Effect of nitrogen form and nutrient solution pH on growth and mineral composition of self-grafted and grafted tomatoes

Scientia Horticulturae 149 (2013) 61–69

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Effect of nitrogen form and nutrient solution pH on growth and mineral composition of self-grafted and grafted tomatoes Daniela Borgognone a , Giuseppe Colla a,∗ , Youssef Rouphael b , Mariateresa Cardarelli a , Elvira Rea c , Dietmar Schwarz d,∗∗ a

Department of Agriculture, Forestry, Nature and Energy, University of Tuscia, via S. C. De Lellis snc, 01100 Viterbo, Italy Department of Crop Production, Faculty of Agricultural Engineering and Veterinary Medicine, Lebanese University, Dekwaneh-El Maten, Beirut, Lebanon c Agricultural Research Council - Research Centre for the Soil-Plant System, via della Navicella 2-4, 00184 Roma, Italy d Leibniz-Institute of Vegetable and Ornamental Crops, Grossbeeren & Erfurt e.V., Theodor Echtermeyer Weg 1, 14979 Großbeeren, Germany b

a r t i c l e

i n f o

Article history: Received 26 November 2011 Received in revised form 1 February 2012 Accepted 7 February 2012 Keywords: Blossom-end rot pH Nitrogen form Ammonium toxicity Carbohydrates Amino acids Solanum lycopersicum L.

a b s t r a c t Three greenhouse experiments were carried out to determine the effect of the nitrogen form and the nutrient solution pH on growth, yield, leaf gas exchange, carbohydrate, N-compound concentrations and mineral composition of tomato cv. Moneymaker (Solanum lycopersicum L.) self-grafted and grafted onto ‘Maxifort’ (S. lycopersicum L. × S. habrochaites S. Knapp and D. M. Spooner) grown in hydroponics. Exp. 1 included five pH levels in the nutrient solution (3.5, 4.5, 5.5, 6.5, and 7.5) while in the Exps. 2 and 3 four different ratios of NO3 − to NH4 + (100:0, 70:30, 30:70, and 0:100) were used. The Exps. 1 and 2 were performed in a short period of time (about 20 days) while Exp. 3 was a long-term experiment. No significant differences among treatments were observed in shoot and root dry biomass of tomato in the pH experiment (Exp. 1), whereas shoot dry biomass, Ca and Mg concentrations decreased sharply when N was exclusively provided as NH4 + (Exp. 2). When averaged over the pH level of the nutrient solution, the highest Ca, Fe, Zn, and Cu concentrations were recorded in grafted than self-grafted plants (Exp. 1), whereas in Exp. 2 shoot and root biomass values recorded in grafted plants were significantly higher than those observed for self-grafted plants, by 20%, and 24%, respectively. In the long-term experiment, the plant growth and yield decreased in response to an increase of NH4 + in the nutrient solution. The decrease in marketable yield with decreasing NO3 − :NH4 + ratio resulted mainly from the increase of blossom-end rot, which reduced the number of marketable fruits per plant. The adverse effects of an increased supply in NH4 + have been associated to a fall in Ca and Mg levels in plant tissues. The carbohydrate concentrations, amino acids and proteins increased under NH4 + in comparison to NO3 − based nutrition. Moreover, NH4 + toxicity was associated with reduced rates of net photosynthesis. Our results also demonstrated that grafting ‘Moneymaker’ into ‘Maxifort’ did not mitigate the negative effects of ammonium nutrition on tomato productivity. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Most plants can make use of either ammonium or nitrate ions. The uptake of these two forms of nitrogen (N) is controlled by genotype, plant development and physiological status, and also by soil properties such as texture, structure, water content and pH (Lea and Morot-Gaudry, 2001; Loulakakis and RoubelakisAngelakis, 2001). Plant growth and development have long been known to benefit from the presence of NO3 − (Marschner, 1995). However, despite the fact that NO3 − assimilation consumes more energy than NH4 + assimilation, only few species perform well

∗ Corresponding author. Tel.: +39 0761 357536; fax: +39 0761 357453. ∗∗ Corresponding author. Tel.: +49 33701 78206; fax: +49 33701 55391. E-mail addresses: [email protected] (G. Colla), [email protected] (D. Schwarz).

when NH4 + is the sole source (Marschner, 1995). Indeed, many plant species develop symptoms of toxicity when subjected to high concentrations of NH4 + , which are not detected when plants are grown with the same concentration of NO3 − or in mixed N nutrition (Britto et al., 2001a,b; Britto and Kronzucker, 2002). Although NH4 + is an important intermediate in many metabolic reactions, it has been reported that high concentrations of NH4 + in the soil or the nutrient solution may lead to leaf chlorosis, net photosynthesis decrease, lower plant yield, lower cation content, changes of several metabolite levels such as amino acids or organic acids and acidification of the rhizosphere (Britto and Kronzucker, 2002). In photosynthesizing tissues, NH4 + can uncouple electron transport from photophosphorylation (Peltier and Thibault, 1983). This has been considered a likely explanation for the reduced photosynthetic rates observed for tomato plants supplied with NH4 +

0304-4238/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2012.02.012

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D. Borgognone et al. / Scientia Horticulturae 149 (2013) 61–69

instead of NO3 − (Horchani et al., 2010). Moreover, the toxic effects of NH4 + nutrition have been often related to an acidification of the rhizosphere or the intracellular medium (Dijk and Grootjans, 1998) due to the excretion of protons via the H+ -ATPase. In contrast, NO3 − uptake is associated with the proton consumptions via 2H+ /NO3 − symport, leading to an increase in the pH of the outer solution (Mengel and Kirkby, 2001). In spite of the large literature about the effects of ammonium and nitrate nutrition in several vegetable crops (Akl et al., 2003; Bonasia et al., 2008; Fallovo et al., 2009; Horchani et al., 2010; Savvas et al., 2010a), no information is available if grafting could alleviate the negative effects of ammonium nutrition in a sensitive crop such as tomato. Grafting vegetable onto appropriate rootstocks is a common practice in Japan, Korea, China, the Mediterranean basin and several European countries. The main purpose of employing this technology is to control soilborne diseases (Crinò et al., 2007; Lee et al., 2010). However, the impact of grafting includes not only a stronger resistance against pathogens but also a higher tolerance to abiotic stress conditions such as salinity, heavy metal, nutrient stress, thermal stress, water stress, organic pollutants, and alkalinity (Colla et al., 2010a,b,c, 2011; Rouphael et al., 2008a,b; Savvas et al., 2009, 2010b; Schwarz et al., 2010), and could improve fruit quality (Proietti et al., 2008; Flores et al., 2010; Rouphael et al., 2010). Moreover, it has been demonstrated on watermelon and melon that the use of certain rootstocks promoted nitrogen assimilation of grafted plants by improving the nitrate uptake and transport to the scion (Colla et al., 2010b, 2011). However, in the former studies only nitrate was used as nitrogen source while there is a lack of information on the response of grafted plants to other nitrogen sources like ammonium alone or in combination with nitrate. Starting from the above considerations, the aim of this study was to investigate the responses of self-grafted and grafted tomato (Solanum lycopersicum L.) plants to nutrient solution pH and nitrogen form at early developmental stage (Exps. 1 and 2, respectively) and to N form on a long term (Exp. 3). To this end, plant growth, yield, photosynthesis, carbohydrate, N-compound concentrations and mineral composition were analyzed.

2. Materials and methods 2.1. Plant material, treatments and growth conditions Three hydroponic experiments were carried out in a greenhouse at the Institute of Vegetable and Ornamental Crops, located at Grossbeeren (Germany): Exp. 1 (short term pH experiment), from 18 November 2008 to 8 December 2008; Exp. 2 (short term nitrogen form experiment), from 7 January 2009 to 28 January 2009; Exp. 3 (long term nitrogen form experiment), from 18 December 2008 to 7 April 2009. All experiments were conducted at the same mean temperature (20 ◦ C), and relative humidity (50–71%). Mean daily photosynthetic active radiation of Exps. 1, 2 and 3 amounted to 5.08, 6.14 and 7.57 Mol m−2 , respectively. Tomato (Solanum lycopersicum L., cv. Moneymaker, British heirloom) plants were either self-grafted, or grafted onto the vigorous rootstock ‘Maxifort’ (S. lycopersicum × S. habrochaites, De Ruiter/Monsanto, Bergschenhoek, The Netherlands). In Exps. 1 and 2 grafted plants were transferred into 2 L glasses filled with aerated nutrient solution when 6 weeks old, 3 weeks after grafting. In Exp. III, 96 plants were transferred into gullies (8 m × 0.2 m × 0.07 m) when 7.5 weeks old, 5 weeks after grafting and supplied continuously with nutrient solution at a flow rate of 2 L min−1 . Distance between plants within a gully was 60 cm, giving a density of 1.6 plants m−2 . The treatments in each experiment were arranged in a randomized

complete-block design. Exp. 1 was designed as a two-factorial combination of five pH levels of the nutrient solution (3.5, 4.5, 5.5, 6.5 or 7.5) and two grafting combinations, with four replications and five plant per experimental plot. Exps. 2 and 3 were designed as two-factorial combinations of four different ratios of NO3 − to NH4 + (100:0, 70:30, 30:70, and 0:100) and two grafting combinations. Each treatment was replicated four times (Exp. 2) and two times (Exp. 3) with six plants in each experimental plot. The treatments in Exp. 3 started 3 weeks after plant transfer into the gullies. In the Exp. 1, the composition of the nutrient solution was: 23 mM NO3 –N, 1.6 mM NH4 –N, 7.1 mM S, 1.0 mM P, 8.0 mM K, 10.0 mM Ca, 4.3 mM Mg, 25 ␮M Fe, 5 ␮M Mn, 0.6 ␮M Cu, 7 ␮M Zn, 50 ␮M B, and 0.5 ␮M Mo. The composition of the nutrient solutions in the experiment 2 and 3 were: 23.0 mM N (as NO3 and/or NH4 according to the experimental treatments), 9.3–10.7 mM S (according to the treatments), 1.0 mM P, 8.8 mM K, 10.0 mM Ca, 3.8 mM Mg, 25 ␮M Fe, 5 ␮M Mn, 0.6 ␮M Cu, 7 ␮M Zn, 50 ␮M B, and 0.5 ␮M Mo. In all experiments, iron was supplied as a chelate with ethylenediaminetetraacetic acid (EDTA). During all experiments, the electrical conductivity was kept constant at 3.8 dS m−1 by adding stock solution or deionized water according to the variations (±10% of the target value). In Exp. 1, the mean pH values of the nutrient solutions at the end of the experiment were 3.8, 4.7, 5.5, 6.5, and 7.3 for solutions with initial pH value of 3.5, 4.5, 5.5, 6.5 and 7.5, respectively. In all nutrient solutions of Exps. 2 and 3 a MES buffer (2-Nmorpholinoethanesulfonic acid) was applied at 1.5 mM to keep the pH in the range of 5.6–5.8. Moreover, phosphoric acid or potassium hydroxide was added when the solution pH drifted above or below the threshold. 2.2. Plant growth measurements At final harvest, 20, 21 and 107 days after transplanting, in Exps. 1, 2 and 3, respectively, all plants per plot were separated into stems, leaves, and roots, and their tissues were dried in a forced-air oven at 80 ◦ C for 72 h for biomass determination. Shoot biomass was equal to the sum of aerial vegetative plant parts (leaves + stems). In Exps. 1 and 2 root to shoot ratio was calculated by dividing root dry weight by the sum of leaf and stem dry weights. Finally, in Exp. 3 harvest index (HI) was calculated as the ratio of dry matter partitioned into the fruit relative to the total plant biomass. 2.3. Yield and yield components In Exp. 3, the harvesting of commercially ripe fruit started 8 weeks after treatment start and was repeated weekly until the end of the experiment. At each harvest, the total number and weight of harvested fruit, as well as the incidence of fruits affected the physiological disorder blossom-end rot (BER), were recorded separately for each treatment. 2.4. Net photosynthesis and carbohydrate analysis Four plants were randomly selected from each treatment for leaf gas exchange measurements. The most recently fully expanded leaf (5th) was employed for measurements with an open gas exchange system (Li-6400, Li-Cor, Inc., Lincoln, NE, USA). The system was calibrated prior to measurement. Net CO2 assimilation rate, was determined between 9:00 and 17:00 at a photosynthetic photon flux density of 800 ␮mol m−2 s−1 . During the measurements, climate conditions in the leaf chamber were set close to conditions in the greenhouse, such as relative humidity at 60%, CO2 concentration at 360 ± 10 ␮mol mol−1 and leaf temperature at 28 ± 0.5 ◦ C.

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Table 1 Main effects of pH and graft combination on macro and microelement concentrations of tomato leaves in the first experiment. Treatment

pH 3.5 4.5 5.5 6.5 7.5 Graft combination Self-grafted Grafted Significancea pH Linear Quadratic Graft combination (G) pH × G a

Macroelements (g/kg)

Microelements (mg/kg)

N

P

K

Ca

Mg

Fe

Mn

Zn

Cu

B

46.2 43.7 41.4 42.7 41.4

6.8 6.7 7.0 6.8 6.4

46.8 49.2 49.1 46.9 47.1

21.9 24.1 25.5 24.9 25.6

5.01 5.05 5.13 5.28 5.84

67.7 59.5 53.6 41.8 38.7

45.9 47.3 46.9 39.5 28.5

44.5 45.5 41.8 28.8 20.3

4.5 5.5 6.5 6.8 6.7

33.9 35.6 35.4 34.0 34.3

43.0 43.2

6.3 7.1

47.8 47.8

23.2 25.6

5.5 5.0

49.9 54.6

41.0 42.3

33.4 39.0

5.5 6.5

34.1 35.1

NS NS NS NS NS

NS NS NS NS NS

NS NS NS NS NS

*** ** *** * NS

*** ** *** *** NS

*** *** * ** NS

*** ** *** NS NS

*** ** *** *** NS

*** ** *** ** NS

NS NS NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

In Exp. 3, samples of the fourth leaf, and roots were collected on 27 March and immediately frozen in liquid nitrogen, then stored at −80 ◦ C for later analyses of soluble sugars. 20 mg of fine-powdered frozen samples were employed to determine the amounts of glucose, fructose and sucrose enzymatically according to the method described by Jones et al. (1977) with a doublebeam/dual-wavelength spectrophotometer (UV-3000, Shimadzu, Kyoto, Japan). 2.5. Nitrate, ammonium, amino acids and soluble-protein assay In Exp. 3, NO3 − , NH4 + , amino acids and total proteins were determined on leaf and root samples taken on 27 March. Nitrate was assayed in plant tissues using the salicylic acid sulfuric acid method (Cataldo et al., 1975). NH4 + in leaf and root samples were extracted in 6% (w/v) TCA and assayed in sample extracts using the phenol–hypochlorite method (Baldet et al., 2002). The soluble amino acids were determined by the ninhydrin method (Moore and Stein, 1948). Total soluble proteins were assayed according to the principle of protein-dye binding (Bradford, 1976). 2.6. Mineral analysis In all experiments (1, 2, and 3) dried leaf tissues taken at the end of the experiment were ground separately in a Wiley mill to pass through a 20-mesh screen, then 0.5 g of the dried plant tissues were analyzed for the following macro- and micronutrients: N, P, K, Ca, Mg, Fe, Mn,. Zn, Cu, and B. Nitrogen concentration in the plant tissues was determined after mineralization with sulfuric acid by “Kjeldahl method” (Bremner, 1965), P, K, Ca, Mg, Fe, Mn, Zn, Cu, and B concentrations were determined by dry ashing at 400 ◦ C for 24 h, dissolving the ash in 1:25 HCl, and assaying the solution obtained using an inductively coupled plasma emission spectrophotometer (ICP Iris; Thermo Optek, Milano, Italy) (Isaac and Johnson, 1998). 2.7. Statistical analysis All data were statistically analyzed by a two way ANOVA using the SPSS software package (SPSS 10 for Windows, 2001). In all experiments, the method of orthogonal polynomials was used to identify functional relationships (linear or quadratic) between response of measured parameters and treatments (pH or

NO3 − :NH4 + ratio) that cover the whole range of treatment levels tested (Gomez and Gomez, 1983).

3. Results 3.1. Exp. 1: short term-pH experiment In Exp. 1, no significant differences among treatments were observed in shoot dry biomass (avg. 5.3 g plant−1 ), root dry biomass (avg. 0.6 g plant−1 ) and root-to-shoot ratio (avg. 0.11). The macro and micro elements concentration in tomato shoots as a function of the grafting combination and pH level are displayed in Table 1. Except for N, P, K, and B, the concentrations of Ca, Mg, Fe, Zn, and Cu were significantly affected by pH of the nutrient solution and grafting combinations, whereas no significant difference was observed on the pH level × grafting interaction. Linear and quadratic relationships were detected for the macro and micro elements, where Ca, Mg and Cu concentrations increased, and Fe, Mn, and Zn concentrations decreased as the pH of the nutrient solution increased from 3.5 to 7.5 (Table 1). Moreover, when averaged over the pH level of the nutrient solution, the highest Ca, Fe, Zn, and Cu concentrations were recorded in grafted compared with self-grafted plants, whereas an opposite trend was observed for the concentration of Mg.

3.2. Exp 2: short term-nitrogen form experiment Our results showed that plant responses varied depending on both nitrogen form and grafting combination. The solution NO3 − :NH4 + ratio had linear and quadratic effects on plant the shoot dry biomass decreased sharply when N was exclusively provided as NH4 + (0:100), whereas an opposite trend was observed for the root-to-shoot ratio (Table 2). Moreover, shoot and root biomass values recorded in grafted plants were significantly higher than those observed for selfgrafted plants, by 20%, and 24%, respectively (Table 2). The N and P concentrations increased and the Ca and Mg concentrations decreased linearly with increasing NH4 + in the nutrient solution (Table 3). Moreover, the following microelements Fe, Mn, Zn, and Cu in the leaves increased linearly and quadratically with increasing the NH4 + in the nutrient solution with the highest values recorded when N was exclusively provided as NH4 + (0:100). Among grafting combinations, the highest P, Mg, Mn, Zn, and Cu concentrations were recorded in self-grafted tomato plants (Table 3).

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Table 2 Main effects of N form and graft combination on plant growth characteristics of tomato grown in the second experiment. Treatments

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Table 4 Main effects of N form and graft combination on dry biomass of leaves, stem, fruits, roots and total, and harvest index (HI) of tomato plants in the third experiment.

Leaf number (n./plant)

Shoot dry biomass (g/plant)

Root dry biomass (g/plant)

Root/Shoot

13.56 14.50 15.00 12.63

7.82 9.03 8.26 5.17

1.02 1.01 1.00 0.90

0.13 0.11 0.12 0.18

13.53 14.31

6.87 8.27

0.88 1.09

0.14 0.14

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted

*** *** *** NS NS

Significancea N-form Linear Quadratic Graft combination (G) N-form × G

*** NS ** * NS

*** ** ** ** NS

NS NS NS ** NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

Treatments

a

Dry biomass (g/plant)

HI

Leaves

Stem

Fruits

Roots

Total

85.5 82.8 70.5 67.8

37.7 33.4 29.3 24.5

81.7 100.5 51.6 51.9

8.1 5.9 7.1 6.1

213.0 222.5 158.5 150.3

0.38 0.44 0.32 0.34

71.3 82.0

30.2 32.3

68.1 74.8

6.9 6.7

176.5 195.8

0.39 0.36

NS NS NS NS NS

* ** NS NS NS

* ** NS NS NS

NS NS NS NS NS

* ** NS NS NS

* ** * NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05 and 0.01, respectively.

3.3. Exp. 3: long term-nitrogen form experiment In Exp. 3, the dry mass of stem, fruits and total biomass values decreased linearly in response to an increase of NH4 + in the nutrient solution (Table 4). No significant difference among treatments was observed for the leaf dry matter (avg. 9.6%; data not shown), whereas, the root dry matter was significantly affected by the nitrogen form with the highest values recorded in the 0:100 ratio treatment (avg. 8%; data not shown). Moreover, the harvest index decreased linearly and quadratically in response to a decrease in the NO3 − :NH4 + ratio (Table 4). Similarly to dry biomass, the yield and its components were highly influenced by nitrogen form but not by grafting combination; there was no N form × grafting interaction (Table 5). Total, marketable yields and the fruit number per plant decreased linearly as NH4 + proportion increased in the solution. The decrease in marketable yield with increasing NH4 + in the nutrient solution resulted mainly from the increase of fruit physiological disorders (BER), which reduced the number of marketable fruits per plant (Table 5). The macro and micro element concentrations of tomato leaves and fruits were only affected by the NO3 − :NH4 + ratio (Tables 6 and 7). The N and P concentrations in leaves increased and the Ca and Mg concentrations decreased with increasing NH4 + in the nutrient solution. Moreover, Fe, Mn, and B concentrations increased linearly with the increase of NH4 + in the nutrient

solution (Table 6). The mineral composition of fruits followed a similar trend, where N, Fe, Zn and B concentrations increased linearly, and Ca and Mg concentrations decreased linearly with the increase of NH4 + in the nutrient solution (Table 7). The gas exchange measurements showed a significant decrease in the photosynthetic activity at the highest NH4 + concentrations (9.7 and 4.3 ␮mol m−2 s−1 , for the 70% and 100% NH4 + treatments, respectively). However, for NH4 + concentrations below 30%, the photosynthetic activity remained unchanged (avg. 12.6 ␮mol m−2 s−1 ). Both glucose and fructose were the predominant sugars in leaves, while sucrose was the predominant soluble sugar in the roots (Table 8). Except for fructose, increasing NH4 + concentration in the nutrient solution did not affect root carbohydrate concentrations. Moreover, leaf carbohydrate (glucose, fructose, sucrose and total) concentrations increased linearly with NH4 + availability, especially when N was exclusively provided as NH4 + (0:100, Table 8). NO3 − , NH4 + , amino acids and total protein concentrations in leaves and roots were only affected by the NO3 − :NH4 + ratio (Table 9). The NO3 − and NH4 + concentrations in both leaves and roots decreased and increased linearly, respectively, with increasing NH4 + in the nutrient solution. Among the four NO3 − :NH4 + ratio, the NO3 − , NH4 + concentrations were always higher and lower in roots than in leaves, respectively (Table 9). Finally, the amino acids

Table 3 Main effects of N form and graft combination on macro and microelement concentrations of tomato leaves in the second experiment. Treatment

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Macroelements (g/kg)

Microelements (mg/kg)

N

P

K

Ca

Mg

Fe

Mn

Zn

Cu

B

39.8 42.0 45.0 45.8

4.8 4.9 5.3 5.7

45.3 46.9 45.0 46.9

26.9 24.9 19.9 14.1

5.8 5.5 4.1 2.8

47.0 52.0 70.1 138.6

36.0 36.1 50.1 79.5

32.2 33.2 41.4 94.2

6.2 5.8 5.8 7.0

30.6 31.1 36.8 52.3

43.7 42.6

5.5 4.9

45.1 46.8

21.5 21.4

4.7 4.4

71.0 82.8

53.3 47.6

52.6 47.9

6.3 6.1

36.1 39.3

** *** NS NS NS

* * NS * NS

NS NS NS NS NS

*** *** NS NS NS

*** ** NS * NS

*** *** ** NS NS

*** *** ** * NS

*** *** ** ** NS

* NS * ** NS

*** *** *** NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

D. Borgognone et al. / Scientia Horticulturae 149 (2013) 61–69

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Table 5 Main effects of N form and graft combination on total and marketable yield, blossom end rot and marketable fruit mean weight and number of tomatoes in the third experiment. Treatments

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Total yield (kg/plant)

Marketable yield (kg/plant)

BER (% of the total)

Marketable fruit Mean weight (g/fruit)

Number (no./plant)

1.22 1.31 0.84 0.77

1.16 1.20 0.74 0.64

5.0 7.5 11.6 18.8

31.0 30.0 30.3 28.7

37.7 40.0 25.6 21.0

1.03 1.05

0.90 0.97

12.9 8.6

28.1 31.8

31.4 30.8

NS * NS NS NS

* ** NS NS NS

** *** NS NS NS

NS NS NS NS NS

* ** NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

Table 6 Main effects of N form and graft combination on macro and microelement concentrations of tomato leaves in the third experiment. Treatment

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Macroelements (g/kg)

Microelements (mg/kg)

N

P

K

Ca

Mg

Fe

Mn

Zn

Cu

B

32.3 36.3 38.5 39.1

4.7 5.4 6.9 7.2

37.2 37.4 37.2 41.1

27.7 16.9 17.5 11.8

4.0 3.1 2.7 2.5

42.4 52.7 59.5 67.4

43.8 61.0 66.7 127.4

47.5 40.4 49.0 74.8

12.5 10.8 10.3 14.0

47.1 45.6 58.6 54.4

37.2 35.9

6.1 6.0

37.1 39.4

18.0 18.9

3.2 2.9

49.5 61.5

75.7 73.8

54.7 51.1

11.9 11.8

50.4 52.5

** ** * NS NS

* *** NS NS NS

NS NS NS NS NS

* ** NS NS NS

** *** NS NS NS

* ** NS NS NS

* ** NS NS NS

NS NS NS NS NS

NS NS NS NS NS

* * NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

and total protein concentrations increased in both organs as NH4 + proportion increased. 4. Discussion The sensitivity of many species to ammonium nutrition is expressed as a suppression of growth and physiological disorders

(Goyal and Huffaker, 1984). Different physiological and biochemical hypothesis may explain the action of ammonium in plants: acidification of the growth medium and NH4 + -toxicity per se leading to antagonism in cation uptake, and/or alterations in osmotic balance (Britto and Kronzucker, 2002; Horchani et al., 2010). The pH of the rooting medium is of paramount importance for plant growth as a large number of processes (e.g. nutrient availability

Table 7 Main effects of N form and graft combination on microelement concentration of tomato fruits in the third experiment. Treatment

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Macroelements (g/kg)

Microelements (mg/kg)

N

P

K

Ca

Mg

Fe

Mn

Zn

Cu

B

14.2 17.8 18.0 21.2

9.2 9.4 10.2 10.4

37.5 38.5 34.8 32.4

10.6 11.6 9.2 7.1

1.6 1.6 1.3 1.2

23.4 32.7 33.7 37.6

8.0 7.0 9.2 9.8

19.8 24.8 25.0 29.5

2.8 2.3 3.2 5.4

8.7 9.2 8.4 10.2

16.6 19.0

10.0 9.6

35.9 35.7

9.4 9.9

1.4 1.5

28.9 34.8

8.4 8.6

23.2 26.4

2.7 4.2

8.8 9.5

* ** NS NS NS

NS NS NS NS NS

NS NS NS NS NS

* ** NS NS NS

* *** NS NS NS

* ** NS NS NS

NS NS NS NS NS

* * NS NS NS

NS NS NS NS NS

* * NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

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Table 8 Main effects of N form and graft combination on glucose, fructose, sucrose and total soluble carbohydrates of tomato leaves and roots in the third experiment. Treatment

Soluble carbohydrates (mmol g−1 FW) Glucose

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

Fructose

Sucrose

Total

Leaves

Roots

Leaves

Roots

Leaves

Roots

Leaves

Roots

9.94 13.79 15.34 21.01

0.41 0.57 0.40 0.71

12.64 14.63 18.79 25.42

0.63 1.32 1.06 1.60

5.89 5.68 7.22 8.48

3.94 3.84 3.34 3.64

28.47 34.09 41.35 54.91

4.90 5.36 4.77 5.74

14.83 15.21

0.56 0.48

17.95 17.79

1.28 1.03

6.79 6.85

3.81 3.57

39.57 39.85

5.49 4.90

*** *** NS NS NS

NS NS NS NS NS

*** ** NS NS NS

NS * NS NS NS

* ** NS NS NS

NS NS NS NS NS

*** *** NS NS NS

NS NS NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

and uptake rate, availability of toxic ion species) are closely related to this parameter (Marschner, 1995). In Exp. 1, the increased pH of the nutrient solution from 3.5 to 7.5 did not affect the plant growth parameters of tomato in accordance with the findings of Islam et al. (1980), who observed that tomato and French bean seem to grow well in solution culture even at pH values up to 8.5. The high pH in the nutrient solution reduced the Fe concentration and suppressed markedly the Mn and Zn contents in the shoot (Table 1). This is a well known effect of high rhizosphere pH on the uptake of Fe, Mn, and Zn (Marschner, 1995). Our results also showed that low pH reduces the uptake of Ca and Mg, in accordance with the key role of the plasma membrane-bound proton efflux as the driving force for ion uptake (Marschner, 1995). When averaged over the pH level of the nutrient solution, the highest Ca, Fe, Zn, and Cu concentrations were recorded in grafted compared with selfgrafted tomato plants (Table 1), indicating that grafted plants facilitated the uptake of nutrients to the shoot even under inappropriate external pH. Our results are in line with our previous observations (Colla et al., 2010a), where we observed that watermelon plants grafted onto pumpkin rootstocks and exposed to an excessively high external pH level (8.1) were capable of maintaining a better plant nutritional status in the shoot tissue in comparison with ungrafted plants. It is often reported that the use of ammonium as sole or dominating N source results in impaired growth of many plant species

(Claussen, 2002; Guo et al., 2007). Exp. 2, confirmed that plant growth characteristics of tomato, such as plant height, leaf number, leaf area, and shoot dry biomass, decreased sharply when N was exclusively provided as NH4 + (0:100, Table 2). In several studies growth inhibition by ammonium uptake has been related to the pH decrease in the root environment leading to an impairment of the root plasma membrane (Marschner, 1995). In Exp. 2, the pH was kept constant between 5.4 and 5.8 and therefore could not have caused growth inhibition. As frequently reported, plants supplied solely with NH4 + have smaller leaf areas and/or lower leaf numbers compared to those supplied with nitrate as sole or dominating N source (Raab and Terry, 1994; Walch-Liu et al., 2000). Our results are in agreement with the above findings showing a reduction in leaf area, leaf number and shoot biomass, when N was supplied exclusively as ammonium (NO3 − :NH4 + ratio of 0:100). This unfavorable effect of ammonium on leaf expansion has been attributed to reduced osmotic regulation (Leidi et al., 1992), concomitantly reduced rates of leaf cell expansion (Raab and Terry, 1994), or hormonal regulation between root and shoot (Walch-Liu et al., 2000). The NO3 − :NH4 + ratio of the nutrient solution influences also the uptake of other ions; the uptake of all ions is involved in maintaining electroneutrality within the plant (Kotsiras et al., 2002). Increased ammonium supply enhanced the levels of N, P, Fe, Mn,

Table 9 Main effects of N form and graft combination on nitrate, ammonium, amino acids, and proteins of tomato leaves and root in the third experiment. Treatment

Nitrogen compounds (␮mol g−1 FM) NO3 −

NO3 :NH4 100:0 70:30 30:70 0:100 Graft combination Self-grafted Grafted Significancea N-form Linear Quadratic Graft combination (G) N-form × G a

NH4 +

Amino acids

Proteins

Leaves

Roots

Leaves

Roots

Leaves

Roots

Leaves

Roots

39.6 31.1 22.1 1.2

23.1 17.6 10.9 0.7

0.45 1.14 3.43 4.89

1.45 5.61 18.75 21.10

27.05 33.02 47.70 51.16

5.44 6.72 9.40 10.54

17.12 19.99 18.37 21.21

1.29 2.09 3.39 3.98

12.4 14

23.5 24.3

2.43 2.53

11.08 12.40

40.75 38.72

9.02 7.03

18.90 19.44

3.21 2.17

*** *** ** NS NS

*** *** ** NS NS

* ** NS NS NS

** ** NS NS NS

*** *** NS NS NS

* * NS NS NS

NS * * NS NS

** ** NS NS NS

NS = not significant; *, **, *** = significant at P ≤ 0.05, 0.01 and 0.001, respectively.

D. Borgognone et al. / Scientia Horticulturae 149 (2013) 61–69

Zn, Cu, and B in leaves and on other hand decreased the Ca and Mg concentrations in leaf tissues (Table 3). The large decrease in N concentration in tomato shoot as the NO3 − :NH4 + ratio increased is in line with the results reported previously for pepper (Bar-Tal et al., 2001). Moreover, our results showed that the uptake of major cations (Ca and Mg) was reduced with increasing external NH4 + concentrations (Wiesler, 1997; Chance et al., 1999), through the mechanism of charge balance in ion uptake, since nitrogen is a dominant macronutrient, its ionic form controls cation and anion uptake as was also reported elsewhere for tomato (Ganmore-Neumann and Kafkafi, 1980). The results of Expts. 1 and 2 carried out at early developmental stage, showed that the effect of NO3 − :NH4 + ratio on crop growth was more pronounced than the effect of the nutrient solution pH. The results also demonstrated that grafted tomato plants exhibited higher crop performance than self-grafted plants. It was reported that grafting directly affects plant growth and yield (Rivero et al., 2003) by interactions of some or all of the following processes: increase of water and nutrient uptake resulting from the vigorous root system of the rootstock (Lee, 1994; Ruiz et al., 1997; Colla et al., 2010a, 2011), and enhanced production of endogenous hormones (Zijlstra et al., 1994). The joint action of some or all of these processes could explain the higher growth of grafted tomato plants observed in the current study. For the above reasons, a third experiment was aimed to confirm the responses of self-grafted and grafted tomato to nitrogen form (NO3 − :NH4 + ratio) on a long term. Our results in Exp. 3 showed, that a decrease in the NO3 − :NH4 + ratio from 100:0 to 0:100 restricted both vegetative growth and fruit yield in tomato (Tables 4 and 5). These results agree with those of Elia et al. (1996) and Savvas et al. (2010a), who reported that the supply of 30% of total-N in the form of NH4 + -N (Elia et al., 1996) and increasing the NH4 + -N concentration in the nutrient solution from 1.0 mM to 4.0 mM (Savvas et al., 2010a) restricted the total plant biomass and yield of eggplants. Similar results have also been reported for other Solanaceous species grown in greenhouses such as tomato (Siddiqi et al., 2002; Akl et al., 2003) and pepper (BarTal et al., 2001). Moreover, the incidence of BER increased as the NO3 − :NH4 + ratio decreased. The effect of NH4 + on BER observed in the current study is consistent with those reported previously for tomato (Ho et al., 1993) and pepper (Bar-Tal et al., 2001). Ammonia toxicity at the intracellular level (Raab and Terry, 1994) due to high rates of NH4 + uptake is a likely explanation for the suppressive effects of the high NH4 + treatment on tomato growth and yield. Another possible cause of growth and yield reduction at high NH4 + level is the well-known suppressive effect of NH4 + ions on the uptake of macronutrients (Marschner, 1995; Kotsiras et al., 2002). In our study, leaf K concentrations were not affected by the increased supply of NH4 + in the nutrient solution, while leaf Ca and Mg levels were significantly reduced (Table 6). The Ca concentration in the tomato leaves dropped below the threshold of sufficiency according to Mills and Jones (1996) (16.0 g/kg DW) when NH4 + was the only source of nitrogen, while the Mg concentration reached a deficiency level (below 3.6 g/kg DW) when the NH4 + proportion was higher than 30% of total nitrogen applied (Table 6). Hence the strong reduction in shoot tissue of Ca and especially Mg contents were presumably responsible of the poor growth and fruit yield of tomato plants grown with high concentration of NH4 + in the nutrient solution. The Ca and Mg concentrations of the fruits decreased with increasing NH4 + in the nutrient solution (Table 7). Calcium deficiency during NH4 + nutrition can induce loss of membrane integrity, which in turn may lower the concentration of Mg and influence the function of chloroplasts and mitochondria (Pill and Lambeth, 1977). In addition, higher rates of organic acid synthesis as a result of NH4 + nutrition may immobilize Ca and Mg within

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the roots (Kotsiras et al., 2002). Although the Ca supply to the fruit is considered to be an important factor in the occurrence of BER, efforts to define critical values or even to correlate BER incidence with Ca concentration in tomato fruits have failed in many experiments (Nonami et al., 1995). However, in the current experiment BER incidence was negatively correlated with Ca content in tomato fruits (r = -0.744**). The photosynthetic activity at the highest NH4 + concentrations decreased sharply, since the NH4 + can be accumulated in plants so as to damage the membrane configuration and uncouple photophosphorylation with non-photophosphorylation. Thereby, the fixation of CO2 is reduced and photochemical efficiency decreases (Guo et al., 2006). Moreover, as the assimilation of NH4 + requires substantial amounts of 2-oxoglutarate obtained from glucose (ultimately sucrose imported from leaves) it has been proposed that tolerance of plants to NH4 + nutrition is associated with adequate carbohydrate status of roots (Schortemeyer et al., 1997). Indeed, most studies showed reduced sugar contents in roots of NH4 + grown plants (e.g. Chaillou et al., 1991), and only occasionally when root growth was severely impaired, were higher sugar levels observed in NH4 + -grown roots (e.g. Walch-Liu et al., 2001). The carbon supply for root growth under NH4 + nutrition is likely to be limiting only when the capacity of shoot to deliver photoassimilate via the phloem is impaired and/or under condition of excessive root respiration (Britto and Kronzucker, 2002). This was not the case since similar levels of glucose and sucrose were recorded in roots grown with different NO3 − :NH4 + ratio, whereas leaf carbohydrate (glucose, fructose, sucrose and total) concentrations increased linearly with NH4 + availability, especially when N was exclusively provided as NH4 + (Table 8). Similarly, Horchani et al. (2010) reported an increasing of carbohydrate content in leaves of tomato plants when ammonium was provided as N source instead of nitrate. The increase of carbohydrate content in leaves of tomato plants grown with high ammonium proportion was probably due to a reduced sink activity resulting from the highest leaf-to-fruit ratio (1.3 vs. 1.0 for 0:100 and 100:0 NO3 − :NH4 + ratio, respectively). As already observed (Cruz et al., 2006; Horchani et al., 2010) root NH4 + and NO3 − concentrations were correlated with NO3 − :NH4 + ratio in the nutrient solution. Root NH4 + increased also for high NO3 − ratio (Table 9). Besides the plant species and the NH4 + concentration in the root medium, plant response to NH4 + nutrition depends on the tissue concentration (Cruz et al., 2006). Our results showed that for the highest NH4 + concentration, root NH4 + content was 21 ␮mol g−1 FM. This root NH4 + concentration has been demonstrated to be associated with significant disturbance in the growth of many plant species, such as lettuce, sunflower (Lasa et al., 2001) and tomato cultivars such as Trust (Cruz et al., 2006). In contrast Horchani et al. (2010) has reported that tomato plants (cv. Rio Grande) grow better under NH4 + compared to NO3 − based nutrition, because the NH4 + tolerance may be related to the efficiency of the plant in sequestering NH4 + in the vacuoles, mainly in root cells. Explanations for this disagreement could be the different environments in which the plants were grown, and variations between tomato cultivars in their sensitivity to NH4 + . Finally, ammonium assimilated in the roots are transported in the plant as amino acids, proteins and other organic N compounds to avoid toxic concentrations of free NH4 + in the xylem sap (People and Gifford, 1997). In the current experiment, the amino acids and total protein concentrations increased in leaves as NH4 + proportion increased. However, the ammonia assimilating enzyme activity in the root was not able to prevent the free NH4 + accumulation in plant organs under high proportions of NH4 + in the nutrient solution leading to ammonium toxicity. The reduction in plant growth and yield by ammonium nutrition in the long term experiment was similar between grafted and self-grafted plants, indicating that the tomato interspecific

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hybrid used as rootstock did not alleviate the negative effects of ammonium nutrition in a sensitive crop such as tomato. 5. Conclusions This study show that in the early growth stages the effect of NO3 − :NH4 + ratio was more pronounced than the effect of the nutrient solution pH on tomato plant growth parameters and mineral composition. In the long-term experiment, the plant growth and yield decreased, whereas the carbohydrate concentrations, amino acids and proteins increased under NH4 + in comparison to NO3 − based nutrition. Moreover, NH4 + toxicity was associated with reduced rates of net photosynthesis. Our results also demonstrated that grafting ‘Moneymaker’ into a vigorous rootstock of tomato interspecific hybrid (‘Maxifort’) did not alleviate the negative effects of ammonium nutrition in a sensitive crop such as tomato. References Akl, I.A., Savvas, D., Papadantonakis, N., Lydakis-Simantiris, N., Kefalas, P., 2003. Influence of ammonium to total nitrogen supply ratio on growth, yield and fruit quality of tomato grown in a closed hydroponic system. Eur. J. Hortic. Sci. 68, 204–211. Baldet, P., Devaux, C., Chevalier, C., Brouquisse, R., Just, D., Raymond, P., 2002. Contrasted responses to carbohydrate limitation in tomato fruit at two stages of development. Plant Cell Environ. 25, 1639–1649. Bar-Tal, A., Aloni, B., Karni, L., Oserovitz, J., Hazan, A., Itach, M., Gantz, S., Avidan, A., Posalski, I., Tratkovski, N., Rosenberg, R., 2001. Nitrogen nutrition of greenhouse pepper. I. Effects of nitrogen concentration and NO3 :NH4 ratio on yield fruit shape, and the incidence of blossom-end rot in relation to plant mineral composition. HortSci 36, 1244–1251. Bonasia, A., Conversa, G., Gonnella, M., Serio, F., Santamaria, P., 2008. Effects of ammonium and nitrate nutrition on yield and quality in endive. J. Hortic. Sci. Biotechnol. 83, 64–70. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Bremner, J.M., 1965. Total nitrogen. In: Black, C.A., Evans, D.D., White, I.L., Ensminger, L.E., Clark, F.E. (Eds.), Methods of Soil Analysis. Agronomy Monograph 9, Part 2, pp. 1149–1178. Britto, D., Kronzucker, H., 2002. NH4 + toxicity in higher plants: a critical review. J. Plant Physiol. 159, 567–584. Britto, D.T., Glass, A.D.M., Kronzucker, H.J., Siddiqi, M.Y., 2001a. Cytosolic concentrations and transmembrane fluxes of NH4+/NH3. An evaluation of recent proposals. Plant Physiol. 125, 523–526. Britto, D.T., Siddiqi, M.Y., Glass, A.D.M., Kronzucker, H.J., 2001b. Futile transmembrane NH4 + cycling: a cellular hypothesis to explain ammonium toxicity in plants. Proc. Natl. Acad. Sci. U. S. A. 98, 4255–4258. Cataldo, D.A., Haroon, M., Schrader, L.E., Youngs, V.L., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6, 71–80. Chaillou, S., Vessey, J.K., Morot-Gudry, J.F., Raper Jr., C.D., Henry, L.T., Boutin, J.P., 1991. Expression of characteristics of ammonium nutrition as affected by pH of the root medium. J. Exp. Bot. 42, 189–196. Chance, W.O., Somda, Z.C., Mills, H.A., 1999. Effect of nitrogen form during the flowering period on zucchini squash growth and nutrient element uptake. J. Plant Nutr. 22, 597–607. Claussen, W., 2002. Growth, water use efficiency, and proline content of hydroponically grown tomato plants as affected by nitrogen source and nutrient concentration. Plant Soil 247, 199–209. Colla, G., Rouphael, Y., Cardarelli, M., Salerno, A., Rea, E., 2010a. The effectiveness of grafting to improve alkalinity tolerance in watermelon. Environ. Exp. Bot. 68, 283–291. Colla, G., Suarez, C.M.C., Cardarelli, M., Rouphael, Y., 2010b. Improving nitrogen use efficiency in melon by grafting. HortSci 45, 559–565. Colla, G., Rouphael, Y., Leopardi, C., Bie, Z., 2010c. Role of grafting in vegetable crops grown under saline conditions. Sci. Hortic. 127, 147–155. Colla, G., Rouphael, Y., Mirabelli, C., Cardarelli, M., 2011. Nitrogen-use efficiency traits of mini-watermelon in response to grafting and nitrogen-fertilization doses. J. Plant Nutr. Soil Sci. 174, 933–941. Crinò, P., Lo Bianco, C., Rouphael, Y., Colla, G., Saccardo, F., Paratore, A., 2007. Evaluation of rootstocks resistance to fusarium wilt and gummy stem blight and effect on yield and quality of a grafted inodorus melon. HortSci 42, 521–525. Cruz, C., Bio, A.F.M., Dominguez-Valdivia, M.D., Aparicio-Tejo, P.M., Lamfus, C., Martins-Louc¸ao, M.A., 2006. How does glutamine activity determine plant tolerance to ammonium? Planta 223, 1068–1080. Dijk, E., Grootjans, A.B., 1998. Performance of four Dactylorhiza species over a complex trophic gradient. Acta Bot. Neerl. 47, 351–368.

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