Vineyard Fertilization Management for Iron Deficiency and Chlorosis Prevention on Carbonate Soil

Abstract

Nitrogen fertilizer efficiency in grapevine production is an important objective for solving the trade-off between improving yield and quality in agroecosystems and reducing environmental impacts. Influence of soil nitrogen fertilization and Fe foliar application on iron dynamics in soil and grapevine leaves of the ‘Graševina’ cultivar on carbonate soil was conducted in a two-year study in 2018 and 2019. The experiment was settled in three replicates on a total of seven fertilization treatments that differed in used form of nitrogen fertilizer and foliar application of Fe before and after the flowering of the grapevine: control (C); calcium ammonium nitrate (KAN); calcium ammonium nitrate + foliar Fe (KAN^+F); ammonium sulfate (AS); ammonium sulfate + foliar Fe (AS^+F); ammonium sulfonitrate + foliar Fe (ASN^+F); urea + foliar Fe (U^+F). Mineral fertilization with acid-forming nitrogen fertilizers (AS and ASN) significantly affected local acidification of alkaline soil, i.e., reducing the actual and exchangeable soil pH reaction, which resulted in increased soil Fe availability. Despite the increase in soil iron availability, no increased iron bioaccumulation in the grapevine leaves was found in the flowering and veraison stages at treatments where foliar fertilization was omitted. Of all the observed treatments, only foliar fertilization had a positive effect on iron concentration in the grapevine leaves, which leads to the conclusion that this is an effective way to solve iron deficiency symptoms and chlorosis occurrence. The use of mineral fertilizers with acid-forming nitrogen fertilizers for many years can result in a reduction of required foliar treatments and thus significantly affect the ecological and economic aspects of grape production. Thus, integrated iron management is needed to meet the needs of the grapevine for this micronutrient and to reduce the occurrence of leaf chlorosis in carbonate soil.

1. Introduction

The grapevine (Vitis vinifera L.) is one of the oldest cultivated plants with exceptional economic importance in Central European countries [1]. According to the Croatian Central Bureau of Statistics (CBS), between 2010 and 2019, the total area of vineyards was reduced from 33 to 20 thousand ha, which consequently halved grape production from 207,743 t in 2010 to 108,206 t in 2019. The reasons for a drastic reduction of native cultivars in Croatia are the requirement of growers for high yields and the introduction of worldwide known cultivars [2].

In Croatia, ‘Graševina’ is the most represented variety of grape, grown on 4589.90 ha, which is 24.7% of the total area under vineyards. ‘Graševina’ is white grape cultivar also known as Welschriesling, Italian Riesling and Olasz Riesling. According to the Ordinance on the National List of recognized grapevine cultivars (OG 159/04), ‘Graševina’ is a recommended cultivar in all subregions of continental Croatia, and is characterized by moderate lushness, medium to above-average yield, and typical wine grapes and berries.

Trace elements such as Fe, Zn, and Mn are important activators of many essential enzymes involved in the metabolism of grapevines [3]. Iron deficiency occurs in about one third of the world’s arable land [4,5]. In Baranja and other subregions of the Croatian Danube basin, chlorosis caused by a deficiency of trace elements (most often Fe-chlorosis) is a major problem in viticulture, which directly affects both grape yield and quality. Iron deficiency in grapevines is manifested by intervascular chlorosis of younger leaves, whereby the leaf ribature remains green, and then marginal necrosis and leaf fall occur [6]. In addition to the primary symptoms, secondary symptoms may appear, which makes visual diagnosis unreliable, therefore chemical analysis is indispensable for determining the true cause, especially in the event of multiple symptoms of nutrient deficiency [7]. Therefore, chemical analysis of plant matter during the growing season is of great importance. It indicates the supply and status of a particular element in a particular phenological phase which allows a proper balance of nutrients reducing the environmental burden caused by agricultural production.

Soil management in vineyards is of fundamental importance for greater sustainability of the production [8]. The basic soil function, in addition to providing the space for rooting, is to supply plants with water and nutrients in sufficient quantities during the growing season. The Earth’s crust is about 5% iron [9]. In the soil, iron originates from primary and secondary minerals which make most of its inorganic reserves, with total content between 0.5–4.0% [10]. Micronutrient availability in the rhizosphere is controlled by soil and plant properties alongside the interactions of roots and microorganisms and the surrounding soil [11]. The total amount of metal in the soil is but a fraction of what is available to the plant, and the bioavailability of metals to the plant is determined not only by the concentration in aqueous solution or the total concentration of heavy metals but also by many other biotic and abiotic factors. The most important factor controlling the availability of iron and other microelements in soil is soil pH [12]. In carbonate soils there is an intensive formation of poorly soluble iron compounds such as Fe(OH)²⁺, Fe(OH)₃ and Fe(OH)⁴⁻, which is the main reason of low iron supply. In addition to the pH reaction of the soil [13], the presence of carbonates (CaCO₃) in the soil, low soil aeration, soil compaction, low temperature in the root zone, low content of organic matter, poor biological properties and inadequate soil nitrogen status are also important factors controlling the supply of microelements.

Nitrogen (N) is an essential nutrient element for plant growth and N deficiency may cause many disorders, leading to overall plant performance reduction. Profitable and sustainable production implies achieving high yields of appropriate quality with environmental sustainability [14], and the quantity and form of nitrogen fertilizer are important factors in achieving these goals. Dynamics of nitrogen uptake and the impact on the physiology and grapevine yield are necessary for the proper application of nitrogen fertilization. It is extremely important to regulate nitrogen fertilization based on the actual needs of the grapevine in a particular phenophase of growth and development [15]. At the same time, the amount and forms of nitrogen affect the availability of trace elements such as iron, indirectly affecting the physiological processes in the grapevine. Thus, the optimal supply and bioavailability of iron can prevent the occurrence of chlorosis which reduces the yield and yield quality. Nitrogen-use efficiency (NUE) by grapevines is an important objective for solving the trade-off between improving yield and quality in agroecosystems and reducing environmental impacts. Nitrogen fertilization, as well as foliar fertilization, is therefore an important factor in viticultural production, but it is necessary to optimize nitrogen fertilization both in terms of total quantities and dynamics and in terms of N form in the fertilizer adapted to the physicochemical properties of the soil. Therefore, achieving a worldwide reduction of the N input or fertilizer efficiency is a major challenge that requires sustained action to improve nitrogen management practices and to reduce nitrogen environmental impact. Adopting nitrogen fertilizers forms on carbonate soil management can provide several benefits, including soil and environment protection, reductions in the appearance of grapevine chlorosis, and enhancements in grape quality and yield. Following of all the above, the aim of the study is to determine the impact of different forms of nitrogen on the availability of iron in the soil and bioaccumulation in grapevine leaves of the ‘Graševina’ cultivar, and to determine the impact of a foliar iron application on grapevine nutrition status on carbonate soil.

2. Materials and Methods

2.1. Field Site and Experimental Design

Field research consisted of opening a soil profile at the study site in order to determine the type of soil, and sampling of soil and plants in the phase of flowering, veraison and harvest. As part of the field research in 2018 and 2019, a two-year vegetation experiment on the ‘Graševina’ cultivar was conducted to optimize the fertilization of permanent grape plantations. A fertilization experiment with seven treatments and three (3) replicates (10 vines per elementary plot) was set up in the wine-growing region of Slavonia and the Croatian Danube region, Baranja vineyards, at the location Zmajevac (45°48′22.1″ N 18°48′12.4″ E) (Supplementary Material Figure S1). The vineyard chosen for the experimental testing was planted in 2011 with a north–west south–east orientation. The experiment was performed on the ‘Graševina’ cultivar (Vitis vinifera L.), grafted on Kober 5BB rootstock. The vine training system was single Guyot, with 12 buds per plant. The vines were planted with 2.2 m spacing between rows and 0.8 m within rows. The vines were grown without irrigation. Types of fertilizers, amounts of nutrients per treatment, as well as applied amounts of fertilizers in basic fertilization and fertilization during vegetation are shown in

Table 1. Fertilizer type, required amounts of nutrients (kg/ha) and applied amounts of fertilizers.

Treatment (Fertilizers Type)	Required Amounts of Nutrients kg/ha			Applied Amounts of Fertilizers kg/ha
	N	P2O5	K2O	Basic Fertilization (NPK 7-20-30)	1st Top Dressing	2nd Top Dressing	Foliar Fe Application
C	14	40	60	200	0	0	NO
KAN	70	40	60	200	142	65.5	NO
KAN+F	70	40	60	200	142	65.5	YES
AS	70	40	60	200	183	83.5	NO
AS+F	70	40	60	200	183	83.5	YES
ASN+F	70	40	60	200	148	67.5	YES
U+F	70	40	60	200	84	38	YES

C—control; KAN—calcium ammonium nitrate; KAN^+F—calcium ammonium nitrate + foliar Fe; AS—ammonium sulfate; AS^+F—ammonium sulfate + foliar Fe; ASN^+F—ammonium sulfonitrate + foliar Fe; U^+F—urea + foliar Fe.

.

Fertilization treatments were determined based on preliminary results of soil analysis, age of plantations, and grapevine cultivation form. Fertilization treatments differed in the form of applied nitrogen fertilizer in top dressings and foliar applied iron. Basic fertilization of the plantation was performed in autumn with granular complex mineral NPK (7-20-30) fertilizer, while nitrogen fertilization was carried out in the spring during the movement of vegetation and in the flowering phase. Foliar fertilization was performed in three applications with an interval of 10 to 14 days (two treatments before and one treatment after the grapevine flowering phase). Foliar fertilization was performed with 0.2% Fe solution of liquid fertilizer (water-soluble 6%, chelated 1.1%) across all treatments that included Fe application.

Sampling of soil and plant material was performed in the phenophase of flowering and veraison and after the end of vegetation (harvest) in each individual year of research. Soil sampling was performed with a pedological (soil) probe to a depth of 30 cm in 5 replicates (subsamples) per treatment (n = 21). In each year of the study, 25 healthy whole leaves per treatment were sampled as opposed to the first bunch in the flowering phenophase and veraison with each fertilization treatment (n = 21).

2.2. Climate Conditions

The consequences of global climate change have appeared during the last decades, with increasing weather variability, therefore climate has an increasing influence on grape development. The average annual amount of precipitation at the meteorological station Osijek during a thirty-year period (1981–2010) was 684.4 mm (

Table 2. Average absolute temperatures and precipitation at the investigated area and relative deviation from the multi-year average (1981–2010).

		Year		Vegetation Period (IV–IX)
		Precipitation (mm)	Temperature (°C)	Precipitation (mm)	Temperature (°C)
1981–2010	absolute	684.3	11.3	390.6	18.2
2018		664.8	12.6	347.7	20.2
2019		650.5	10.8	522	18.9
1981–2010	relative	100	100	100	100
2018		97.2	111.5	89.0	111.0
2019		95.1	95.6	133.6	103.8

).

In the vegetation period (April–September) the average amount of precipitation is 390.6 mm, which is 57% of total precipitation. The maximum monthly amount of precipitation during the two-year study was recorded in May 2019 (141.2 mm). The minimum recorded monthly precipitation was 1.8 mm (March 2019), while the driest month in the vegetation period was April 2018, with 26.7 mm of precipitation. The average amount of precipitation in both research years was lower compared to the multi-year average; in 2018 it was recorded 664.8 mm, and at 650.5 mm in 2019, which is a decrease of 2.8 and 4.9%, respectively. In the vegetation period of 2018 (from April to September), a decrease in precipitation by 11% (347.7 mm of precipitation) was determined in relation to the multi-year average (390.6 mm of precipitation), while in the 2019 vegetation period 522 mm of precipitation was determined, which was an increase of 33.6% compared to the multi-year average. Air temperature, in addition to precipitation, is one of the most important climatic elements for vegetation. Favorable temperature regimes allow plants to grow in optimal conditions without the consequences of stress caused by high or low temperatures. Plants require optimal temperatures for their development, and any deviation from this leads to a certain level of stress. Average annual temperature at the measuring station Osijek in the period from 1981 to 2010 was 11.3 °C. The highest recorded mean monthly air temperature was 23.9 °C in August 2018, while the lowest average monthly temperature was measured in January 2019 and was 0.2 °C. The lowest average monthly temperature in the vegetation period was recorded in April 2019 with a determined 12.7 °C. Average monthly air temperatures in the vegetation period in 2018 and 2019 had an increase of 11% (20.2 °C in 2018) and 3.4% (18.9 °C in 2019) compared to the multi-year average (18.2 °C).

2.3. Soil Analysis

Laboratory analysis of soil chemical properties were performed on soil samples of fertilization treatments (n = 21) as well as on samples from diagnosed horizons of soil profiles for the purpose of determining soil type (n = 3): electrometric determination of actual and exchangeable soil reactions [16], organic matter content in soil by sulfochromic oxidation [17], the concentration of AL-available phosphorus and potassium [18] and volumetric determination of carbonate content in soil [19].

On soil samples from determined profiles’ horizons (n = 3), in addition to the above-mentioned agrochemical analysis, additional analysis of physical properties was conducted: content of hygroscopic soil moisture, stability of soil microstructural aggregates, soil density, soil porosity, soil field water capacity, soil air capacity [20] and soil texture composition determined by the ISO method [21]. An extraction method with a solution of ethylenediaminetetraacetic acid (EDTA) [22] was used to determine the available Fe in the soil, which is also the most commonly used method in the Republic of Croatia. In the analysis, 25 g of soil was extracted with 50 mL of EDTA solution (mixture of 1 M (NH₄)₂CO₃ and 0.01 M EDTA (pH 8.6)). After shaking on a rotary shaker for 30 min, the suspension was filtered, and Fe concentrations were directly measured in a clear filtrate by an absorption technique on atomic absorption spectrometer (AAS-) Shimadzu Scientific Instruments-AA-7000) with calibration of the device with a series of standard solutions.

2.4. Plant Analysis

All plant samples for measuring Fe concentrations were digested with 8 mL of a 3:1 mixture of HNO₃ and H₂O₂ at 180 °C for 60 min in microwave oven (CEM Mars 6) according to the following procedure [23]: 0.5 g of sample was weighed into a cuvette and poured with 6 mL of HNO₃ and 2 mL of H₂O₂. After digestion with the MARS 6 microwave system, the extracts were filtered into 50 mL flasks which were then filled with distilled water to the measuring mark. The concentrations in solutions of digested plant samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES; PerkinElmer Optima 2100 DV; 238.205 nm wavelength). The whole series of plant samples were analyzed with an internal pooled plasma control and with the reference material (Hay powder; NIST BCR 129) prepared in the same way as the other samples.

2.5. Statistical Analysis

The obtained results were statistically processed by using Minitab 17 Statistical Software (2010, State College, PA: Minitab, Inc. (www.minitab.com, accessed on 20 July 2021)). Statistical analysis of the impact of nitrogen fertilization and foliar fertilization was processed using the analysis of variance (ANOVA) with the Tukey method of significant differences. The relationships between variables were studied using linear regression methods and Pearson correlation coefficients. Correlation and regression analysis was performed using the computer program Microsoft Excel.

3. Results and Discussion

3.1. Soil Pedomorphological Characteristics

The experiment was settled on anthropogenic soil, formed from eutric cambisol on loess (

Table 3. Morphological features of the investigated soil profile.

Soil Profile	Depth (cm)	Horizon Designation	Morphology Characteristics
	0–25	Ap	Soil color: 10YR 4/4 (dark yellowish-brown) Texture: Silt loam Structure: Granular-Medium (GR-ME) Carbonate reaction: Moderately calcareous, visible effervescence
	25–45	B	Soil color: 7YR 4/2 (dark brown) Texture: Silt loam Structure: Granular-Fine/thin (GR-FI) Carbonate reaction: Strongly calcareous, strong visible effervescence. Bubbles form a low foam.
	45–100	C	Soil color: 2.5 Y 5/6 (light olive-brown) Texture: Silt loam to loam Structure: Granular-Very fine and fine (GR-FF) Carbonate reaction: Strongly calcareous, strong visible effervescence. Bubbles form a low foam.

Horizon designation according to: Guidelines for soil description—FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS Rome, 2006.

). National classification of soil was used [24], which is based on the properties of soils that are morphologically visible or easily measurable, and also serves as the basis for soil production and ecological assessment.

The origin of this soil is associated with specific combinations of pedogenetic factors that enable the transformation of the mineral part of the soil in the zone below the developed humus-accumulating horizon alongside the formation of secondary clay minerals and the formation of cambic horizon [25]. An alkaline soil reaction (pH KCl) was found on all identified horizons ranging between 7.68 in the arable horizon and 7.85 determined on the parent material (

Table 4. Basic chemical, physical and hydrological properties of the investigated soil.

		Depth (cm)
		0–25	25–45	45–100
Chemical properties
Actual soil acidity	pH H2O	8.64	8.66	8.71
Exchangeable soil acidity	pH KCl	7.78	7.71	7.85
Available phosphorus (P2O5)	mg/100 g	31.53	7.46	1.62
Available potassium (K2O)	mg/100 g	27.1	8.64	6.74
Organic matter content	%	2.03	0.72	0.45
Total carbonates (CaCO3)	%	20.97	31.67	32.53
Physical properties
Coarse sand	2.0–0.2 mm	3.80	3.53	3.50
Fine sand	0.2–0.063 mm	4.24	3.81	4.49
Coarse silt	0.063–0.02 mm	44.92	41.21	46.95
Fine silt	0.02–0.002 mm	26.26	33.36	29.55
Clay	<0.002 mm	20.78	18.09	15.51
Texture		Silt loam	Silt loam	Silt loam
Soil aggregate stability	%	88.20	-	-
Total porosity	% vol.	40.55	47.24	-
Packing density	g/cm3	2.22	1.86	-
Hydrological properties
Field capacity	% vol.	38.46	39.98	-
Air capacity	% vol.	2.09	7.26	-

). According to research [26], the maximum determined value of exchangeable acidity on the eutric cambisol of Eastern Croatia is 7.91 pH. The determined content of organic matter in the arable horizon was 2.03%, belonging to the category of soils that are poor in organic matter. According to the various authors [27,28], in the Republic of Croatia, there is a large presence of soils with 2% of organic matter on average. The low level of organic matter in the soil was largely contributed to by agriculture and insufficient intake of organic matter into the soil [28]. Soil supply with available P₂O₅ in the arable horizon is rich [29], with 31.53 mg P₂O₅/100 g, while potassium supply is good [29], with 27.1 mg K₂O/100 g soil (Table 2). The dominant pedogenetic process typical for this type of soil is argylosynthesis, during which three-layer clay minerals are mostly formed [24]. However, due to anthropogenization of the soil, the content of clay particles in the cambic horizon did not increase. Terrestrial anthropogenic soils are characterized by the presence of an anthropogenic (P) horizon that arises as a result of the application of various agrotechnical interventions affecting the surface and subsurface horizon, and often the parent substrate [25]. The determined porosity of the arable horizon of eutric cambisol, compaction, water capacity and air capacity, are not in accordance with the statements of other authors [25,30] that this soil is characterized by good drainage and optimal water and air regime.

3.2. The Effects of Nitrogen Fertilization on Soil Properties

The amount of nutrients available to plants is closely related to the soil chemical properties. The soil pH reaction affects the formation of soil nutrients, and its accessibility has a significant impact on fertilization efficiency [31]. Therefore, the choice of mineral fertilizers of different residual (physiological) reactions can significantly affect the availability of macro and microelements, which is especially pronounced in adverse agroecological conditions (e.g., dry periods, high temperatures or depleted soils). The influence of nitrogen fertilization on the actual and exchangeable acidity in both years of research in the stages of flowering, veraison and harvesting is shown in

Table 5. Influence of nitrogen fertilization on the actual soil acidity (pH H₂O).

Treatment	pH H2O
	2018			2019
	Flowering	Veraison	Harvest	Flowering	Veraison	Harvest
C	8.64 a	8.59 a	8.57 a	8.56 a	8.54 a	8.59 a
KAN	8.51 ab	8.32 bc	8.55 ab	8.27 bc	8.30 ab	8.44 ab
KAN+F	8.56 ab	8.31 bc	8.45 bc	8.39 ab	8.30 ab	8.42 ab
AS	8.44 b	8.21 c	8.34 de	8.29 bc	8.17 b	8.36 b
AS+F	8.47 ab	8.23 bc	8.36 cd	8.16 c	8.27 b	8.36 b
ASN+F	8.51 ab	8.32 bc	8.24 e	8.19 bc	8.26 b	8.38 b
U+F	8.62 ab	8.37 b	8.46 bc	8.27 bc	8.30 ab	8.40 ab

Values marked with different letters refer to statistically significant differences (p < 0.05) depending on treatment; n.s. (not significant).

and

Table 6. Influence of nitrogen fertilization on the exchangeable soil acidity (pH KCl).

Treatment	pH KCl
	2018			2019
	Flowering	Veraison	Harvest	Flowering	Veraison	Harvest
C	7.78 n.s.	7.78 a	7.88 n.s.	7.91 a	7.82 a	7.81 a
KAN	7.77	7.65 ab	7.83	7.82 ab	7.69 ab	7.72 abc
KAN+F	7.73	7.61 b	7.83	7.79 ab	7.61 ab	7.77 a
AS	7.65	7.65 ab	7.80	7.83 ab	7.59 b	7.55 c
AS+F	7.63	7.63 ab	7.84	7.78 ab	7.62 ab	7.58 bc
ASN+F	7.61	7.69 ab	7.84	7.64 b	7.66 ab	7.56 c
U+F	7.78	7.69 ab	7.85	7.87 a	7.69 ab	7.75 ab

Values marked with different letters refer to statistically significant differences (p < 0.05) depending on treatment; n.s. (not significant).

. The actual acidity (pH H₂O) in the flowering stage in 2018 on the AS treatment significantly decreased as compared to the control treatment. Differences in the nitrogen fertilizers applied were not significant in 2018; however, after the second nitrogen application, i.e., in the veraison phase, a significantly higher value of actual acidity was determined on the control treatment in comparison to other fertilization treatments. In the same year, at harvest stage the highest value of actual acidity was determined in control (pH H₂O-8.57) which did not significantly differ from the value established in KAN treatment.

KAN contains 4.5–5.5% MgO and 6.5–8.5% CaO, and it is not an acid-forming fertilizer, i.e., the acidifying residual effect is neutralized by the present cations Ca and Mg [29]. In 2019, in the flowering phase higher values of actual acidity were determined in control (pH H₂O-8.56) and KAN^+F treatment. After the second nitrogen fertilization in 2019 in both the veraison and harvest phase, treatments with AS and ASN significantly lowered soil pH as compared to the control and treatments with KAN and urea. AS is a fast-acting and physiologically very acidic fertilizer, because NH⁴⁺ is adsorbed on the soil colloidal fraction by substituting Ca²⁺ [29]. The same author also states that ASN is a fertilizer with a slightly reduced acid-forming residual effect of AS due to the addition of ammonium nitrate. After the two-year study, urea did not show any effect on actual acidity in the harvest phase. The first reaction of urea in the soil is weakly alkaline because it turns into ammonium carbamate. With the appropriate humidity, temperature and oxygen it transforms into nitrate in a short time, resulting in weak transient acidification considering it as a weakly acidic fertilizer [29]. Ammonium sulfate-based nitrogen fertilizers will lower pH value more significantly in comparison to urea [32].

The effect of nitrogen fertilization on the exchangeable acidity (pH KCl) is shown in Table 6. In the first year of the research, a significant difference between fertilization treatments was found only in the veraison phase where treatment with KAN^+F significantly lowered exchangeable acidity as compared to the control. In the second year of the research, in the harvest phase significantly lower values of substitution acidity were found in treatments with AS and ASN, which is analogous to the determined differences for the actual acidity.

The influence of nitrogen fertilization on the dynamics of available Fe (EDTA) in the soil is shown in Figure 1. In both years of the research, the influence of fertilization on the availability of Fe in the soil during the flowering phase of grapevine was absent, i.e., after first topdressing of nitrogen fertilizers. Soil containing less than 4.9 mg/kg of available iron belong to the category of low availability [33]. In both years of the research in the veraison phase, significantly higher values of available iron were found in treatments where acid-forming nitrogen fertilizers AS and ASN were used. As compared to the control, an increase of 18% (AS) and 23% (ASN) in 2018 and 26% (AS) and 29% (ASN) in 2019 was determined. Increased availability of Fe in the soil is visible in the harvest in both years of research, with a more pronounced effect in 2019 when increase in available Fe was found in all treatments using acid nitrogen fertilizer (AS, ASN and U) (Figure 1). However, the concentration of iron available to plants is very low in relation to the total amount of iron present in the soil. While plant nutrition with iron is conditioned by its solubility, it is affected by a decrease in soil pH due to increased Fe³⁺ ion activity in soil solution [34]. The results of the influence of nitrogen fertilization on the soil microbial activity (spore-forming bacteria) are presented in Supplementary Materials.

3.3. Influence of Nitrogen Fertilization and Foliar Fertilization on the Nutritional Status of Grapevine Leaves

Numerous authors [35,36,37,38,39] state that plant iron deficiency is not caused by iron deficiency in the soil, but more likely due to soil conditions that reduce availability of minerals. Therefore, the analysis of the mineral composition of plant leaves is a more reliable method to determine iron supply. The impact of nitrogen fertilization and foliar fertilization on the dynamics of Fe in grapevine leaves in 2018 and 2019 is shown in Figure 2 and Figure 3.

In the flowering and veraison phases in 2018 and 2019, lower values of Fe in the grapevine leaf were found in treatments where foliar fertilization was omitted. Fe content in leaves was below the recommended amounts in the flowering and veraison phases [40,41]. Foliar fertilization increased the concentration of Fe in the grapevine leaf in the flowering phase in the treatment with KAN^+F for 42% in 2018 and 90% in 2019 as compared to the treatment without foliar application (KAN), while the increase in the veraison phase was 70% in 2018 and 139% in 2019. A similar trend was found by comparing AS treatments with and without foliar fertilization, where in the flowering phase an increase of 49.9% (2018) and 117% (2019) was found, and an increase of 65% (2018) and 117% (2019) was found in the veraison phase. Leaf contents of individual elements are not a constant, they keep changing during the growing period. Furthermore, element contents depend on the variety, soil chemical properties, and weather conditions, as well as on the fertilization impact [42].

The correlation between the Fe content in the leaf in the flowering phase and in the veraison phase was analyzed by correlations, i.e., using the Pearson linear correlation coefficient (r) for both years of research. In both years of research, very significant positive correlation was found between the Fe content in the leaf in the flowering and veraison phase (p = 0.01) with the determined values r = 0.77 in 2018 and r = 0.91 in 2019. Based on regression equations (Figure 4) or models based on data on the concentration of Fe in the grapevine leaf in the flowering phase, the concentration of Fe can be predicted in the veraison phase without additional analysis of plant matter.

3.4. Influence of Nitrogen Fertilization and Foliar Fertilization on Some Quality Variables of Grapes and Musts

Influence of nitrogen fertilization and foliar fertilization on some quality variables of grapes (cluster weight and weight of 100 berries) and grape must density are presented in

Table 7. Influence of nitrogen fertilization and foliar fertilization on cluster weight, weight of 100 berries and grape must density.

Treatment	Cluster Weight (kg)		Weight of 100 Berries (kg)		Must Density (°Oe)		Cluster Weight (kg)		Weight of 100 Berries (kg)		Must Density (°Oe)
	2018						2019
C	0.109	c	0.178	n.s.	83.67	b	0.087	c	0.142	b	81.99	b
KAN	0.117	bc	0.19		89.67	ab	0.096	bc	0.152	ab	87.87	a
KAN+F	0.136	bc	0.187		88.67	ab	0.113	ab	0.155	ab	87.78	a
AS	0.138	bc	0.201		90.67	a	0.116	a	0.167	a	89.76	a
AS+F	0.143	a	0.188		87.33	ab	0.122	a	0.158	ab	86.46	ab
ASN+F	0.143	ab	0.188		87.33	ab	0.117	a	0.169	a	87.87	a
U+F	0.132	abc	0.184		87.67	ab	0.117	a	0.157	ab	88.46	a

Values marked with different letters refer to statistically significant differences (p < 0.05) depending on treatment; n.s. (not significant).

.

Numerous studies show that N applications can cause changes in the chemical composition of grape must, and it reflect both positive and negative implications for wine quality [43,44]. In our case, a statistically significant difference was found between the treatment with nitrogen fertilization and the control treatment. Obtained results are in accordance with the results of other authors’ studies [45,46]. Further, some authors [15,46] report in their research that cluster weight values were not so responsive to fertilization treatments but were generally higher in fertilization treatments. Additionally, the same authors have not determined the influence of nitrogen fertilization on qualitative parameters on the cultivar ‘Graševina’, while in the same study fertilization with N affected the reduction of sugar content in Chardonnay must, and also increased the content of sugar in the must of the Riesling Rhine. Influence of soil nitrogen fertilization and foliar fertilization on other pomological characteristics are presented in the Supplementary Materials (Table S1).

4. Conclusions

Based on a two-year study of the impact of soil nitrogen fertilization and foliar Fe application on soil dynamics and grapevine leaf Fe concentration on carbonate soil, it can be concluded that the low soil Fe supply (<10 mg/kg) was found to be due to alkaline soil reaction. The performed mineral fertilization with acid-forming nitrogen fertilizers (ammonium sulfate, ammonium sulfate-nitrate) significantly affected a local acidification of alkaline soil, i.e., a lowering of the actual and exchangeable pH values, which resulted in increased Fe availability in the soil. Based on the established results on the dynamics of iron in the grapevine leaves, it can be concluded that foliar fertilization significantly affected the amount of Fe in the grapevine leaves both in the flowering and veraison phase, i.e., in foliar application in two and three terms. Of all the observed treatments, only foliar fertilization had a positive effect on iron concentration in the grapevine leaves, which leads to the conclusion that this is an effective way to solve chlorosis occurrence and iron deficiency symptoms in carbonate soil. Therefore, the use of mineral fertilizers with acid-forming nitrogen fertilizers for many years can result in a reduction in the number of required foliar treatments and thus significantly affect the ecological and economic aspect of grape production, without affecting the reduction of yield and quality of grapes and wine.