Influence of Fertigation Regimes on Nitrogen Concentration in Apple (Malus × domestica Borkh.) Leaves at Different Age Stages

Abstract

Nitrogen (N) deficiency in apples (Malus × domestica Borkh.) leads to characteristic physiological symptoms, including leaf and fruit discoloration. Fertigation, i.e., the application of dissolved fertilizers, can significantly improve the growth and fruit quality of apples while optimizing nutrient uptake through a more precise and better timed application than conventional fertilization. This study therefore investigates how different fertilization treatments affect the N concentration of different age categories of apple leaves. Apples of the variety ‘Braeburn’ were grown hydroponically on the low-vigorous rootstock M9. Four fertilizer treatments were used: (1) Hoagland solution (HS); (2) HS nitrogen excluded; (3) HS iron excluded; and (4) HS magnesium excluded. Through vegetation, leaf samples were taken from three shoot positions representing different leaf ages (young, semi-young and old) and then chemically analyzed. The lowest N concentrations across all leaf ages and sampling moments were found in the treatment with N excluded (1.69–2.07% N), while the highest values occurred in the treatments where iron (2.00–2.49% N) or magnesium (1.98–2.37% N) were excluded. The seasonal changes in N concentration reflect interactions between the leaf age and the sampling moment. These results show that the N concentration of apple leaves strongly depends on the type of fertilization.

1. Introduction

The apple (Malus × domestica Borkh.) is one of the most widely cultivated fruit species in the world, valued for its flavor, nutritional properties and adaptability to different agro ecological conditions [1,2]. Global production amounts to over 97 million tonnes per year, with Croatia contributing around 60 thousand tonnes, which corresponds to around 0.06% of the global supply [2].

Although apple trees have favorable natural conditions and strong genetic potential, a balanced supply of important nutrients is required for a stable and high yield. An imbalance, whether due to deficiency or excess, can reduce photosynthetic activity, compromise fruit quality and storability, and increase sensitivity to abiotic and biotic stress, ultimately reducing yield and profitability [3,4]. Apple orchards are often insufficient in important macronutrients such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulfur (S) [5]. Such deficiencies often lead to leaf discoloration, stunted growth, smaller fruits and greater risk of pathogen infection. The nutrient status of the growing medium directly affects the concentration and availability of nutrients in the plants, maintaining a complete and appropriate balance with plant requirements, especially nutrients for optimal physiological processes [4,6]. Consequently, careful planning of fertilization and irrigation is crucial for maintaining tree health, maximizing fruit yield and ensuring the production of high-quality apples [7,8,9].

For many years, apple orchards were fertilized by spreading or incorporating solid fertilizers into the soil. Today, fruit production is shifting toward precision agriculture, where nutrient management is controlled by digital monitoring and remote sensing. As part of this concept, fertigation, the supply of dissolved fertilizers via an irrigation system, is becoming increasingly important. This allows nutrients to be applied directly to the root zone with each irrigation, at a far more precise time and dosage than conventional fertilization [5].

Of all nutrients, N is a key biogenic element that plays a particularly important role in plant defense. It is also essential for metabolism as a structural component of amino acids, proteins, nucleic acids, coenzymes, photosynthetic pigments and polyamines [6,10,11,12,13]. Although N in the molecular form (N₂) makes up around 78% of the Earth’s atmosphere, plants cannot utilize it directly due to the exceptional stability of the triple bond between the two N atoms. The conversion of atmospheric N₂ into plant-available ammonium (NH₄⁺) or nitrate (NO₃⁻) ions requires a considerable amount of energy, and the limited supply of these ions often limits plant growth and yield [4,14,15]. Nitrogen deficiency slows down overall plant growth and often limits yield, as N is easily converted into its molecular form and quickly lost from the soil [14,16]. Under deficient conditions, older leaves become pale green or chlorotic because proteins and chlorophyll pigments in the chloroplasts are degraded and N is remobilized. This loss of chlorophyll lowers photosynthetic intensity, accelerates leaf ageing, and ultimately reduces yield [11,16]. In contrast, excess N promotes vigorous, dark green vegetative growth, but increases susceptibility to pests, diseases, drought and low temperatures and can lead to NO₃⁻ leaching into groundwater [10].

At the beginning of the vegetation period, N is remobilized from internal nutrient reserves and older leaves into young leaves and actively growing tissues, ensuring an adequate supply for the rapid development of buds, fruit and biomass production [4]. Furthermore, as growth continues into the mid-season, the plant increasingly relies on N delivered through fertigation to maintain development. Towards the end of the growing season, it replenishes its nutrient stores, which remain over winter and serve as a critical N source for the following growth cycle [17,18].

Since nutrient concentrations change as the shoots develop and the leaves mature, the age of the leaves is an essential criteria for the precise diagnosis of a plant’s nutrient status, as the symptoms of deficiency also change with leaf development [19]. For example, N and Mg deficiency typically appear first in older leaves, while iron (Fe) deficiency is most evident in young leaves [13,20]. Apple leaves generally contain N concentration between 2 and 5% N in dry matter, depending on leaf age and vegetation stage [14,21,22].

Previous studies have extensively examined the relationship between N management, tree growth, and fruit quality in apple orchards, highlighting the importance of optimizing nutrient supply to balance vegetative vigor and fruit production. Research on fertigation and irrigation practices has shown that proper nutrient and water management can enhance early tree establishment and improve yield [9,23]. According to Dominguez and Robinson [9], irrigation and fertigation affected leaf nutrient levels in five apple cultivars, resulting in leaf N concentrations ranging from 2.63 to 2.85% N. Furthermore, Jang et al. [24] observed that the excessive, adequate, and insufficient treatment groups in leaves had N concentration ranges of 2.48–3.32%, 2.15–2.84%, and 1.85–2.51%, respectively. As the growth period progressed, N concentration decreased regardless of the treatment group. Moreover, some investigations have explored how the rate and timing of N application influence apple tree nutrition and fruit characteristics, revealing that excessive N generally promotes vegetative growth and increases tissue N concentration, but can negatively affect fruit color, firmness, and storability [25,26,27].

Other studies have focused on N uptake efficiency and internal N distribution, indicating that adequate pre-harvest N supply can enhance N uptake and translocation to actively growing organs without compromising fruit quality. Authors reported that leaf N concentration in pre-harvest trees during the N application period reached maximum values between 2.0 and 2.4% N [18]. Environmental conditions, such as soil temperature and tree developmental stage, have also been identified as critical factors regulating N uptake, particularly during early spring [28].

However, most of these studies have focused on young seedlings grown under controlled or hydroponic systems [29,30], providing limited insight into mature field-grown trees. Maintaining this optimal N concentration is a challenge, however, as the nutritional status of a plant is determined by complex interactions between the plant and its growing medium. The availability of nutrients depends on factors such as soil pH, antagonistic ions, biological activity of soil and temperature of both soil and air. This environmental complexity often makes the timely and accurate diagnosis of nutritional disorders difficult [4,13,31].

Therefore, the aim of this study was to determine how different fertilization regimes affect the N concentration in apple leaves of different ages (young, semi-young and old leaves). In addition, the effects of sampling moment, leaf age and their interaction on the N concentration was analyzed.

2. Materials and Methods

The study was conducted during the 2024 growing season at the Maksimir experimental station of the University of Zagreb Faculty of Agriculture, located in Zagreb, Croatia (45°49′36″ S; 16°01′47″ E). The experimental orchard was established in an outdoor hydroponic system to enable precise control of nutrient supply and minimize the influence of external soil variability. The apple variety ‘Braeburn’, grown on low-vigorous rootstock M9, were cultivated in 30 L vegetative pots filled with perlite, an inert and nutrient neutral substrate (Figure 1).

After the acclimatization period, trees were randomly assigned to four fertilization treatments based on Hoagland nutrient solution [32]:

• Treatment 1 (H1): Hoagland solution, all macronutrients and micronutrients;
• Treatment 2 (H2): Hoagland solution N excluded;
• Treatment 3 (H3): Hoagland solution Fe excluded;
• Treatment 4 (H4): Hoagland solution Mg excluded.

Each row represented one treatment (Figure 1A) and consisted of 40 trees. Within each row, trees were divided into three replicates of 12 trees each (first, second, and third segments of the row). Two trees at each end of every row were used as isolation to minimize border effects between treatments. This design provided within-treatment replication and controlled for spatial variability along the row. Due to fertigation requirements, each row represented a single treatment, and all trees within the row were fertilized with the same nutrient solution. Daily fertigation (Figure 1B) was applied, with each tree receiving one liter of nutrient solution specific for each treatment, depending on meteorological conditions (e.g., precipitation). Leaf sampling was conducted once a month from June to September (vegetation season). Within each fertilization treatment, fully developed, mechanically undamaged leaves were randomly sampled from a each segment of the row (12 trees) and arranged in three replicates for each leaf age category (young, semi-young and old), resulting in 36 samples per sampling date. The leaf age category was determined based on leaf position on the shoot, with young leaf located at the top of the shoot, semi-young in the middle section, and old at the base of the shoot. Each sample was analyzed for 12 leaves.

Chemical analysis of plant material samples was carried out at the Analytical Laboratory of the Department of Plant Nutrition at University of Zagreb Faculty of Agriculture, accredited according to the HRN EN ISO/IEC 17025:2017 standard [33]. Prior to analysis, leaf material was homogenized, oven-dried at 105 °C, and finely ground. Total nitrogen was determined according to AOAC Official Method 978.04—Nitrogen (Total) (Crude Protein) in Plants—Kjeldahl Methods [34], and results were expressed as percentage of N (% N) in dry matter.

All statistical analyses were performed using SPSS statistical software (version 22.0; SPSS Inc., Chicago, IL, USA). One-way ANOVA was used to assess the effect of the different fertilization treatments on leaf N concentration within each leaf age category and to evaluate the effect of treatments on N concentration across the four sampling moments. In addition, a two-way ANOVA was conducted to examine the main effects of leaf age category and sampling moment and, importantly, to test the significance of their interaction on leaf N concentration. The significance level for all statistical tests was set at α = 0.05. When significant main effects or interactions were detected, Tukey’s honestly significant difference (HSD) post hoc test was applied to identify specific differences between treatment means.

3. Results

Nitrogen concentration in dry matter (% N) in apple leaves in different leaf age categories is shown in Figure 2. There was no statistical difference in concentrations of N in treatment H1 (2.08–2.18% N) and H2 (1.86–1.91% N). However, treatment H3 showed a difference in N concentration between young and old leaves. Statistically, the highest N concentration was recorded in old leaves (2.36% N), while the lowest was recorded in young leaves (2.08% N). Furthermore, a significant decrease in N concentration was observed in treatment H4 in old leaves (2.10% N) compared to semi-young leaves (2.31% N). Overall, these results indicate that N concentration decreases with leaf ageing in H1, H2 and H4 treatments, in contrast to H3.

Moreover, Figure 3 shows total N concentration in apple leaves during four sampling moments under different fertilization treatments. From June to September, there was no statistical significance of N concentration in H1 and H4 treatments. Significant increase in N concentration was observed in treatment H2 during July, August and September (1.95; 2.02 and 1.94% N, respectively) in comparison to June (1.77% N). Also, statistically the highest N concentration was observed in July (2.29% N) in H3 treatment, while a statistical decrease occurred during August and September (2.17 and 2.16% N, respectively). Overall, these results show that the seasonal variation in N concentration was most pronounced in treatment H3, where concentrations reached their maximum in mid-summer before declining in late summer, while H2 treatment showed a steady increase in mid-summer.

The changes in N concentration in the apple leaves were evaluated considering the effects of the leaf age category, the sampling moment and their interaction (

Table 1. ANOVA table with p values for effects of leaf age and sampling moment.

Source	Df	H1	H2	H3	H4
		p Value
Age	2.24	0.054	0.065	<0.001	0.001
Sampling moment	3.24	0.586	<0.001	0.013	0.073
Age × sampling moment	6.24	0.022	0.547	0.367	0.878

Note. H1—Hoagland solution; H2—Hoagland solution N excluded; H3—Hoagland solution Fe excluded; H4—Hoagland solution Mg excluded.

).

Although neither leaf age nor sampling moment showed a significant main effect in H1 treatment, their interaction was significant (p = 0.022). This indicates that the differences in N concentration between leaf age categories varied depending on the time of sampling.

Furthermore, in treatment H2, sampling moment had a highly significant main effect (p < 0.001), while leaf age and the interaction were not significant. This suggests that N concentration changed across the four sampling moments regardless of leaf age.

Both main factors, leaf age (p < 0.001) and sampling moment (p = 0.013), were significant in H3 treatment. These results highlight that N concentrations differed between leaf age categories as well as between sampling moments, even though the differences between leaf ages did not change over time.

Differences in N concentration were primarily due to the effects of leaf age rather than sampling moment, as only leaf age had a significant effect (p = 0.001) in treatment H4.

Figure 4A–D illustrate how N levels in young, semi-young, and old leaves varied from June to September, revealing treatment-specific patterns of N dynamics.

In the H1 treatment (Figure 4A), at the start of leaf sampling in June, N concentration in all leaf age categories varied in narrow range from 2.08 to 2.24%. Furthermore, a clear downward trend in N concentration was observed in the young leaves from July towards the end of the vegetation period. From June to September, the N concentration fluctuated between 1.96 and 2.31%, regardless of the age of the leaves.

In the H2 treatment, N concentration fluctuated within a narrow range (1.69–1.81% N) during June (Figure 4B) and was consistently lower than in treatments where N supply was not excluded. Across all leaf age stages, it ranged from 1.69 to 2.07%, except in June, when young leaves had lower concentrations. Generally, the lowest concentrations throughout the season were found in the old leaves compared to the other leaf age categories.

On the other hand, in the H3 treatment (Figure 4C), N concentrations varied greatly in all leaf age categories (2.07 to 2.49%), especially at the beginning of leaf sampling. In addition, the lowest values were consistently found in the young leaves throughout the growing season, as the N concentration varied between 2.00 and 2.49% N in all leaf age categories.

In contrast, N concentrations in all leaf age categories in the H4 treatment (Figure 4D) remained within the range of 1.98 to 2.37%. The lowest concentrations were recorded in the old leaves throughout the entire vegetation period. Additionally, during first sampling, N concentration varied in narrow range across all leaf age categories (2.22–2.30% N).

4. Discussion

The results of this study (Figure 2) clearly demonstrate that the response of plants to nutrient availability varies with fertilization treatment. Also, the distribution of the N among the leaves depends on the leaf age category. According to Neubert et al. [35] and Crassweller [36], the N status of apple leaves is divided into three categories: low (<1.80% N), optimal (1.80–2.40% N), and high (>2.40% N).

In H1 treatment, plants received a balanced nutrient solution containing all essential macro- and micronutrients throughout the growing season. This lead to an optimal N supply, promoting a relatively homogeneous distribution of N within the plant regardless of leaf age. Studies [4,37] showed that, with a stable and adequate external N supply, N metabolism in older and younger leaves remains at a comparable level, minimizing the differences between leaf age even across different stages of plant growth.

On the other hand, the plants activated internal remobilization mechanisms during H2 treatment as a result of N limitation by remobilizing organic and mineral forms of N from older, partially ageing organs to younger, metabolically active leaves and meristematic tissues [38,39]. Sakuraba et al. [40] also stated that yellowing of leaves due to remobilization of N sources from older leaves to younger leaves and reproductive organs is one of the representative responses to N deficiency. Such redistribution helps to maintain the required minimum N concentrations throughout the canopy, which can lead to relatively uniform concentrations between leaf age categories even during a prolonged growing season [4]. In addition, sampling was conducted during the active vegetation phase, when both old and young leaves are photosynthetically active, which also contributes to the lack of significant differences.

Furthermore, the lowest N concentrations were found in young leaves, while semi-young and old leaves contained increasingly higher amounts of N in the H3 treatment where Fe was excluded. Iron deficiency typically occurs first in the youngest leaves, as Fe is only weakly mobile in the phloem [4]. Limited Fe availability disrupts chlorophyll synthesis and the function of numerous Fe-dependent enzymes within the photosynthetic electron transport chain. As a result, photosynthetic activity and N assimilation are reduced in newly developing tissues, leading to a temporary imbalance in internal N distribution [4,41]. Therefore, N accumulation is relatively lower in young leaves, while older leaves, in which Fe reserves are already stored and whose metabolic activity is less dependent on the current Fe supply, have comparatively higher N concentrations.

In H4 treatment where Mg was excluded, the lowest N concentrations were observed in old leaves, while semi-young leaves had significantly higher values. Magnesium is very mobile in the phloem, as well as in the xylem of the plant, and in the case of Mg deficiency it is preferentially remobilized from older to younger leaves. Because of that, Mg deficiency symptoms (interveinal chlorosis) typically occur first in older leaves [4,37]. Since Mg is the central atom of the chlorophyll molecule and an essential cofactor in ATP formation, its deficiency reduces photosynthetic capacity and ATP production [42]. The lower ATP availability limits the energy-consuming processes of N uptake and assimilation (such as nitrate reduction and amino acid synthesis), which further contributes to the decrease in N concentration in the oldest leaves, as was the case in this study. In contrast, semi-young leaves, which still receive Mg through remobilization, maintain higher photosynthetic activity and N assimilation and therefore have a higher N concentration [4,42], as shown in H4 treatment.

The N concentration in the apple leaves at the beginning of sampling (June) in the H2 treatment was the lowest (1.77% N) compared to the other sampling moments and treatments (Figure 3). According to Aguirre et al. [43], at the beginning of the season (usually early June), the N concentration in the leaves may be lower because the growth of the fruit, rapid shoot expansion, and the formation of the flower buds take place simultaneously, forming strong, competing sinks that dilute and reduce the limited N pool. In this study, trees without adequate N fertilization are mainly dependent on small stocks of remobilized N. About half of the N used for spring growth comes from internal reserves [17,44], with reserves providing most of the N for shoot growth [45]. This reserve-driven phase generally lasts until the end of May/June, after which rapid root uptake must maintain further growth, emphasizing the need for new N supply [46]. Even without external N supply and an inert perlite substrate, a small increase in leaf N can be observed later, with minor atmospheric inputs due to nitrate and ammonium supplies via rain, with irrigation water and aerosol also contributing. Taken together, these incidental inputs potentially affect measurable leaf N even without N supply [47], as observed in this study. In combination with the initial limited availability, followed by later increases in N release and uptake, they led to a statistically significant increase (2.0% N) in leaf N concentration by the end of this experiment [4,47,48,49,50].

Furthermore, nutrient concentrations are not stable within a season, as the nutrient supply and the internal cycle in the tree change during leaf and shoot development [51]. In this study, the seasonal dynamics of total N in the leaf differed between all treatments, with treatments H2 and H3 showing significant increase or slight fluctuations, while H1 and H4 remained stable throughout the observation period.

In accordance with established physiology, N is very mobile and is gradually remobilized from older to younger, actively growing tissues and storage organs, so that leaf age and season strongly influence leaf N concentration [4]. In addition, the stage of development, irrigation/fertilization regime and nutrient interactions can change leaf N concentration during the season [4,52].

The seasonal patterns of N concentration observed in the four treatments show clear treatment-specific effects on leaf N status and illustrate how leaf age influences N dynamics (Figure 4). Additionally, the reference values reported for apples for old leaves (1.80–2.50% N) and semi-young leaves (1.80–3.0% N) [4,53] are in good agreement with our values (Figure 4).

Against this background, H2 treatment (N excluded) consistently had the lowest leaf N concentration (1.69–2.07%), often close to or below the lower optimal range [35,36], especially in old leaves. Remobilization and age-related decline made the deficiency more pronounced, which is consistent with the literature on N mobility and deficiency symptoms in leaf ageing [4,18,53]. In contrast, treatments with adequate N supply (H1, H3, H4) maintained leaf N concentration largely within the optimal range [35,36], reflecting reports that timely supply maintains the mid-season N concentration of semi-young leaves and supports shoot growth [4,16,53]. During the mid-season, N concentration in semi-young leaves in H1, H3 and H4 treatments were in line with values in the literature, while in H2 it remained below 2.2% as expected.

Treatment contrasts further emphasize the nutrient interactions in H3 treatment (Fe excluded); leaf N concentration varied the most, with some values higher than 2.4% N, entering the high category [35,36]. Concentrations in young leaves were the lowest across all sampling moments; this pattern is consistent with Fe limitation affecting chlorophyll formation and altering N metabolism, as has been noted for interactions between micronutrients and N [4,52]. In H4 treatment (Mg excluded), N concentration remained within the optimal range [35,36], with old leaves having the lowest value, consistent with the role of Mg in photosynthesis and cation–N interactions that may affect N status [4], as mentioned.

Finally, the youngest leaves typically have the highest N concentration (1.80–3.50%) because N is actively mobilized to support rapid growth [54], and half of spring N requirements can come from internal reserves [17], highlighting why the timing and form of supply strongly influence the seasonal pattern of leaf N concentration [13,18]. In practice, our results show that supplying apples with N (H1, H3, H4 treatments) keeps N status within the recommended ranges across leaf age. In contrast, excluding N from fertilization (H2 treatment) leads N values into the lower range, underlining the importance of nutrient management and standardized sampling of leaf age categories for valid diagnosis. The changes in total N concentration in apple leaves (Table 1) confirm that both the age of the leaves and the time of sampling are important determinants of leaf N status and that their relative influence varies depending on the fertilizer treatment. The significant interactions observed in some treatments indicate that the differences in N concentration between leaf age categories cannot be interpreted independently from the time of sampling. In other words, the differences between young, semi-young and old leaves are not constant, but shift over the course of the season. This result underlines the need for standardized and precisely timed leaf sampling when assessing the nutritional status of apple trees.

For this reason, a balanced fertilization regime is essential for efficient N assimilation and maintaining appropriate concentrations in plant tissues from the start to end of vegetation [7,55,56]. An oversupply of fertilizer can lead to N accumulation in young leaves, while insufficient fertilization can cause N depletion in older leaves [54,57]. Therefore, careful nutrient management is required to maintain optimal N levels across different leaf ages.

5. Conclusions

This study shows that the N concentration in apple leaves varies predictably with the age of the leaf and sampling moment and is strongly influenced by the fertilization regime. Nitrogen levels remain relatively stable in H1 treatment (1.96 and 2.31%), suggesting that balanced fertilization results in optimal N concentration in apple leaves. Therefore, an optimal N concentration in leaves is required to promote optimal apple production by ensuring higher yields, better overall fruit quality and higher market value. Additionally, N deficiency triggers the remobilization of stored reserves, highlighting the importance of characterizing internal N pools and their distribution, resulting in different concentrations of N in H2 treatment. Interactions with other nutrients, such as Fe and Mg in H3 and H4 treatment, modulate these patterns of N concentration in leaves. Unlike previous studies that mainly examined N rate and timing, this work highlights how leaf age and sampling moment interact with fertilization regimes under outdoor hydroponic conditions. These results address a key knowledge gap and provide a basis for refining fertigation strategies to improve N use efficiency and sustainable orchard management. Future research should focus on tracking N dynamics over multiple seasons, accurate quantification of internal N forms and the physiological mechanisms of nutrient buffering. Such integrated findings will support more effective, efficient and ecologically sustainable N management in apple orchards.