1. Introduction
´Valencia´ orange (Citrus sinensis L. Osbeck) is one of the main citrus species cultivated in northeastern Mexico, where calcareous soils predominate. Most crops established in these soils show zinc (Zn) deficiency (Bolan et al., 2023), which affects citrus production in soils with high calcium carbonate (CaCO3) content and, occasionally, in acidic soils (Fu et al., 2016).
Zn is an essential element for plants, involved in the synthesis of chlorophyll, nucleic acids, and auxins. Additionally, it acts as a regulatory cofactor in various enzymes and participates in protein metabolism (Asadi et al., 2020). A deficient Zn concentration is critical in crops, as it leads to reduced tree vigor, lower photosynthetic activity, poor fruit setting, and smaller fruit size and quality (Fei et al., 2016).
The fertilizers commonly used to manage Zn deficiency are monohydrated zinc sulfate (ZnSO4H2O), heptahydrated zinc sulfate (ZnSO47H2O), and synthetic chelates such as EDTA-Zn and DTPA-Zn. In calcareous soils, these fertilizers exhibit low efficiency when applied directly to the soil or through fertigation, as Zn is rapidly retained by the CaCO3 and MgCO3 present in these soils (Srivastava & Singh, 2005).
Foliar fertilization is considered the most efficient method for correcting Zn deficiency in fruit crops grown in alkaline soils, compared to soil applications (Xie et al., 2020). However, the metal salts and synthetic chelates commonly used for this purpose exhibit low absorption by leaves (Souri & Hatamian, 2019). Their translocation to other plant organs such as the stem, roots, and fruit is limited, due to the low permeability of the cell membrane and the mobility of Zn within the plant (Gupta et al., 2016). For these reasons, Zn deficiency persists in citrus crops. An alternative approach to mitigate Zn deficiency is the application of alternative fertilizers to traditional ones, such as amino chelates and micro-fertilizers (flowable sources) (Macedo et al., 2021; Souri & Hatamian, 2019).
Amino chelates are newly formulated fertilizers that have been evaluated in various crops. They exhibit high efficiency of use and do not cause environmental pollution (Souri & Hatamian, 2019); additionally, they improve the translocation of micronutrients such as Zn within the plant (Ghasemi et al., 2013). Zinc oxide (ZnO) is poorly soluble in water, which limits Zn absorption by plant leaves when applied as a foliar fertilizer. However, this limitation can be overcome by reducing the particle size of the oxide to microparticle level (0.2-20 µm) (Du et al., 2015).
This research evaluated the effect of foliar application of amino-chelates and Zn microparticles on the nutritional status and growth of ´Valencia´ orange trees grown in calcareous soil, aiming to determine the most effective Zn source for correcting Zn deficiency.
2. Methodology
2.1 Growing conditions and experimental design
The experiment was conducted in a greenhouse at the Colegio de Postgraduados, Montecillo, Texcoco, State of Mexico, Mexico, located at latitude 19° 27' 30'' N and longitude 98° 54' 14'' W, at an altitude of 2,240 meters, with an average annual temperature of 15.9 °C and annual precipitation of 680 mm. Nine-month-old ´Valencia´ orange trees (Citrus sinensis L. Osbeck), grafted onto C35 rootstock (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf.), were used. The average plant height at the time of transplanting was 75 cm. The trees were transplanted into 21 L capacity pots, which contained 18 kg of calcareous soil. The physical and chemical characteristics of the soil at the beginning of the experiment were as follows: pH = 8.1, clay texture, electrical conductivity (EC) of 0.36 dS m⁻¹, organic matter content of 3.2%, extractable Olsen phosphorus of 8.5 mg kg⁻¹, Kjeldahl nitrogen of 0.2%, and exchangeable potassium of 1.2 cmol(+) kg⁻¹. The concentrations of Zn, Cu, Mn, and Fe in the soil were 1.2, 0.5, 17, and 2.4 mg kg⁻¹, respectively, while the CaCO3 concentration was 46.5%.
At the time of tree transplanting, the soil was fertilized with N, P, K, Mg, Fe, Cu, Mn, and B (30.0, 21.6, 30.0, 5.0, 1.4, 1.4, 1.4, and 0.1 g per tree, respectively), except for Zn, which was the element being evaluated. Urea and diammonium phosphate were used to supply N and P, while the respective sulfates provided K, Mg, Fe, Cu, and Mn, and B was supplied as borax. The doses of macro and micronutrients were determined according to the guidelines reported by Obreza et al. (2020). One-third of the nitrogen (N) was applied at transplanting, and the remaining amount was divided into equal parts and applied during the first ten irrigations, with an approximate interval of 5 days, without any loss of water or fertilizers. A topping pruning was performed 20 days (d) after transplanting, at 35 cm above the grafting point, to induce new vegetative growth and achieve greater tree uniformity. Irrigation was applied each time the Irrometer ISR-300® tensiometer (USA) recorded soil tension between 15 and 20 kPa. The average minimum and maximum temperatures inside the greenhouse were 9 °C and 37 °C, respectively, with an average relative humidity of 57%.
The treatments (T) consisted of the foliar application of four Zn sources, each at two doses: the amino chelates Aton Zn® at 0.3% and 0.5% (T1 and T2) and Kelatex Zn Forte® at 0.5% and 1.0% (T3 and T4); Zn microparticles Basfoliar Zn 75 Flo® at 0.1% and 0.2% (T5 and T6); ZnSO4H2O at 0.3% and 0.5% (T7 and T8); and the control (T9), with no Zn application (Table 1).
The foliar application of treatments began on September 1, 2022, 50 days after transplanting the trees. Seven applications were made, two in September, October, and November 2022, with a final application in mid-March 2023. The amount of solution used for spraying was proportional to the tree canopy to ensure good coverage. The pH of the solution for each treatment was adjusted to 5.5 with DAP-PLUS®, and INEX-A® was added as a surfactant at a dose of 0.1% (v/v).
Table 1
Treatments evaluated on young ´Valencia´ orange trees (Citrus sinensis L. Osbeck) grafted onto C35 rootstock (Citrus sinensis L. Osbeck x Poncirus trifoliata L. Raf) and transplanted into calcareous soil
Nº Treatment | Source of Zn | Dose (%)a |
1 | Aton Zn | 0.3 (v/v) |
2 | Aton Zn | 0.5 (v/v) |
3 | Kelatex Zn Forte | 0.5 (w/v) |
4 | Kelatex Zn Forte | 1.0 (w/v) |
5 | Basfoliar Zn 75 Flo | 0.1 (v/v) |
6 | Basfoliar Zn 75 Flo | 0.2 (v/v) |
7 | ZnSO4H2O | 0.3 (w/v) |
8 | ZnSO4H2O | 0.5 (w/v) |
9 | Control | - |
a(v/v) = volume/volume ratio; (w/v) = weight/volume ratio.
Tree growth was evaluated by stem diameter and tree height, number of sprouts, number of leaves per sprout, sprout length, leaf area, and aerial biomass dry weight. The nutritional status was analyzed based on the chlorophyll index (SPAD), concentrations of N, P, K, Zn, Fe, Mn, Cu, and B in leaves, and the Zn concentration in roots. All variables were determined in six trees per treatment, except for the Zn concentration in roots, which was evaluated at the end of the experiment in three trees per treatment.
2.2 Stem diameter and tree height
The stem diameter of the trees was measured using a Steren® HER-411 digital caliper (USA) at a height of 8 cm above the grafting point of the variety, and the tree height was measured with a tape measure from the soil level to the apex. Both variables were evaluated every 30 d throughout the experiment, with a total of eight measurements recorded.
2.3 Number of sprouts and leaves per sprout
The number of sprouts and leaves per sprout was counted, considering the spring, summer, and fall growth periods. Each sprout was identified and monitored. These determinations were made eight times, with a 30-day interval throughout the experiment.
2.4 Sprout length
The sprout length was measured using a tape measure, starting 80 d after the initiation of treatments. During the experimental period, five measurements were made, with a 40-day interval between each determination.
2.5 Leaf area
Leaf area was estimated on five occasions. In the first four, it was obtained using a non-destructive method, measuring the length and width of the leaf with a 30 cm ruler. In the fifth and final measurement, a leaf area integrator (LI-COR-3000A; Lincoln, NE, USA) was used. This variable was assessed at 48-day intervals during the experimental period.
2.6 Aerial biomass dry weight
The dry weight of aerial biomass was determined at the end of the experiment, 240 d after the initiation of treatments. The stem and leaves of each tree were dried in a forced-air oven (Riossa, HCF-125, Mexico) at 70 °C until reaching a constant weight (approximately 72 h), then weighed using an analytical balance (Adventurer Ohaus Pro, AV213C; Parsippany, NJ, USA). The rootstock stem of the trees was not included.
2.7 Chlorophyll index
The chlorophyll index was determined based on SPAD readings, which provide a relative index closely correlated with chlorophyll content in leaves. Eight evaluations were made throughout the experimental period using a SPAD Plus Minolta 502 (USA), which measures light transmission at wavelengths between 650 and 940 nm over an area of 0.06 cm² (Kulig et al., 2024). The readings were taken at three points on a fully developed leaf, between the middle portion and the apex of three buds of the same age on each tree, and the average was calculated (Intrigliolo et al., 2000). The leaves were marked, and measurements were taken every 30 d during the experimental period.
2.8 Concentration of macro and micronutrients in leaves
Leaf sampling for macro- and micronutrient analysis was conducted twice: at the beginning of the treatments and at the end of the experiment, that is, 240 d after transplanting. One day before the first application of the treatments, the initial leaf sampling was performed on 20 trees out of the 54 that made up the experiment, considering that uniform management conditions were applied to all plants at the start. Six leaves were collected per tree from spring 2022 shoots that were four to six months old. The leaves were successively washed in running water, a 0.1M HCl acid solution, and distilled water in the laboratory. They were then left at room temperature on kraft paper for 30 min and subsequently dried in a forced air oven (Riossa, HCF-125, Mexico) at 70 °C until they reached a constant weight (approximately 72 h). The samples were ground using a Thomas-Wiley stainless steel mill (NJ, USA). For the analysis of P, K, Ca, Mg, Zn, Fe, Mn, and Cu, 0.5 g of leaf tissue was weighed and then digested with a mixture of HNO3/HClO4 (v/v, 2:1). The concentration of K, Ca, Mg, Zn, Fe, Mn, and Cu in the extracts was measured using an atomic absorption spectrometer (Varian SPECTRAA 220 FS, Australia). The concentration of P was determined using a HACH UV/VIS DR 5000 (Iowa, USA) by visible spectrophotometry. The determi-nation of N was performed using the modified semi-micro Kjeldahl method (Bremner, 1965). For digestion, 0.1 g of the sample was weighed, and a mixture of H2SO4/C7H6O3 was added. The concentration of B was determined by the azomethine-H method (Gaines & Mitchell, 1979). A second sampling and analysis were performed at the end of the experiment. Twenty leaves, four to six months old, were collected from the autumn 2022 flush, in each of the six trees per treatment. The processing of the foliar tissue samples and their analysis were conducted in the same manner as the first, as described above. The same nutrients determined at the start of the treatments were analyzed. The nutrient concentrations were expressed relative to dry matter (DM).
2.9 Concentration of Zn in the root
Root sampling for the analysis of Zn was conducted at the end of the experiment, that is, 240 d after the application of treatments. To obtain the roots, each tree was removed from the pot. The aerial part was separated from the root ball, which was treated with successive volumes of running water to remove the adhered soil. The cleaning, drying, and grinding of the samples were performed in the same way as the foliar tissue samples described earlier. Half a gram of the dry, ground root was digested with the HNO3/HClO4 (v/v, 2:1) mixture, and the extract was then ana-lyzed using an atomic absorption spectropho-tometer (Varian SPECTRAA 220 FS, Australia).
2.10 Statistical analysis
The effect of treatments on the response variables: stem diameter and tree height, number of shoots and leaves per shoot, shoot length, leaf area, dry biomass of the aerial part, chlorophyll index, concentration of macro and micronutrients in leaves, and concentration of Zn in the root, was evaluated by performing an ANOVA (Analysis of Variance). The Shapiro-Wilk (q-q plot) and Bartlett tests were used to verify the assumptions of nor-mality and homogeneity of variances, respectively. The means were compared using the Tukey test at a probability level of 0.05. The statistical program R-studio version 4.1.1 was used.
3. Results and discussion
3.1 Tree Growth
No significant effect was observed from the foliar Zn applications on tree growth at any evaluation date (Table 2). Zn deficiency affects plant metabolism and consequently growth, as it is involved in the regulation and activation of genes, protein synthesis, and carbohydrate metabolism (Clemens et al., 2022; Hamzah et al., 2022). Moreover, Zn is involved in the regulation of hormones and participates in the synthesis of tryptophan (precursor of indoleacetic acid, the predominant auxin in plants) (Sourati et al., 2022).
Auxins are involved in apical dominance, photo-tropism, senescence, response to pathogens, response to abiotic stress and fruit formation, thus regulating plant growth and development (Gao et al., 2024). In young ´Washington navel´ orange trees grown in alkaline soil, foliar application of solutions containing 0.2, 0.4, 0.6, 0.8, and 1.0% ZnSO4 increased diameter and height after two years of evaluation (Dawood et al., 2001). Similarly, foliar applications of Zn increased the leaf area in pecan trees (Carya illinoinensis) established in soil with 30% CaCO3 (Ojeda et al., 2014). The authors report positive effects on plant growth after two years of evaluation in soils with a 30% concen-tration of CaCO3. However, in this experiment, the CaCO3 concentration of the soil was 46.5%, and the study period was eight months, conditions that may have influenced the lack of response.
3.2 Chlorophyll Index
No statistical differences were found in the chlorophyll index in response to the application of the different Zn sources, at any evaluation date, compared to the control. However, the results of the last measurement show that SPAD values tended to decrease in most treatments, except for trees treated with ZnSO4H2O at a low dose, which showed a slight increase of 1.7 SPADs (Table 3). Trees sprayed with the aminoquelate Kelatex Zn Forte at a high dose showed a minimal decrease of 1.0 SPADs (Table 3). Applications of Zn increase the chlorophyll content in plants (Umair et al., 2020), because Zn regulates the amount of iron (Fe) and magnesium (Mg) in the cytoplasm, which are essential nutrients in chlorophyll molecule synthesis (Makhasha et al., 2024). In ´Fengwan´ navel orange trees (Citrus sinensis L. Osbeck), chlorophyll index in leaves increased when sprayed with ZnSO4, ZnCl2, and Zn(NO3)2 (Fu et al., 2016).
3.3 Concentration of macronutrients in leaves
The macronutrient concentrations at the beginning of the treatments were low: N (22.0 g kg⁻¹), P (1.1 g kg⁻¹), and K (11.3 g kg⁻¹), compared to the concentrations indicated by Obreza et al. (2020). The concentration of N in the leaves sprayed with ZnSO4H2O at 0.5% increased significantly compa-red to the control. At the end of the experiment, this concentration was 43.0 g kg⁻¹ (Figure 1A).
Similar results were obtained by Nasir et al. (2016) when they applied moringa extract, K, and Zn to the foliage of ´Kinnow´ mandarin, increasing the foliar N concentration. ZnSO4H2O applied at the high dose (0.5%) also induced a higher level of foliar Zn (Table 4), demonstrating the synergism between Zn and N (Prasad et al., 2016). In all treatments, the N concentration in leaves was excessive at the end of the evaluation (>30.0 g kg⁻¹), but no toxicity symptoms due to this element were observed.
The concentration of P in leaves did not show a significant increase compared to the control (Figure 1B). Although the P level was high in all treatments (0.17 – 0.30 g kg⁻¹), it did not affect the Zn concentration in some treatments, where an optimal Zn status was observed in leaves (25 – 100 mg kg⁻¹ Zn) (Obreza et al., 2020). An excess of P causes a Zn deficiency in plants (Bouain et al., 2014). However, in the present experiment, the trees sprayed with ZnSO4H2O at the high dose (0.5%) showed the highest concentration of both Zn and P in the leaves.
Significant differences were observed in the leaf K concentration (Figure 1C). At the end of the experiment, Aton Zn increased the K concentration to 27.9 and 30.7 g kg⁻¹ for trees sprayed with 0.3% and 0.5%, respectively. Similarly, Basfoliar Zn 75 Flo (0.1%) and ZnSO4H2O (0.3%) increased the K concentration in leaves to 30.0 and 29.0 g kg⁻¹, respectively.
Mosa et al. (2021) published similar results concerning aminochelates, i.e., the application of tryptophan and glycine to the leaves caused an increase in the leaf content of N, P, K, and Ca in ´Anna´ apple trees (Malus domestica L. Borkh). At the end of the experiment, the K concentration in leaves was high (18.0 – 24.0 g kg⁻¹) in the control trees and those sprayed with Kelatex Zn Forte (0.5%), while the rest of the treatments showed excess levels (>24.0 g kg⁻¹) (Obreza et al., 2020). The trees sprayed with Kelatex Zn Forte (0.5%) had the lowest concentrations of N, P, and K in the leaves at the end of the experiment; however, these levels remained above the optimal level for ´Valencia´ orange trees (Figure 1A, 1B y 1C). The low concentrations of N, P, and K in the same treatment may be due to interactions between these nutrients. A low K level leads to reduced N absorption by plants because K is essential for the absorption and transport of nitrate (NO3⁻) (Fageria, 2001). Synergism between N and P in plants has been documented, with N promoting the absorption of P (Krouk & Kiba, 2020). It is believed that the low K concentration in the leaves led to a lower N level, which resulted in a similar P content due to the synergism between these nutrients.
The soil fertilization carried out at the time of transplanting adequately provided the required amounts of N, P, and K in all treatments, including the control. The leaf concentrations of these elements at the beginning of the treatments were low, and by the end of the experiment, they were high and/or excessive (Obreza et al., 2020).
3.4 Micronutrient Concentration in Leaves
Regarding the micronutrient concentration in leaves, at the beginning of the treatments, Zn (19.3 mg kg⁻¹) and Fe (45.9 mg kg⁻¹) were at low levels, Mn (7.1 mg kg⁻¹) was deficient, while Cu (4.8 mg kg⁻¹) and B (83.8 mg kg⁻¹) were at optimal levels (Obreza et al., 2020).
Foliar applications of Zn had a significant effect on Zn concentration in the leaves. Trees sprayed with Aton Zn (0.3%), Kelatex Zn Forte (0.5% and 1.0%), and ZnSO4H2O (0.3% and 0.5%) showed a significant increase in foliar Zn concentration by the end of the experiment, placing them within the optimal range for ´Valencia´ orange trees (25–100 mg kg⁻¹ Zn) Obreza et al., 2020) (Table 4).
Aton Zn (0.3%) increased the Zn concentration 2.35-fold compared to the control. Similarly, Kelatex Zn Forte enhanced the Zn concentration by 2.05 and 3.77 times relative to the control for doses of 0.5% and 1.0%, respectively (Table 4).
A study on the effect of foliar applications of Zn complexed with the amino acids methionine and lysine, and ZnSO4 on pistachio (Pistacia vera L.) ´Akbari´ reported a higher Zn concentration in leaves sprayed with ZnSO4 (58 mg kg⁻¹ Zn), followed by the treatment with Zn complexed with methionine (45 mg kg⁻¹ Zn) (Najizadeh & Khoshgoftarmanesh, 2019).
ZnSO4H2O applied at doses of 0.3% and 0.5% increased the foliar Zn concentration by 3.57 and 5.43 times, respectively, compared to the control (Table 4). Boaretto et al. (2023) reported an increase in foliar Zn concentration up to 70 mg kg⁻¹ in leaves of ´Pera´ orange trees (Citrus sinensis L. Osbeck) after foliar sprays with ZnSO47H2O at a dose of 2 kg ha⁻¹, while the control showed concentrations ranging from 10 to 27 mg kg⁻¹ Zn. Frequent foliar sprays with ZnSO₄ can correct Zn deficiency in leaves; however, its translocation to other plant organs is limited (Zekri & Obreza, 2003).
3.5 Zinc concentration in the root
The zinc concentration in the roots of trees treated with the amino chelate Aton Zn (0.5 %) increased to 23.59 mg kg⁻¹, and this increase was significant. Trees that received this treatment also had an optimal foliar zinc concentration (45.7 mg kg⁻¹) (Obreza et al., 2020) (Table 4).
No significant differences were observed in the zinc concentration in the roots for the other treatments. Mirboloock et al. (2021) reported that foliar applications of zinc complexed with glycine, phenylalanine, tryptophan, and alanine increased the zinc concentration in the roots and stems of bean plants (Phaseolus vulgaris L). Similarly, Rafie et al. (2023) reported that foliar applications of zinc chelated with lysine in onions (Allium cepa L.) increased the zinc level in the bulbs compared to ZnSO4 and the control (no zinc application). These results are consistent whit those obtained in the present study. However, limited information is available on the translocation of zinc after applying amino chelates in fruit trees.
Amino chelates improve the translocation of elements such as zinc within the plant (Souri & Hatamian, 2019). Therefore, when the leaves are sprayed with this form of zinc, it can increase the concentration of the nutrient in the fruit, stem, or roots. This gives an advantage to these fertilizers compared to metal salts (sulfates).
4. Conclusions
The foliar application of different Zn sources did not cause, over the eight-month evaluation period, a significant effect on the growth of young ´Valencia´ orange trees established on calcareous soil. Regarding nutritional status, only the amino-chelate Kelatex Zn Forte at a 1.0% dose proved to be a promising Zn source, as it increased the Zn concentration in roots and leaves and caused a minimal decrease in the chlorophyll index. The foliar application of this Zn source in citrus trees grown on calcareous soil shows potential to mitigate Zn deficiency; however, its evaluation over a longer period is warranted.
Conflict of interest
The authors declare that they have no conflict of interest.
Author contributions
O. Martínez-Ríos: Methodology, Research, Writing–draft, Resources. J. I. Cortés-Flores: Methodology, Conceptualization, Writing–draft, Resources. A. López-Jiménez: Writing–review, Editing, Validation, Resour-ces. J. D. Etchevers-Barra: Methodology, Writing–review, Formal analysis, Editing. M.B. Contreras-Soto: Writing–review, Editing, Software, Conceptualization.
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