1 Introduction

Microelement deficiency is an important health problem, and more than 25% of the world’s population is adversely affected (Ramzan et al. 2023; White and Broadley 2009). Among these microelements, zinc (Zn) deficiency stands out, with a staggering 17.3% of the global population facing the risk of insufficient Zn intake. Zinc is a trace metal vital for human health. Currently, about two billion people suffer from Zn malnutrition, the majority of whom live in developing countries where cereals, such as wheat, are used as staple foods (Saha et al. 2017; Praharaj et al. 2021). Zinc is involved in the structure of more than 300 enzymes in the human body and also has important roles in protein synthesis, DNA and RNA metabolism, the structure of some proteins involved in gene transfer, and the inhibition of free oxygen radicals (Cakmak and Marschner 1993; Cuajungco et al. 2021; Liu et al. 2023). Zinc fertilization is important to increase Zn concentration in cereals biofortified Zn increases the uptake and absorption of this element in human body (Hotz and Brown 2004; Praharaj et al. 2021). Wheat varieties have shown an increase in yield of up to 175% over the last 120 years, but with a decrease in Zn concentrations about 25% (Murphy et al. 2008). The reason for this is not fully understood but is thought to be due to a possible "dilution effect" resulting from an increase in grain size in modern varieties and a change in the ratio of bran to endosperm (Murphy et al. 2008). Biofortification seeks to address this imbalance not only by focusing on breeding for enhanced agronomic performance but also by making concerted efforts to elevate the concentrations of zinc and other micronutrients in newly developed varieties. In general, the Zn concentrations in wheat grains varied between 20 and 35 mg kg−1(Cakmak and Kutman 2018; Ivanović et al. 2021) The range of grain Zn levels in cereal-based diets should be 40–60 mg kg−1 (Cakmak 2008a; Zhao et al. 2022) and for these reasons, biofortification of wheat with Zn has become an important tool to combat Zn deficiency in humans.

Zinc deficiency is commonly observed in plants grown in calcareous soils of arid and semi-arid climatic zones. This is generally related to reduced Zn availability due to factors such as high pH, excess free calcium carbonate, low organic matter, inadequate and poor drainage, and unbalanced application of fertilizers (Alloway 2009; Younas et al. 2023). Calcareous soils contribute to more than one-third of agricultural land worldwide. Additionally, it can lead to deficiencies in various nutrients beyond Zn (Sánchez-Rodríguez et al. 2021). In addition, high phosphorus (P) content in soil or the use of excessive amounts of P fertilizers adversely affect the availability of Zn in soils (Zhang et al. 2021). Phytic acid (PA), the primary storage form of P in grains, binds Zn at a rate of 65–85%, particularly in cereal group plants. Consequently, this binding results in zinc deficiency in human nutrition (Brouns 2021).

Agronomic biofortification in staple foods to increase crop yields and nutrient levels of edible portions is an effective and sustainable strategy to overcome food insecurity (Akca et al. 2023; Taskin and Gunes 2022; Velu et al. 2014). In general, Zn was applied in the form of zinc sulfate (Yu et al. 2021; Zou et al. 2012), or as Zn-EDTA (Doolette et al., 2018; Wang et al. 2020), and amino acid-chelated Zn (Akça et al. 2022; Ghasemi et al. 2013; Souri et al. 2017). In practice, various zinc sources are used for foliar fertilization. However, there are not many studies comparing their effectiveness. This study compares different Zn sources that have the potential for foliar application. Foliar Zn application has been accepted as an effective strategy for the biofortification of wheat grains with Zn. Zinc accumulation in wheat grains is the highest at the early stages of grain development (Ozturk et al. 2006), and foliar Zn application at late growth stages increases the amount of Zn in wheat grain, especially in the endosperm and thus in flour (Cakmak et al. 2010; Li et al. 2016). Many studies have shown that effective biofortification of wheat grain with Zn is largely dependent on adequate N supply (Cakmak et al. 2010; Gonzalez et al. 2019), therefore, the application of Zn from leaves together with urea results in good outcomes in increasing the zinc content of grains. Urea-N promotes the mobility of Zn in the phloem, facilitating its transportation to the grain (Ghasemi et al. 2013; Gupta et al. 2016).

In Turkey, as well as globally, wheat plants cultivated in arid-semi-arid climatic zones, where the soil tends to be alkaline and irrigation is not applied, typically exhibit low grain Zn concentrations. The literature provides diverse Zn sources and application methods aimed at boosting the grain zinc concentration in wheat plants, elucidating the mechanisms of uptake and transport of applied Zn by plants (Adrees et al. 2021; Kamaral et al. 2022; Stangoulis and Knez 2022). There are studies that determine the effects of different Zn sources (Liu et al. 2023; Xu et al. 2021, 2022), and foliar Zn fertilization is also practiced. A significant portion of these studies has been conducted in pots and under controlled conditions. In our study, however, the effectiveness of zinc sources such as ZnSO4.7H2O, Zn-Methionine, Zn-Polyol, and Zn-EDTA was compared, and in addition to their effectiveness, their relationship with PA was also examined in wheat plants grown under field conditions. The hypothesis of this study investigates whether applying varying doses of Zn, either by dividing them at different growth stages or all at once, sourced from the soil and applied through the leaves, will enhance both wheat grain zinc content and yield in real field conditions. This study aims to enhance the zinc content of bread wheat grains through two distinct fertilization strategies: Experiment I involves applying zinc from ZnSO4.7H2O source in the soil at various stages and quantities, spanning from the sowing period to top-dressing periods such as tillering. Experiment II entails foliar application of zinc using ZnSO4.7H2O, Zn-Methionine, Zn-Polyol, and Zn-EDTA from different sources and at different developmental stages, including tillering and booting periods, both with and without urea.

2 Material and Methods

2.1 Experimental Site

The experiments, denoted as Experiment I and Experiment II, were carried out in two distinct areas under field conditions at the Haymana Research and Experiment Station, Faculty of Agriculture, University of Ankara, located in Ankara, Turkey. These experiments were conducted during the 2017–2018 season (first year, latitude:39.612971, longtitude:32.689273, altitude:1057 m) and the 2018–2019 season (second year, latitude:39.611625, longtitude:32.688437, altitude 1056 m), both of which were conducted under rain-fed conditions. For both years, the experimental areas were deliberately chosen from regions known to be deficient in Zn, as indicated in Table 1. The soil in the areas where the experiments took place was classified as Typic Haploxerept soil in both years. Moreover, the average rainfall recorded was 45.2 mm during the first year (from November 2017 to August 2018) and 32.3 mm during the second year (from November 2018 to August 2019) (Table S1).

Table 1 Effect of soil applied zinc on grain phytic acid (PA) and phosphorus (P) concentrations of Experiment I
Full size table

2.2 Soil Sampling and Analyses

Soil samples were taken from 0–20 cm depth (Jackson 1962), air dried and sieved through a 2 mm sieve before sowing. All soil analyses have been conducted according to Page and Keeney (1982). Available P, NH4OAc-extracted K, Ca, Mg and DTPA-extracted Zn, Fe, Cu and Mn were determined by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry, Perkin Elmer Model DV 2100) (Boss and Fredeen 2004). The results of some physical and chemical analyses of the soils of the experimental field in both cropping years are given in Table S2.

2.3 Source of Zinc Fertilizers

Zinc sulfate (ZnSO4.7H2O, Merck), Zn-Methionine (2.70% Zn, SCHWARP), Zn-Polyol (3.30% Zn, SCHWARP), and Zn-EDTA (Merck) chemicals were used in the experiments. Zinc Methionine was obtained by linking Zn with the amino acid L-methionine. Zn-Polyol is a chemical in which poly-oligo compounds produced by reducing raffinose, galactose, and mannose from eutrophic algae species are used as Zn carriers.

2.4 Experimental Design

The experiments were conducted in plots of 5.30 m in length and 1.20 m in width with five replications according to the randomized block design. Wheat cultivar Bezostaja-1 seeds were sown with five rows using an experimental drill (HEGE 75–90 Ulrich-Hege, Waldenburg, Germany) in the Diammonium phosphate (DAP, 18%N and 46% P2O5) at the rate of 150 kg ha−1 (27 kg N ha−1 and 70 kg P2O5 ha−1) was applied to all plots as basal fertilizer at the time of sowing and NH4NO3 (33% N) at the rate of 60 kg N ha−1 was applied as top dressing fertilizer in both cropping years. The experiments were established on November 3, 2017 for first year and November 4, 2018 for second year. The treatments of Experiment I were as follows; (i) control (Zn0), (ii) 5 kg Zn ha−1 with sowing (Zn1), (iii) 2.5 kg Zn ha−1 with sowing + 2.5 kg Zn ha−1 top dressing period (Zadoks 30–32) (Zn2), and (iv) 5 kg Zn ha−1 top dressing period (Zadoks 30–32) (Zn3). The applied Zn source was ZnSO4.7H2O, which was sprayed on the soil surface in solution form and mixed with the help of a rake. Control plots (Zn0) were sprayed with pure water only. The first year, plants were harvested on July 16, 2018 and the second year was harvested on August 6, 2019 with a plot harvester.

The treatments of Experiment II were (i) control, (ii) foliar urea (2%, w/v), (iii) foliar ZnSO4.7H2O (0.02% Zn, w/v), (iv) foliar ZnSO4.7H2O (0.02% Zn, w/v) + urea (2%, w/v), (v) Zn-Methionine (0. 02% Zn, w/v), (vi) Zn-Methionine (0.02% Zn, w/v) + urea (2%, w/v), (vii) Zn-Polyol (0.02% Zn, w/v), (viii) Zn-Polyol (0. 02% Zn, w/v) + urea (2%, w/v), (ix) Zn-EDTA (0.02% Zn, w/v) and (x) Zn-EDTA (0.02% Zn, w/v) + urea (2%, w/v). In the first year (2017–2018 sowing period), the tillering stage (Zadoks 30–32) foliar Zn application was made on March 27, 2018, and the booting (pre-spike) stage foliar Zn application was made on May 16, 2018., in the second year (2018–2019 sowing period), the tillering stage foliar Zn application was made on April 3, 2019 and the booting (pre-spike, Zadoks 45) stage foliar Zn application was made on May 23, 2019. In both cropping years, a spreader-adhesive preparation was added to the prepared solution to prevent the Zn applied in solution form from flowing off the leaves. In both experiments, weed killer (Hektas Resital Dio) was applied using a pulverizer at a rate of 700 ml ha−1 at the stage of tillering. The field experiments in the first year was harvested on July 16, 2018, while the second year was harvested on August 6, 2019, using a plot harvester.

2.5 Grain Yield, Determination of Grain and Leaf Element Concentrations

In both cropping years, grain samples were collected from each plot during the harvest period of the experiments. Subsequently, after determining grain weight, the grains were ground into flour. As part of Experiment II, leaf sampling was conducted both before and after the foliar application of Zn. In the pre-tillering period, leaf samples were taken before foliar Zn application from different sources and after fertilization after the tillering stage. In the booting stage, leaf samples were taken before foliar Zn application from different sources and after fertilization after the booting period, including old and flag leaves. Within the scope of Experiment II, leaf samples were taken at the tillering and booting stages and brought to the laboratory in paper bags, washed with 0.02 M HCl and distilled water, and subsequently dried in a drying chamber with air circulation at 65οC until they reached constant weight and ground for analyses. The ground leaf and grain samples were digested in a microwave sample digester using an HNO3-H2O2 mixture (Berghof-MWS-2 Model 24) as described by Boss and Fredeen (2004). Total Zn and P were determined by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry, Perkin Elmer Model DV 2100) (Boss and Fredeen 2004). Certified tomato plant leaf (Standard Reference Material 1573a) and wheat flour (Standard Reference Material 1567a) samples obtained from the National Institute of Standards and Technology (NIST) of the United States of America were used to determine the accuracy of the analyses performed on leaf and grain samples. Grain yield was determined with grain samples taken after harvest.

2.6 Determination of Grain Phytic Acid Concentration and Phytic Acid/Zn Molar Ratio

Sixty milligrams of flour samples were weighed, and 10 mL of 0.2 N HCl was added, followed by shaking for 2 h and filtered through Whatman No 42 filter paper. 0.5 ml of the samples taken from this filter were transferred to capped tubes, and 1 ml of 0.4 mM ammonium ferric solution was added, and the tubes were sealed. The samples were kept in a water bath at 105 °C for 30 min, then cooled on ice for 15 min and kept at room temperature. A 1% bipyridine solution was added to the samples at room temperature, mixed, and read at 519 nm in a spectrophotometer (Shimadzu model UV 1201) (Wolfang and Lantsch 1983). Based on the information that the molecular weight of phytic acid (PA) is 660 g and the molecular weight of Zn is 65 g, the Phytic Acid/Zn (PA/Zn) values on mole basis were determined by calculating the molar values of PA determined in mg kg−1 and Zn determined in mg kg−1 and determining their ratio to each other with the equation given below Eq(ı);

$$PA/Zn molar ratio=((PA content in mg/kg)/660)/((Zinc content in mg/kg)/65)$$
(1)

2.7 Statistical Analysis

The experimental design was a randomized complete block design with five replications. The significance of treatments was determined with one-way ANOVA in Experiment I and two-way ANOVA in Experiment II, using ORIGINPRO 2021software (OriginLab Inc., Northampton, MA, USA). The significant differences among the treatment means were calculated by Tukey’s honestly significant difference (HSD) test at p < 0.05 and descriptive statistics (± SE). Boxplots and principal component analysis (PCA) were created using ORIGINPRO 2021 software.

3 Results

3.1 Grain Yield of Wheat Plant

The soil applied Zn at varying periods and quantities demonstrated a significant enhancement in wheat growth and yield during both cropping years (Fig. 1 A,B).

Fig. 1
figure 1

Effect of soil applied zinc on grain yield in Experiment I. The values are means of five replicates ± standard errors. Different letters in each column represent significant difference at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test. Zn0:control; Zn1:5 kg Zn ha−1 applied as basal fertilizer; Zn2: 2.5 kg Zn ha−1 applied as basal fertilizer + 2.5 kg Zn ha−1 applied as top dressing; Zn3: 5 kg Zn ha−1 applied as top dressing

Full size image

In the initial year, the grain yield of wheat in the absence of Zn treatment (control) was 4254 kg ha−1 (Fig. 1 A). As the application dose of Zn increased, the yield demonstrated a proportional ascent, reaching 4459 kg ha−1 with Zn1, 4615 kg ha−1 with Zn2, and reaching its pinnacle at 4775 kg ha−1 with Zn3 treatments. Moving on to the second year, the grain yield of wheat plants subjected to the Zn0 treatment was recorded at 2739 kg ha−1. In line with the previous year's pattern, grain yields exhibited a consistent increase corresponding to the applied levels of Zn, resulting in yields of 3246 kg ha−1 with Zn1 treatment, 3513 kg ha−1 with Zn2 treatment, and a peak yield of 3676 kg ha−1 with Zn3 treatment (Fig. 1 B). Applying varying quantities of Zn to the soil during different growth stages resulted in enhanced grain yield over the control in both years. Notably, the most effective treatment was the application of Zn during the tillering stage (top dressing period) or in a split manner (combining sowing and tillering stages), rather than applying the entire zinc amount with the initial soil sowing. Specifically, a substantial 12.4% increase in yield was observed in the first year, and an even more impressive 34.3% increase was noted in the second year when the entire Zn amount was applied during the tillering stage, as compared to the control. Likewise, a significant 8.47% increase in yield was recorded in the first year, along with a notable 28.1% increase in the second year, when comparing the entire zinc application during the tillering stage to the control. In contrast, applying the entire zinc quantity during the sowing period resulted in a comparatively lower yield increase of 4.94% in the first year and 18.6% in the second year compared to the control.

The effects of foliar Zn sources with or without urea on grain yield were found to be statistically insignificant in both cropping years (Fig. 2A,B). Foliar application of Zn from different sources increased grain yield. When Zn sources were evaluated together, foliar Zn with or without urea was effective on the grain yield of wheat plants in both years, and the grain yield of wheat plants increased significantly (Fig. 2A,B). In the first year, the average grain yield was 4640 kg ha−1 when Zn was applied without urea, increased to 4850 kg ha−1 when Zn was applied with urea. In the second year, the average grain yield was 3070 kg da−1 when Zn was applied without urea, but it increased to 3330 kg ha−1 when Zn was applied together with urea. The simultaneous application of Zn with urea from various sources proved to be effective in enhancing yield, with the highest yields achieved through this co-application in both years. In the first year, the highest grain yield was observed with Zn in the form of Zn Polyol combined with urea (5078 kg ha−1) (Fig. 2A), while in the second year, the highest grain yield was obtained with Zn in the form of ZnSO4.7H2O in combination with urea (3539 kg da−1) (Fig. 2B).

Fig. 2
figure 2

The effect of different zinc-containing fertilizers applied through foliar with and without urea on total grain yield in Experiment II. The values are means of five replicates ± standard errors. Different letters in each column represent significant difference at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test

Full size image

3.2 Total Zn Concentration in Wheat Grain

Soil applied Zn at varying periods and quantities significantly increased grain total Zn concentration in both years (Fig. 3A,B). The grain Zn concentration obtained in the first year was above the critical level, and in the second year, it was within the critical level (5–15 mg Zn kg−1).

Fig. 3
figure 3

Effect of soil applied zinc on total Zn concentration in Experiment I. The values are means of five replicates ± standard errors. Different letters in each column represent significant difference at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test. Zn0:control; Zn1:5 kg Zn ha−1 applied as basal fertilizer; Zn2: 2.5 kg Zn ha−1 applied as basal fertilizer + 2.5 kg Zn ha−1 applied as top dressing; Zn3: 5 kg Zn ha−1 applied as top dressing

Full size image

In both cropping years, the treatments with the highest grain total Zn concentration were those receiving Zn3 treatment (5 kg Zn ha−1 during the top-dressing period), with concentrations reaching 22.4 mg kg−1 and 14.8 mg kg−1, respectively. Zinc application at different growth stages significantly increased the total Zn concentration of grain compared to the control, and it was more appropriate to apply the whole amount of Zn during the tillering stage instead of the whole amount of Zn during the sowing period. Indeed, the increases in grain total Zn concentrations obtained with the application of Zn at the tillering stage compared to the control application were 8.81%, 14.6% and 25.2% for Zn1, Zn2, and Zn3, respectively, in the first year and 9.79%, 18.4%, and 31.9% for Zn1, Zn2, and Zn3, respectively, in the second year.

The effects of foliar applied Zn from different sources with or without urea on the total Zn concentration of wheat grain were found to be statistically insignificant in both years (Fig. 4A,B). However, the individual effects of Zn sources and Zn with or without urea on the total Zn concentration of wheat grain were statistically significant. In the first year, when conditions with and without urea were collectively assessed, the average grain total Zn concentration was 18.2 mg kg−1 in the absence of foliar Zn application (control) (Fig. 4A). However, it increased with Zn applications from different sources, with average Zn concentrations of 20.3 mg kg−1 in ZnSO4.7H2O treatment, 21.5 mg kg−1 in Zn-Methionine, 19.9 mg kg−1 in Zn-Polyol, and 21.5 mg kg−1 in Zn-EDTA treatment. Grain total Zn concentration significantly increased with Zn application compared to the control, the difference between ZnSO4.7H2O and Zn-Polyol treatments and between Zn-Methionine and Zn-EDTA treatments were not found to be statistically significant and they were in the same group in terms of their effects (Fig. 4A,B).

Fig. 4
figure 4

The effect of different zinc-containing fertilizers applied through foliar with and without urea on total Zn concentrations of Experiment II. The values are means of five replicates ± standard errors. Different letters in each column represent significant difference at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test

Full size image

In the first year, when the conditions with and without urea were evaluated together, the average grain total Zn concentration was 12.1 mg kg−1 in the absence of foliar Zn application (control treatment), but increased depending on the Zn applied from different sources. The average Zn concentration was 14.4 mg kg−1 in ZnSO4.7H2O, 14.9 mg kg−1 in Zn-Methionine, 13.5 mg kg−1 in Zn-Polyol, and 11.7 mg kg−1 in Zn-EDTA application. Grain total Zn concentration increased statistically with Zn application compared to the control except for Zn-EDTA application. When Zn sources were grouped in terms of their effects on total Zn concentration of wheat plants, it was found that control and Zn-EDTA were in the same group, ZnSO4.7H2O, Zn-Methionine and Zn-Polyol were in the same group (Fig. 4A,B). The total Zn concentration in grain increased significantly when Zn from different sources was applied with urea compared to Zn without urea. Indeed, the highest total Zn concentration was obtained in both years when Zn was co-applied with urea. In the first year, the highest grain total Zn concentration was obtained with Zn applied in the form of Zn-Methionine and Zn-EDTA with urea (22.1 mg kg−1), while in the second year, the highest grain total Zn concentration was obtained with Zn given in the form of Zn-Methionine with urea (15.3 mg kg−1).

3.3 Total Phosphorus, Phytic Acid Content, and PA/Zn Molar Ratio in Wheat Grain

In the first year, the grain total P concentration was 2.57 g kg−1 under the control treatment, and this concentration consistently decreased with increasing Zn dose. However, in the second year, the impact of Zn application on grain total P concentration did not show statistical significance (Table 1). Soil applied Zn at varying periods and quantities decreased the grain PA concentration of wheat grains in both cropping years, but this decrease was not statistically significant (Table 1). In both cropping years, the lowest grain PA concentration was obtained in Zn3 as 7.28 mg g−1 and 7.09 mg g−1, respectively. The decreases in grain PA concentration obtained with soil Zn treatments during the top dressing period compared to the control treatment were 0.93%, 3.31% and 3.58% for Zn1, Zn2 and Zn3, respectively, in the first year; and 2.05%, 1.23% and 3.01% for Zn1, Zn2 and Zn3, respectively, in the second year. In this study, the PA/Zn molar ratio, which is used as an index to evaluate the Zn bioavailability of wheat grain, was calculated. Soil applied Zn at varying periods and quantities decreased the calculated grain PA/Zn molar ratio in both cropping years, and these decreases were found to be statistically significant (Fig. 5A,B). In both years, the lowest grain PA/Zn molar ratio was obtained in Zn3 treatment (32.5 and 48.1, respectively). The differences in the grain PA/Zn molar ratio depending on Zn treatments (Zn0, Zn1, Zn2, and Zn3) were found statistically significant in the first year (Fig. 5A), while in the second year, the difference in PA/Zn molar ratio between Zn1 and Zn2 treatments and the difference in PA/Zn molar ratio between Zn2 and Zn3 treatments were not statistically significant (Fig. 5B).

Fig. 5
figure 5

Effect of soil applied zinc on PA/Zn molar ratio in Experiment I. The values are means of five replicates ± standard errors. Different letters in each column represent significant difference at p < 0.05 level based on Tukey’s honestly significant difference (HSD) test. Zn0:control; Zn1:5 kg Zn ha−1 applied as basal fertilizer; Zn2: 2.5 kg Zn ha−1 applied as basal fertilizer + 2.5 kg Zn ha−1 applied as top dressing; Zn3: 5 kg Zn ha-1 applied as top dressing

Full size image

The individual effects of foliar applied Zn sources with or without urea on P concentration and total PA concentration of wheat grains were statistically insignificant in both cropping years (Table 2). The PA/Zn molar ratio of grains decreased with Zn applied from different sources compared to the control and this decrease was found to be statistically significant (Fig. 6A,B). When the Zn sources were evaluated together, Zn applied without urea and with urea was effective on PA/Zn molar ratio of wheat plants in both years and decreased grain PA/Zn molar ratio of wheat plants (Fig. 6A,B). The simultaneous application of Zn with urea from various sources proved effective in reducing the grain PA/Zn molar ratio, with the lowest ratio observed in both cropping years under this co-application. When the conditions with and without urea were evaluated together, the average grain PA/Zn molar ratio was 41.2 molar in the first year and 61.1 molar in the second year in the absence of foliar Zn application (control treatment); the average grain PA/Zn molar ratio decreased depending on the Zn applied from different sources and ZnSO4.7H2O application decreased to 35.8 molar in the first year (Fig. 6A) and 50.6 mol in the second year (Fig. 6B); Zn-Methionine application decreased to 33.5 molar in the first year and 48.3 molar in the second year; Zn-Polyol application decreased to 37.3 molar in the first year and 54.1 molar in the second year; Zn-EDTA application increased from 34.4 molar in the first year to 63.7 molar in the second year.

Table 2 The effect of different zinc-containing fertilizers applied through foliar with and without urea on grain phytic acid (PA) and phosphorus (P) concentrations of Experiment II
Full size table
Fig. 6
figure 6

The effect of different zinc-containing fertilizers applied through foliar with and without urea on on PA/Zn molar ratio in Experiment II. The values are means of five replicates±standard errors. Different letters in each column represent significant difference at p<0.05 level based on Tukey’s honestly significant difference (HSD) test

Full size image

3.4 Total Zn and P Concentrations of the Shoot

Total Zn concentrations in wheat plants increased in both years due to zinc applied with and without urea foliarly from different sources during the tillering stage. In the first year, the interactions between Zn sources applied before and after foliar application and Zn applied without ureaand combined with urea were found significant (Table S3). In the second year, this interaction was only effective on the post-application old leaf Zn concentration. The interactions of Zn sources applied and Zn without urea and Zn with urea on flag leaf Zn concentration taken after Zn application during the booting stage were found statistically significant only in the first year before application. The application of Zn without urea and in combination with urea was effective on flag leaf Zn concentration, and Zn in combination with urea increased flag leaf Zn concentration from 10.5 mg kg−1 to 12.4 mg kg−1 in the first year and from 8.89 mg kg−1 to 9.97 mg kg−1 in the second year. Flag leaf Zn concentration was increased by co-application of Zn with urea in all Zn sources. Co-application of Zn with urea was more effective in increasing leaf Zn concentration in wheat plant than Zn alone. Total P concentrations of shoot at tillering stage before and after the foliar application, old and flag leaf at booting stage after foliar application was given in Tables S4.

3.5 Principle Component Analysis

In the Experiment I, principle component analysis (PCA) showed a total variability of 84.62% (PC1 = 63.25%; PC2 = 21.37%) for the first year while total variability of 78.92% (PC1 = 57.32%; PC2 = 21.60%) for the second year (Fig. 7A, B). According to PCA analysis, Zn2 and Zn3 treatments were effective on the grain yield and Zn concentration in both cropping years. The P concentration, PA content and PA/Zn molar ratio increased in both years under non-zinc treated conditions (Zn0). A significant positive correlation was found between the total Zn and yield in first year while positive relationships were observed among Zn, P, and yield in second year. Phosphorus, PA, PA/Zn molar ratio showed a negative correlation with Zn concentration in the first year (Fig. 7A), while in the second year all parameters except yield and P were negatively correlated with Zn concentration (Fig. 7B).

Fig. 7
figure 7

The biplots of principal component analyses (PCA) of in Experiment I in first year (A) and second year (B). Zn0:control; Zn1:5 kg Zn ha−1 applied as basal fertilizer; Zn2: 2.5 kg Zn ha−1 applied as basal fertilizer + 2.5 kg Zn ha−1 applied as top dressing; Zn3: 5 kg Zn ha−1 applied as top dressing

Full size image

In the Experiment II, PCA showed a total variability of 73.73% (PC1 = 51.12%; PC2 = 22.61%) for the first year while total variability of 70.01% (PC1 = 49.25%; PC2 = 20.51%) for the second year (Fig. 8A, B). Foliar application of Zn with urea positively affected grain yield and Zn concentrations of wheat plants in both growing years. A significant positive correlation was found between the total Zn, P and grain yield in both cropping years. Phytic acid and PA/Zn molar ratio showed a negative correlation with Zn concentration in both cropping years (Fig. 8A, B).

Fig. 8
figure 8

The biplots of principal component analyses (PCA) of in Experiment II in first year (A) and second year (B). 1: control (pure water only); 2: ZnSO4.7H2O; 3: Zn Methionine; 4: Zn Polyol; 5: Zn EDTA; 6: urea; 7: ZnSO4.7H2O + urea; 8: Zn Methionine + urea; 9: Zn Polyol + urea; 10: Zn EDTA + urea. Zn: Zinc, P: Phosphorus; PA: Phytic acid; PA/Zn: PA/Zn molar ratio

Full size image

4 Discussion

In this study, the effectiveness of Zn sources (ZnSO4.7H2O, Zn-Methionine, Zn-Polyol, and Zn-EDTA) in real-world field conditions to understand their performance in practical agriculture were investigated. We also explored the relationship between Zn bioavailability in wheat grains and PA. Our findings offer valuable insights into Zn fertilization strategies and emphasize the importance of field-based studies in validating nutrient management approaches developed under controlled conditions.

Soil Zn applications at different growing periods and quantities were effective in increasing grain yield and grain Zn concentration. Zinc application (5 kg Zn ha−1) during the top fertilization period (tillering stage, Zadok 30–32) was more effective in increasing grain yield than that of the other periods. Spring-applied soil Zn enhances solubility, facilitated by elevated soil moisture, leading to increased Zn transport to the root zone via diffusion and/or mass flow compared to applications in other seasons. Numerous research has reported that Zn application to the soil in the form of ZnSO4.7H2O increased the grain yield of wheat plants (Yassen and Alwan 2022). Akram et al. (2020) reported an increase in grain yield, especially in wheat plants, with the application of 5 kg Zn ha−1 in the form of ZnSO4.7H2O to the soil. In both Experiment I and II, the decrease in grain yield in the second year was attributed to unfavorable climatic conditions. Meteorological data for related years revealed irregular and variable rainfall. Analyzing May precipitation during wheat booting and flowering periods, the monthly value dropped from 152 mm in the first year to 18.6 mm in the second year, resulting in decreased wheat yield. Gomez-Coronado et al. (2016) noted that drought, particularly during the flowering period, significantly reduces wheat yield.

Foliar application of Zn, with or without urea, significantly increased grain yield. The enhancements in grain yield were influenced by both the control group and the specific Zn sources. This increase is attributed to the positive effects of Zn application, including the stimulation of various enzymes, elevated chlorophyll content, and increased activity of the IAA hormone in the plant (Lian et al. 2023). Additionally, Zn plays a crucial role in converting nitrogen from N-NH4+ to N-NO3− form (Hacisalihoglu et al. 2003). Foliar applied Zn sources with or without urea was effective on grain total Zn concentration of wheat plants and grain total Zn concentration continuously increased. It has been reported in the studies that grain Zn concentration increased with Zn fertilization (Lian et al. 2023; Martínez Cuesta et al. 2023; Nayak et al. 2023a; Zhang et al. 2012). Different foliar Zn sources combined with urea resulted in higher grain Zn concentrations compared to those without urea. Foliar application of Zn with urea increases grain Zn concentration by elevating total free amino acid levels in the leaf and facilitating Zn transport to the phloem through these amino acids (Caputo and Barneix 1997). Zinc is transported to the grain in the phloem by forming ligands with sulfur-containing amino acids (Haydon and Cobbett 2007; Torrance et al. 2008). Since methionine is a sulfur-containing amino acid, it was more effective in the transport and accumulation of Zn in the grain compared to other Zn sources. Nicotianamine synthesized from methionine is also involved in the transport and storage of Fe and Zn in the phloem (Waters et al. 2009). Nitrogen applications also provide Zn transport and accumulation in the endosperm by promoting the formation of Zn-protein complexes (Kutman et al. 2011; Xue et al. 2014a). To a certain extent, N application is an effective way to increase endosperm Zn concentration without causing yield reduction. This is important for human nutrition because wheat flour (mainly endosperm) is an important component of the daily diet in many countries. Thus, foliar application of Zn in wheat can be taken as a measure to solve the problem of dietary Zn deficiency. This suggests that vegetative organs represent important Zn or N reserves and that increasing Zn and N pools in vegetative tissues, especially leaves, by foliar Zn and soil N application before anthesis is a promising agronomic strategy to increase grain N and Zn levels under field conditions. Considering that Zn concentration of grain should be greater than 22 mg kg−1 (Cakmak 2008b) in terms of its effect on human health, it was determined that Zn concentration of grain exceeded 22 mg kg−1 with some the treatments (5 kg Zn ha−1 applied as top dreesing from soil and foliar Zn-Methionine, and Zn-EDTA with urea) in our study.

The determination of grain PA concentration and the PA/Zn molar ratio is crucial for assessing the nutritional quality and potential impact on mineral bioavailability in grains. By understanding these parameters, researchers can develop strategies to improve the nutritional value of grains and mitigate the risk of micronutrient deficiencies in populations reliant on grain-based diets. Phytic acid, which is found as an organic P compound in the grains of cereals, contains 70–80% of the total P in the grain and prevents the absorption of these elements by forming a complex with Zn and other elements in humans (Nolan et al. 1987). Many research on grain crops such as bread wheat and maize has shown that PA is positively correlated with nutrients such as grain protein, total Zn, Fe, P, Mg, and Ca (Pramitha et al. 2021; Yu et al. 2022; Wang et al. 2015). Soil Zn applications varying periods and quantities decreased PA concentration in general, although it was statistically insignificant on grain PA concentration. It was concluded that Zn application (5 kg Zn ha−1) during the top fertilization period (tillering stage, Zadok 30–32) may be an effective method to reduce grain PA concentration. The decreases in PA concentration were greater in treatments in which foliar Zn was co-applied with urea. This decrease in PA concentration compared to the control was due to the application of Zn in the form of Zn-Methionine. Graham et al. (2001) reported that amino acids formed as a result of protein assimilation in plants bind both PA and Zn, thereby enhancing Zn absorption in plants. In addition to the total Zn content of foods, the bioavailability of Zn is one of the most crucial criteria for food quality. The bioavailability of Zn is directly associated with the PA content of cereal grains (Arafsha et al. 2023; Perera et al. 2018). Studies indicate that both soil and foliar applications of Zn reduce grain phytic acid (PA) concentration and enhance Zn bioavailability in cereal crops (Kumar et al. 2023; Nayak et al. 2023b; Wang et al. 2023). Co-application of Zn with N has also been shown to decrease grain PA concentration (Li et al. 2015; Sharafi 2023; Xue et al. 2014b). While (Zhang et al. 2012) reported a non-statistically significant decrease in wheat grain PA concentration with foliar Zn and urea applications, Li et al. (2015) and Wang et al. (2019) found similar non-significant decreases in grain PA concentration with foliar Zn applications, consistent with the results of this study.

Determining the bioavailability of grain Zn from various fertilization approaches is a key focus in plant nutrition research and vital for human nutrition (Li et al. 2015). In this study, all foliar fertilizer treatments significantly reduced the PA/Zn ratio, underscoring the importance of foliar fertilizers in enhancing Zn bioavailability. Considering that high protein and amino acid concentrations in cereals contribute to greater bioavailability of dietary micronutrients (Lonnerdal 2000). In soil Zn applications of varying periods and quantities, the PA/Zn molar ratio in grain decreased due to reduced grain PA concentration and increased Zn concentration. Different foliar zinc sources (Zn-Methionine and ZnSO4.7H2O)combined with urea also decreased the PA/Zn molar ratio. Although the study found the PA/Zn molar ratio was higher than the molar ratio associated with high Zn bioavailability, it's reported that Zn-amino acid complexes are more effective in reducing the PA/Zn molar ratio compared to ZnSO4.7H2O, impacting both Zn concentration and bioavailability (Ghasemi et al. 2013). Despite a significant decrease in the PA/Zn molar ratio, indicating improved Zn bioavailability in Experiment I and II compared to the control, this decrease exceeded the accepted limit value for the PA/Zn molar ratio (PA/Zn molar ratio 25–30, Oberleas and Harland 1981). These results highlight the low bioavailability of Zn in the Bezostaja-1 bread wheat cultivar.

5 Conclusion

This study demonstrated that the top-dressing application zinc and foliar application of zinc sulfate with urea led to a significant increase in grain zinc levels in bread wheat. Fertilizing with Zn emerged as the most effective strategy for enhancing zinc content in grains. Nevertheless, the dose, timing, and source of zinc application play crucial roles. The effectiveness of zinc sulfate, particularly in comparison to other tested sources, is pivotal in addressing the issue without escalating fertilizer input costs. The incorporation of zinc into fertilization programs, especially in arid and semi-arid regions, holds paramount significance for human nutrition and well-being, given the vital role of zinc in human health.