625 Pise and Gaikwad.vp Acta Bot. Croat. 72 (2), 295–310, 2013 CODEN: ABCRA 25 ISSN 0365–0588 eISSN 1847-8476 DOI: 10.2478/v10184-012-0021-9 Interactions between leaf macronutrients, micronutri- ents and soil properties in pistachio (Pistacia vera L.) orchards PRODROMOS KOUKOULAKIS1, CHRISTOS CHATZISSAVVIDIS2*, ARISTOTELIS PAPADOPOULOS1, DIMITRIOS PONTIKIS3 1 National Agricultural Research Foundation, Soil Science Institute, 57001 Thermi, Greece 2 Democritus University of Thrace, Department of Agricultural Development, Pantazidou 193, Orestiada 68200, Greece 3 Agromichaniki Ltd, Anthilis 13, 35100 Lamia, Greece Abstract – The interactions between: (i) leaf dry matter macronutrietns, micronutrients and soil chemical properties, (ii) leaf macro- and micronutrients, (iii) soil macro- and micronutrients and (iv) soil chemical properties, and soil micro- and macronutrients in 50 pistachio orchards were investigated in leaves and soils by means of regression analysis. Most of the soils were deficient in plant-available P, Zn, Mn, Fe, and B, while they were ex- cessively supplied with Cu. Leaf analysis showed that most of the trees were sufficient in K, Mg, Mn and B, but deficient in N, P and Fe, and excessive in Zn and Cu. It was found that almost all the significant elemental interactions occurring in pistachio leaves or soils were synergistic, contributing considerable quantities of available nutrients and, therefore, improving the nutrient status of pistachio trees, and the level of soil fertility. On the other hand, the interactions between K and Mg in leaves, and between soil pH and leaf N or soil Fe, Mn and B, were antagonistic. It is suggested that these results must be taken into ac- count during fertilization of pistachio trees, in order to avoid nutritional disorders and to promote plant growth, productivity and nut quality. Keywords: Pistacia vera, plant nutrition, leaf, macronutrients, micronutrients, soil Introduction In Greece, pistachio (Pistacia vera L.) trees are cultivated on about 5,000 ha and pro- duce 9,000 tons of fruit (FAO 2008). It is an economically important species that produces nuts of high nutritional value. The proper nutrition of pistachio trees is a basic prerequisite for the production of nuts of excellent quality and high commercial value. The high produc- ACTA BOT. CROAT. 72 (2), 2013 295 * Corresponding author, e-mail: cchatz@agro.duth.gr Copyright® 2013 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. tivity of pistachio trees makes necessary, among other factors, knowledge of: a) the interac- tions between leaf dry matter (d.m.) nutrient content, and chemical soil characteristics and b) the interrelations among nutrient elements in leaves. The interactions between leaf macro- and micronutrients, also between leaf nutrients and the soil properties, as well as between soil macro- and micronutrients, and between nu- trients in soil and soil properties have been studied sporadically and in a very general way (BRADY and WEIL 2002). According to relatively recent work, there is a direct relationship between the plant nutrient content and soil properties (KIZILGOZ et al. 2001). Usually, the in- teractions between soil characteristics and nutrient elements contribute negatively to the available level of nutrients in pistachio, with serious consequences to nut yield and quality. The low pH is correlated positively with the availability of most micronutrients (e.g. Zn, Mn, Fe) and P (OLSEN 1972). A high CaCO3 concentration in combination with a highly al- kaline soil accentuates plant growth and yields, notwithstanding any reduction in nutrient availability (MENGEL and KIRKBY 1987). Organic matter in soil is related positively to an in- crease of nutrient availability due either to the favorable effect on decomposition of the rocks and minerals or to the increase of cation exchange capacity (CEC) (TISDALE et al. 1993, KOUKOULAKIS et al. 2000). Moreover, clay positively affects the increase of available cations (Ca++, Mg++, K+) (BRADY and WEIL 2002). The interactions between nutrients have been studied to a certain extent in several crops, but not in pistachios. There is a particular shortage of relevant data concerning the elemental contribution of these interactions in pistachio cultivation, at least under the conditions of Greece.. According to DIBB and THOMPSON (1985), the nutrient concentrations in plants depend on the rate of their absorption, translocation and accumulation, and during these processes the elemental interactions within the plants may play an important role (KALA- VROUZIOTIS and KOUKOULAKIS 2009a). The aim of this work was to investigate the elemental contribution of the interactions be- tween nutrients in leaves, and in soils, as well as between soil properties and nutrients, and their effect on soil fertility and on the nutrient status of pistachio trees. Materials and methods Planting material and sampling procedure Fifty productive pistachio (Pistacia vera cv. Aegenes) orchards were selected in various locations of the Fthiotida district (south Greece). The trees were grafted on the rootstock 'Tsikoudia' (Pistacia terebinthus cv. Tsikoudia), trained as a vase and they were mostly planted at a spacing of 6.5–7.0 m between and within rows. Standard commercial cultural practices were followed during the survey. Representative soil samples from a depth of 0 to 0.30 m were collected from 20 cores in each orchard. The sampling points (four cores per tree) were located 2 m from the trunk, along and between tree rows. At the end of July, ten mature leaves per tree were collected from the middle node of non-bearing 1-year-old shoots and from all around the periphery of the canopy. Soil analyses Each sample was air-dried, crushed, sieved through a 2-mm mesh sieve and tested for pH, free CaCO3 and organic matter (OM). The basic soil characteristics were determined 296 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. according to the accepted methods (JACKSON 1958). The clay content of the soil samples was determined using the Bouyoucos method (BOUJOUCOS 1962). Most soils of the orchards studied (92%) had a high clay content, which ranged between 44 and 74%. The remaining 8% of the soils contained clay 30–38%, and they are character- ized as sand-clay-loamy (SCL) and clay-loamy (CL) soils. As regards pH, 82% of the stud- ied soils may be characterized as moderately alkaline, with 74% of them presenting pH val- ues between 7.5 and 8.0. Interestingly, 79% of the studied orchards presented the optimum range of pH values (7.5–8.5) for pistachio tree growth. Seventy four percent of the soils had a high CaCO3 content (above 4%), while the organic matter content was low (below 1%) for 54% of the studied soils and medium (1–2%) for 38% of the studied soils. Moreover, soil electrical conductivity was low and ranged between 0.06 and 0.70 mS cm–1 with a mean value of 0.40 mS cm–1. The available soil nutrients were extracted by the following methods: P by Olsen’s pro- cedure, exchangeable K and Mg by ammonium acetate, B by hot water, and the micronutri- ents (Mn, Zn, Fe and Cu) by DTPA (Diethylenetriamine pentaacetic acid). Analytical deter- mination of the elements K, Mg, Fe, Mn, Zn and Cu in the soil samples was performed by atomic absorption spectroscopy (Perkin-Elmer 2340) using standard methods (CHAPMAN and PRATT 1961). Phosphorus was analyzed by the vanado-molybdo-phosphate yellow complex (CHAPMAN and PRATT 1961). Finally, B was determined by the Azomethine-H method (WOLF 1971). Leaf tissue analyses The leaf samples were washed twice with distilled water, air-dried at 85 oC for 48h (till constant weight was obtained) and ground in a mill to pass through a thirty-mesh screen. Tissue B extraction was made by dry ashing of a 0.5 g sample in a muffle furnace at 500 oC for 6 h. The ash was dissolved in 10 mL of 0.1 N hydrochloric acid (HCl) and B was deter- mined colorimetrically (420 nm) by the Azomethine-H method (WOLF 1971). The analyti- cal determination of N was performed by the Kjeldahl method (CHAPMAN and PRATT 1961). Phosphorus, K, Mg, Fe, Mn, Zn and Cu analyses were conducted by dry ashing of 0.5 g of dried tissue for 6h at 550 oC. Subsequently, the ash was dissolved in 3 mL of 6N HCl and the solution was diluted with deionised water to 50 mL final volume. Phosphorus concentration was determined by the vanado-molybdo-phosphate yellow complex method, while K, Mg, Fe, Mn, Zn and Cu were determined by atomic absorption spectroscopy (Perkin-Elmer 2340) using standard methods (CHAPMAN and PRATT 1961). Statistical analysis In each orchard, thirty uniform size pistachio trees were selected and they were sepa- rated in five blocks. Each replicate block consisted of six trees. Regression models were de- veloped for each interaction found among soil properties, i.e. pH, CaCO3, clay, organic mat- ter and leaf N, P, K, Mg, Zn, Fe, Mn, B and Cu concentrations, as well as among leaf and soil nutrients. The estimators in the models were tested by t-test and the overall regression model by F-test at a level of significance of 0.05 and 0.001. Moreover, regression coeffi- cients of the models were determined. All the regression models were obtained by using the statistical package of SPSS version 17. ACTA BOT. CROAT. 72 (2), 2013 297 NUTRIENT ELEMENTS IN PISTACHIO TREES In order to quantify the percent elemental contribution (PEC) of an interaction, the pro- cedure developed by KALAVROUZIOTIS et al. (2010), was modified with respect to the calcu- lation of PEC. More specifically, the quantification procedure and the modification men- tioned are explained below in four steps. 1) Regression analysis is run between the concentrations of elements of soil or of plant dry matter, as given by the available analytical experimental data, and the statistically significant regression equations are chosen. 2) The interactions corresponding to the statistically significant regression equations, are classified on the basis of the same dependent variable, i.e. x0*y, x1*y, x2*y etc., where x and y represent a different element, for example if x0 is P, x1 is Zn, x2 is Mn, and y Fe, then the interactions will be P-Fe, Zn-Fe, and Mn-Fe. As can be seen, the dependent variable is written on the right to avoid confusion. Obviously, each one of the elements studied can be a dependent or independent variable according to which one is to be contributed. The number of interactions with the same dependent vari- able is not constant, but it varies according to the interactive capacity of the elements that participate in the interactions, and their respective concentration levels. In calcu- lating the PEC, the mean value of the calculated dependent variables is found by di- viding their total sum by the number of interactions involved. 3) The statistically significant equations are solved for the maximum (xmax) and the minimum (xmin) values of the independent variable, as given by the analytical data. Thus, the calculated maximum and minimum values are the dependent variables ymaxcl and ymincl, respectively, are found, and the difference between them is deter- mined. 4) Also, the maximum analytical value of the dependent variable (ymaxan) and minimum values (yminan) are taken respectively from the analytical experimental data, and the PEC is calculated by means of the relation 1: PEC = (ymaxcl – ymincl) ´ 100/(ymaxan – yminan) (1) where ymaxcl represents the calculated maximum value of the dependent variable in mg kg–1, ymincl represents the calculated minimum value of the dependent variable in mg kg–1, ymaxan represents the maximum value of the dependent variable obtained from the set of the exist- ing analytical data of soil or plant tissue analysis (in mg kg–1), and yminan represents the mini- mum value of the dependent variable, obtained from the set of the existing analytical data of soil or plant tissue analysis (in mg kg–1). It is noted that the relation 1 can be used for the cal- culation of PEC for interactions occurring either in the soil or in the plant tissues. It should be mentioned at this point that the mathematical relation for the calculation of PEC, given in the publication of KALAVROUZIOTIS et al. (2010), was the following: PEC= (ymaxcl– ymincl) ´100/Emc (2) where ymaxcl, ymincl represent the maximum and minimum calculated values of the depend- ent variable in mg kg–1, and Emc represents the mean total plant dry matter or soil content of the element representing the dependent variable. As it can be seen, relation 2 did not take into account the observed actual difference (ymaxan – yminan), but only the difference between the calculated values of the dependent variables, and the mean dry matter concentration (Eec), of the dependent variable, giving not very satisfactory results. Therefore, after a de- 298 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. tailed consideration, we modified the calculation of PEC and replaced the above relation 2 with the relation 1, as mentioned above. Results Interactions between soil properties and leaf nutrient concentration Regression equations of the statistically significant interrelations between leaf nutrient concentration and soil properties (pH, organic matter (OM), CaCO3 (CC), and soil clay con- tent (C)), are given in table 1. The interactions pH ´ N (Fig. 1c) and CaCO3 ´ Cu (Fig. 1d) and C ´ Mn (Fig. 1e) are antagonistic, suggesting that the increase of pH or of CaCO3 or of C decreases the leaf N, Cu and Mn concentrations, respectively. On the other hand, the interac- tions OM ´ Zn (Fig. 1a), and OM ´ B (Fig. 1b) are synergistic contributing to pistachio leaf Zn and B, respectively. The results of the interaction quantification procedure, as expressed by the percent elemental contribution (PEC), are shown in table 2. The negative figures, be- ing the result of antagonistic interactions, reflect a decrease of the respective element in the soil or in the plant respectively. On the other hand, the positive figures show an increase of the nutrient level. It is noted that most of the figures are positive, a fact that emphasizes the synergistic nature of most of the interactions that occurred in the soil and in plants, underlining the importance of the elemental interactions. Interactions between leaf nutrients Seventeen interactions (regression equations) among pistachio leaf macro- and micro- nutrients were statistically significant out of a total 81 interactions (9 ´ 9) (Tab. 3). From these 17 interactions 12 were synergistic (S), meaning that they contributed to leaves the re- spective quantities of the interacting elements, one was antagonistic (A), and four synergis- tic-antagonistic (S-A). According to interactions between macro- and micronutrients occurring in pistachio leaves (Figs. 2 a–f), it is obvious that the elemental interactions among leaf nutrients are mainly described by quadratic, and secondarily by logarithmic regression equations. In ad- dition, concerning the above interactions, the respective PEC values are presented in table 2. ACTA BOT. CROAT. 72 (2), 2013 299 NUTRIENT ELEMENTS IN PISTACHIO TREES Tab. 1. Interactions between pistachio leaf nutrients and soil properties. No Interactions Regression equations R Sig. Type 1 pH ´ N N = 0.275 ´ (pH)2 – 3.969 ´ (pH) + 16.258 0.439 0.007 Aa 2 CC ´ Cu Cu = –0.048 ´ (CC)2 – 0.101 ´ (CC) + 5.73 0.356 0.041 A 3 OM ´ Zn Zn = –7.593 ´ (OM)2 + 21.67 ´ (OM) + 8.037 0.363 0.036 Sb 4 OM ´ B B = –8.287 ´ (OM)2 + 18.054 ´ (OM) + 78.905 0.341 0.055 S 5 C ´ Mn Mn = 0.037 ´ (C)2 – 4.501 ´ (C) + 162.202 0.350 0.046 A CC=CaCO3, OM=Organic matter, C= Clay a Antagonistic b Synergistic 300 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. 10 20 30 40 50 60 0 0.5 1 1.5 2 2.5 L e a f Z n ( µ g g - 1 ) Organic matter (%) (a) 60 80 100 120 140 0 0.5 1 1.5 2 2.5 L e a f B ( µ g g - 1 ) Organic matter (%) (b) 1.4 1.9 2.4 2.9 6.5 7 7.5 8 8.5 L e a f N ( % ) Soil pH (c) 0 20 40 60 80 0 5 10 15 20 25 L e a f C u ( µ g g - 1 ) Soil CaCO 3 (%) (d) 0 20 40 60 80 25 35 45 55 65 75 85 L e a f M n ( µ g g - 1 ) Soil clay (%) (e) Fig. 1. Interactions of pistachio leaf Zn, B, N, Cu and Mn concentrations with soil organic matter, organic matter, pH, CaCO3 and clay, respectively. Elemental contribution to soil by the interactions between soil nutrients, and soil properties According to regression equations of the interactions between soil nutrients and soil properties (Tab. 4) the interactions OM ´ K, C ´ K, C ´ Mg, CaCO3 ´ Zn and OM ´ Zn were synergistic while those of CaCO3 ´ Fe, pH ´ Fe, CaCO3 ´ Mn, pH ´ Mn, pH ´ B, CaCO3 ´ B ACTA BOT. CROAT. 72 (2), 2013 301 NUTRIENT ELEMENTS IN PISTACHIO TREES Tab. 2. Percent elemental contribution values by the interactions among i) soil properties and leaf nutrients, ii) leaf nutrients, iii) soil properties and soil nutrients, and iv) soil nutrients. Elements i ii iii iv N –7.45 41.80 0.0 0.0 P 0.0 0.0 0.0 15.21 K 0.0 –14.33 29.20 47.09 Mg B Fe Mn Zn Cu 0.0 5.18 0.0 –26.80 13.53 –8.16 12.90 22.73 4.64 3.14 20.07 23.07 –3.67 31.15 –31.97 –18.61 4.56 0.0 17.76 0.0 13.83 2.10 13.59 13.15 Tab. 3. Interactions among nutrients in pistachio leaves. No Interaction Regression equations R Sig. Type 1 K ´ N N = –0.37 ´ (K)2 + 1.046 ´ (K) + 6.169 0.350 0.046 Sa 2 Mg ´ N N = 0.173 ´ (Mg) + 1.717 0.286 0.044 S 3 Mn ´ N N = 8.11 ´ 10–5 ´ (Mn)2 + 0.009 ´ (Mn) + 1.645 0.371 0.031 (S-Ab) 4 Mg ´ K K = ln(Mg) ´ (–0.184) + 0.905 0.278 0.050 A 5 N ´ Mg Mg = ln(N) ´ (0.863) + 0.125 0.280 0.049 S 6 Zn ´ Fe Fe = 0.018 ´ (Zn)2 + 0.261 ´ (Zn) + 50.438 0.440 0.006 S 7 Mn ´ Fe Fe = 0.002 ´ (Mn)2 + 0.403 ´ (Mn) + 46.988 0.535 0.000 S 8 Mg ´ Zn Zn = –13.297 ´ (Mg)2 + 35.126 ´ (Mg) + 3.827 0.375 0.028 (S-A) 9 Fe ´ Zn Zn = 0.147 ´ (Fe) + 10.373 0.429 0.002 S 10 Mn ´ Zn Zn = 0.008 ´ (Mn)2 – 0.105 ´ (Mn) + 18.822 0.496 0.001 S 11 Cu ´ Zn Zn = –0.003 ´ (Cu)2 + 0.371 ´ (Cu) + 10.781 0.377 0.027 S 12 Fe ´ Mn Mn = –0.002 ´ (Fe)2 + 0.835 ´ (Fe) – 8.556 0.540 0.000 S 13 Zn ´ Mn Mn = 0.033 ´ (Zn)2 – 0.621 ´ (Zn) + 31.975 0.470 0.003 S 14 B ´ Mn Mn = –0.021 ´ (B)2 + 4.415 ´ (B) – 186.661 0.368 0.033 S 15 Mg ´ Cu Cu = –29.57 ´ (Mg)2 + 72.103 ´ (Mg) + 3.038 0.416 0.011 (S-A) 16 Zn ´ Cu Cu = –0.021 ´ (Zn)2 + 1.889 ´ (Zn) + 6.997 0.400 0.017 S 17 Mn ´ B B = –0.003 ´ (Mn)2 + 0.471 ´ (Mn) + 75.14 0.196 0.006 (S-A) a Synergistic b Antagonistic and C ´ B were antagonistic. Most of the interactions between soil properties and soil nutri- ents are described by quadratic regression equations. In order to have a more concrete idea about the elemental contribution to soil, by the in- teractions mentioned in Table 4, a quantification procedure was applied and the results are shown in table 2. 302 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. 0 20 40 60 80 100 120 140 60 70 80 90 100 110 L e a f M n ( m g g - 1 ) Leaf B ( mg g -1 ) (a) 0 20 40 60 80 100 120 140 0 50 100 150 L e a f M n ( m g g - 1 ) Leaf Fe ( mg g -1 ) (b) 0 40 80 120 160 0 40 80 120 L e a f F e ( m g g - 1 ) Leaf Mn ( mg g -1 ) (c) 0.5 1 1.5 2 0 0.5 1 1.5 2 2.5 L e a f K ( % ) Leaf Mg (%) (d) 1.4 1.6 1.8 2 2.2 2.4 0 0.5 1 1.5 2 2.5 L e a f N ( % ) Leaf Mg (%) (e) 1.4 1.6 1.8 2 2.2 2.4 0.5 1 1.5 2 L e a f N ( % ) Leaf K (%) (f) Fig. 2. Interactions among nutrients in pistachio leaves. Elemental contribution to soil by the interactions between soil nutrients Most of the statistically significant interactions between soil macro- and micronutrients (Tab. 5) were described by quadratic (Fig. 3 a, d, e, g, h) and some of them by logarithmic equations (Fig. 3 b, c, f). It can be seen that almost all of these interactions were synergistic with the exception of P ´ Zn (Fig. 3 d), which was 'synergistic-antagonistic' (S-A). There- fore, the elemental contribution of these interactions was positive, i.e. significant quantities ACTA BOT. CROAT. 72 (2), 2013 303 NUTRIENT ELEMENTS IN PISTACHIO TREES Tab. 4. Interactions among soil properties and nutrients occurring in pistachio orchard soils. No Interactions Regression equations R Sig. Type 1 OM ´ K K = –23.505 ´ (OM)2 + 195.03 ´ (OM) + 86.12 0.549 0.000 Sa 2 C ´ K K = In(C) ´ 410.1 – 1325.823 0.500 0.000 S 3 C ´ Mg Mg = 0.714(C)2 – 61.268(C) + 1759.68 0.432 0.007 S 4 CC ´ Fe Fe = 0.01 ´ (CC)2 – 0.365 ´ (CC) + 4.977 0.373 0.029 Ab 5 pH ´ Fe Fe = 6.616 ´ (pH)2 – 95.376 ´ (pH) + 371.1 0.653 0.000 A 6 CC ´ Mn Mn = –0.079 ´ (CC) + 2.219 0.286 0.044 A 7 pH ´ Mn Mn = ln(pH) ´ (–9.86) + 21.802 0.339 0.016 A 8 CC ´ Zn Zn = 0.023 ´ (CC)2 – 0.34 ´ (CC) + 2.932 0.359 0.039 S 9 OM ´ Zn Zn = 0.394 ´ (OM)2 + 2.04 ´ (OM) – 0.219 0.548 0.000 S 10 pH ´ B B = 0.066 ´ (pH)2 – 1.03 ´ (pH) + 4.47 0.447 0.005 A 11 CC ´ B B = –0.003 ´ (CC) + 0.480 0.398 0.004 A 12 C ´ B B = ln(C) ´ (–0.079) + 0.76 0.342 0.015 A C=Clay, CC=CaCO3, OM =Organic matter aSynergistic bAntagonistic Tab. 5. Interactions among soil nutrients occurring in pistachio orchards. No Interactions Regression equations R Sig. Type 1 Fe ´ P P = –0.204 ´ (Fe)2 + 3.579 ´ (Fe) + 10.076 0.363 0.036 Sa 2 P ´ K K = –0.315 ´ (P)2 + 20.165 ´ (P) + 33.757 0.525 0.001 S 3 Cu ´ K K = ln(Cu) ´ 67.828 + 214.352 0.657 0.000 S 4 P ´ Mg Mg = ln(P) ´ 114.341 + 212.76 0.274 0.054 S 5 Fe ´ Mn Mn = 0.03 ´ (Fe)2 – 0.108 ´ (Fe) + 1.511 0.529 0.000 S 6 P ´ Fe Fe = ln(P) ´ 1.105 + 0.038 0.303 0.033 S 7 P ´ Zn Zn = –0.009 ´ (P)2 + 0.434 ´ (P) – 1.505 0.386 0.023 (S-Ab) 8 K ´ Zn Zn = –1.81 ´ 10–5 ´ (K)2 + 0.021 ´ (K) – 1.255 0.461 0.004 S 9 Cu ´ Zn Zn = –0.01 ´ (Cu)2 + 0.509(Cu) + 0.811 0.481 0.002 S 10 K ´ Cu Cu = –0.0000472 ´ (K)2 + 0.043 ´ (K) – 2.944 0.465 0.003 S a Synergistic b Antagonistic of macro- and micronutrients have been contributed to soil. More specifically, in terms of various nutrients, the PEC values are presented in table 2. 304 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. 0 100 200 300 400 500 600 0 20 40 60 S o il K ( m g g - 1 ) Soil P ( mg g -1 ) 0 200 400 600 800 1000 1200 0 10 20 30 40 50 S o il M g ( m g g - 1 ) Soil P ( mg g -1 ) 0 5 10 15 0 10 20 30 40 50 S o il F e ( m g g - 1 ) Soil P ( mg g -1 ) 0 5 10 15 20 0 10 20 30 40 50 S o il Z n ( m g g - 1 ) Soil P ( mg g -1 ) 0 10 20 30 40 50 0 100 200 300 400 500 600 S o il P ( m g g - 1 ) Soil K (mg g -1 ) 0 100 200 300 400 500 600 700 0 10 20 30 40 S o il K ( m g g - 1 ) Soil Cu ( mg g -1 ) 0 5 10 15 20 0 500 S o il Z n ( m g g - 1 ) Soil K (mg g -1 ) 0 5 10 15 20 0 10 20 30 40 S o il Z n ( m g g - 1 ) Soil Cu ( mg g -1 ) (a) (b) (c) (d) (e) (f) (g) (h) Fig. 3. Interactions among soil available nutrients Discussion The critical ranges of soil nutrients are the following (in mg g–1): K 140–280, P 15–25, Ca 300–750, Mg 50–100, B 0.50–1.0, Fe 4–25, Mn 15–25, Zn 1.0–2.5 and Cu 0.9–1.5 (KOUKOULAKIS 1995). Regarding the previous values, most of the studied soils were de- ficient in P, Zn, Mn, Fe and B content (46%, 44%, 100%, 82% and 100%, respectively). On the other hand, the soils of 62% of the orchards were excessively supplied with available Cu. Interestingly, according to BASLAR et al. (1999) Pistacia terebinthus subsp. palaestina, a species related to the rootstock used in the present work, generally prefers neutral soils and thrives in the presence of variable CaCO3 and P concentrations. Therefore, lime and P con- tent in soil are not expected to be limiting factors for pistachio trees growth in the Fthiotida district. On the other hand, the recommended critical values for pistachio nutritional status (CRANE and MARANTO 1988, MILLS and BENTON JONES 1996) are the following: N 2.5–2.9%, P 0.14–0.17%, K 1.0–2.0%, Mg 0.6–1.2%, B 50–230 mg g–1, Fe 30–125 mg g–1, Mn 30–80 mg g–1, Zn 7–14 mg g–1and Cu 3–4 mg g–1. Based on the previous values, leaf analysis showed that K, Mg, Mn and B were sufficient in 60%, 52%, 54% and 100% of the studied orchards, respectively, while, N, P and Fe were deficient in 100%, 96% and 44% of the or- chards, respectively. On the other hand, Zn and Cu were excessive in 68% and 100%, re- spectively, of the orchards under consideration. Some soil properties seem to correlate significantly with the concentration of nutrients in the leaves. Such antagonistic interaction CaCO3 ´ Cu (Fig. 1 d) has also been reported by KALAVROUZIOTIS et al. (2010) for this soil which was planted with Brassica oleracea var Gemmifera (Brussels sprouts). In another study with Pistacia lentiscus trees, CaCO3 con- centration in soil was found to be negatively correlated with leaf N and K concentrations (DOGAN et al. 2003). Also, the interaction OM ´ B (Fig. 1b) is in line with findings of STEVENSON and COLE (1999), who found that B is the only non metal which can combine with OM, primarily as organic complexes, with compounds that contain cis-hydroxyl groups such as saccharides, the plant-available B being released after mineralization of the organic matter. Similarly, in relation to Zn (Fig. 1a), it has been stated by HODGSON et al. (1966) that 28–90% of this element may be organically bound in the soil. It has generally been found in the present work that the elemental interactions between leaf nutrients are mainly described by quadratic, and secondarily by logarithmic regression equations. Similar results have been reported by KALAVROUZIOTIS and KOUKOULAKIS (2009b) and KALAVROUZIOTIS et al. (2010) experimenting with Brussels sprout plants. Similar interactions between leaf nutrients (Tab. 3) have also been found by other re- searchers. More specifically, experiments with broccoli (Brassica oleracea var Italica) plants resulted in the following synergistic interactions in leaves: N ´ K, Mg ´ N, Fe ´ Mn, Mn ´ B and B ´ Mn (KALAVROUZIOTIS et al. 2008, 2009). In accordance with the above find- ings, KALAVROUZIOTIS and KOUKOULAKIS (2009b) reported the synergistic interactions of N ´ K and Zn ´ Fe occurring in the leaves of Brussels sprout plants. On the other hand, HALDAR and MANDAL (1981) showed that the application of Zn decreased Fe concentration in shoots, but they mention that this decrease was not due to dilution effect or to reduced rate of translocation from roots to tops. ACTA BOT. CROAT. 72 (2), 2013 305 NUTRIENT ELEMENTS IN PISTACHIO TREES In relation to the interaction of Mn ´ Fe, contrary to our results, SRIVASTAVA and GUPTA (1996) state that the chlorosis of Fe, which is induced by increased levels of Mn, is related to the enzymatic disturbance due to the fact that Mn may compete with Fe for binding sites of the enzymes. Similarly, Mn may interfere with the translocation of Fe from roots to shoots. The interaction Zn ´ Cu found in the present study to be synergistic was said by HEWITT (1983) to be antagonistic, each of the interacting elements affecting their respective concen- tration in the plant, competing for common carrier sites. Also, during metabolism they re- place each other in some metalloenzymes. HALDAR and MANDAL (1981) found that high Zn concentration in soil accentuates Cu deficiency, each element competitively inhibiting the uptake of the other (GIORDANO et al. 1974). These workers also stated that the interaction Zn ´ Fe is mutually antagonistic, because excess Zn or Fe may cause reduction in the absorp- tion of Fe or Zn, respectively. This mutual effect of Zn and Fe increases the possibility of their deficiency in the growing plants (KAUSAR et al. 1976). A careful study of the results about PEC points out that the elemental synergistic interac- tions occurring within the pistachio leaves may contribute significant quantities of nutrients, while the antagonistic interactions can also deprive the plants of these nutrients, as in the case of K, which was found to be negative, suggesting a 14.33% decrease of K contribution to leaves (Tab. 2). Moreover, the generally high PEC to soil found, suggests that the synergistic interac- tions mobilize from non-available sources significant quantities of nutrients, while the an- tagonistic interactions immobilize available nutrients to plants, unfavorably affecting soil fertility. Studying these results, the following may be concluded: the interactions between soil nutrients and soil properties play a significant role in supplying or decreasing the soil available plant nutrients, and therefore, they determine to a great extent the level of soil fertility. Obviously, the interactions among soil nutrients favored significantly soil fertility, and hence, plant growth and nut yield. In detail, the synergistic and statistically significant inter- action P ´ Mg (Fig. 3b) found in the soil of pistachio orchards has also been found to occur between soil P and leaf Mg (MERHAUT 2007). This interaction seems to be associated with the ionic balance, related to cation and anion uptake by plants as well as to the increased root growth, sometimes observed with increased P fertilization. The effect of P fertilization in- creasing Mg uptake has also been documented in rice (Oryza sativa L.), wheat (Triticum aestivum L.), bean (Phaseolus vulgaris L.), and corn (Zea mays L.) (FAGERIA et al. 1995). In relation to K, it has been suggested that the interaction of this element with other ele- ments presents a great variability among plant species (DIBB and THOMPSON 1985). This in- teraction should be studied more scrupulously, considering that many other factors, such as the level of available Mg (very high in the present study), fertilization and age of plants, moisture and temperature in soil, could affect the above interaction (ARNON 1975). The macronutrient K is a very strong competitor, when it is present in high concentration. It par- ticularly affects the uptake of Mg. However, in the present work, the interaction Mg ´ K was found to be antagonistic due to the high level of Mg in the leaves (2.4%); the mean K con- centration in leaves was only 1.2%, close to the low limit of the critical range (1.0–2.0%) (CRANE and MARANTO 1988). It must be noted that, generally, the statistically significant in- teractions are usually biphasic, that is, the elements involved affect each other negatively or positively but at high concentrations. For example KOUKOULAKIS et al. (1988) observed that increasing the supply of K reduced leaf Mg concentration in tomato and cucumber, with an 306 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. increase of K availability in the soil, due to K ´ Mg antagonism. However, increasing K concentrations of nutrient solutions in hydroponically grown tomatoes resulted in increased Mg concentration in fruits and seeds (MENGEL and KIRKBY 1987). In the present work, it has been shown that in general, the increase of soil Cu concentra- tion significantly increased K level in the soil, suggesting that the interaction CuxK is syner- gistic (Fig. 3f). Similar results have been reported by KALAVROUZIOTIS and KOUKOULAKIS (2009b) according to which the interaction Cu ´ K was synergistic in the roots, leaves and sprouts of Brussels sprouts. In contrast to the above findings, SONMEZ et al. (2007) reported that increasing Cu applications resulted in a decline of leaf and root K concentration in to- mato seedlings. On the other hand, ALVA et al. (1999) found that the application of increased levels of Cu decreased the Zn content of leaves and roots of Citrus seedlings, grown in a sand substrate. However, in the present work, the interaction Cu x Zn was synergistic and statistically significant in both, soil and pistachio leaves (Tabs. 3, 5). The availability of nu- trients, especially of micronutrients, in soil was strongly influenced by pH, CaCO3 and or- ganic matter (Tab. 4). It is known that Zn solubility increases at low pH values (LINDSAY 1991, BRADY and WEIL 2002). In particular, the optimum availability of Zn in soil is ob- served at a pH range 5–7. Indeed, we found that the interactions pH ´ N, CaCO3 ´ Cu (Tab. 1), and CaCO3 ´ Fe, pH ´ Fe, CaCO3 ´ Mn, pH ´ Mn, pH ´ B, and CaCO3 ´ B (Tab. 4) were antagonistic, as they were negatively interrelated. On the other hand, the interactions OM ´ B and OM ´ Zn (Tab. 1) as well as OM ´ K and OM ´ Zn (Tab. 4) were synergistic, suggest- ing the favorable effect of organic matter on the availability of soil plant nutrients. The fact that the interaction CaCO3 ´ Zn was synergistic was unexpected, as most of the soils studied in this work were calcareous with high pH. A possible explanation could be the formation of ZnCO3 due to the high Zn level in 13 cases of orchards out of a total of 50, varying from 3–17.5 mg kg–1 soil. The ZnCO3 is a relatively soluble compound, which could even be used as fertilizer (TISDALE et al. 1993). In conclusion, almost all the significant elemental interactions occurring in pistachio leaves or soils were synergistic. They contribute considerable quantities of available macro- and micronutrients and therefore improve the nutrient status of pistachio leaves, and the level of soil fertility. On the other hand, any antagonistic or synergistic interactions must be taken into account during fertilization of pistachio, because among other consequences, they may possibly lead to plant deficiencies. Soil analytical data showing the nutrient con- centration levels, and their relationships, may be helpful in predicting possible antagonistic or synergistic interactions. Furthermore, the significant interactions between soil physical or chemical properties and nutrient content of the leaves or soils found in the present study, suggest that altering these properties (e.g. improving the organic matter or pH), could favor- ably modify the level of soil fertility, and therefore enhance plant growth, productivity and nut quality. References ALVA, A. K., HUANG, B., PRAKASH, O., PARAMASIVAM, S., 1999: Effects of copper rates and soil pH on growth and nutrient uptake by citrus seedlings. Journal of Plant Nutrition 22, 1687–1699. ARNON, I., 1975: Mineral nutrition of maize. International Potash Institute, Bern, Switzerland. ACTA BOT. CROAT. 72 (2), 2013 307 NUTRIENT ELEMENTS IN PISTACHIO TREES BASLAR, S., DOGAN, Y., MERT, H. H., 1999: Studies on the ecology of Pistacia terebinthus L. subsp. palaestina (Boiss.) Engler in West Anatolia. Journal of the Faculty of Science, Ege University 22, 1–12. BOUJOUCOS, G. J., 1962: Hydrometer method improved for making particle size analysis of soils. Journal of Agronomy 54, 464–465. BRADY, N. C, WEIL, R. R., 2002: The nature and properties of soils. Prentice Hall, N.J., USA. CHAPMAN, H. D., PRATT, P. F., 1961: Methods of analysis for soils, plants and waters. Divi- sion of Agricultural Sciences, University of California, Riverside, USA. CRANE, J. C., MARANTO, J., 1988: Pistachio production. Cooperative Extension University of California, Division of Agricultural and Natural Resources. Publication 2279, Oak- land, CA. DIBB, D. W., THOMPSON, Jr. W. R., 1985: Interaction of potassium with other nutrients. In: MUNSON, R. D. (ed.), Potassium in agriculture, 515–533. SSSA, Madison, Wisconsin. DOGAN, Y., BASLAR, S., AYDIN, H., MERT, H., 2003: A study of the soil-plant interactions of Pistacia lentiscus L. distributed in the western Anatolian part of Turkey. Acta Botanica Croatica 62, 73–88. FAO (Food and Agriculture Organization), 2008. Retrieved September 5, 2012, from http://faostat.fao.org. FAGERIA, N. K., ZIMMERMANN, F. J. P., BALIGAR, V. C., 1995: Lime and phosphorus interac- tions on growth and nutrient uptake by upland rice, wheat, common bean, and corn in an oxisol. Journal of Plant Nutrition 18, 2519–2532. GIORDANO, P. M., NOGGLE, J. C., MORTVENDT, J. J., 1974: Zinc uptake by rice as affected by metabolic inhibitors and competing cations. Plant Soil 41, 637–646. HALDAR, M., MANDAL, L. N., 1981: Effect of phosphorus and zinc on the growth and phos- phorus, zinc, copper, iron and manganese nutrition of rice. Plant Soil 59, 415–425. HEWITT, E. J., 1983: The essential and functional mineral elements. In: ROBINSON, J. B. D., BOULD, C., HEWITT, E. J., NEEDHAM, P. (eds.), Diagnosis of mineral disorders in plants, vol 1, 7–53. Her Majesty’s Stationary Office, London, UK. HODGSON, J. F., LINDSAY, W. L., TRIERWEILER, J. F., 1966: Micronutrient cation complexing in soil solution. II. Complexing of zinc and copper in displacing solution from calcare- ous soils. Soil Science Society of America Proceedings 30, 723–726. JACKSON, M. L., 1958: Soil chemical analysis. Prentice Hall Inc., Englewood Cliffs, N.J., USA. KALAVROUZIOTIS, I. K., KOUKOULAKIS, P. H., 2009a: Environmental implications of soil properties and essential nutrient interactions, under the effect of treated municipal wastewater. Water Air Soil Pollution 197, 267–276. KALAVROUZIOTIS, I. K., KOUKOULAKIS, P. H., 2009b: Distribution of elemental interactions in Brussels sprout plants, under the treated municipal wastewater. Journal of Plant Inter- actions 4(3), 219–231. KALAVROUZIOTIS, I. K., KOUKOULAKIS, P. H., ROBOLAS, P., PAPADOPOULOS, A. H., PANTAZIS, V., 2008: Macro- and micronutrient interactions in soil under the effect of Brassica oleracea var Italica, irrigated with treated municipal wastewater. Fresenius Environ- mental Bulletin 17, 1–15. 308 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D. KALAVROUZIOTIS, I. K., KOUKOULAKIS, P. H., SAKELLARIOU-MAKRANTONAKI, M., PAPANIKO- LAOU, C., 2009: Effects of treated municipal wastewater on the essential nutrients inter- actions in the plant of Brassica oleracea var Italica. Desalination 242, 297–312. KALAVROUZIOTIS, I. K., KOUKOULAKIS, P. H., MEHRA, A., 2010: Quantification of elemental interactions effects on Brussels sprouts under treated municipal wastewater. Desalina- tion 254, 6–11. KAUSAR, M. A., CHAUDHARY, F. M., RASHID, A., LATIF, A., ALAM, S. M., 1976: Micronu- trient availability to cereals from calcareous soils. I. Comparative Zn and Cu deficiency and their mutual interaction in rice and wheat. Plant Soil 45, 397–410. KIZILGOZ, I., KIZILKAYA, R., ACAR, I., KAPTAN, H., 2001: Nutrient contents of pistachio trees (Pistacia vera L.) growing in district of Sanliurfa and the relationship between their microelement deficiency and some soil properties. Cahiers Options Méditerranéennes 56, 47–52. KOUKOULAKIS, P., 1995: Principles of rational fertilization of crops (in Greek). Agriculture and Husbandry 9, 43–61. KOUKOULAKIS, P. H., SIMONIS, A. D., BLADENOPOULOU, S., 1988: Potassium-magnesium an- tagonism in tomato and cucumber, grown in plastic greenhouse. Proceedings of Athens Academy 63, 130–139. KOUKOULAKIS, P. C., SIMONIS, A. D., GERTSIS, A., 2000: The organic matter in soils. The problem of Greek soils (in Greek). Stamoulis Publications, Athens. LINDSAY, W. L., 1991: Inorganic equilibria affecting micronutrients in soil. In: MORTDVEDT, J. J., GIORDANO, M., LINDSAY, W. C. (eds.), Micronutrients in agriculture, 90–111. SSSA, Madison, Wisconsin, USA. MENGEL, K., KIRKBY, E. A., 1987: Principles of plant nutrition. International Potash Insti- tute, Berne, Switzerland. MERHAUT, D. J., 2007: Magnesium. In: BARKER, A. V., PILBEAM, D. J. (eds.), Handbook of plant nutrition, 146–181. CRC Press, Boca Raton, FL, USA. MILLS, H. A., BENTON JONES Jr, J., 1996: Plant analysis handbook. MicroMacro Publishing Inc., Athens, USA. OLSEN, S. R., 1972: Micronutrient interactions. In: MORTDVEDT, J. J., GIORDANO, M., LINDSAY, W. C. (eds.), Micronutrients in agriculture, 243–264. SSSA. Madison, Wiscon- sin, USA. SONMEZ, S., KAPLAN, M., SOMNEZ, N. K., KAYA, H., UZ, I., 2007: Effect of both soil copper applications and foliar copper application frequencies on macronutrients contents of to- mato plants. Asian Journal of Chemistry 19, 5372–5384. SRIVASTAVA, P. C., GUPTA, U. C., 1996: Trace elements in crop production. Science Publish- ers Inc., Lebanon, USA. STEVENSON, F. J., COLE, M. A., 1999: Cycles of soil. Carbon, nitrogen, phosphorus, sulfur, micronutrients. John Wiley and Sons Inc., New York, USA. TISDALE, S. L., NELSON, W. L., BEATON, J. D., HAVLIN, J. L., 1993: Soil fertility and fertiliz- ers. MacMillan Publishing Co, New York, USA. ACTA BOT. CROAT. 72 (2), 2013 309 NUTRIENT ELEMENTS IN PISTACHIO TREES WOLF, B., 1971: The determination of boron in soil extracts, plant materials, composts, ma- nures, water and nutrient solutions. Communications in Soil Science and Plant Analysis 2, 363–374. 310 ACTA BOT. CROAT. 72 (2), 2013 KOUKOULAKIS P., CHATZISSAVVIDIS C., PAPADOPOULOS A., PONTIKIS D.