed also in the average of the four years.
Acidity values in varieties from Spain were significant different in some of the tested varieties between the treatments in years 2003 (Arbequina and Picudo), 2004 and 2005
Arbequina and Picual.
58 Significant differences between treatments were found in Kalamata in years 2003, 2004 and 2005 respectively and only in 2004 acidity in Koroneiki was found significant different.
Finally in Picholin and Picholin de Morocco significant differences were found in 2003 and 2004.
Table 3.1 A. Comparison of free fatty acid (% of oleic) of oils obtained from all tested olive varieties irrigated whit saline and fresh water.
Salinity Acidity ( % of oleic acid) Variety Average (dS m -1) 2001 2003 2004 2005 Israeli Origin Barnea 1.2 0.50 a 0.57 b 0.35 a nd 0.46 a x y z Barnea 4.2 0.62 a 0.65 a 0.27 b nd 0.48 a x y z Souri 1.2 0.62 a 0.62 a 1.28 a 0.60 a 0.91 a x y z Souri 4.2 0.79 a 0.46 b 1.00 b 0.38 b 0.63 a x y z Maalot 1.2 nd 0.96 a 1.01 a 0.72 b 0.99 a z Maalot 4.2 nd 0.79 b 0.83 b 1.12 a 0.88 b z Italian Origin Frantoio 1.2 0.93 a nd 0.52 a nd 0.75 a y z Frantoio 4.2 0.84 a nd 0.82 a nd 1.06 a z Leccino 1.2 0.62 a 0.33 a 0.15 b 0.23 b 0.27 a x Leccino 4.2 0.61 a 0.29 a 0.29 a 0.28 a 0.26 a x Spanish Origin Arbequina 1.2 0.71 a 0.84 a 0.30 b 0.33 a 0.59 a y z Arbequina 4.2 0.83 a 0.60 b 0.46 a 1.24 b 0.77 a y z Picual 1.2 0.33 a 0.37 a 0.13 b 0.27 a 0.26 a x Picual 4.2 0.39 a 0.38 a 0.24 a 0.14 b 0.32 a x Picudo 1.2 0.56 a 0.81 b 0.18 a nd 0.57 a x y z Picudo 4.2 0.44 a 0.97 a 0.29 a nd 0.53 a x y z Greek Origin Kalamata 1.2 nd 0.59 a 0.19 b 0.21 b 0.36 a x y Kalamata 4.2 nd 0.41 b 0.25 a 0.46 a 0.36 a x y Koroneiki 1.2 0.89 a 0.89 a 0.35 b nd 0.72 a x y z Koroneiki 4.2 0.99 a 0.82 a 0.50 a nd 0.64 a x y z French Origin Picholin 1.2 0.51 a nd 0.11 a nd 0.37 a x Picholin 4.2 0.56 a nd 0.33 b nd 0.22 a x Morocco Origin P. Morocco 1.2 nd 0.44 a 0.29 b nd 0.33 b x y P. Morocco 4.2 nd 0.38 b 0.38 a nd 0.41 a x y Tukey Kramer test (p=0.01) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x, y and z). Levels not connected by same letter are significantly different. Nd: not available.
B. Tukey Kramer analysis of variance of olive oil acidity.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant effect at p=0.01; NS: non significant effect. 59 C. Grouping of olive varieties based on percentage of free fatty acid.
Group Range ( % oleic acid) Olive Varieties A 0 – 0.35 Picholine, Picual, Leccino B 0.35 – 0.80 P. Morocco, Kalamata, Koroneiki, Picudo, Souri, Barnea C 0.80 – 1.20 Maalot, Frantoio, Arbequina The grouping is based on the response of variety to saline irrigation.
In the average of four studied years, only in the cases of Maalot and P. Morocco
significant differences were found in between oils obtained from both treatments, with
opposite pattern. According to The International Olive Oil Council the threshold of
acidity accepted is 0.8 % expressed as oleic acid (IOOC, 1995); averages of acidity
values of four studied years were under 0.8 % in the majority of the analyzed oils in the
present study, except Maalot in both treatments (0.99% and 0.88% respectively), Souri
in oils from controls trees (0.91%) and Frantoio in saline treatment (1.06 %).
However, a Tukey Kramer analysis shows a significant difference in acidity values
between oils obtained from trees irrigated with fresh or saline water, these differences
are may be partially related to genetics differences between the varieties and can only
hardly explained by the presence of NaCl in the irrigation water as showed in Table
3.1B. Salvador (2001) has reported that free fatty olive oil acidity can be increased
probably as consequence of several years of drought followed by heavy rainfall shortly
and low temperatures registered before harvesting in oils obtained from trees cv
Cornicabra; on the other hand, Stefanoudaki (2001) report that acidity values of oils
obtained from trees cv Koroneiki subjected to different water regimes was not affected.
Finally we can divide the twelve analyzed olive varieties in accordance with their
response to saline irrigation in terms of oil acidity and according to three different
ranges of percentage of free fatty acid as showed in Table 3.1C. 60 3.3 2 Peroxides Value
Peroxide value reflects the oxidation of the fatty acid chains building the triglyceride of the olive oil. As report by Cimato (1990), oil extraction processes originate some degradation extra and intra-cellular causer of two important alterations in olive oil: acidification and oxidation.
Table 3.2 A. Comparison of peroxide concentrations (milleq. peroxide / Kg oil) of oils obtained from all tested olive varieties irrigated whit saline and fresh water.
Salinity Peroxides (Milleq. peroxide/Kg oil) Variety -1 Average (dS m ) 2001 2003 2004 2005 Israeli Origin Barnea 1.2 6.0 a 10.0 a 3.7 a nd 5.72 a x Barnea 4.2 7.3 a 6.7 b 4.2 a nd 5.76 a x Souri 1.2 15.3 a 14.4 a 5.4 a 4.8 a 7.38 a x Souri 4.2 18.1 a 15.0 a 3.6 b 4.1 b 5.45 a x Maalot 1.2 nd 14.5 a 5.1 a 5.0 b 5.65 a x Maalot 4.2 nd 14.0 a 6.0 a 6.3 a 6.99 a x Italian Origin Frantoio 1.2 11.2 a nd 5.8 a nd 6.88 a x Frantoio 4.2 10.8 a nd 4.3 b nd 7.16 a x Leccino 1.2 10.1 a 8.2 b 5.1 b 5.0 a 6.58 a x Leccino 4.2 9.7 a 11.5 a 2.6 a 3.9 b 5.57 a x Spanish Origin Arbequina 1.2 6.2 a 15.0 a 3.7 a 2.6 b 4.68 a x Arbequina 4.2 7.4 a 16.6 a 3.2 a 4.5 a 5.25 a x Picual 1.2 4.0 a 8.6 a 3.6 b 4.7 a 5.16 a x Picual 4.2 4.1 a 8.0 b 4.3 a 3.3 b 6.72 a x Picudo 1.2 4.3 a 13.5 a 3.4 a nd 5.93 a x Picudo 4.2 3.5 a 12.0 a 3.2 a nd 4.13 a x Greek Origin Kalamata 1.2 nd 9.7 a 4.3 a 2.3 b 4.67 a x Kalamata 4.2 nd 5.7 b 3.1 b 7.2 a 5.51 a x Koroneiki 1.2 14.4 a 17.1 a 4.5 a nd 7.51 a x Koroneiki 4.2 16.0 a 15.0 b 4.9 a nd 9.75 a x French Origin Picholin 1.2 6.4 a nd 3.3 a nd 4.10 a x Picholin 4.2 7.5 a nd 3.5 a nd 4.54 a x Morocco Origin P. Morocco 1.2 nd 12.2 a 3.1 b nd 6.96 a x P. Morocco 4.2 nd 8.8 b 3.5 a nd 5.72 a x Tukey Kramer test (p=0.01) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letter a), and between all the tested varieties in both treatments (letter x). Levels not connected by same letter are significantly different. Nd: not available
B. Tukey Kramer analysis of variance olive oil peroxides concentration.
Source DF Significance Variety 11 NS Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; NS: non significant effect. 61 These alterations could be produced by hydrolysis with liberation of free acids, detectable like acidity, and enzymatic auto-oxidation resulting as peroxides. In the present study, in accordance with previous reports, Wiesman (2004) and Royo (2005) how didn’t found differences in peroxide value in oils obtained from olives irrigated with saline water, no significant differences were found in the average of peroxides
-1 value in the four studied years, and all the values were under 20 meq O 2 kg , which is the maximum accepted by the International Olive Oils Council (IOOC, 1995), (Table
3.2A). Clearly we can observe that salinity doesn’t affect the peroxide value of oils as showed in Table 3.2B.
Previous reports (Salvador et al., 2001; Stefanoudaki et al., 2001) dealing with different olive cultivars, irrigation regimens and environmental conditions, indicated that peroxides values was not affected by genetic or differences in the amount of supplied water to the trees. It is world wide accepted that peroxide values as free fatty acid level, are mainly affected by fruit ripening (Salvador et al., 2001), olive oil extraction methodology and oil mild practices (IOOC, 1995) (Figure 3.1). 62
A
BGU Extra Virgin olive Oil (b)
The olive oil mill (a)
Cake exit
Olive’s fruit receiving Olive fruit washer Crusher and Malaxer
Olive oil extraction process (c)
Oil exit Olive Oil receiving
Figure 3.1 The olive oil mill (a); real photographs of the used mill to oil extraction (b) and sequence of the extraction process (c). 63 3.4.3 Fatty Acid Composition.
Fatty acids are the fundamental and major component of olive oil; variations in fatty acid profile rates can influence the organoleptic characteristics, especially when these variations modify the unsaturated / saturated acid ratio, because oils with high content of saturated fatty acids are more viscous and persistent in the mucous of the oral cavity, giving the “fatty sensation” (Solinas, 1988).
According to the literature, salinity can affect the fatty acid composition (Zarrouk et al.,
1996) and also modify the unsaturated / saturated (uns/sat) ratio, decreasing significantly at moderate and high salinity levels, (Stefanoudaki, 2004; Wiesman et al.,
2004; Zarrouk et al., 1996); salinity can influence the oleic/linoleic ratio, decreasing by the increase of salt concentration present in the used irrigation water (Cresti et al., 1994;
Stefanoudaki, 2004).
In the present study no significant differences were observed in the oleic acid percentage (Table 3.3A), in agreement with Royo (2005) who analyzed oils from c.v
Arbequina irrigated with three different levels of salinity, and Bouaziz (1990), who didn’t found changes in fatty acid composition after 12 years of studies in oil obtained from trees irrigated with brackish water. Oleic acid, the main fatty acid present in olive oil, was found in the same rate in both treatments without significant differences as can be seen for the others fatty acids. As can be seen in Figure 3.2 fatty acid profiles of the main acids present in olive oil (Palmitic, Stearic, Oleic and Linoleic) of six different varieties (Barnea, Picual, Leccino, Koroneiki, Picholin and P. Morocco) were not modified in their sequence by the fact that the trees are irrigated with saline water.
64 Uns/sat ratios were found no uniform along the studied oils; some of them were smaller in comparison to the control (Maalot and Picudo) in oils obtained from trees irrigated with saline water, in good agreement with previous reports (Stefanoudaki, 2004;
Wiesman et al., 2004; Zarrouk et al., 1996). Some ratios were almost the same in both treatment (Souri, Kalamata, Koroneiki, Picholin and P. Morocco) and finally in Barnea,
Maalot, Arbequina, Frantoio, and Leccino the uns/sat ratio was bigger in oils obtained from olives irrigated with saline water in contradiction with the literature (Stefanoudaki,
2004; Wiesman et al., 2004; Zarrouk et al., 1996; Bouaziz, 1990).
65
Table 3.3 A. Comparison of fatty acid composition of oils obtained from all tested olive varieties irrigated whit saline and fresh water. Fatty Acid Profile (%) Salinity Variety Palmitic Stearic Oleic Linoleic Saturated Unsaturated Unsat /Sat * (dS m -1) Others 16:0 18:0 18:1 18:2 Israeli Origin Barnea 1.2 13.18 a 2.66 a 68.00 a x y 14.52 a 0.75 a 15.84 82.52 5.20 Barnea 4.2 14.52 a 2.79 a 65.30 a x y 16.30 a 0.91 a 17.31 81.60 4.71 Souri 1.2 17.12 a 2.06 a 63.74 a x y 17.44 a 0.75 a 19.18 81.18 4.23 Souri 4.2 16.49 a 2.32 a 63.00 a x y 17.47 a 0.75 a 18.81 80.47 4.27 Maalot 1.2 16.44 a 2.96 a 62.20 a x y 17.65 a 1.87 a 19.40 79.85 4.33 Maalot 4.2 15.78 a 1.85 a 60.00 a x y 19.00 a 2.90 a 17.63 79.00 4.50 Italian Origin Frantoio 1.2 18.90 a 3.07 a 55.42 a y 18.64 a 3.00 a 21.97 74.06 3.37 Frantoio 4.2 19.32 a 1.89 a 59.22 a x y 16.94 a 1.80 a 21.21 76.16 3.60 Leccino 1.2 16.22 a 3.38 a 65.14 a x y 14.00 a 1.93 a 19.60 79.14 4.03 Leccino 4.2 16.20 a 1.01 a 65.62 a x y 14.06 a 1.33 a 17.21 79.68 4.62 Spanish Origin Arbequina 1.2 19.10 a 1.32 a 55.54 a y 20.67 a 3.43 b 20.42 76.21 3.73 Arbequina 4.2 20.17 a 1.70 a 54.10 a y 19.61 a 4.57 a 21.87 73.71 3.37 Picual 1.2 10.01 a 1.84 a 70.17 a x 10.45 a 3.64 a 11.85 80.62 6.80 Picual 4.2 15.35 a 3.18 a 70.55 a x 8.92 a 1.27 a 18.53 79.47 4.28 Picudo 1.2 18.16 a 1.00 a 64.23 a x y 14.84 a 0.75 a 19.16 79.07 4.12 Picudo 4.2 15.47 a 1.31 a 58.49 a x y 21.78 a 2.35 a 16.78 80.27 4.78 Greek Origin Kalamata 1.2 12.90 a 1.27 a 68.20 a x y 17.30 a 1.23 a 14.17 85.50 6.03 Kalamata 4.2 12.73 a 1.27 a 68.91 a x y 15.34 a 0.75 a 14.00 84.25 6.01 Koroneiki 1.2 15.17 a 2.20 a 67.95 a x y 10.73 a 4.00 a 17.37 78.68 4.52 Koroneiki 4.2 14.82 a 2.52 a 71.53 a x 10.12 a 1.11 b 17.34 81.65 4.70 French Origin Picholin 1.2 14.43 a 1.80 a 73.60 a x 7.05 a 2.26 a 16.23 80.65 4.96 Picholin 4.2 14.82 a 1.48 a 71.30 a x 9.04 a 2.60 a 16.30 80.34 4.92 Marocco Origin P. Marocco 1.2 11.94 a 3.70 a 65.90 a x y 16.83 a 0.90 a 15.64 82.73 5.38 P. Marocco 4.2 13.87 a 0.95 a 65.92 a x y 17.44 a 1.15 a 14.82 83.36 5.62 Tukey Kramer test (p=0.01) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b) in oleic acid (18:1), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different. * Unsat /Sat = ratio unsaturated/sutured. 66 B. Tukey Kramer analysis of variance of olive oil fatty acid composition.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant effect at p=0.01; NS: non significant effect
C. Grouping of olive varieties based on percentage of Oleic Acid (18:1) according to the response of the variety to saline water irrigation.
Group Range (% of oleic acid) Olive Varieties A 70 – 75 Picholine, Picual, Koroneiki Kalamata, Barnea, Leccino, P. Morocco, Picudo, Souri, Maalot, B 55 – 70 Frantoio C 50 - 55 Arbequina
All the above exposed can be attributed to genetic differences existing between the
analyzed varieties and no to the fact that the trees were irrigated with saline water
(Table 3.3B).
Based on oleic acid percentage in oils coming from olives irrigated with saline water
and according to three different ranges of oleic acid percentage, we can group the
twelve analyzed varieties as showed in Table 3.3C.
67 18:1 18:1 18:0 18:1 18:0 18:0 16:0 18:2 18:2 16:0 16:0 a 18:2 c e
A C E
b d f
B D F
(Min) (Min) (Min) 5 10 15 20 5 10 15 20 5 10 15 20
Figure 3.2: Olive oil GC fatty acid profile of six different varieties irrigated with fresh water (a-f) and saline water (A-F).Barnea (a/A), Picual (b/B), Leccino (c/C), Koroneiki (d/D), Picholin (e/E) and P. Morocco (f/F). 16:0: Palmitic acid; 18:0 Stearic acid; 18:1 Oleic acid; 18:2 Linoleic acid. 68 3.4.4 Polyphenols
Phenolic compounds present in olive oil affects its stability and contribute to oil flavour and aroma (Gutierrez Gonzales- Quijano, 1977; Vazquez Roncero 1978). The relationship between phenolic compounds and oils stability have been demonstrated
(Montedoro et al., 1992; Papadopoulos and Boskou 1991). Polyphenols also play an important role in human health since they have a wide range of biochemical and pharmaceutical effects, including anticarcinogenic, antiatherogenic, antimicrobial and antioxidant activities (Kohyama, 1997; Visioli and Galli, 1998).
Small number of works about the effect of salinity on the total polyphenols present in olive oil has been done, showing that total phenol concentration increased with high
NaCl levels supplied or present in the irrigation water (Stefanoudaki, 2004; Wiesman et al., 2004).
Olive oil polyphenol concentrations based on colorimetric assay (Gutfinger, 1981) are showed in Table 3.4A; in accordance with Royo (2005) Stefanoudaki (2004) and
Wiesman (2004), Frantoio, Leccino, Picudo, Koroneiki, Picholin and P. Morocco total polyphenol contents shows no significant differences in both treatments in the average of three studied years, but evidence an increase in there total polyphenol contents in the saline treatment (ranged this increase from 15 % to 40%); significant differences was found between the treatment only in oils obtained from Arbequina and again, with an increase in the saline treatment ( 102 ppm).
69 Table 3.4 A. Comparison of total polyphenols concentrations (ppm of caffeic acid) of oils obtained from all tested olive varieties irrigated whit saline and fresh water.
Polyphenols Concentration Salinity Variety (ppm of caffeic acid) Average (dS m -1) 2001 2002* 2003 Israeli Origin Barnea 1.2 142 a 39.5 a 173.0 a 139.80 a x Barnea 4.2 155 a 68.8 a 131.6 a 118.97 a x Souri 1.2 138 a 44.6 a 131.0 a 105.81 a x y Souri 4.2 165 a 45.1 a 72.6 b 73.63 a x y Maalot 1.2 nd 78.5 a 148.2 a 125.00 a x Maalot 4.2 nd 42.2 b 115.9 b 115.90 a x Italian Origin Frantoio 1.2 29 a 28.2 a nd 28.44 a y Frantoio 4.2 62 a 23.6 a nd 33.19 a y Leccino 1.2 171 a 39.4 a 122.9 a 107.37 a x y Leccino 4.2 699 b 39.6 a 154.6 a 170.02 a x Spanish Origin Arbequina 1.2 40 a 24.3 b 92.6 b 66.89 b y Arbequina 4.2 83 b 68.3 a 122.9 a 102.57 a x y Picual 1.2 150 a 38.7 a 115.1 a 100.2 0 a x y Picual 4.2 209 b 23.7 a 102.7 a 92.68 a x y Picudo 1.2 96 a 22.1 a 87.9 a 75.95 a x y Picudo 4.2 101 a 34.6 a 110.9 a 92.55 a x y Greek Origin Kalamata 1.2 nd 46.4 a 107.1 a 86.91 a x y Kalamata 4.2 nd 57.1 a 71.7 b 66.82 a y Koroneiki 1.2 120 a 33.6 141.2 a 106.85 a x y Koroneiki 4.2 143 a nd 144.2 a 144.06 a x French Origin Picholin 1.2 77 a 21.3 a nd 35.23 a y Picholin 4.2 81 a 42.8 a nd 52.40 a y Morocco Origin P. Morocco 1.2 nd 33.8 b 122.2 a 80.78 a x y P. Morocco 4.2 nd 67.4 a 131.7 a 94.31 a x y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different. * The oil in 2002 was over washed with water. Nd: not available.
B. Fisher analysis of variance of olive oil polyphenols content.
Source DF Significance Variety 11 *** Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; *** high significant effect at p=0.05; NS: non significant effect.
C. Grouping of olive varieties based on polyphenols concentrations according to the response of the variety to saline water irrigation.
Range (ppm of Group Olive Varieties polyphenol) A 150-190 Leccino B 90-150 Barnea, Maalot, Arbequina, Picual, Picudo, Koroneiki, P. Morocco C 30-90 Souri, Frantoio, Kalamata, Picholin 70 Uceda and Hermoso (1997) suggested that the antioxidant (including polyphenols) levels in olive oil depend to the degree of maturation of the olive fruits. Considering the fact that not all the variety fruits mature at the same time, and analyzing the total polyphenol concentration of the three studied year by Fisher analysis of variance its possible to conclude that the variety is the source of variations in the oil total polyphenol concentrations and no the salt supplied by the water irrigation or the interaction Varity/ Treatment (Table 3.4B).
In accordance with the total polyphenol concentrations in oils obtained from the saline treatment is possible to group the varieties as exposed in Table 3.4C.
3.4.5 Phenol Composition
The polyphenol olive oil composition is highly complex and includes a large number of phenols (Figure 3.3). Tyrosol, hydroxytyrosol, simple phenolic acids and esterified derivates of tyrosol and hydroxytyrosol are the most representative compounds
(Tsimidou, 1998). Evidences of the antioxidant proprieties of hydroxytyrosol are reported by Manna (1999) who demonstrated its antioxidant activities in different models. 71
Syringic Acid Caffeic Acid
Vanillic Acid Vanillin Cinnamic Acid Hydroxy Tyrosol Apigenin
Coumaric Galic Acid Tyrosol Luteolin
5 10 15 20 25 30 35
Min Figure 3.3 LC-MS Phenol Chromatogram mix standards.
The major shortcoming of the qualitative/quantitative evaluation of olive oil phenolic compounds lies in the currents luck of an appropriate methodology. At the present the most widely employed methods for evaluating the polyphenolic content of olive oil are the Folin-Ciocalteau colorimetric assay (Visioli et al., 1995) and high performance liquid chromatography (HPLC) (Montedoro et al., 1992). The former is simple and economical to perform but is limited by the low specificity of the reagent toward phenolic compounds; further, it does not provide qualitative information of singles phenolics.
Conversely, HPLC is very sensitive and specific, but it is time-consuming and does not provide information on phenolic molecules for which reference standards are unavailable (Visioli et al., 2002). In the last few years an increasing number of publications have contributed to the development of very effective Liquid 72 Chromatography Mass Spectra (LC/MS) techniques for the analysis of various classes of organic compounds. The main advantage over consolidated GC/MS alternatives is the possibility of injecting the extract directly into the system without further analyte derivatization, allowing for consistent time saving in routine analyses ( Cappiello et al.,
1994).
The olive oil phenol concentrations obtained by LC/MS technique of oils from different varieties are exposed in Table 3.5A. As could be seen, Hydroxytyrosol concentrations were significantly different in Barnea and Souri oils, with an important increase of this phenol in oils obtained from trees irrigated with saline water, 102 and 78 ppm respectively for each of them in the saline treatment. The same pattern was found in
Leccino (324 ppm), Picual (201 ppm), Picudo (44.2 ppm) and Koroneiki (786 ppm). No significant differences in Hydroxytyrosol concentrations between treatments were found in Arbequina and Kalamata oils.
Tyrosol concentrations were found no significant different between treatments in almost all the studied varieties, despite that some increase in tyrosol concentration was observed in oils obtained from the saline treatment, like Barnea (59 ppm), Leccino (131 ppm), and Kalamata (75 ppm). In Koroneiki and Picual, tyrosol concentration was significant different with bigger values in the saline treatment, 131 ppm more in
Koroneiki and 51 ppm in Picual.
Luteolin concentrations were not significantly different in the majority of the studied oils and, again, the saline treatment shows bigger values. Significant differences were found only in Barnea (40-253 ppm), Kalamata (14-29 ppm) and Koroneiki (8-32), where the values were bigger in the saline treatments. 73 Few ppm of Apigenin, Cinnamic acid, Cumaric acid, Vanillic acid and Caffeic acid were found in the studied oils; in the majority of the studied phenols, no significant differences were found between treatments, and again in some of them the saline treatment value was bigger, like Apigenin concentration in Leccino (15-20 ppm) and
Vanillic acid in Arbequina (44-46 ppm).
To the best of our knowledge no previous works were found in the literature about the effect of saline water irrigation on the polyphenols profile composition of olive oil, consequently we can not contrast trustworthily our results. Stefanoudaki, (2004) reported that separation of the phenolic compounds by HPLC analysis of the Koroneiki oil extract showed an increasing tendency for the second fraction of the secoiridoid derivatives with salinity levels. In addition Cimato (1990), reported that significant differences in the polyphenol composition were pointed out among analysis of samples of oils from different cultivars (‘Leccino’, ‘Coratina’, ‘Frantoio’), in three different harvesting periods and two production areas; particular observations of these components show that with ripening of fruits, an important hydrolysis of the components with higher molecular weight happens, with formation of tyrosol and hydroxytyrosol.
Tukey Kramer analysis of variance of the seven studied phenols are resumed in Table
3.5B; as can be seen , hydroxytyrosol, luteolin and vanillic acid the concentration of each of them was high affected by genetic; while saline water irrigation has a high affect in luteolin concentration, intermediate effect in hydroxytyrosol and low effect in
Vanillic acid. Finally the interaction between variety and NaCl present in the water irrigation has an intermediate effect in luteolin, and low effect in hydroxytyrosol and vanillic acid. 74 Tyrozol, apigenin and coumaric acid concentrations were intermediate affected by genetic; saline water irrigation in these phenols has a low effect in tyrozol and coumaric acid, but affect significantly the apigenin concentration. In the interaction
(variety*treatment), low significant effect were found in tyrozol and coumaric acid and intermediate in apigenin. Finally cinnamic acid was not affected by any of the two analyzed parameters and the interaction of them.
As discussed in 3.4.4 the total polyphenol concentration is not affected by saline irrigation, but may be an unknown mechanism of compensation taking place in the polyphenols profile, as exposed in Table 3.5B, after Tukey Kramer analysis of variance, saline irrigation has a significant influence in the phenol concentrations in all the studied phenols, altering the polyphenol profile but no the total concentration present in the oil (Table 3.4A)
75
Table 3.5 A. Effect of saline irrigation water on Phenol composition (ppm) in subjected to different varieties.
Phenol (ppm) Variety Salinity Hydroxytyrozol Tyrozol Luteolin Apigenin Cinnamic acid Cumaric acid Vanillic acid Caffeic acid (dS m -1) Israeli Origin Barnea 1.2 44.1 ± 2.9 b t 47.5 ± 3.4 a z 40.6 ± 8.6 b z 1.3 ± 0.7 b 20.3 ± 1.1 a x nd nd ± Barnea 4.2 102.0 ± 7.9 a s 59.0 ± 2.6 a z 253.0 ± 7.9 a x 17.7 54.6 ± 3.5 a x 20.6 ± 1.9 a x nd nd ± Souri 1.2 23.7 ± 4.0 b t 144.3 ± 9.0 a x ± 1.8 z 3.9 ± 0.5 t nd 45.0 ± 4.2 a x 11.3 ± 2.7 a z nd Souri 4.2 78.6 ± 7.8 a s 110.2 ± 7.4 b y nd nd nd 10.3 ± 2.2 b z 6.8 ± 0.9 a z nd Italian Origin Leccino 1.2 134.5 ± 17.2 b s 119.8 ± 13.1 a x 27.1 ± 5.5 a 15.2 ± 1.5 a z nd nd 11.4 ± 1.5 b z ± Leccino 4.2 342.9 ± 19.9 a z 131.3 ± 15.9 a x 29.9 ± 4.6 a 20.6 ± 1.7 a z 20.0 ± 2.3 x 0.4 ± 0.2 z 31.4 ± 3.7 a y 0.4 ± 0.1 x Spanish Origin Arbequina 1.2 213.0 ± 13.0 a r 49.4 ± 6.5 a z 108.3 ± 8.0 a y 17.6 ± 0.9 b z 20.7 ± 1.5 a x 14.3 ± 1.6 a y 44.8 ± 2.4 a x nd Arbequina 4.2 210.0 ± 5.5 a r 32.9 ± 6.3 a z 131.4 ± 7.9 a y 29.8 ± 3.8 a y 20.6 ± 2.1 a x 5.7 ± 2.0 b z 46.2 ± 1.7 a x nd Picual 1.2 83.1 ± 6.28 b s 100.0 ± 4.7 b y 29.7 ± 7.5 a z 4.4 ± 0.9 a t 20.1 ± 1.9 a x 24.1 ± 1.7 a y 29.7 ± 1.8 a y nd Picual 4.2 201.1 ± 9.23 a r 151.7 ± 12.1 a x 34.4 ± 7.1 a z 7.8 ± 1.0 a t 19.6 ± 1.9 a x 21.8 ± 1.7 a y 23.4 ± 1.8 a y 0.6 ± 0.2 a x Picudo 1.2 21.0 ± 4.9 b t 93.3 ± 10.5 a y 11.6 ± 0.9 a z 2.3 ± 0.7 b t nd 9.5 ± 1.5 b z 8.4 ± 1.3 a z nd Picudo 4.2 44.2 ± 5.1 a t 53.9 ± 8.7 b z 15.2 ± 2.0 a z 7.5 ± 0.8 a t nd 15.5 ± 0.7 a y 2.2 ± 0.7 b z 1.4 ± 0.4 x Greek Origin Kalamata 1.2 59.4 ± 5.0 a t 57.2 ± 6.1 a z 14.1 ± 1.7 b z 0.9 ± 0.2 t nd 8.2 ± 1.2 a z 29.6 ± 2.0 a y nd Kalamata 4.2 45.0 ± 5.5 a t 75. 0 ± 8.4 a z 29.6 ± 4.0 a z nd nd 3.4 ± 1.0 b z 21.6 ± 1.2 b z nd Koroneiki 1.2 632.3 ± 23.5 b y 30.3 ± 8.5 a z 8.4 ± 1.3 b z 4.2 ± 0.7 b t 20.0 ± 1.1 a x nd 22.3 ± 1.2 b y nd Koroneiki 4.2 786.2 ± 33.2 a x 161.7 ± 17.0 b x 32.1 ± 6. 1 a z 9.5 ± 1.0 a t 20.9 ± 2.0 a x nd 30.7 ± 2.0 a y nd Tukey Kramer test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters s, t, x and z). Levels not connected by same letter are significantly different Levels of each column not connected by same letters are significantly different (Tukey Kramer statistic analysis P ≥ 0.05. Values are the mean of three ± SE. Nd: no available.
76
B. Tukey Kramer analysis according to each of the analyzed phenol concentration subjected to different varieties.
Phenol Source Hydroxytyrozol Tyrozol Luteolin Apigenin Cinnamic acid Cumaric acid Vanillic acid DF Significance DF Significance DF Significance DF Significance DF Significance DF Significance DF Significance Variety 7 *** 7 ** 6 *** 5 ** 3 NS 3 ** 6 *** Treatment 1 ** 1 * 1 *** 1 *** 1 NS 1 * 1 * Variety*Treatment 7 * 7 * 6 ** 5 ** 3 NS 3 * 6 * DF: degree of freedom; *** high significant effect; ** intermediate significant effect; * low significant effect. 77 3.3.7 Vitamin E
Four tocopherols are present at different concentrations and ratios in olive oil: α, β, γ and δ; tocopherols are methyl-substituted chromanols with a three-isoprene moiety side chain (Psomiadou et al., 2000). Studies of α –tocopherol, the most important tocopherol present in olive oil, concentrations and levels has been carried out mainly for Italian
(Ranalli and Angerosa, 1996; Conte et al., 1993; Fedeli and Cortesi, 1993;) olive oils and, to a lesser extent, for olive oils from other producing countries (Salvador et al.,
1998; Cert et al., 1996;).
There are a large number of publications on the antioxidant effect of α-tocopherol on lipids (Pongracz et al., 1995; Cort, 1974). This activity is dependent on both concentration (Cillard el al., 1980) and temperature (Lee and Montag, 1992; Marinnova and Yanishlieva; 1992). In addition Blekas and Boskou (1998) has been demonstrated the antioxidant activity of alpha tocopherol at 100, 500 and 1000 ppm, pointed out that the lowest concentration was the most effective.
As well reported in previous studies different stresses situation can affect the tocopherol concentration such as saline irrigation, which can increase or modify the content of aliphatic or triterpenic alcohol and volatile compounds, produced through the lipoxygenase pathway, due to the stress caused to the cells by NaCl and/or environmental changes (Cresti et al., 1994), in addition Maranz and Wiesman (2004) have demonstrated that α-tocopherol concentration can be modify by drought and temperature in shea butter ( Vitellaria paradox a G.). Shea butters obtained from the hot and dry climates of Africa had the highest levels of -tocopherol, in contraposition with the lowest concentrations of -tocopherol found in samples from cool highland areas in the same continent. 78 Alpha tocopherol average concentrations of four years study are showed in Table 3.6A.
Table 3.6 A. Comparison of Vitamin E concentration (µg/g oil) of oils obtained from all tested olive varieties irrigated whit saline and fresh water.
Salinity Alpha Tocopherol concentration (µg/g) Variety (dS m -1) Average 2001-2005 Israeli Origin Barnea 1.2 57.72 a x y Barnea 4.2 64.92 a x y Souri 1.2 27.34 a y Souri 4.2 28.83 a y Maalot 1.2 16.86 a y Maalot 4.2 16.84 a y Italian Origin Frantoio 1.2 19.63 a y Frantoio 4.2 18.79 a y Leccino 1.2 114.33 a x Leccino 4.2 179.39 a x Spanish Origin Arbequina 1.2 10.63 a x y Arbequina 4.2 60.08 a x y Picual 1.2 53.87 a x y Picual 4.2 58.69 a x y Picudo 1.2 19.39 a y Picudo 4.2 27.10 a y Greek Origin Kalamata 1.2 17.87 b x y Kalamata 4.2 50.11 a x y Koroneiki 1.2 40.74 a y Koroneiki 4.2 35.10 a y French Origin Picholin 1.2 77.13 a x Picholin 4.2 233.46 a x Morocco Origin P. Marocco 1.2 19.18 a y P. Marocco 4.2 21.24 a y Fisher test (p=0.05) was done between two saline levels treatments (1.2 dS m-1 and 4.2 dS m-1) (letters a and b), and between all the tested varieties in both treatments (letters x and y). Levels not connected by same letter are significantly different.
B. Fisher analysis of variance of olive oil Vitamin E concentration.
Source DF Significance Variety 11 * Treatment 1 NS Variety*Treatment 11 NS DF: degree of freedom; * low significant effect at p=0.05; NS: non significant effect.
79 C. Grouping of olive varieties based on Alpha Tocopherol concentration according to the response of the variety to saline water irrigation.
Group Range (Vit. E µg/g oil) Olive Varieties A 160-240 Picholine, Leccino B 50-160 Barnea, Arbequina, Picual, Kalamata C 10-50 Souri, Maalot, Frantoio, Picudo, Koroneiki, P. Morocco
In the present study significant differences were found in oils obtained from Barnea,
Leccino and Kalamata; in all the cases the saline treatment shows higher α-tocopherols concentrations: 65 µg/g in Barnea, 179 µg/g in Leccino and 50 µg/g in Kalamata, this result are in good agreement with Wiesman (2004), who reported significant differences of Vitamin E concentrations in oils obtained from trees irrigated with two saline levels water. In brief and as showed in Table 3.6B differences in alpha tocopherol concentrations between the varieties are given by genetics differences and not by the fact that the trees are irrigated with saline water.
In accordance with the response to the variety to saline water irrigation and its alpha tocopherol concentration in the saline treatment we can group the varieties as showed in
Table 3.6C.
3.4.8 Sterol concentrations
Plant sterols, or phytosterols, belong to the group of the desmethylsterols steroid alcohols present in all living organisms (Rivera et al., 2004).
It has been proposed that sterol profiles can be used to classify virgin olive oils according to their fruit variety (Bucci et al., 2002; Ranalli et al., 2002; Aparicio et al.,
1997). Sterol profiles are also used to characterize virgin olive oils and especially to detect the adulteration of olive oil, especially with hazelnut oil (Mariani et al., 1999,
Vichi et al., 2001). Since the sterol profile differs from one variety to another, these 80 characteristics are known to be affected by many factors. Many studies were done aimed to elucidate the agronomic (climate, soil, water), geographic (altitude, longitude), harvesting (cultivar, ripeness), technological (conservation of the fruit or of the oil, extraction systems), and processing (refining, solvent extraction) effect in the sterol olive oil profile (Rivera et al., 2004). Phytosterols also, apparently help to reduce the total plasma and LDL-cholesterol, and as a result these compounds are being considered as ingredients of functional foods (Ostlund et al, 2002). Gas Chromatography separation of common olive oil phytosterols is demonstrated in Figure 3.4; the analysis carried out based on the standard methodology suggested by the IOOC (IOOC, 1995). In our study, minimum and maximum sterol values of oils from four different varieties irrigated with fresh water (1.2 dS m-1) and moderate saline water (4.2 dS m-1) are exposed in Table
3.7. Table 3.8 shows the minimum and maximum sterol values of oil obtained from olives c.v Barnea, irrigated with three different EC in the drip water irrigation (1.2 dS m-1, 4.2 dS m-1 and 7.5 dS m-1).
c e i
n
j
m h k
l f a g d
b
5 10 15 20 25 30 Min
Figure 3.4 GC Chromatogram of sterol mix standards. a Cholesterol, b Brassicasterol, c Campesterol, d Campestanol, e Stigmasterol, f 7 Campesterol, g 5.23 Sigmastadienol, h Cloresterol, i Sitostanol, j b- sitosterol, k Avenasterol, l 5,24 sigmastadienol, m 7- stigmastenol, n 7- avestanol.
81 All the five analysed oils contain more than 1000 µg/kg of total sterols, the minimum value established by EU Regulation for olive oil (EC Regulation 2568, 1991).
As showed in Table 3.7, in some of the studied oils extracted from olives irrigated with saline water, the total phytosterol value was smaller than the control, like Koroneiki
(3813 µg/kg) and Arbequina (2752 µg/kg). These results disagree with Royo (2005) who found an increase, but no significant different, in the total sterol content in oil obtained from Arbequina irrigated with moderate saline water (5.5 dS m-1).
Total sterol concentration was higher in oils obtained from c.v Picual trees irrigated with saline water (3892 against 3200 in the control), while in oil obtained from the saline treatment of Leccino shows the same patter, an increase in their total phytosterol concentration (1624 – 3197 µg/kg), in good agreement with Royo (2005).
In oil obtained from c.v Barnea trees, the total phytosterol value increased by the increasing of salt concentration supplied with the irrigation water, 1791, 1810 and
2243µg/kg for each of mentioned above saline water irrigation levels, this results, as mentioned for cv Leccino and Picual, are in good agreement with Royo (2005).
Rivera del Alamo, et al., (2003) reported an increase in the campesterol concentration after four olive crop season, this also sterol could have major differentiating power as it is insensitive to variations in factors such as hydric stress (Stefanoudaki et al., 2001) and geographical location (Gracia, 2001; Christopoulou et al., 1996; Duarte and
Martins,1976).
82 In our results, campesterol concentrations were higher in oils from c.v Picual and
Leccino irrigated with 4.2 dS m-1 EC saline water (Table 3.7) and Barnea irrigated with
7.5 dS m-1 (Table 3.8); while in Arbequina and Koroneiki (Table 3.7) the concentration of campesterol in the control was higher compared to the saline treatment; confirming that genetics variations alter the phytosterol profile (Bucci et al., 2002; Ranalli et al.,
2002; and Aparicio et al., 1997) and probably also the salt present in the irrigation water.
Stigmasterol is related to various parameters of the quality of virgin olive oil, high levels correlate with high acidity and low organoleptic quality (Gracia, 2001; Gutierrez et al., 1999). Only in oils obtained from c.v Arbequina (irrigated with moderate saline water) and c.v Barnea (irrigated with 7.5 dS m-1 saline water) stigmasterol was bigger in comparison with the control.
83
Table 3.7 Effect of moderate saline water irrigation on olive oil sterol profile concentration (µg/kg oil) in different olive varieties.
Arbequina Picual Leccino Koroneiki Salinity Level 1.2 dSm -1 4.2 dSm -1 1.2 dSm -1 4.2 dSm -1 1.2 dSm -1 4.2 dSm -1 1.2 dSm -1 4.2 dSm -1 Sterol Min Max Min Max Min Max Min Max Min Max Min Max Min Max Min Max Cholesterol 0.16 1.39 1.50 1.82 0.35 1.20 0.80 1.94 1.15 1.66 0.70 1.28 0.56 0.82 0.66 0.75 Brassicasterol 0.09 0.18 0.04 0.11 0.11 0.56 0.08 0.23 0.11 0.19 0.31 0.81 0.27 0.50 0.12 0.53 24- methylene - 0.56 3.22 0.54 2.17 1.61 4.73 0.75 1.33 1.30 4.20 1.40 4.62 0.70 6.95 0.80 4.98 cholesterol Campesterol 12.90 39.14 10.69 25.10 5.04 22.4 12.35 35.20 13.32 26.91 16.68 37.30 15.20 18.35 10.24 14.30 Campestanol 0.09 0.38 1.10 0.30 1.20 3.51 2.00 4.10 1.10 1.46 0.33 0.90 0.85 1.43 2.60 3.75 Stigmasterol 2.42 12.30 2.46 14.70 10.30 18.34 0.52 11.40 1.35 17.40 0.89 11.80 4.1 17.80 1.08 12.50 Delta-7-campesterol 0.36 0.59 nd nd 0.57 2.40 0.61 0.91 0.35 0.88 0.53 1.47 0.85 1.45 0.73 1.20 Delta-5,23- 0.21 1.36 0.42 1.01 0.37 2.35 0.52 2.45 0.18 0.30 0.09 0.25 2.17 2.67 1.17 1.93 sigmastadienol Clerosterol 6.10 40.50 14.10 43.20 6.25 10.20 10.04 14.65 2.18 14.60 3.03 15.00 8.78 14.81 3.05 14.70 Beta-sitosterol 1289.0 3190.0 1470.0 2461.8 1780.0 3046.2 1923.03 3705.6 1128.0 1334.3 1217.0 1697.3 2250.0 4351.2 2373.8 3690.00 Sitostanol 2.41 3.80 3.44 5.14 3.48 5.38 0.57 2.75 4.74 6.91 2.22 4.80 0.49 3.21 2.67 7.59 Delta-5-avenasterol 21.70 94.40 75.20 182.00 3.19 49.70 8.70 97.33 77.70 196.40 58.70 161.50 20.90 84.60 2.19 43.20 Delta-5,25- 2.35 8.29 2.33 7.90 1.98 7.21 1.85 6.52 4.02 10.60 1.29 8.70 7.5 14.25 1.41 9.71 sigmastadienol Delta-7-stigmastenol 2.12 3.19 1.54 2.85 1.10 3.40 1.11 3.95 1.76 3.77 0.76 3.24 0.74 2.23 0.90 3.40 Delta-7-avenasterol 3.42 4.05 2.11 4.00 1.75 4.26 1.44 3.70 2.69 5.39 0.93 5.23 0.49 3.36 1.30 5.29 Total Sterol 1343.89 3402.70 1585.47 2752.10 1817.30 3200.18 1964.36 3892.03 1239.95 1624.97 1305.56 1954.20 2313.60 4523.63 2402.72 3813.83 *Min: Minimum; Max: Maximum; nd= no available. 84 Table 3.8 Effect of moderate saline water irrigation on Barnea olive oil sterols concentration (µg/kg oil) oil at three different saline levels.
Salinity level Sterol 1.2 dS m -1 4.2 dS m -1 7.5 dS m -1 Min* Max* Min Max Min Max Cholesterol 0.35 0.87 0.26 0.95 0.18 0.99 Brassicasterol 0.16 0.20 0.10 0.35 0.15 0.46 24- methylene - cholesterol 1.29 1.35 0.30 1.20 0.41 0.42 Campesterol 9.63 75.03 3.12 70.25 7.01 97.51 Campestanol 0.30 0.31 0.13 0.68 1.02 0.17 Stigmasterol 5.10 16.65 2.50 11.34 2.67 19.86 Delta-7-campesterol 0.20 1.84 0.10 4.46 0.92 5.15 Delta-5,23-sigmastadienol 0.10 1.97 0.10 4.75 0.22 3.34 Clerosterol 16.16 30.40 11.95 13.00 18.92 76.60 Beta-sitosterol 1392.32 1791.20 1318.99 1810.00 1898.55 2243.8 Sitostanol 3.19 6.59 1.08 4.81 3.57 5.23 Delta-5-avenasterol 57.00 72.24 21.12 41.88 25.40 80.83 Delta-5,25-sigmastadienol 0.95 2.34 0.41 4.13 0.96 3.46 Delta-7-stigmastenol 0.55 8.57 0.23 2.88 0.90 16.59 Delta-7-avenasterol 0.63 6.05 0.31 6.35 0.93 10.18 Total 1487.93 2015.61 1360.7 1977.03 1961.81 2564.59 *Min: Minimum; Max: Maximum.
Due to the lack of data in the literature about the effect of salinity in olive oil phytosterols, all our results were discussed and compared in light of previous reports dealing with the influence of different irrigation regimens (Stefanoudaki et al., 2001), location (Gracia, 2001; Christopoulou et al., 1996; Duarte and Martins, 1976); or variety
(Bucci et al., 2002; Ranalli et al., 2002 and Aparicio et al., 1997) in the total concentration and profile phytosterol. Outstanding the fact that in the present study trees were irrigated with saline water, the observed changes in the total phytosterol concentration and phytosterol profile could be also attributed to the presence of salt in the supplied drip water irrigation. Effects of salinity on olive oil quality are not being contrasted till today and only few studies have been published dealing in this field (Royo et al. 2005; Wiesman et al., 2004; Wiesman et al., 2002).
85 To summarise and in accordance with the obtained data concerning the olive oil quality of the eleven saline water irrigated cultivars, it is possible to conclude after four years of studies that the salt supplied to the olives in the irrigation water, does not affect the oil free fatty acid (Table 3.1A, B and C), peroxide value (Table 3.2A and B), fatty acid profile (Table 3.3A, B and C) and alpha tocopherol content (Table 6A, B and C). In the total polyphenol contents only Arbequina evidence a significant effect in the average of the four studied years by salinity; total polyphenol contents in trees irrigated with saline was higher than the control, while in the others cultivars no saline effect was observed
(Table 3.4A, B and C).
As can be observed in Tables 3.5A and 3.5B saline water irrigation and genetic variability, affects significantly the concentration in the most important polar phenolic compounds presents in the six analyzed oils. At the end, sterol concentrations (Table 3.7 and Table 3.8) were found variable along to the present study. 86 4. Olive c.v Barnea root development under saline water drip irrigation
4.1 Introduction
Plant roots serve a multitude of functions; they anchor and supply plants with water and nutrients and exchange various growth substances with the shoots. At the root-soil interface, numerous interactions between plants and their environment take place. The diversity of functions and broad range of interactions with the environment render the biology of roots complicated.
Roots are a major sink for photoassimilates and their primary task is water and nutrient uptakes. Consequently, variations in root system dimensions lead to variations in the efficiency of water and nutrient uptake.
Roots mediate between certain external growth factors and the plant as a whole.
Considerable variation exists in the efficiency of different root plant systems. Extensive root systems are not a prerequisite for maximum plant production if water and nutrients are supplied with out limitations (Willigen and van Noordwijk, 1987).
Exposure to NaCl may affect root metabolism by an osmotic effect, causing water deficit, or by a specific ion effect, causing excessive accumulation of Na + or C1 -, or inadequate uptake of essential nutrients (Munns and Termaat, 1986). Under saline conditions, it is important to maintain and/or improve soil water availability to crops (Van Hoorn and Van
Alpen, 1990). This can be accomplished through several strategies such as leaching of salts from the soil profile, maintaining high soil water content in the root zone, selecting more salt tolerant plants and improving crop systems (Meiri, 1984). 87 Root weight has been the root character most frequently studied as a response to salinity, but root distribution parameters such as volume, length, diameter, depth of penetration, degree of branching and numbers of root hairs have also been the subject of comparative studies. Variation among root systems has also been found for characters (i.e. root length, root length density, root surface area, root length index) other than those related to the quantity of roots (Glinski et al, l993; Cramer et a1, 1990; Waisel and Breckle, l987;
Mclntosh and Miller, l980; Schenk and Barber, l979; Gerard, l978; Bohm et al, 1977) .
Root growth distribution, using any character, may result from interaction of genetically and environmentally induced factors.
Many of the least destructive approaches to observing roots and soil organisms involve transparent materials placed in the soil. Once installed, transparent-wall techniques permit repeated, non-destructive observation of individual roots for growth, phenology and demography (Fahey et al., 1999). With the development of miniature video cameras, boroscopes and fiberscopes, minirhizotrons are becoming the most used method to study individual root demography both in pot studies and in the field (Johnson et al., 2001;
Joslin and Wolfe, 1999; Hendrick and Pregitzer, 1996; McMichael and Taylor, 1987;). As well demonstrated, minirhizotrons are used as a non-destructive tool for investigating root system dynamics and consists of a miniature video camera installed in clear tubes which have been installed into the soil. Root images are captured on video and then later analyzed with a root tracing computer program (Box, 1996; Hendrick and Pregitzer,
1996).
The principal assumption regarding the estimation of root growth dynamics with minirhizotrons is that the observed roots next to the tubes are behaving in a manner 88 similar to those in the bulk soil, but little research has been devoted to potential effects of the minirhizotron material on the data collected (Ephrath et al., 1999)
Although olive is a moderately salt tolerant plant, olive root development under saline condition has not been yet characterized. In this study, root growth of olive (cv. Barnea) was monitored using the Minirhizotron system.
89 4.2 Material and Methods
4.2.1 Field Experiment
In this experiment, root development of Barnea olive cultivar under saline drip irrigation water (4.2 dS m-1) and control (1.2 dS m-1) was studied. For measuring root development the minirhizotron system (BTC-2 model, Bartz Technology Corp., Santa Barbara, CA,
USA) was used.
Clear acrylate tubes (52 mm inner, and 60 mm outer diameter), were placed vertically in the soil at three distances: 50; 100 and 150 cm from the trunk (Figure 4.1). The tubes were inserted to a depth of 2 m.
The tubes were numbered in order to facilitate their identification according to their position and the treatment which it belong, as follow:
Tube 1: fresh water irrigation, 50 cm from the trunk, Tube 2: fresh water irrigation, 100 cm from the trunk, Tube 3: fresh water irrigation, 150 cm from the trunk, Tube 4: saline water irrigation, 50 cm from the trunk, Tube 5: saline water irrigation, 100 cm from the trunk, and Tube 6: saline water irrigation, 150 cm from the trunk
The tubes were placed vertically to ensure a good contact between the soil and the tube wall surface according to Johnson et al., 2001 and Ephrath et al., 1999. The tubes were installed on May 5, 2005 and the measurements started one month later, determined as the beginning of this part of the experiment. The observations were carried out once a month for a period of 13 months (412 days).
In order to correctly install the tubes two consideration were taken into account: (1) during the installation, the disturbance to the soil surrounding the tube was minimized and
(2), the soil was perforated with a spiral auger with a diameter somewhat smaller than the tube diameter in order to prevent air gaps between the tube wall and the soil. 90
50 cm
100 cm
150 cm
Figure 4.1 Picture showing the tube disposition in the Minirhizotron olive experiment.
Video images of pre-set positions were recorded at 12.5 mm intervals along the tubes with a marked rod. As the camera was aimed at the same position along the tube wall on successive measurements, changes in the root length and surface area were inferred by comparing homologous video frames in the laboratory using the Win Rhizotron 2003a,b,c software (Regent Instruments Inc, Canada).
Number of roots at each location was counted manually in the obtained video images.
Root length was measured using the “Straight lines” method, described by Win Rhizotron
2003a, b, c software manual: root length was measured by drawing in the image exactly as doing with a pencil tool paint programs. A path was started by clicking one start of the measured root (A-start, Fig 4.2). The cursor was moved along the central line of the root in the same direction of the root growth (dashed line in Fig 4.2). When the cursor was over to the opposite end of the root (B-end, Fig 4.2), the mouse button was released. This procedure allows the program to calculate the morphological measurements, length and surface area, of the path.
91
D C
A-start
B-end
A-1 B-1
Figure 4.2 “Straight lines” used method for tip number, length and diameter root determinations in the Minirhizotron olive experiment.
Due to the fact that roots are not homogeneous in their area, direction and shape, measurement of roots, using the “Straight lines” method need to be divided into short segments with different length and diameter, according to the variation in root morphology. The described procedure was done many times on the same root, following the root variations (diameter or orientation), as many “start” and “end” points were determinate (A-1 and B-1, Figure 4.2) in the same analyzed root. The final total length was calculated by the software adding the calculated sub-lengths (Figure 4.2). Surface area was calculated by the program using the measured root length, the diameter line (C-
D, Figure 4.2) perpendicular to the length line and with the assumption that root are circulars (Figure 4.2).
4.2.2 Soil salt concentration
Soil samples for electrical-conductivity (EC) measurement were taken three times during the experiment. Samples were taken at the same distance from the trunk as the tubes were placed. Samples were taken at (14-12-2005; 23-03-2006 and 17-07-2006) at four depths 92 (0-30 cm, 30-60 cm, 60-90 cm, 90-120 cm). Measurements were taken both at the control
(1.2 dS m-1) and at the saline (4.2 dS m-1) treatments.
The EC of the soil saturated extract was measured at the soil laboratory of the Gilat
Experimental Station. Electrical conductivity and chloride concentration were determined in saturated soil extract (Sparks, 1996). Sodium was measured using flame photometer
(Corning).
93 4.3 Results and Discussion
Studying plant root development under field conditions irrigated with saline water drip irrigation is rather difficult, which is reflected in the lack of information in the literature, especially in commercial orchards destined to fruit productions and it derivates. Olive root system development under saline water irrigation will be discussed in this chapter, based on four parameters: number of roots, root length, root diameter and root surface area.
Total root number of trees irrigated with fresh water (1.2 dS m-1) and saline water (4.2 dS m-1), at different depths in each of the three analyzed distances from the trunk are presented in Table 4.1. In all the three analyzed positions and four soil depths, total root number of trees irrigated with fresh water was almost three times higher than trees irrigated with saline water, 6556 compared to 2104 respectively. Similar to the present study, Mamo et al (1996) also found a reduction in the number of roots with increasing salinity in chickpea ( Cicer arietium L ) and lentil ( Lens culinaris Medic.). Sane et al.,
(2005) found the same pattern working in seedlings of date palm ( Phoenix dactilifera L.) cultivar Tijib under salinity and drought stresses.
In the upper layer (0-30 cm depth) of the soil of the tube close to the trunk (tube 1), the maximal number of roots was 235 after 56 days from tube installation, and decrease to 21 roots 412 days after installation, while in the saline treatment the number of roots remain almost constant throughout the experimental period. In the remaining soil depths in the position of tube 1, root number remained constant and higher compared to the same position in the saline treatment (Table 4.1). The number of roots measured throughout the experimental period at a distance of 100 cm from the trunk in the saline water irrigated treatment was constant both in time and in depth: 56 days after tube installation, the 94 number of roots at each of the measured depth remains almost constant with somewhat higher number of tips at the uppermost soil layer compared to the deeper soil layers. At the same location (100 cm from the trees) in the fresh water treatment, the number of roots counted in the 60-90 cm and 90-120 cm soil column layers was higher than at the two upper soil layers.
Table 4.1 Total root number at different depths in each of the three analyzed distances from the trunk (50 cm, 100 cm and 150 cm) of the two treatments: fresh water 1.2 dS m-1 (t) and saline water 4.2 dS m-1 (s) irrigation.
Distance Days Depth from the (cm) 30 56 82 156 213 304 365 412 trunk (cm) 0-30 21 235 168 45 19 34 27 21 30-60 8 179 165 270 96 146 107 110 50 (f) 60-90 3 150 136 276 178 241 169 169 90-120 1 139 128 147 143 153 140 133 0-30 11 11 14 18 14 11 20 20 30-60 35 44 48 45 41 38 44 45 50 (s) 60-90 23 28 28 19 19 25 30 25 90-120 15 17 15 17 18 17 17 21 0-30 9 9 15 10 11 8 10 6 30-60 0 31 31 9 32 28 27 23 100 (f) 60-90 0 37 221 211 238 164 180 166 90-120 1 3 8 226 279 168 161 183 0-30 35 53 67 42 50 39 53 40 30-60 14 33 31 23 24 26 30 29 100 (s) 60-90 5 11 24 37 29 39 37 36 90-120 23 35 35 34 29 32 33 34 0-30 0 0 0 0 0 1 1 0 30-60 0 0 0 0 0 17 16 15 150 (f) 60-90 0 0 0 0 0 14 19 5 90-120 0 0 0 0 0 3 11 0 0-30 2 7 8 6 4 14 7 5 30-60 12 17 18 11 13 7 18 15 150 (s) 60-90 3 2 6 7 7 10 6 4 90-120 0 4 6 2 6 10 3 3
Number of roots at a distance of 150 cm from the trunk in trees irrigated with saline water was small throughout the growing season. Unlike the saline treatment, in the fresh water irrigation treatment, the first roots were detected only 304 days after tube installation. 95 As is well know that roots posses a high degree of morphological plasticity for an exploitation of water and nutrients. This morphological plasticity comprises biomass partitioning between root and shoot and architecture of the roots system (Zang and
Forde 1998; Drew, 1973). There is evidence that soil salinity can induce modifications in morphology of the root system (Reinhardt and Rost 1995; Gersani et al., 1993 ; Wisel and Breckle 1987). Probably in response to saline water irrigation, olive roots explore the soil moisture at larger distance from the trunk where soluble salts are in lower concentrations so roots can grow (Figure 4.7). Different response was found in non- halophytes plants; Waisel and Breckle (1987) have reported that Raphanus sativus new lateral roots were found to be most salt tolerant process and growth of the lateral roots.
In G. hirsutum elongation of roots was more inhibited by salinity by their initiation and emergence (Reinhardt and Rost., 1995); while in the halophyte Plantago maritima
Rubbing (2004) found lateral root length variations in response to duration and soil concentration NaCl levels.
In the present study number of roots, root length, root diameter and root surface area, in both treatments at each of the measured dates are presented in Figs 4.3, 4.4 and 4.5
(saline water irrigation) and Figs 4.6, 4.7 and 4.8 ( fresh water irrigation). A
“compensation pattern”, between diameter, length and root number in response to saline water irrigation was observed in root system in the entire observed soil depths column at 100 cm from the trunk (tube 5) after 213 days (Figures 4.2 and 4.3).
96
a b c 180 160 180 160 160 140 ) 2 140 140 120 120 120 100 100 100 80 80 80 60 60 60 Number of roots 40 40 40 Total Surface area (cm Total Root Lenght (cm) 20 20 20 0 0 0 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 Days Days Days
180 d 160 e 180 f 160 160 140 ) 2 140 120 140 120 120 100 100 100 80 80 80 60 60 60
Number of Roots 40 40 40 Total Root Lenght (cm) 20 20 Total Surface area (cm 20 0 0 0 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 Days Days Days
Figure 4.3 Olive (cv Barnea) root development under saline water irrigation at 50 (a, b and c) and 150 (d, e and f) cm distance from the trunk; Total root number (a and d), Total root length (b and e) and Total root surface area (c and f). 97 An increase in root diameter and a parallel decrease in the root length (figure 4.2) as a response to saline water irrigation were found, while a parallel change in root number was not observed (Figure 4.3)
200 1.8 180 1.6 160 1.4 140 1.2 120 1 100 0.8 80 0.6 60 Total root lenght (cm) lenght root Total 40 Lenght 0.4
20 Diameter 0.2 Totalroot diameter (mm) 0 0 0 50 100 150 200 250 300 350 400 450
Days
Figure 4.4 Total root diameter (mm) and total root length (cm) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk.
180 180 160 160 140 140 120 120 100 100 80 80
60 60 40 40 TotalRoot Number Root Number 20 20 (cm2) area surface Totol Surface Area 0 0 0 50 100 150 200 250 300 350 400 450 Days
Figure 4.5 Total root number and total surface area (cm 2) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk.
98
500 2.0 450 1.8
400 1.6 350 1.4 300 1.2 250 1.0 200 0.8 150 0.6 100 Length 0.4
Totalroot lenght (cm) 50 Diameter 0.2 Totalroot diameter (mm) 0 0.0 0 50 100 150 200 250 300 350 400 450
Days
Figure 4.6 Total root diameter (mm) and total root length (cm) development under fresh water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk.
600 300
500 250
400 200
300 150
200 100 Root Number Total Root Number Total 100 Surface Area 50
(cm2) area surface root Total 0 0 0 50 100 150 200 250 300 350 400 450
Days
Figure 4.7 Total root number and surface area (cm 2) development under saline water irrigation of olive cv Barnea during the growing year at 100 cm distance from the trunk.
This compensation allows the tree to maintain an almost constant root surface area
(Figure 4.3) during the growing year. Neumann (1994) reported that salinity can modify root diameter in Zea mays . A decrease in root length caused by salinity was reported by
Hussain et al., (2002) in Barley. Other studies, however, report that in some halophytes 99 such as Suaeda maritima or Chloris gayana , salinity increased root length (Hajibagheri et al., 1985; Waisel, 1985).
Roots of different sizes or orders within broadly defined classes probably perform different functions (e.g., nutrient uptake, water uptake or transport to other tissues), which should result in different rates of metabolic activity. Similarly, roots of a given size might differ in function or degree of metabolic activity in deeper, nutrient and water absorption (Pregitzer et al., 1997).
This compensation pattern at a distance of 100 cm from the trunk in the saline water irrigated olive was observed in the soil zone where root number was 50% (1062) of the total roots observed in the treatment during the experiment (2104). Saline EC levels at the same soil zone were found between 7.05 and 3.92 dS m-1 in the lower soil layers
(Figure 4.7) at 23-06-06 after soil leaching (described in materials and methods, chapter
1). This range include the 6 ds/m -1, which is the limit where olive can grow and produce with no significant differences in comparison whit olives irrigated with fresh water, as demonstrated in chapter 2 (tables 2.3 to 2.9) and in chapter 3 (tables 3.1 to 3.8), in accordance with previous reports (Royo et al, 2005; Weisman et al., 2004). Trees irrigated with fresh water evidence a non uniform pattern in the four studied parameters
(figures 4.4 and 4.5), allowing the plant to waste the resources in root development instead of express all the genetic productivity potential. 100
800 300
700 a b ) c 700 2 250 600 600 200 500 500 400 400 150
300 300 100
Number of roots 200 200 50 100 100 Total Surface area (cm Total Root Lenght (cm) 0 0 0 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 Days Days Days
800 700 300
700 ) d 600 e 2 250 f 600 500 200 500 400 400 150 300 300 100 200 200
total Number of roots
Total Root Lenght (cm) 50 100 Total Surface area (cm 100 0 0 0 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 Days Days Days
Figure 4.8 Olive (cv Barnea) root development under fresh water irrigation at 50 (a, b and c) and 150 (d, e and f) cm distance from the trunk; Total root number (a and d), Total root length (b and e) and Total root surface area (c and f). 101
Tree
Hose Drip
Depth Salinity (cm) (ds/m -1) Salinity 14/12/05 23/03/06 17/07/06 (ds/m -1) 0-30 4.05 11.00 12.3 Salinity 14/12/05 23/03/06 17/07/06 (ds/m -1) 8.86 13.80 16.7 30-60 6.76 8.35 12 14/12/0523/03/06 17/07/06
9.46 16.80 13.1 8.59 10.60 11.7 60-90 6.26 7.80 7.1 7.69 9.25 9.05 4.68 8.40 9.9 90-120 3.43 6.50 7.15 6.35 8.60 7.7 4.13 6.35 7.65 Tube 1
5.45 4.82 5.1 Tube 2
Tube 3
Figure 4.9 Schematic diagrams showing the position of minirhizotron acrylic tubes in olive irrigated with fresh water (1.2 dS m-1) and soil salt concentrations at different depths (cm). 102
Tree Hose Drip
Depth Salinity (cm) (ds/m -1) 14/12/05 23/03/06 17/07/06 Salinity -1 0-30 16.18 5.70 19.4 (ds/m ) 14/12/05 23/03/06 17/07/06 Salinity (ds/m -1) 7.44 17.10 43.6 14/12/0523/03/06 17/07/06 30-60 19.35 6.55 22.2 16.61 37.00 19.6 12.45 7.05 26.2 60-90 13.1 4.92 17.2 12.92 15.80 17.1 9.64 5.75 19.5 90-120 10.7 4.37 9.9 10.43 8.90 14.5 Tube 1 13.41 3.92 11.5
10.28 7.95 11.6 Tube 2
Tube 3
Figure 4.10 Schematic diagrams showing the position of minirhizotron acrylic tubes in olive irrigated with saline water (4.2 dS m-1) and soil salt concentrations at different depths (cm). Summarizing, total root number in the saline treatment was considerably smaller in comparison with the control (Table 4.1). Total root length was shorter in trees irrigated with saline drip water irrigation. Root surface area was smaller in the saline water treatment compared to the fresh water treatment. Unlike root number and root length, root surface area was more o less constant throughout the measurement period in the saline treatment at 100 cm distance from the tree as a result of the interaction between root number, root length and root diameter (Figure 4.2 and Figure 4.3). Root distribution with depth and distance from the trunk varied between the two water quality treatments. This study suggests that salinity plays an important role on root distribution and development in olive trees. 104 5. Conclusions
Although olive tree cultivation is expanding to many parts of the world during the last decades, its main production is concentrated in the Mediterranean region, where 97% of the world’s olive oil is produced. Irrigation of olives with saline water will inevitably increase in the future in the Mediterranean due to negative effects of population growth and climate change on the availability and quality of existing fresh water supplies.
As a consequence, the risk land salinization will exacerbate threading the agricultural production particularly in countries with a semi-arid or arid climate.
The salt tolerance on olive tree, like most glycophytes, is associated with the restriction of Na + and/or Cl - transport from the root to the shoot. This inclusion/exclusion trait for both Na + and C - is heritable (Sykes, 1992), suggesting that breeding and selection for
Na + and Cl - excluding genotypes will continue to be a potentially rewarding area of research.
One of the most important problems in irrigation under saline conditions involves determining of the irrigation scheduling which allows obtaining, on the one hand, good crop yields and, on the other hand, adequate leaching of the soil. Successful saline irrigation requires a new production functions that relates crop yield to water consumption with acceptable irrigation intervals for the various crops.
This study demonstrate that, leaching is the key by which soil salinity can be maintained at acceptable levels without undue damage to the olive trees; the irrigation system was cautiously adjusted in their practices in order to keep the Na + and Cl - levels in the root zone as low as possible.
104 105 According to the results obtained after four field studied years of the effect of moderate saline water, it would seem that olive can be cultivated and produce satisfactorily in arid areas like in the Ramat Negev environment for a sustainable and reasonable period of time. However this work leaves open the question about how many years this cultivation olive practices can be applied without environmental or soil damages.
105 106 6. References:
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116 אפיון זני זית חדשים (Olea europea L) להשקיה במים מליחים באזור רמת הנגב
סבסטיאן וייסביין אוניברסיטת בן - גוריון בנגב תשרי , תשס ז" ז"
חיבור זה מהווה חלק מהדרישות לקבלת תואר מוסמך בלימודי מדבר (.M.A. / M.Sc ) )
הזית ( . Olea europaea L ) הינו עץ ירוק עד , הנפוץ באגן הים התיכון ופריו משמש בתעשיית המזון
לייצור שמן איכותי ולתעשיית השימורים כפרי כבוש . ארצות אגן הים התיכון מפיקות -כ 95 % מיבול הזית
העולמי . -כ 90 % מהשטח הנטוע משמשים לייצור שמן זית . עץ הזית דורש מנות רקו משמעותי מ"ע, מ"ע,
להניב פרי באיכות טובה , אך העץ רגיש לטמפרטורות הנמוכות -מ c° 10 . עץ הזית נחשב כעמיד ליובש ,
ולכן מתאים לגידול בתנאי האקלים באגן הים התיכון . התרחבות שטחי הנטיעה כיום לאזורים מדבריים
מציבה שתי בעיות ואחת מהן היא שמאגר המים הגדול הזמין להשקיה הינו של מים מליחים . השוואת
עמידות למליחות של עץ הזית למינים אחרים , מראה כי הזית עמיד יותר מעצי הדר אך עמיד פחות מדקל
ותגובתו למליחות טרם אופיינה . על מנת לתמוך בהתפתחות תעשיית גידול עצי הזית באזור המדברי
בישראל ובאזורים אחרים בעולם , הוחל ב - 1997 במחקר שמטר תו לאפיין תגובת הזית לעקת מלח . .
בניסוי ניטעו שנים ברמת נגב עשר זני זיתים שונים שמקורם בישראל ובאגן הים התיכון . ניתנו שני שני
טיפולים של השקיה בטפטוף באחד הושקו העצים במים שפירים ( dS / m 1.2 ) כביקורת , ובשני במים
מליחים ברמת מליחות מתונה ( dS / m 4.2 ). פעמיים בשנה , לפני ואחרי החורף , נשטפה הקרקע למניעת
הצטברות מלחים . במסגרת ה מחקר הוערכו התפתחות וגטטיבית , רפרודוקטיבית ואיכות השמן . .
בשלוש השנים הראשונות בטיפול מליחות הקף הגזע היה קטן , באופן לא מובהק סטטיסטית בהשוואה
לעצים שהושקו במ ים שפירים . תוצאות אלו תואמות לדיווחים ממקורות אחרים . בשנתיים הבאות ( -2004
2005 ) , התבנית השתנתה ונראה שקצב התפתחות היקף גזעי העצים שהושקו במים מליחים היה מהיר יותר
מעצי ביקורת , אך לא נמצא הבדל סטטיסטי מובהק בין הטיפולים . במשקל הפרי של ה זני ם השונים , לא לא
נמצאה השפעה מובהקת לטיפול המליחות . בספרות מופיעים דיווחים סותרים שבהם נמצאה השפעה מליחות
על גדילת הפרי . תנודות כלליות בפוריות העצים נצפו בכל זני הזיתים , לאורך כל שנות המחקר . במשך
ארבע שנות המחקר לא נמצאה השפעה מובהקת של המליחות על היבול . ישנה הסכמה כללית בספרות , כי כי ,
קיימת ירידה משמעותית בתנובת עצי זית אשר גודלו תחת השקיה עם מים מליחים . נראה כי המידע אשר 118 הושג במחקר הנוכחי סותר הנחה .זו בדיקת אחוז ה שמן ה ממוצע במשך ארבע שנות הניסוי ( -2001 2004 )
מראה כי אין הבדל משמעותי בתכולת השמן בין הטיפולים . .
בהערכת איכות השמן בהת אם לספים אשר נקבעו על ידי International Olive Oil IOCC)
(Council( סף החומציות המרבי של שמן הזית -ל 0.8 % המבוטא כחומצה אולאית ) נמצא שממוצע
החומציות בארבע שנות המחקר היה קטן -מ 0.8 % ברוב הזנים ובכל הטיפולים . בהתאמה לתוצאות מחקרים
-1 קודמים , לא נמצאו הבדלים משמעו תיים בערך אפר וקסידים כאשר ברובם היה מתחת -ל meq O 2 kg 20 ,
כ שנקבע י"ע IOCC . חומצה אולאית , חומצת השומן העיקרי ת בשמן זית , נמצאה באותו שיעור בשני
הטיפולים ללא הבדל משמעותי . ניתוח הרכב חומצות השומן מראה לגבי שאר חומצות השומן העיקריות (
חומצה פלמיטית , סטיארית , אולאית ולינולאית ) שאין הבדל משמעותי בהרכב . לא נמצא הבדל משמעותי
בתכולה הכוללת של פוליפנולים בין הטיפולים אך נמצאה השפעה מובהקת על הרכב הפוליפנולים
העיקריי ם ל אינטראקציה בין הזנים והטיפולים שמקורה בשונות בין הזנים . בטיפולים בניהם . נמצא הבדל
בריכוז α- tocopherol בין הזנים השונים הנובע מהבדלים גנטיים ולא מהעובדה שהעצים הושקו במים
מליחים . בנוסף , נמצא כי היו הבדלים בריכוז הכולל של פיטוסטרולים ובהרכבם אשר עשוי לנבוע
מההשקיה במים מליחים . .
מספרם הכולל של השורשים נמדד , בעצים אשר הושקו עם מים רגילים היה גבוה כמעט פי שלושה מאשר
עצים שהושקו עם מים מליחים . נראה שקיים " מנגנון פיצוי " במערכת השורשים בין הקוטר לאורך ומספר
השורשים בתגובה להשקיה עם מים מליחים . פיצוי זה מאפשר לשורשים לשמור על שטח פנים כמעט קבוע
במשך שנת הגידול . .
השקיית עצי זית במים מליחים צפויה להתרחב בעתיד הקרוב , כתוצאה משינויים אקלימיים וירידה בזמינות
ואיכות של מי ההשקיה . כתוצאה מכך , אדמת הגידול תהפוך למליחה ותפגע בתוצרת , במיוחד במדינות עם עם
אקלים מדברי או מדברי למחצה . עמידות למלח נובעת ממניעת קליטה של יוני נתרן וכלוריד בצמח י"ע י"ע
הרחקתם במערכת השורשים . אחת הב עיות המרכזיות בהשקיה במים מליחים היא קביעת משטר השקיה
המאפשר מצד אחד תנובה נאותה , ומצד שני שטיפת קרקע יעילה המונעת הצטברות מלחים . השקיה מליחה
מוצלחת דורשת פונקצית ייצור חדשה המקשרת בין תנובת היבול לצריכת מים עם מרווחי השקיה מקובלים
לכל אחד מהגידולים השונים . מחקר זה מדגים כי שטיפת קרקע נכונה היא המפתח לשמירה על רמות
118 119 מליחות סבירות של הקרקע מבלי לגרום נזק לעצי הזית . מערכת ההשקיה הותאמה בזהירות על מנת לשמור
את רמות יוני הנתרן והכלוריד באזור מערכת השורשים נמוכה ככל האפשר . בהתאם לתו צאות שהתקבלו
מארבע שנות המחקר ש בחנו השפעת השקיה במים מליחים ברמה מתונה על עצי זית , נראה כי ניתן לגדלם
בהצלחה באזורים צחיחים כגון רמת נגב . למרות זאת , עבודה זו אינה עונה על השאלה , כמה שנים ניתן לגדל
עצים אלו ללא נזק לגידול , לסביבה ולקרקע.
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