Abstract
BACKGROUND:
Nutrient solution composition affects plant growth, yield and fruit quality. Nitrate, ammonium and urea are absorbable forms of nitrogen for plants however each form can have different effects on plant growth aspects. Urea is a low cost source of nitrogen and urease enzyme is activated by nickel (Ni), so using these nutrients may be beneficial for strawberry plant growth. The influence of different nutrient solution on strawberry cv. “Paros” growth, enzymatic activity and leaf mineral content was studied in this research at Shiraz University, Shiraz, Iran.
OBJECTIVES:
This study aimed to understand the role of Ni and different forms of nitrogen in the hydroponic culture on growth, nutrient composition and enzyme activity of strawberry plants, to identify whether a combination of different nitrogen form, especially urea and Ni, would improve plant growth and quality.
METHODS:
The effects of nitrate: ammonium: urea ratios (75:25:0, 50:25:25, 25:25:50, 25:0:75 and 0:0:100) and nickel sulfate concentration (0, 1 and 2 mg/L) in nutrient solutions were evaluated on strawberry cv. ‘Paros’ growth, mineral nutrient, and enzymatic activity. The trial was laid out as a factorial experiment with a completely randomized design. Rooted strawberry daughter plants were potted in 3 liter pots filled with cocopeat: perlite (1:1). Application of treatments started after establishment of the plants and continued for 5 months.
RESULTS:
Urea (25% and 50%) and nickel (1 mg/L) adding in the nutrient solution improved plant shoot and root dry weights. However, greater urea concentrations in the nutrient solution decreased nitrogen, potassium and phosphorous contents in leaves, but increased iron concentration. Urea concentration in strawberry leaves decreased when nickel concentration in the nutrient solution increased. Nitrate content in leaves of urea-fed plant decreased when urea concentration in nutrient solution increased. Urease activity increased with adding urea to the solution; however nitrate reductase activity was low at a high level of urea in nutrient solution.
CONCLUSION:
In addition to nitrate, adding nickel and urea in the nutrient solution improved growth and the enzymatic activity of strawberry plants, however it appeared that a higher rate of nickel and using urea as the only source of nitrogen was less beneficial. Overall, the best result was obtained from nickel at 1 mg/L and N source ratio of nitrate (50): ammonium (25): urea (25).
Introduction
Strawberry (Fragaria × ananassa Duch.) is one of the small fruits widely grown around the world [1], both in soil and hydroponic systems. Nutrient solution composition could noticeably have an impact on strawberry yield andfruit quality [2]. Nitrate and ammonium are the absorbable forms of nitrogen (N) by plants. Also urea is hydrolyzed to ammonium in the soil by the microorganism, but the plant can also take up urea directly [3, 4]. The preferred amount of each form depends on the plant species and environmental conditions [5]. Nitrate ions are not adsorbed by soil colloids and are easily leached from the soil [6]. Furthermore, the amount of nitrate absorption by vegetable plants is high [7]. Nitrate in high concentration causes various concerns and human diseases like methemoglobinemia [8]. It appears that applying ammonium can solve this problem, but an important disadvantage is the sensitivity of plants to the high amount of ammonium, which causes toxicity in plants. The uptake of ammonium and nitrate can affect the uptake of other cations and anions by changing the rhizosphere pH [9]. Changes in the rhizosphere pH can affect both nutrient solubility and nutrient uptake [9]. There are some reasons to explain ammonium toxicity. For instance, analyzing the ammonium-fed plant’s tissue shows an increase in ammonium, chloride, sulfate, and phosphate contents. In contrast, the concentration of essential cations such as K +, Ca2 + and Mg2 + is decreased [10]. Another absorbable form of N is urea. It can be suitable for the cultivation of field crops. In addition, urea is one of the low cost fertilizers that has high N content (46%). But assimilation and detoxification of urea in plants depend on urease enzyme. Urease is a metalloenzyme, catalyzes the hydrolysis of urea to form ammonia and carbon dioxide [11]. Urea seems to be assimilated in the root [12]. It is probably stored in vacuoles, whereas the urease is located in the cytoplasm. So the concentration of urea in shoots is higher than would be predicted from the rates of potential urease activity [4].
Nickel (Ni) is one of the essential micro elements [12] that is involved in N metabolism as a metal component of the enzyme urease [9]. Ni is an important element for those plants using urea as the sole source of N [13]. In pecan, deformed leaves are created as a result of Ni deficiency [14]. Jamali et al. [15] reported the promoting effect of Ni in combination with salicylic acid on strawberry growth. Mihucz et al. [16] reported that applying Ni in the urea solution decreased urea toxicity symptoms in cucumber leaves and increased root dry weight. Eshghi and Ranjbar [1] studied the effect of Ni and urea on the growth of strawberry, reporting NiSO4 at 150 and 300 mg L–1 along with urea at 2 g L–1as the best treatments. Growth of lettuce was increased by decreased leaf urea content of urea-fed plants in the presence of Ni in the applied solution, and the health quality of nitrate-fed plants was improved too [17]. Leon et al. [18] reported that the low concentration of Ni had a positive effect on the growth of Cunonia macrophylla. There are few studies, however, regarding the effect of Ni and urea on trace elements, and strawberry has received much less attention at this point. In this respect, the present study was designed to investigate the interaction between ammonium, nitrate, urea ratios and Ni concentrations on nutrient uptake, growth and the enzymatic activity of strawberry plants in a soilless culture.
Materials and methods
Growing conditions and treatment
This study was conducted in the greenhouse of Shiraz University, Shiraz, Iran. The experiment was a factorial with a completely randomized design involving four replicates and two pots in each replication. The factors included five ratios of nitrate: ammonium: urea (75:25:0, 50:25:25, 25:25:50, 25:0:75 and 0:0:100) and three rates of Ni of 0, 1 and 2 mg/L in the form of nickel sulfate. The concentrations of other mineral nutrient elements are shown in Table 1. All other nutrients were added to the solution according to Hoagland solution [19]. Rooted daughter plants of strawberry (Fragaria × ananassa Duch.) cv. ‘Paros’ were planted in 3 liter pots filled with cocopeat and perlite (1:1). Light intensity was >800 μmol·m2.s–1; relative humidity was set at 50±5% and day and night temperatures were 25/15±3°C during the experimental period. The solution pH was adjusted to 6.2 by adding HCl as a pH buffer. Treatment application was performed when plants were well established and each produced 5-6 fully expanded leaves (September 2015). Solutions were given to strawberry plants every other day and after each week; pots were leached with tap water to prevent salt accumulation in pots. The experiment lasted for 5 months (to March 2016).
Concentration of salts in nutrient solutions
Concentration of salts in nutrient solutions
At the end of the experiment, plants were harvested. Plants were separated into roots and shoots. Each part was dried for 48 hours in the oven at 70°C for the dry weight of each part. Then, leaves were ground to a fine powder to measure elements. The Total-N was measured using Kjeldahl method [20]. The remaining dried leaves were ashed at 480°C for 12 hours and dissolved in 10 ml HCl (2M). Micronutrients (Fe, Zn and Ni) were analyzed with atomic absorption spectrophotometry (Atom-Absorption-Spectrometer FMD4). Potassium was measured by flame photometry (model- JENWAY PFP7). Phosphorous concentration was determined with colorimetric assay [21].
Determination of Urea-N and NO3-N
To determine the concentration of urea-N and NO3-N, the samples were extracted with hot water. Glass tubes containing 0.1 mg of the dried leaf sample were placed in boiling water, and then centrifuged for 60 min. The extraction solution was passed through Whatman 42 filter paper. The concentration of urea-N was determined using the method developed by Cline and Fink [8], and the concentration of NO3-N was determined by colorimetric assay [23].
Enzyme activity
To measure urease activity, mature leaves with no damage symptoms were selected and dried at 20°C. Then, they were ground to a fine powder. Urease activity was measured as described by Frankenberger & Tabatabaei [24]. For nitrate reductase (NR) assay 0.5 g leaf powder was extracted in 5 ml buffer (0.1M phosphate buffer, pH = 7.5) containing 0.5 mM EDTA. The nitrate reductase activity in the leaves of experimental plants was measured by using the method developed by Stewart et al. [25]. Superoxide dismutase activity was measured based on enzyme ability to stop photochemical reduction of NBT by superoxide radical in the presence of riboflavin [26].
Statistical analysis
All data were subjected to analysis of variance using Statistix 10. The treatment means were tested for significance differences using the LSD at P < 0.05.
Results
Plant growth
The effects of N-sources and different Ni concentrations on the root and shoot dry weight (RDW, SDW) are shown in Table 2. Comparing all N ratios, the nitrate (25%): ammonium (0%): urea (75%) resulted in greater RDW than the other treatments. Plants not treated with Ni and plants treated with 1 mg/L Ni had the highest RDW, whereas 2 mg/L Ni decreased root dry weight. In nitrate and urea (100%) fed plants supplemented with Ni, RDW was decreased. The highest RDW was obtained in nitrate (25%): ammonium (25%): urea (50%) and 0 or 1 mg/L Ni.
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on root and shoot dry weight of strawberry cv. ‘Paros’
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on root and shoot dry weight of strawberry cv. ‘Paros’
Means followed by different small letters indicate significant differences at P < 0.05 within the interaction. Means followed by different capital letters indicate significant differences at P < 0.05 for main effects.
Independent of Ni absence or presence in the nutrient solution, solution with 50% urea resulted in the highest SDW. Similarly to RDW, adding 1 mg/L Ni had no effect on SDW and 2 mg/L Ni decreased shoot dry weight. Only in plants treated with nitrate (50%): ammonium (25%): urea (25%) and nitrate (25%): ammonium (25%): urea (50%) did the addition of 1 mg/L Ni elevate the SDW compared with other treatments and highest SDW was observed with the combination that had 50% urea and 1 mg/L Ni.
Urease activity was significantly influenced by N sources and Ni concentrations (Table 3). In urea-fed plants, urease activity was significantly higher than plants with no urea as an N source. Comparing all Ni treatments, the highest urease activity was obtained when 1 mg/L of Ni was applied (Table 3).
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on enzymatic activity of strawberry cv. ‘Paros’
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on enzymatic activity of strawberry cv. ‘Paros’
Means followed by different small letters indicate significant differences at P < 0.05 within the interaction. Means followed by different capital letters indicate significant differences at P < 0.05 for main effects.
The highest NR activity was found in the treatments with solutions containing nitrate (75%): ammonium (25%): urea (0%). NR activity was highest at the highest Ni rate. Table 3 showed that for some N-source combinations, nickel addition increased NR activity; in the case of urea (100%), Ni addition had no effect on enzyme activity.
The effect of ammonium: nitrate: urea ratios and Ni on SOD was significant (Table 3). SOD activity in plants fed with high nitrate (75%) was much higher than other N ratios. SOD activity was significantly higher at the highest Ni rate. The highest SOD activity was from the treatment nitrate (75%): ammonium (25%): urea (0%) and 2 mg/L Ni. In contrast, the lowest enzyme activity was observed in nitrate (25%): ammonium (25%): urea (50%) and 2 mg/L Ni.
Leaf total N content
Leaf total N content was affected by different N sources and Ni concentrations. Data showed that N concentration was decreased with increasing urea concentration in solution (Table 4). Increasing the Ni supply in plants significantly decreased the N in the leaf tissue (Table 4). The highest N concentration was in the absence of Ni with the solution that contained nitrate (75%): ammonium (25%): urea (0%).
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on macro element content of strawberry cv. ‘Paros’
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on macro element content of strawberry cv. ‘Paros’
Means followed by different small letters indicate significant differences at P < 0.05 within the interaction. Means followed by different capital letters indicate significant differences at P < 0.05 for main effects.
The effect of Ni concentrations and nitrate: ammonium: urea ratios on the concentration of Ni in leaves can be seen in Table 5. The concentration of Ni was increased in plants when Ni concentration was increased in the nutrient solution. Supplying 75 and 100 percent of the plant N requirement in urea form decreased the leaf Ni content.
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on micro element contents of strawberry cv. ‘Paros’
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on micro element contents of strawberry cv. ‘Paros’
Means followed by different small letters indicate significant differences at P < 0.05 within the interaction. Means followed by different capital letters indicate significant differences at P < 0.05 for main effects.
Plants treated with 75% or 50% nitrate had greater K content than those grown in high urea content. Ni application had no effect on K content (Table 4). The effect of Ni supplement on the P content was not significant, but adding a high urea content in nutrient solution reduced the P content (Table 4). Only in solutions with nitrate (25%): ammonium (0%): urea (75%) P content was less than that of other treatments. In all treatments, iron concentration was increased with added Ni in the nutrient solution, and applying urea increased the iron content in the plants (Table 5). Zn was not influenced by Ni or different N sources (data not shown).
Leaf urea content
Accumulation of urea in plants receiving high urea content was much greater than in those grown in the solution without urea or with the lowest urea concentration (Table 6). Adding Ni to the nutrient solution reduced urea content in all plants, even in plants receiving all N requirements in the form of nitrate (Table 6).
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on urea-N and NO3
- -N contents of strawberry cv. ‘Paros’
Effect of Nitrate: Ammonium: Urea ratios and nickel concentration and their interaction supplied in nutrient solution on urea-N and NO3 - -N contents of strawberry cv. ‘Paros’
Means followed by different small letters indicate significant differences at P < 0.05 within the interaction. Means followed by different capital letters indicate significant differences at P < 0.05 for main effects.
Leaf nitrate concentration was significantly affected by N sources (Table 6). Nitrate content was much greater in plants grown in solutions that contained nitrate than those receiving urea. Applying Ni in the nutrient solution decreased nitrate content in all plants (Table 6).
Discussion
Generally, the highest shoot dry weight was observed in plants that received 50% of their N from urea. Decreasing nitrate concentration to 25% and increasing urea concentration to 50% of N-source promoted the growth. Urea in high concentrations decreased growth, probably as a result of urea or ammonium toxicity [27]. Nitrate reduction in plants is an energy-intensive progress and 15 moles Adenosine triphosphate [ATP] are required for the reduction of 1 mol nitrate and additional 5 moles ATP for ammonium assimilation [28]. In barley, up to 23% of energy from root respiration is required for nitrate assimilation [29]. So growth reduction at a high rate of nitrate could be expected.
High concentration of Ni decreased shoot and root dry weight. Recent reviews by Yusuf et al. [30] noted that Ni toxicity on plants can limit growth of all plant organs (i.e. root, shoot, leaves). Ni plays role as a trace element (≥1 mg. kg–1DW and ≤1000 mg. kg–1DW) in many species and as an ultra-trace element (>1 mg. kg–1 DW) in others [31]. A high concentration of Ni has been observed in plants grown in nutrient solutions with ammonium, however plants fertigated with high urea ratios (75% and 100%), Ni content decreased significantly. Ni absorption is facilitated when plants are grown in solutions with acidic pH, especially lower than 6 [28].The presence of ammonium in the solution might increase nickel absorption more than that in urea-fed plants because of decreasing pH, by releasing H + to the rhizosphere as a result of its absorption. Also, the high amount of Ni in plants might be a result of nitrate existence. Hu et al. [32] reported that the greater expression of the IRT1 gene could be responsible for the coding transporter of divalent cation including Ni2 + in Arabidopsis in the presence of nitrate.
N concentration was decreased in plants grown in a high concentration of urea and these plants had a lower content of N than nitrate-fed plants. Urea not only decreased N absorption, but disrupted protein assimilation and accumulated asparagine in a considerable concentration [33]. The growth of sunflower plants grown in 2 mM nitrate was much higher than those grown in 8 mM urea [33]. Urea is taken up by plant in a molecular form [3, 4]. The relationship between the diameter of uncharged molecules and the rates at each permeate membrane was inverse. The reflection coefficient is defined as between 0.00 and 1.00, where 1.00 indicates that the membranes are impermeable to the solute. For urea, molecule reflection coefficient was 0.76 [9].
The lowest P content was in plants grown in the ammonium free solution. Phosphate ions absorption depended on the nutrient solution pH. Hendrix [34] reported that the absorption of phosphate in a solution with pH 4 was 4 times higher than that at pH 8.7 in bean. Presumably, ammonium elimination from solution increased pH, resulting in less phosphate absorption. Rayer et al. [35] observed the stimulatory effect of ammonium on the uptake and content of P in soybean.
K content in strawberry plants was enhanced with increasing nitrate concentration in the nutrient solution. Applying urea in the solution fed to zucchini plants depressed K content in the plants [36]. The role of potassium ions in anion-cation balance is clear during nitrate metabolism [9]. The high concentration of K in nitrate grown plants may be explained by the role of K as a counter-ion in the nitrate transport through the xylem to the shoot [9]. Through nitrate reduction in shoots, charge balance has to be maintained by the corresponding net increase in organic acid anions (e.g., malate). K plays a role as an accompanying cation for re-translocating organic acid in the phloem to the root [9]. Anion (e.g. nitrate) absorption increases the rhizosphere pH; in contrast, the rhizosphere pH is decreased by cation absorption. Increasing OH– not only occurs by nitrate ion absorption, but also as a result of nitrate reduction [9].
Urea and Ni significantly increased Fe content of the strawberry plants. In an experiment with Arabidopsis, the leaves of ammonium-fed plants had a higher Fe concentration than leaves of nitrate-fed plants (37). Assimakopolou [38] showed that high pH in the rhizosphere owning to NO3-N supply reduced the shoot P concentration and suppressed markedly the Fe, Mn, Zn and Cu content of spinach. Islam et al [39] showed plant growth reduction from microelements deficiency as a result of the increasein the rhizosphere pH. Nisupplementationinfenugreek resulted in a decrease in iron concentration [40], whereas there was a strong positive correlation between iron and Niin barley shoots [41]. In this research urea content increased in strawberry plants with low Ni content. In our trial, supplying all N in urea-N form decreased nitrate concentration to about 61% of that when N was supplied as nitrate (75): ammonium (25): urea (0). Application of Ni in the nutrient solution decreased nitrate concentration in plants. A lower amount of urea in the nitrate fed plant showed urea was produced in plants from other activity (e.g. protein degradation in mature leaves), at the onset of reproductive growth and cytokinins degradation [42].
Application of Ni in all plants decreased urea content. Urea addition to the solution caused a high urease activity. Urea is hydrolyzed to ammonium and CO2 by urease enzymes [3]. Ni deficiency causes lower urease activity and accumulation of urea to toxic level [42]. Nitrate reductase activity was greater inform the nitrate (50): ammonium (25): urea (25) treatment than other treatments. There are some different reports regarding the effect of Ni on NR activity. Khoshgoftarmanesh et al. [17], for example, reported thatNi had a positive effect on the growth of lettuce as a result of its role in NR activity, whereas Ni addition to bean growth medium decreased NR activity [44]. The effect of Ni on nitrate assimilation is still unclear and needs further study. Superoxide dismutase activity is elevated in the presence of Ni. Ni is involved in the function of at least 9 proteins including SOD [45]. There has not been much study regarding the effect of Ni and N-sources ratios on SOD activity and our knowledge about it is limited.
Conclusion
The data presented in this study showed the significant influence of Ni and ammonium: nitrate: urea ratios on mineral nutrient concentrations, growth and the enzymatic activity of strawberry plants. Urea applied in the nutrient solution increased shoot and root dry weight, and Ni (1 mg/L), as an essential element, improved growth of urea-fed (25 or 50% of N ratio) plants. Application of urea as a common and old form of Nin the nutrient solution led to the low accumulation of nitrate in plants. The positive role of urea and Ni in reducing nitrate content in the human diet could be important in terms of human health safety. In the presence of Ni, some elements’ absorption was increased, such as iron, and urea concentration in plants was low. All three enzymes (SOD, NR and Urease) had a high activity in the presence of Ni. This trial focused on vegetative growth and plant health, but the next step would be to examine the effects of strawberry yield and fruit quality. Therefore, further investigation is needed to find the optimum N-sources ratio and Ni concentration for the best growth and fruit yield and quality.
Conflict of interest
The authors have no conflict of interest to report.
Footnotes
Acknowledgments
The work was supported by Shiraz University.