Evaluating zinc biofortification strategies in wheat: soil application, foliar spray, and combined approaches

Introduction

An estimated 17.3% of the global population is at risk of inadequate zinc intake, making it the fifth leading risk factor for disease in developing countries [1] . Wheat provides roughly 20% of daily caloric intake globally, yet most commercial wheat varieties contain only 15-25 mg Zn kg⁻¹ in grain well below the 40 mg kg⁻¹ target set by HarvestPlus for meaningful nutritional impact [2,3] . Agronomic biofortification increasing grain micronutrient content through fertilizer management offers a faster route to zinc-enriched wheat than genetic breeding, which requires 10-15 years of variety development [4] . Three main approaches exist: soil application of zinc fertilizer to increase root uptake, foliar spraying to deliver zinc directly to leaves for phloem transport to grain, and combining both methods [5,6] . Soil application is simpler but typically achieves lower grain Zn enrichment because of fixation in calcareous soils, while foliar spraying is more efficient per unit Zn applied but requires precise timing during the grain-filling period [7] . Despite growing interest, few studies have compared all three strategies under identical field conditions in Western European wheat systems, where high-yielding varieties and fertile soils create different dynamics than the zinc-deficient soils of South Asia where most biofortification research has been conducted [8] . This research filled this gap by testing soil, foliar, and combined Zn application on winter wheat at Wageningen, Netherlands, over two consecutive seasons [9] .

Soil quality analysis

The experimental site at Wageningen University's Droevendaal farm (51.99°N, 5.66°E) had a sandy loam soil (Typic Fluvaquent) with pH 7.2, organic carbon 1.87%, DTPA-extractable Zn 0.74 mg kg⁻¹, available P (Olsen) 23.4 mg kg⁻¹, CEC 18.6 cmol(+) kg⁻¹, and CaCO₃ content 4.2%. The relatively low DTPA-Zn (below the critical level of 0.8 mg kg⁻¹) indicated marginal zinc availability, making the site suitable for evaluating biofortification responses. Soil texture was 48% sand, 37% silt, and 15% clay. The water table was maintained at approximately 80 cm depth through controlled drainage [10] .

Quality control and calibration

Grain zinc analysis followed rigorous quality assurance protocols. Grain samples were washed three times with deionized water to remove surface contamination, oven-dried at 65°C for 48 hours, and ground in a titanium blade mill to avoid metal contamination. Zinc concentration was determined by inductively coupled plasma optical emission spectrometry (ICP-OES, Thermo Fisher iCAP 7400) after microwave-assisted nitric acid digestion (MARS 6). Certified reference material (NIST SRM 1567b wheat flour, certified Zn = 11.6 ± 0.4 mg kg⁻¹) was analyzed alongside each batch, with measured values falling within 3% of certified values. All analyses were run in triplicate. Blank corrections and matrix-matched calibration standards were used throughout [11,12] .

Materials and Methods

Materials

Winter wheat variety Benchmark (a high-yielding bread wheat registered in the Netherlands) was sown at 350 seeds m⁻² in October of each year. The experiment used a randomized block design with four replications and four treatments: control (no Zn), soil ZnSO₄•7H₂O at 25 kg ha⁻¹ (incorporated before sowing), foliar Zn-EDTA at 0.5% (w/v) applied twice (at booting and heading stages, 400 L ha⁻¹ spray volume each), and combined (soil + foliar). Plot size was 6 m × 3 m. A uniform basal fertilizer of 200-60-80 kg ha⁻¹ N-P₂O₅-K₂O was applied, with nitrogen split into three applications. Crop protection followed standard Dutch wheat protocols [13] .

Methods

Grain Zn concentration was measured at harvest by ICP-OES after acid digestion. Zinc uptake was calculated as grain Zn concentration × grain yield. Zinc use efficiency was computed as (Zn uptaketreatment -Zn uptakecontrol) / Zn applied × 100. Yield components (ears per m², grains per ear, 1000-grain weight) and grain yield were recorded at harvest. Grain samples were also collected at five growth stages (tillering through maturity) to track Zn accumulation dynamics. Data were analyzed by two-way ANOVA (treatment × season) with Tukey's HSD at 5% using GenStat 22 [14] . The combined treatment achieved the highest grain Zn (47.1 mg kg⁻¹), followed by foliar alone (41.8 mg kg⁻¹) both exceeding the 40 mg kg⁻¹ biofortification target (Table 1). Soil application alone reached only 26.3 mg kg⁻¹. Grain yield was unaffected by Zn treatments (differences non-significant), indicating that biofortification does not compromise productivity. Zinc use efficiency was dramatically higher for foliar application (27.89%) than soil application (1.51%) due to the much smaller quantity of Zn applied. The temporal pattern of zinc accumulation differed markedly between strategies (Table 2). Soil-applied Zn peaked at heading (31.2 mg kg⁻¹) then declined during grain filling, likely due to dilution. Foliar Zn continued increasing through grain fill (42.3 mg kg⁻¹) because the second spray at heading delivered Zn directly during active grain loading. The combined approach showed the steepest accumulation curve, reaching 48.2 mg kg⁻¹ at grain fill.

Results

Comprehensive interpretation

The accumulation curves (Figure 1) demonstrate why foliar timing is so effective: zinc delivered during the heading-tograin-fill window enters the phloem transport pathway at precisely the time when grain is actively accumulating minerals. Soil-applied zinc must travel through the entire plant before reaching grain, and much is diluted by continued biomass production. The biofortification timeline (Figure 2) shows that the entire enrichment strategy can be implemented within the normal wheat management calendar without additional field operations beyond two spray passes.

Discussion

The 170% increase in grain Zn from the combined strategy confirms that soil and foliar applications work through complementary mechanisms root uptake provides a baseline elevation while foliar spray delivers a late-season boost directly to the grain-loading pathway [5,6] . The foliar-only treatment was nearly as effective (140% increase) at a fraction of the zinc cost, making it the most economically efficient option. This aligns with findings from Turkey and Pakistan, where foliar ZnSO₄ consistently outperformed soil application for grain Zn enrichment [4,7] . The decline in grain Zn between heading and maturity in the soil-only treatment reflects the well-documented dilution effect: as grain starch accumulates rapidly during filling, the fixed pool of translocated Zn is spread across increasing grain mass [3] .

Foliar application bypasses this bottleneck by providing zinc during the filling period itself. The absence of any yield penalty across treatments is encouraging for farmer adoption biofortification adds nutritional value without sacrificing productivity [8] . One limitation was that the Wageningen site, while marginally zinc-deficient, had a calcareous soil where zinc fixation is high.

Results may differ on acidic soils where soil-applied zinc remains more available [9] . Testing across a pH gradient and with different wheat varieties varying in zinc translocation efficiency would strengthen these recommendations [15] .

Conclusion

Foliar Zn-EDTA application at booting and heading stages was the most zinc-efficient biofortification strategy, achieving grain Zn concentrations above the 40 mg kg⁻¹ target while using only 0.80 kg Zn ha⁻¹. The combined soil + foliar approach reached the highest absolute grain Zn (47.1 mg kg⁻¹) but required 8× more zinc input. Neither strategy affected grain yield, confirming that biofortification and productivity are compatible goals.

For practical implementation in European wheat systems, two foliar sprays of 0.5% Zn-EDTA during the reproductive phase represent the simplest and most cost-effective pathway to zincbiofortified grain. Integrating this approach with existing fungicide spray schedules would minimize additional operational costs. Multi-site validation across different soil types and wheat varieties, along with assessment of zinc bioaccessibility in processed wheat products, should guide future scaling efforts.

References

1. Wessells KR, Brown KH. Estimating the global prevalence of zinc deficiency. PLoS One. 2012;7(11):e50568.
2. Cakmak I. Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant Soil. 2008;302(1-2):1-17.
3. HarvestPlus. Biofortification progress briefs. Washington (DC): International Food Policy Research Institute; 2020.
4. Zou CQ, Zhang YQ, Rashid A, Ram H, Savasli E, Arisoy RZ, et al. Biofortification of wheat with zinc through zinc fertilization in seven countries. Plant Soil. 2012;361(12):119-130.
5. Cakmak I, Kalayci M, Kaya Y, Torun AA, Aydin N, Wang Y, et al. Biofortification and localization of zinc in wheat grain. J Agric Food Chem. 2010;58(16):9092-9102.
6. Phattarakul N, Rerkasem B, Li LJ, Wu LH, Zou CQ, Ram H, et al. Biofortification of rice grain with zinc through zinc fertilization. Plant Soil. 2012;361(1-2):131-141.
7. Yilmaz A, Ekiz H, Torun B, Gultekin I, Karanlik S, Bagci SA, et al. Effect of different zinc application methods on grain yield and zinc concentration in wheat cultivars grown on zinc-deficient soils. J Plant Nutr. 1997;20(4-5):461-471.
8. White PJ, Broadley MR. Biofortification of crops with seven mineral elements often lacking in human diets. New Phytol. 2009;182(1):49-84.
9. Alloway BJ. Zinc in soils and crop nutrition. 2nd ed. Brussels: International Zinc Association; 2008.
10. Lindsay WL, Norvell WA. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J. 1978;42(3):421-428.
11. Wheal MS, Fowles TO, Palmer LT. A cost-effective acid digestion method using closed polypropylene tubes for ICPOES analysis of plant essential elements. Anal Methods. 2011;3(12):2854-2863.
12. National Institute of Standards and Technology. Certificate of analysis SRM 1567b wheat flour. Gaithersburg (MD): NIST; 2014.
13. Commissie Bemesting Akkerbouw/Vollegrondsgroenteteelt. Handboek bodem en bemesting. Lelystad: CBAV; 2022.
14. VSN International. GenStat for Windows. 22nd ed. Hemel Hempstead: VSN International; 2022.
15. Ram H, Rashid A, Zhang W, Duber AA, Stirber J, Arisoy RZ, et al. Biofortification of wheat, rice and common bean by applying foliar zinc fertilizer along with pesticides. Plant Soil. 2016;403(1-2):389-401.
16. Gomez-Becerra HF, Yazici A, Ozturk L, Budak H, Peleg Z, Morgounov A, et al. Genetic variation and environmental stability of grain mineral nutrient concentrations in bread wheat. J Cereal Sci. 2010;52(2):251-256.