Effect of foliar application of microelements on chlorophyll content, canopy architecture indicators, and physiological parameters of Hordeum vulgare L. plants

Introduction

The use of micronutrients is an important aspect in plant nutrition. All higher plants require six essential micronutrients such as manganese, iron, copper, zinc, boron, and molybdenum. These elements are essential components, required in small amounts for proper plant growth and development (Welch & Shuman, 1995; Ram et al., 2017; Kaur et al., 2023). Microelements are involved in various metabolic processes. The use of micronutrients increases antioxidant metabolism in plants and helps alleviate environmental stresses (Ahmed et al., 2020; Tavanti et al., 2021; Vatansever, Ozyigit & Filiz, 2017). However, too high doses of micronutrients can be toxic to plants (Gupta, 2010; Cruz et al., 2022). Optimizing plant nutrition with mineral compounds is a sustainable approach to improve plant health and yields. The supply of nutrients through foliar fertilization is a faster and more environmentally friendly method compared to soil fertilization. Nutrients can be applied in controlled amounts and at a specific time of plant growth (Niu et al., 2021). Furthermore, foliar fertilization is particularly effective in situations where nutrient uptake by the root system is limited, for example, in conditions of drought, excessive soil compaction, or oxygen deficiency in the rhizosphere. Currently, the foliar fertilization strategy is used in agriculture as a form of complementary plant nutrition, especially in crops with a high potential for yield (Niu et al., 2021; Oliveira et al., 2022; Januszkiewicz, Kulczycki & Samoraj, 2023). Barley (Hordeum vulgare L.) ranks fourth among all cereal crops and is widely grown for human consumption, as feed for livestock and for brewing and malting purposes around the world (Meints & Hayes, 2019; FAO, 2025).

Barley requires micronutrients such as copper (Cu), manganese (Mn), molybdenum (Mo), and zinc (Zn) for optimal growth and development, although the significance of specific elements may vary depending on soil properties and environmental conditions (Afzal et al., 2020; Assunção et al., 2022; Nyiraguhirwa et al., 2022). In some agroecosystems, boron (B) or iron (Fe) may also be critical. Effective micronutrient management in barley involves a combination of soil and foliar applications, balanced with macronutrient fertilization. Regular soil testing and tailored fertilization strategies are essential to address specific nutrient deficiencies and optimize barley production (Mazur et al., 2024; Grzanka et al., 2024).

Micronutrients such as copper (Cu) and manganese (Mn) play key roles in plant metabolism, participating in electron transport chains during photosynthesis and respiration and acting as cofactors for numerous antioxidant and defense enzymes (Yruela, 2009; Mir, Pichtel & Hayat, 2021). Manganese plays a crucial role in the functioning of photosystem II, where it catalyzes water splitting and supplies electrons to the photosystem. Although the importance of these elements is well documented, much less is known about their effects under foliar application conditions in the field, particularly with respect to photosynthetic parameters and gas exchange in cereals. Manganese deficiency negatively affects plants by limiting the functioning of key enzymes responsible for the removal of reactive oxygen species, seed germination, and photosynthesis (Waldron et al., 2009; Stoltz & Wallenhammar, 2014; Schmidt & Husted, 2019).

Also, selective enzymes use molybdenum to carry out redox reactions. Plant molybdenum enzymes are involved in nitrogen reduction and assimilation, and sulfate metabolism. Molybdenum fertilization by foliar sprays can effectively replenish internal molybdenum deficiencies and support molybdenum enzyme activity (Kaiser et al., 2005; Rana et al., 2020). Zinc is a catalytic and structural protein cofactor in hundreds of enzymes and performs key structural functions in protein domains that interact with other molecules. The largest class of Zn-binding proteins in organisms is the zinc finger domain (Broadley et al., 2007; Rudani, Vishal & Kalavati, 2018; Cabot et al., 2019; Stanton et al., 2022). Photosynthesis is one of the most important biochemical pathways by which plants convert solar energy into chemical energy and grow. It is the most basic and complex physiological process in all green plants (Evans, 2013; Janssen et al., 2014; Ashraf & Harris, 2013). It is the basic physiological activity that determines the formation of plant yield, and the strength of photosynthesis is closely related to the level of yield (Lawlor, 1995; Long et al., 2006; Zhang et al., 2022). Analysis of chlorophyll fluorescence parameters is a useful technique that reflects the condition of plants. It plays an important role in understanding the basic mechanisms of photosynthesis and the response of plants to environmental changes. Chlorophyll content and chlorophyll fluorescence are important markers of the photosynthetic state of the plant (Maxwell & Johnson, 2000; Murchie & Lawson, 2013; Goltsev et al., 2016; Kalaji et al., 2016, 2018). Previous studies have rarely comprehensively assessed the effects of foliar micronutrient applications on barley physiology in intensive cropping systems typical of temperate climate zones. In particular, data on the effects of these treatments on chlorophyll fluorescence and gas exchange parameters are lacking, limiting the ability to fully understand their mechanisms of action in agricultural practice. Therefore, there is growing interest in assessing the physiological responses of cereal plants to foliar micronutrients, particularly in the context of photosynthetic efficiency and adaptation strategies in temperate climates. The foliar application of micronutrients has the potential to precisely and rapidly replenish element deficiencies in critical plant development stages (Fernández & Brown, 2013). However, the application of this technique in malting barley cultivation requires a thorough assessment of plant physiological responses, such as photosynthesis rate, chlorophyll content, transpiration rate, and chlorophyll fluorescence parameters. These parameters are rarely studied in field studies. Previous publications indicate that adequate micronutrient supply can significantly influence photosynthesis rate, water management efficiency, enzyme activity, and plant resistance to abiotic stresses (Niu et al., 2021; Kaya & Ashraf, 2024). However, comprehensive studies linking these aspects to foliar fertilization in malting barley cultivation are lacking. The study was carried out to investigate the effect of the foliar application of selected microelements on the chlorophyll content, canopy architecture, and physiological parameters of barley plants. The research hypothesis assumes that the application of microelements will have a positive effect on the course of the analyzed photosynthesis parameters of barley plants.

Materials and Methods

A three-year field experiment (2019–2021) was conducted in Reczpol (49°47′03″N, 22°34′37″E), in southeastern Poland, according to the methodology described by Stadnik, Tobiasz-Salach & Migut (2024). The experiment was set up in a split-block design with four replications. The main factor (block) was a spring barley cultivar (Baryłka, KWS Irina, RGT Planet), while the effect of a subfactor, i.e., variants of foliar fertilization with micronutrients (Cu, Mn, Mo, Zn and the non-fertilization control), was analyzed within each cultivar. The area of a single crop plot was 15 m². Agrotechnical treatments were conducted according to intensive spring barley cultivation technology, including full protection of the plant against weeds, diseases, and pests. Winter oil seed rape was the preceding crop.

In each study year, certified source seed material was seeded in the first ten days of April (6 April 2019, 2 April 2020, 8 April 2021), at a rate of 165 kg ha⁻¹, in rows 12 cm apart, at a depth of 3 cm. Harvest was carried out at full grain maturity (BBCH 89) (Lancashire et al., 1991) in the third decade of July (July 22, 2019, July 30, 2020, July 25, 2021).

Before the experiment, the physicochemical properties of the soil were analyzed in an accredited laboratory. The soil pH was determined potentiometrically (PN-ISO 10390:1997, 1997), organic carbon content using the Tiurin method (PN-ISO-14235:2003, 2003), available phosphorus and potassium using the Egner-Riehm method (PN-R-04023:1996, 1996; PN-R-04022:1996+AZ1:2002, 1996) and magnesium using the Schachtschabel method (PN-R-04020:1994+AZ1:2004 (1994)). The trace elements (Cu, Mn, Zn, and Fe) were determined after mineralization in HCl using flame absorption spectrometry (PN-R-04017:1992, 1992; PN-R-04019:1993, 1993; PN-R-04016:1992, 1992; PN-R-04021:1994, 1994). The soil was slightly acidic, containing approximately 1% organic carbon. Cu content was low in the first year and average in subsequent years, while other trace elements were present at average levels. Mineral (pre-sowing) fertilization included 50 kg ha⁻¹ N, 60 kg ha⁻¹ P₂O₅, and 90 kg ha⁻¹ K₂O. Polifoska® 6 was applied at a rate of 300 kg ha⁻¹ and ammonium nitrate (32% N) at 100 kg ha⁻¹. During the tillering phase (BBCH 25–27), a multi-component foliar fertilizer, Opti Zboża (3 kg ha⁻¹), containing macro- and micronutrients, was additionally applied.

The experimental factors were the following:

• I.

  Barley varieties: Baryłka (Hodowla Roślin Strzelce Sp. z o.o., IHAR Group), KWS Irina (KWS Lochow GmbH), RGT Planet (RAGT 2n);

• II.

  Foliar fertilization with micronutrients: Control (no fertilization), Cu (MIKROVIT® COPPER), Mn (MIKROVIT® MANGANESE), Mo (MIKROVIT® MOLYBDENUM), Zn (MIKROVIT® CYNK) (Table 1).

The application was performed once during the stem elongation phase (BBCH 30–32) (Lancashire et al., 1991) in windless conditions, using a pressure sprayer. Grain yield was assessed separately for each plot.

The mean temperatures and total rainfall during the growing season were determined based on data from the meteorological station in Zadąbrowo (50°19′09″N, 21°48′19″E). The Sielianinow hydrothermal coefficient (k) was calculated to assess water-thermal conditions. Based on the k value, meteorological conditions were characterized as dry, optimal, humid, or extreme (Stadnik, Tobiasz-Salach & Migut, 2024).

Results

Pearson’s correlation coefficient

The barley grain yield of the field experiment was presented in the publication by Stadnik, Tobiasz-Salach & Migut (2024). All correlation coefficients refer to aggregated data for application, operation, and year, with a sample size of n = 45. The table also presents the results of the correlations extracted from independent users and Fisher’s tests, which are distributed between the data sets (p ≤ 0.05 between all users). The strongest positive correlations with yield were recorded for transpiration rate (r = 0.81), chlorophyll content index (0.69), intercellular CO₂ concentration (0.62), and net photosynthesis rate (0.57). This relationship between MTA and yield (r = −0.28) was not statistically significant (Fig. 1).

Discussion

Plant growth and development depend on environmental conditions, including the availability of macro and microelements. Differences in nutrient content significantly affect the photochemical process of photosynthesis, therefore, they play a key role in biomass production (Kalaji et al., 2018; Cakmak & Engels, 2024). Micronutrients, although needed by plants in small amounts, play an important role in metabolic processes related to photosynthesis, chlorophyll formation, cell wall development, and respiration (Ram et al., 2017; Therby-Vale et al., 2022). Scientific studies on the effect of the foliar application of important micronutrients for plants focus primarily on their effect on mitigating environmental stresses and improving yield and its components (El-Magid, 2001; Ahanger et al., 2016; Kaur et al., 2023; Singhal et al., 2023). The aim of our study was to determine the effect of foliar fertilization with selected microelements on chlorophyll content, canopy architecture parameters, and the course of photosynthesis in spring barley under field conditions.

Leaf chlorophyll is a key indicator of leaf greenness, often used to determine nutrient deficiencies in leaves (Schlichting et al., 2015; Shah, Houborg & McCabe, 2017; Liu et al., 2019). In the field experiment conducted, the application of each of the microelements tested increased CCI in barley leaves, and the highest mean values were recorded in the plots fertilized with Cu and Zn (Table 1). Copper is an essential transition metal with redox activity, and it is involved in many physiological processes in plants. Cu deficiency affects young leaves, causing chlorosis and reduced photosynthetic activity. The positive effect of the foliar application of copper can be attributed to an important metabolic function, since this metal participates in photosynthesis and chloroplast development (Amberger, 1974; Yruela, 2005; El-Metwally et al., 2010; Yruela, 2013; Harangozo et al., 2017). Also, the study by Tobiasz-Salach & Augustyńska-Prejsnar (2020) conducted on barley, copper fertilization had a positive effect on chlorophyll content and, similarly to our study, the effect of copper was more visible than that of manganese. Improvements in physiological functions and an increase in chlorophyll content compared to control were also observed in Syuhada et al. (2014) in maize leaves sprayed with Cu. Noreen et al. (2021) indicate that the foliar application of Zn in barley cultivation increases the content of photosynthetic pigments, which was also confirmed by the results of our own studies.

The LAI index describes the size of the leaf area per unit of horizontal ground surface. This index is an important biophysical variable to understand the efficiency of the use of radiation by field crops and their potential yield. The LAI value depends on many factors, including genetic traits of plants, their habitat, and agrotechnical factors such as sowing density and the phenological stage of the plant (Chen & Black, 1992; Zou et al., 2018). LAI and MTA indices are primarily used to assess the growth rate and biomass accumulation (Feledyn-Szewczyk, 2009; Lepiarczyk et al., 2005; Dereń et al., 2018; Fang et al., 2019). In our study, the foliar application of each of the microelement fertilizers tested had a positive effect on the LAI and MTA values of barley plants, and the best results were obtained on plots fertilized with Mo (Table 2). Significant genotypic differences were also demonstrated. In the study by Tobiasz-Salach, Jańczak-Pieniążek & Bobrecka-Jamro (2018) in which the foliar application of microelements was used in four barley cultivars, the LAI index also depended on the cultivar and the application of foliar microelement fertilization. There are no reports in the scientific literature on the effect of single-component microelement fertilizers on the MTA value.

Carbon assimilation in photosynthesis and water loss through transpiration are key physiological processes that affect biomass production (Lambers, Oliveira & Pons, 2019; Hatfield & Dold, 2019). The analysis of the effect of fertilization with the microelements tested shows that each of the fertilizers used improved gas exchange parameters in H. vulgare L. The highest increases in values were observed after the application of Cu and Zn. In the conducted experiment, C_i and E were significantly higher after the application of Zn. Zinc plays an important role in the activation of enzymes, N metabolism, and is involved in photosynthesis and DNA reproduction during cell division (Ram et al., 2017). In the studies by Liu et al. (2016), the use of Zn at an optimal dose (30 kg ZnSO₄ 7H₂O) in maize caused an increase in P_N, had a positive effect on the chlorophyll content in the leaves, and increased grain yield. Studies conducted on various species of crop plants report that zinc deficiencies in plants cause disorders in the functioning of the photosynthetic apparatus (Mattiello et al., 2015; Rudani, Vishal & Kalavati, 2018; Li et al., 2020). The particularly visible improvement in gas exchange parameters in the experiment carried out after the use of zinc may indicate that the plants were provided with an adequate supply of this nutrient and, consequently, increased photosynthetic efficiency.

In the research conducted, among the microelements tested, the highest C_i and P_N values were recorded after the application of Cu (Table 2). The increase in photosynthetic parameters may indicate the optimal dose of Cu for barley plants. In the study by Moraes et al. (2025) on coconut seedlings, in which Cu was applied foliarly at low concentrations, an improvement was observed in the rate of photosynthesis, transpiration, and stomatal conductance. In addition, Cu at lower concentrations increased the efficiency of photosystem II. Copper is potentially harmful when present at concentrations that exceed optimal ones. Excess accumulation can destabilize membrane integrity, reduce photosynthesis, and change enzyme activity, consequently inhibiting plant development (Moustakas et al., 1997; Shabbir et al., 2020; Mir, Pichtel & Hayat, 2021). The study also noted a beneficial effect of foliar Mo spraying on plant gas exchange. Oliveira et al. (2022) showed that the application of Mo to soybeans and corn increased net photosynthesis. The study authors indicated that Mo-based foliar fertilization could effectively improve nitrogen metabolism and plant response to carbon fixation, resulting in improved yields.

The analysis of the values of the chlorophyll fluorescence parameters indicates a particularly significant effect of molybdenum and zinc on the shape of the values of individual indicators. After the application of fertilizers based on these microelements, the highest values of F_v/F_m, RC/ABS, F_v/F₀ and PI were recorded (Table 3). Decreases in chlorophyll fluorescence parameters are recorded in plants both under conditions of Zn deficiency (Wang & Jin, 2005) and at too high doses (Andrejić et al., 2018). Studies have indicated a positive effect of foliar application of Zn on the accumulation of photosynthetic pigments and chlorophyll fluorescence, especially in stress conditions (Narimani & Sharifi, 2020; Klofac, Antosovsky & Skarpa, 2023). An increase in F_v/F₀ by 6.7% compared to the control was recorded in studies with sugar beet after foliar fertilization with Zn, and the total chlorophyll content increased by more than 25% (Zhao et al., 2024). There are no reports in the literature on the effect of the foliar application of Mo on the chlorophyll fluorescence parameters. Han et al. (2020) investigated the effect of soil Mo application under stress from cadmium (Cd) on rapeseed. In this study, the application of Mo alleviated the negative effect of Cd on plants, among others, by improving F_v/F₀ and F_v/F_m.

The response of crop plants to microelement foliar fertilization varies by species and genotype. Barley cultivars, similar to studies by other authors, showed differences in the values of measured physiological parameters (Tobiasz-Salach & Augustyńska-Prejsnar, 2020; Moshfeghi et al., 2019). In a laboratory study conducted by Jarecki, Lachowski & Migut (2024) on soybean where various microelements were applied in foliar form, differences in the response of the cultivars were also shown. In our own studies, a significant increase in CCI, E, and P_N was noted in the RGT Planet cultivar compared to the Baryłka and KWS Irina cultivars (Tables 1, 2). In the study by Jalakas et al. (2018) on malted barley, the genotype had a significant effect on the P_N and g_s values. The chlorophyll fluorescence parameters analyzed from the KWS Irina cultivar showed statistically lower values of the F_v/F_m and F_v/F₀ indexes compared to the RGT Planet and Baryłka cultivars (Table 3).

Pearson’s correlation analysis showed the influence of the parameters analyzed on grain yield. The strongest correlations were shown between yield and CCI, E, P_N, and PI. A better understanding of the physiological conditions of crop yield formation and the identification of traits related to grain yield help improve the efficiency of crop breeding and cultivation. However, in long-term experiments, physiological parameters are relatively rarely measured in relation to yield (Araus et al., 2008; Jalakas et al., 2018). Jalakas et al. (2018) reported that the correlation between leaf gas exchange traits and grain yield depends on the climatic conditions during the growing season. In studies on wheat and barley, stomatal conductance and CO₂ assimilation rate were reported to correlate with grain yield (Fischer et al., 1998; Gonzalez, Bermejo & Gimeno, 2010). In the study by Wasaya et al. (2021), strong positive correlations between P_N, g_s, and E were observed with grain yield in wheat subjected to drought stress and irrigation. Zhu, Long & Ort (2010) reported that increased photosynthesis in the crop under standard field cultivation conditions resulted in higher yields. Parry et al. (2011) reported an improvement in wheat yield due to increased photosynthesis. Our results also confirm the relationship between the course of plant photosynthesis and the efficiency of barley cultivation.

Conclusions

The studies carried out demonstrated a positive effect of foliar application of fertilizers containing Cu, Mn, Mo, and Zn on the relative content of chlorophyll in leaves, the parameters of the canopy architecture, and the selected parameters of chlorophyll fluorescence and gas exchange in barley plants. The cultivars studied were characterized by a varied response to foliar application of microelements. The values of selected parameters were strongly correlated with grain yield. The analysis carried out indicates that the measurement of physiological parameters during plant vegetation may be useful in forecasting crop productivity. The conducted experiment provides information on the importance of foliar application of micronutrients in the cultivation of an economically important crop species. The research results may be useful in optimizing barley grain production for brewing purposes. Increasing crop efficiency through fertilization without interfering with the soil environment supports the achievement of sustainable development goals. The varied response of the cultivars indicates the need for more research on the optimization of fertilization strategies based on genotypic traits. The research results have significant application implications for precision agriculture and environmentally friendly agriculture. The use of foliar microelement fertilization may contribute to increasing the efficiency of nutrient management, improving the condition of plants under stress conditions, and optimizing yield. This can find practical application in modern crop management systems, supporting farmers in adapting fertilization strategies to changing site conditions and cultivar specificity.

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