Influence of Biochar Foliar Application on Malvazija Istarska Grapevine Physiology

Abstract

Biochar has attracted interest in viticulture for its potential to enhance nutrient uptake and improve grapevine physiology under changing climatic conditions, particularly in Mediterranean regions. However, the widespread adoption of biochar has been limited due to economic and logistical constraints associated with its large-scale application. To address these barriers hindering the widespread adoption of biochar, this study investigates the effects of foliar-applied water suspensions of biochar at concentrations of 300 mg/L (B300), 600 mg/L (B600), and 1200 mg/L (B1200), compared to a water-only control (C), as a practical alternative application method. The research focused on Malvazija istarska (Vitis vinifera L.), an indigenous Croatian grapevine variety, conducted in an experimental vineyard in Poreč, Croatia. The key physiological parameters examined included photo-synthetic activity, leaf water potential, the elemental composition of the grapevine leaves, and grape yield. Foliar applications were administered three times during the growing season, with five replicates per treatment. The results indicated that biochar treatments had no significant impact on photosynthetic activity, suggesting that foliar application did not cause leaf shading. However, higher biochar concentrations (B600 and B1200) led to increased leaf concentrations of nitrogen (2.1–3.8%), potassium (10.1–18.4 g/kg), sulfur (2.2–2.5 g/kg), boron (65.1–83.6 mg/kg), and manganese (42.4–69.8 mg/kg) compared to B300 and C treatments. Conversely, magnesium content decreased (2.1–2.7 g/kg), likely due to potassium–magnesium antagonism. Furthermore, the B600 treatment produced the highest grape yield (2.67 kg/vine), representing up to a 37% increase compared to other treatments. These findings suggest that the foliar application of biochar can be an effective and sustainable strategy to enhance vineyard productivity. Moreover, it offers a circular economy approach by valorizing grapevine pruning waste as a biochar source.

1. Introduction

Viticulture faces significant challenges due to climate change, including increased drought stress and soil degradation [1]. These threats are particularly severe in the Mediterranean region, where rising global temperatures, erratic precipitation, and persistent drought have dire consequences for grape cultivation. These changes lead to reduced grape yield and variations in wine quality [2]. As these environmental pressures intensify, the strain on grape production and consistency in wine quality will likely increase, underscoring the urgent need for innovative management strategies to adapt and sustain viticulture under changing climatic conditions [3,4,5]. Climate variability also affects soil nutrient availability, necessitating the development of innovative vineyard management practices to maintain sustainability and productivity.

A promising material in the field of sustainable viticulture is biochar, a carbon-rich material produced by the pyrolysis of organic biomass under low-oxygen conditions [6]. Biochar has been studied for its potential to enhance soil fertility, increase water retention, and improve plant resilience to environmental stress [7]. Its application in viticulture has gained attention due to its potential benefits for soil health and plant productivity. Research indicates that biochar produced from grapevine pruning residues can influence grapevine growth and soil properties, depending on factors such as rootstock selection and the pyrolysis temperature used during production. Anđelini et al. [8] reported that the composition of biochar derived from grapevine residues was predominantly influenced at the initial level but not significantly affected by different rootstocks. Moreover, Beatrice et al. demonstrated that long-term biochar applications tend to enhance soil characteristics by increasing pH and nutrient availability while also promoting root development in grapevines [9]. Similarly, Lucchetta et al. [10] found that biochar improved water retention and soil pH without negatively impacting vine productivity or grape quality. Other studies have reported yield increases of up to 66% across multiple harvests due to improved water availability and reduced drought stress in biochar-amended soils [11,12]. These benefits are especially pronounced in low-rainfall conditions, where the inverse correlation between precipitation and yield underscores the water-retention capabilities of biochar-treated soils [13]. These findings support the integration of biochar into vineyard management as a sustainable agricultural practice that enhances soil conditions for vine growth.

The rate of biochar application significantly influences grapevine physiological responses and yield outcomes. Studies report application rates typically ranging from 5 to 50 tons per hectare, although specific guidelines for optimal doses remain limited [14]. The same authors suggest that even lower dosages—around 1% by weight or less—can be effective in enhancing crop performance, including in grapevines. Traditionally, biochar is incorporated into the soil, where it has shown promising results in improving soil structure and microbial activity. However, the high cost and logistical challenges associated with applying large quantities of biochar (ranging from 1 to 40 t/ha) have hindered its widespread adoption [15].

An emerging alternative is foliar biochar application, which is considered a more cost-effective and efficient method to enhance nutrient uptake while minimizing soil-related complications. The foliar application delivers essential nutrients directly to the plant, potentially improving photosynthetic efficiency, water-use efficiency, and overall vine health [16]. For instance, experiments with lettuce showed that foliar sprays prepared from biochar extracts significantly enhanced growth under both natural light and greenhouse conditions [17]. Treatments using biochar from pine wood and wheat straw led to increased levels of chlorophyll a, chlorophyll b, and carotenoids, indicating improved photosynthetic capacity. Furthermore, Shahzadi et al. [18] reported that a 3% nano-biochar foliar solution improved plant pigments such as chlorophyll and carotenoids, as well as the relative water content in tomato leaves under both stressed and non-stressed conditions.

Despite its potential, limited research has investigated the effects of foliar biochar application in viticulture. This study aims to evaluate the impact of foliar-applied biochar at varying concentrations on key physiological and agronomic parameters in Malvazija istarska (Vitis vinifera L.), a grape variety widely cultivated in the Istrian Peninsula, Croatia [19]. Specifically, the effects of biochar foliar application on photosynthetic activity (net assimilation rate and CO₂ exchange), leaf water potential, the elemental content in leaves, and grape yield were evaluated. Abd Elwahed et al. [15] reported that the concentration of 300 mg/L of biochar foliar fertilizer improved the growth and yield characteristics of wheat. This concentration was used in the current study and then doubled and quadrupled to explore the dose–response relationship between biochar concentration and grapevine physiological responses. Understanding this relationship is crucial for optimizing foliar application rates to maximize growth and yield while minimizing environmental impact and material waste.

This research aims to confirm that foliar biochar applications can positively influence photosynthesis, leaf water potential, and elemental composition, ultimately increasing grape yield. Gaining insight into grapevine physiological responses to foliar biochar application can support its integration into sustainable viticultural practices, especially in regions facing increasing climatic variability.

2. Materials and Methods

The experiment with biochar-based foliar treatments was conducted during the 2023 vegetative season in the experimental vineyard of the Institute of Agriculture and Tourism in Poreč, Istria, Croatia (45°13′22″ N, 13°36′02″ E; 15 m above sea level). The trial involved 12-year-old vines of the indigenous Malvazija istarska grapevine variety, clone VCR4, trained using the single Guyot system and grafted onto SO4 rootstock (Selection Oppenheim; V. berlandieri × V. riparia). Grapevines were planted at a spacing of 2.5 m between rows and 0.8 m within rows, resulting in a planting density of 5000 vines per hectare. These spacings are typical for the region and the selected cultivar, as they facilitate complete mechanization, align with the chosen training system, and ensure adequate airflow, thereby reducing the risk of fungal diseases. The vineyard is established on terra rossa soil, characteristic of the Istrian peninsula. A randomized complete block design was established using selected vineyard rows. Each treatment was replicated five times, with seven consecutive vines per replicate, resulting in a total of 35 vines per treatment. To prevent foliar spray drift and cross-contamination, isolation vines (seven per set) were placed between treatment groups within the same row, and isolation rows were positioned between treated rows. All standard grapevine cultivation practices typical of the region were carried out, with the exception of foliar or any other form of fertilization. No irrigation was applied to the vineyard.

Meteorological data for the vegetative season are presented in Figure 1. According to existing research, a minimum of approximately 50 mm of precipitation per month is generally considered sufficient to prevent significant water stress in grapevine cultivation [20]. Sufficient precipitation was recorded during all vegetative months, except in June, when it was slightly below the threshold at 45 mm. In contrast, August experienced a significantly higher amount of rainfall, reaching 239 mm—nearly five times the minimum requirement. Average monthly air temperatures did not exceed 25 °C throughout the season.

The biochar used to prepare foliar suspensions was produced from the grapevine-pruning residues of Malvazija istarska (Vitis vinifera L.) at a temperature of 400 °C, using a muffle furnace and the method reported by Anđelini et al. [8]. Grapevine-pruning residues were placed in ceramic crucibles with ceramic covers to minimize oxygen contact. The following peak temperature program was used: after a 10 °C/min ramp-up, the maximum temperature of 400 °C was reached and held for one hour. Subsequently, the samples were left to cool to room temperature. Biochar samples were produced in three replicates and analyzed separately.

Prior to the preparation of foliar suspensions, the produced biochar was crushed into a very fine powder using a ceramic mortar and analyzed in triplicate, and it presented the following parameters (

Table 1. Overview of the physicochemical properties of the biochar used in the experiment.

Parameter	Unit	Value
pH	/	9.79 ± 0.05
EC	µS/cm	792 ± 65.9
ash	%	8.36 ± 0.01
total carbon (TC)	%	73.1 ± 0.43
specific surface area (SSA)	m2g−1	2.07 ± 0.14
N	%	1.06 ± 0.01
P	g/kg	27.2 ± 0.21
K	g/kg	22.8 ± 0.78
Mg	g/kg	27.5 ± 1.67
S	g/kg	12.4 ± 0.30
Ca	g/kg	187 ± 9.61
Cu	mg/kg	4.65 ± 0.25
Mn	mg/kg	6.56 ± 1.22
Mo	mg/kg	0.11 ± 0.00
Zn	mg/kg	2.69 ± 0.01

Analyses were performed as follows: pH, EC—DIN ISO 10390 [21]; ash—by mineralizing 1 g of the sample in ceramic crucibles using a muffle furnace (Nabertherm Muffle Furnace L9/11/B410, Nabertherm GmbH, Lilienthal, Germany); TC—by burning 50 mg of grounded sample in the Solid Sample Combustion Unit (SSM-5000A) on a TOC-L analyzer (Shimadzu Corporation, Kyoto, Japan); SSA—BET method; N—Kjeldahl method; all macro- and microelements used the ICP-OES (Shimadzu Corporation, Kyoto, Japan) technique after microwave digestion (Ethos UP, Millestone Srl, Milan, Italy).

).

The produced biochar characteristics met the requirements defined by the European Biochar Certificate [22], with the exception of SSA values, which should be higher.

The study focused on four foliar treatments: a control (C) treated with water only and a water suspension containing 300 mg biochar/L (B300), 600 mg biochar/L (B600), and 1200 mg biochar/L (B1200). Foliar suspensions were prepared 24 h prior to application, without the addition of any surfactant due to the small size of the biochar particles (in the micro- to nano-scale range), which ensured a stable suspension in water. Additionally, surfactants were omitted to avoid potential interactions with biochar and to isolate the effects of biochar alone. Prior to foliar application, the pH and electrical conductivity (EC) of each suspension were measured in triplicate using a pH meter (inoLab Multi 9310 IDS, Xylem Inc., Washington, WA, USA) and an EC meter (FiveGo F3, Mettler Toledo AG, Columbus, OH, USA), respectively. The measured values are presented in

Table 2. Values of pH and EC of the prepared biochar-based foliar suspensions.

Foliar Suspension	pH	EC (µS/cm)
C	6.15 ± 0.45	1.80 ± 0.30
B300	9.21 ± 0.26	36.0 ± 1.04
B600	9.42 ± 0.32	57.5 ± 1.82
B1200	9.34 ± 0.04	92.0 ± 2.27

Applied treatments: control (C) with water only; water suspension with 300 mg biochar/L (B300); water suspension with 600 mg biochar/L (B600); water suspension with 1200 mg biochar/L (B1200).

.

The foliar biochar-based suspensions exhibited an alkaline pH. Although a pH value of around 9 is generally not recommended for grapevines due to the potential risk of phytotoxicity, no such effects were observed in this study. While maximum acceptable EC (electrical conductivity) values for foliar application in grapevines are not clearly defined, the EC levels recorded in this study did not result in any negative effects. All foliar treatments were applied in the early morning hours using an electric battery-powered backpack sprayer (V.black Electron, Davide e Luigi Volpi S.p.A., Casalromano, Italy) to ensure uniform coverage. Approximately 140 mL of suspension was applied per vine for each treatment, corresponding to an application rate of 700 L/ha. Foliar suspensions were applied three times during the growing season at the flowering (S19 [23], 31 May 2023), setting (S27, 20 June 2023), and veraison stages (S35, 2 August 2023). No nozzle clogging was reported during the applications. Precipitation and temperature conditions were observed after application, and no precipitation levels were observed that would lead to wash-off, nor were there any excessively high temperatures that could lead to phytotoxicity.

Photosynthetic activity was assessed by measuring net photosynthetic assimilation (A) and internal CO₂ concentration (Ci) using a portable photosynthesis system LI-6800 (LI-COR Biosciences, Lincoln, NE, USA). Fully developed young leaves were randomly selected from the cluster zone on both sides of the vine. Three leaves per replicate were measured for each treatment.

Leaf water potential was determined using a Scholander-type pressure chamber (Model 1000, PMS Instrument Company, Albany, OR, USA). Fully developed leaves were sampled in triplicate from each replicate opposite the grape cluster. Each leaf was cut from the shoot and placed in the chamber with the petiole protruding. Pressure was applied until xylem sap appeared at the petiole cross-section. The recorded pressure at sap emergence indicated the leaf water potential, with more negative values reflecting higher water stress levels.

The leaf-washing and sampling procedures were standardized and consistently applied throughout the trial to minimize cross-contamination and ensure the integrity of the analytical results. To control for potential contamination from prior applications, grapevine leaves were sampled from the cluster zone immediately before each treatment and prior to harvest. Samples were washed with distilled water to remove any pesticide residues. Leaf samples were collected in triplicate from each replicate for all treatments.

Washed leaves were dried at 105 °C and ground into a fine powder. For elemental analysis, 250 mg of dry sample was digested in a microwave system using 6 mL of HNO₃ and 2 mL of H₂O₂. Elemental concentrations were quantified using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES, Shimadzu Corporation, Kyoto, Japan).

Leaf nitrogen content was determined using the Kjeldahl method. One gram of ground leaf material was digested at 420 °C for one hour with 12 mL H₂SO₄ and two KJTabs™ catalyst tablets (VELP Scientifica, Usmate, Italy). Following digestion and cooling, the sample was distilled with 30 mL H₃BO₃ and 50 mL NaOH using a UDK 149 nitrogen analyzer (VELP Scientifica Srl, Usmate Velate, Italy). Titration was carried out using 0.1 N HCl.

Grape yield was measured at harvest (phenological stage S38). All clusters from each replicate of each treatment were hand-harvested and weighed. The yield per vine was calculated by dividing the total cluster weight by the number of vines sampled per replicate.

Statistical analysis was conducted using Statistica 12 software (Tibco, Inc., Palo Alto, CA, USA). The normality of the data was confirmed prior to analysis. One-way analysis of variance (ANOVA) was used to assess the effects of treatments. Differences between treatment means were evaluated using Tukey’s HSD post-hoc test at a 95% confidence level.

3. Results

3.1. Photosynthetic Activity of Grapevine Leaves

During the growing season and following each treatment application, net photosynthetic assimilation (A) and intercellular carbon dioxide concentration (Ci) of the grapevine leaves were measured (

Table 3. Net photosynthetic assimilation rate (A) and intercellular carbon dioxide concentration (Ci) in grapevine leaves measured across different phenological stages.

	Net Photosynthetic Assimilation Rate (A) and Intercellular Carbon Dioxide Concentration (Ci) in Grapevine Leaves
Treatment	Phenological Stage
	Flowering (S19)		Setting (S27)		Veraison (S35)		Harvest (S38)
	A	Ci	A	Ci	A	Ci	A	Ci
	µmol m−2 s−1	µmol mol−1	µmol m−2 s−1	µmol mol−1	µmol m−2 s−1	µmol mol−1	µmol m−2 s−1	µmol mol−1
C	2.72 ± 0.13	377 ± 2.14	3.00 ± 0.27	376 ± 0.88	3.02 ± 0.34	350 ± 5.25	2.23 ± 0.41	500 ± 8.63
B300	2.73 ± 0.20	378 ± 0.80	4.10 ± 0.08	375 ± 4.95	2.93 ± 0.19	333 ± 8.51	1.97 ± 0.50	538 ± 32.3
B600	2.92 ± 0.44	371 ± 5.64	3.37 ± 0.39	372 ± 0.41	3.99 ± 0.33	356 ± 3.23	3.45 ± 0.49	315 ± 13.9
B1200	2.01 ± 0.41	376 ± 5.97	3.35 ± 0.09	374 ± 0.28	3.66 ± 0.36	348 ± 8.07	2.81 ± 0.71	308 ± 4.07
p value	0.283	0.609	0.925	0.783	0.122	0.168	0.294	0.051
significance	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.	n.s.

Applied treatments: control (C) with water only; water suspension with 300 mg biochar/L (B300); water suspension with 600 mg biochar/L (B600); and water suspension with 1200 mg biochar/L (B1200). Net photosynthetic assimilation rate (A) and intercellular carbon dioxide concentration (Ci) were measured at four phenological stages: flowering (S19), setting (S27), veraison (S35), and harvest (S38). The results are presented as the mean value of five replicates per treatment ± standard error. The data were subjected to one-way ANOVA. n.s.—not significant. For statistically significant results, Tukey’s post-hoc test was performed.

). No statistically significant differences were observed among the treatments for either parameter at any of the assessed phenological stages. Although no statistically significant differences were detected, certain trends were observed across treatments. The B600 treatment exhibited higher net photosynthetic assimilation (A) values during the flowering, veraison, and harvest stages, while the B300 treatment showed slightly higher values at the setting stage. Intercellular carbon dioxide concentration (Ci) values were similar across treatments at the flowering and setting stages. However, at veraison, the B600 treatment showed a higher Ci value, whereas, at harvest, the highest Ci value was observed in the B300 treatment.

3.2. Leaf Water Potential of Grapevine Leaves

In the flowering phenological stage, leaf water potential was measured prior to the first treatment in order to assess the initial physiological state. Although there were no statistically significant differences among treatments during the phenological stages of flowering, setting, and veraison, a large variation and certain trends in the measured values were observed (

Table 4. Leaf water potential of grapevine leaves measured across different phenological stages.

	Leaf Water Potential (MPa)
Treatment	Phenological Stage
	Flowering (S19)	Setting (S27)	Veraison (S35)	Harvest (S38)
C	−0.47 ± 0.08	−0.70 ± 0.03	−0.77 ± 0.04	−0.55 ± 0.00 b
B300	−0.47 ± 0.03	−0.73 ± 0.06	−0.80 ± 0.10	−0.68 ± 0.03 a
B600	−0.53 ± 0.09	−0.68 ± 0.06	−0.85 ± 0.08	−0.58 ± 0.02 ab
B1200	−0.77 ± 0.09	−0.80 ± 0.08	−0.98 ± 0.07	−0.68 ± 0.04 a
p value	0.116	0.544	0.282	0.022
significance	n.s.	n.s.	n.s.	*

Applied treatments: control (C) with water only; water suspension with 300 mg biochar/L (B300); water suspension with 600 mg biochar/L (B600); water suspension with 1200 mg biochar/L (B1200). The results are presented as the mean value of five replicates per treatment ± standard error. The greater the pressure required to exude the xylem sap from the petiole, the more negative the leaf water potential value, indicating a higher water-induced stress level. The data were subjected to one-way ANOVA. Significance markers indicate statistically significant differences as follows: * at p < 0.05; n.s.—not significant. For statistically significant results, Tukey’s post-hoc test was performed, and different letters within a column next to the mean values indicate statistically significant differences between treatments.

). At the flowering stage, the B600 treatment showed a relatively higher leaf water potential. During the setting and veraison stages, higher values were recorded in the B1200 treatment. At harvest, significant differences were noted, and the most negative leaf water potential—indicating higher water stress—was observed in the B300 and B1200 treatments, and these values were comparable to B600. The control treatment exhibited the least negative pressure, which was also comparable to B600.

3.3. Elemental Composition of Grapevine Leaves

Table 5. Macroelement content of grapevine leaves measured across different phenological stages.

	Macroelement Content of Grapevine Leaves
Element	N	P	K	Mg	Ca	S
Unit	%	g/kg	g/kg	g/kg	g/kg	g/kg
	Flowering (S19)
C	3.53 ± 0.02 c	15.3 ± 0.97 b	11.8 ± 0.58 b	2.38 ± 0.11 ab	15.3 ± 0.97 b	2.24 ± 0.11
B300	3.45 ± 0.02 b	18.3 ± 0.41 a	12.6 ± 0.3 b	2.62 ± 0.07 a	18.3 ± 0.41 a	2.4 ± 0.09
B600	3.8 ± 0.02 a	16.3 ± 0.07 ab	17.7 ± 0.18 a	2.3 ± 0.02 b	16.3 ± 0.07 ab	2.52 ± 0.02
B1200	3.81 ± 0.01 a	17.2 ± 0.2 ab	17.3 ± 0.08 a	2.11 ± 0.01 b	17.2 ± 0.2 ab	2.55 ± 0.01
p value	<0.001	<0.001	<0.001	0.004	0.025	0.058
significance	***	***	***	**	*	n.s.
	Setting (S27)
C	3.01 ± 0.01 a	2.38 ± 0.01 a	13.2 ± 0.07 c	3.01 ± 0.01 a	22.8 ± 0.13 c	2.39 ± 0.01 a
B300	2.84 ± 0 b	2.34 ± 0.02 a	11.1 ± 0.15 d	2.73 ± 0.05 b	20.8 ± 0.35 d	2.4 ± 0.01 b
B600	2.86 ± 0.04 b	2.24 ± 0 b	18.4 ± 0.1 a	2.22 ± 0.02 c	25 ± 0.2 a	2.34 ± 0.01 b
B1200	2.91 ± 0.03 ab	2.34 ± 0.02 a	16.1 ± 0.32 b	2.31 ± 0.04 c	24 ± 0.1 b	2.46 ± 0.02 a
p value	0.004	<0.001	<0.001	<0.001	<0.001	0.003
significance	**	***	***	***	***	**
	Veraison (S35)
C	2.14 ± 0.01 b	2.00 ± 0.04	9.82 ± 0.27 c	2.29 ± 0.03 c	27.5 ± 0.67 b	1.87 ± 0.06 b
B300	2.00 ± 0.00 c	1.91 ± 0.08	8.44 ± 0.38 d	3.41 ± 0.15 a	32.4 ± 1.74 a	1.94 ± 0.13 ab
B600	2.17 ± 0.02 ab	2.00 ± 0.01	14.1 ± 0.3 a	2.38 ± 0.00 bc	32.9 ± 0.01 a	2.08 ± 0.02 ab
B1200	2.22 ± 0.02 a	1.89 ± 0.03	12.1 ± 0.07 b	2.74 ± 0.03 b	34.2 ± 0.27 a	2.21 ± 0.03 a
p value	<0.001	0.306	<0.001	<0.001	0.005	0.051
significance	***	n.s.	***	***	**	*
	Harvest (S38)
C	2.02 ± 0.01 bc	2.99 ± 0.03	7.25 ± 0.03 b	2.46 ± 0.02 b	29.63 ± 0.67	1.78 ± 0.02
B300	2.01 ± 0.02 c	3.32 ± 0.01	6.95 ± 0.02 b	3 ± 0.03 a	35.83 ± 0.41	1.83 ± 0.01
B600	2.11 ± 0.01 a	2.95 ± 0.03	10.23 ± 0.04 a	2.36 ± 0.12 b	36.04 ± 1.32	2.05 ± 0.03
B1200	2.07 ± 0.01 ab	3.25 ± 0.41	10.13 ± 0.48 a	2.45 ± 0.08 b	30.33 ± 8.04	2.22 ± 0.20
p value	0.002	0.536	<0.001	0.001	0.573	0,050
significance	**	n.s.	***	**	n.s.	n.s.

Applied treatments: control (C) with water only; water suspension with 300 mg biochar/L (B300); water suspension with 600 mg biochar/L (B600); water suspension with 1200 mg biochar/L (B1200). The results are presented as the mean value of five replicates per treatment ± standard error. The data were subjected to one-way ANOVA. Significance markers indicate statistically significant differences as follows: * at p < 0.05; ** at p < 0.01; *** at p < 0.001; n.s.—not significant. For statistically significant results, Tukey’s post-hoc test was performed, and different letters within a column next to the mean values indicate statistically significant differences between treatments.

presents the macronutrient content in grapevine leaves measured at different phenological stages during the growing season.

At the flowering stage, nitrogen content was highest in the B600 and B1200 treatments. Phosphorus content peaked in the B300 treatment and was comparable to B600 and B1200. Potassium content was highest in B600 and B1200, while magnesium content peaked in B300 and was comparable to the control. Calcium content was also highest in B300 and comparable with B600 and B1200. No significant differences were observed for sulfur at this stage.

At the setting stage, nitrogen content was highest in the control treatment and comparable to B1200. Phosphorus content was elevated in the control, B600, and B1200 treatments. Potassium content peaked in B600, while magnesium and sulfur were highest in the control. Calcium content was highest in B600.

At the veraison stage, nitrogen and sulfur contents were highest in B1200, with nitrogen also comparable in B600 and sulfur also comparable in B300 and B600. Potassium content again peaked in B600, while magnesium content was highest in B300. All biochar treatments showed elevated calcium levels compared to the control. No significant differences were observed for phosphorus at this stage.

At the harvest stage, nitrogen content was highest in B600 and comparable to B1200. Potassium content remained highest in B600 and B1200. Magnesium content was, again, highest in B300. No significant differences were observed for phosphorus, calcium, and sulfur content at this stage.

Table 6. Microelement content of grapevine leaves measured across different phenological stages.

	Microelement Content of Grapevine Leaves
Element	B	Cu	Fe	Mn	Mo	Na	Si	Zn
Unit	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg	mg/kg
	Flowering (S19)
C	46.5 ± 0.94 d	7.10 ± 0.29 b	76.1 ± 2.72	33.8 ± 0.69 b	0.84 ± 0.03	15.3 ± 0.97 b	379 ± 12.5 b	12.0 ± 0.29 b
B300	60.9 ± 1.41 b	8.16 ± 0.19 a	80.5 ± 2.01	44.4 ± 1.02 a	0.92 ± 0.04	18.3 ± 0.41 a	672 ± 22.5 a	14.6 ± 0.35 a
B600	54.0 ± 0.43 c	8.17 ± 0.04 a	75.9 ± 0.17	36.0 ± 0.20 b	0.93 ± 0.02	16.3 ± 0.07 ab	402 ± 2.62 b	13.0 ± 0.14 b
B1200	65.1 ± 0.2 a	8.52 ± 0.02 a	78.6 ± 0.74	42.4 ± 0.17 a	0.91 ± 0.04	17.2 ± 0.2 ab	407 ± 21.3 b	14.7 ± 0.05 a
p value	<0.001	0.002	0.268	<0.001	0.226	0.027	<0.001	<0.001
significance	***	**	n.s.	***	n.s.	*	***	***
	Setting (S27)
C	44.3 ± 0.13 c	6.90 ± 0.02 ab	82.6 ± 1.21 ab	48.3 ± 0.22 c	0.89 ± 0.03	26.3 ± 0.23 b	403 ± 18.3 b	12 ± 0.06 a
B300	45.8 ± 0.4 c	6.41 ± 0.03 b	83.2 ± 1.41 ab	41.5 ± 0.51 b	0.84 ± 0.02	22 ± 0.26 d	412 ± 12.9 b	9.02 ± 0.12 b
B600	56.7 ± 0.13 b	6.18 ± 0.06 b	79.7 ± 0.28 ab	53.4 ± 0.19 a	0.86 ± 0.01	23.6 ± 0.28 c	680 ± 16.1 a	10.7 ± 0.12 a
B1200	67.5 ± 0.74 a	7.48 ± 0.37 a	77.5 ± 1.5 b	52.2 ± 0.5 a	0.92 ± 0.02	28.0 ± 0.35 a	695 ± 34.9 a	12.0 ± 0.61 a
p value	<0.001	0.006	0.033	<0.001	0.089	<0.001	<0.001	<0.001
significance	***	**	*	***	n.s.	***	***	***
	Veraison (S35)
C	69.6 ± 1.42 b	233 ± 4.67 b	107 ± 2.57 a	52.4 ± 0.82 c	69.6 ± 1.42 b	77.1 ± 1.88 b	592.7 ± 9.02	9.99 ± 0.3 c
B300	72.4 ± 3.94 b	292 ± 14.43 c	97.5 ± 6.07 ab	62.3 ± 3.04 b	72.4 ± 3.94 b	93.2 ± 4.68 a	637.9 ± 38.5	10.4 ± 0.49 c
B600	74.7 ± 1.07 ab	465 ± 2.82 a	95.8 ± 1.01 ab	64.6 ± 0.36 ab	74.7 ± 1.07 ab	96.7 ± 2.76 a	643.5 ± 47.2	14.9 ± 0.25 a
B1200	83.6 ± 1.61 a	486 ± 9.70 a	88.3 ± 0.75 b	69.8 ± 0.73 a	83.6 ± 1.61 a	87.4 ± 3.70 ab	718.1 ± 33.8	13.0 ± 0.24 b
p value	0.012	<0.001	0.031	<0.001	0.041	0.016	0.167	<0.001
significance	*	***	*	***	*	*	n.s.	***
	Harvest (S38)
C	67.3 ± 0.67	99.5 ± 5.31	86.3 ± 0.51 a	52.2 ± 0.12 b	0.87 ± 0.02 ab	73.4 ± 2.16	852 ± 3.63 a	11.2 ± 0.19 b
B300	79.2 ± 0.15	133 ± 2.86	93.8 ± 4.72 a	61.9 ± 0.17 ab	0.91 ± 0.01 a	65.6 ± 0.72	893 ± 16.2 a	12.4 ± 0.06 b
B600	73.3 ± 4.23	257 ± 25.68	63.3 ± 8.57 b	65.3 ± 0.64 a	0.78 ± 0.01 c	69.8 ± 2.54	407 ± 16.9 b	14.2 ± 1.43 ab
B1200	82.7 ± 5.49	206 ± 98.73	79.9 ± 8.57 ab	59.6 ± 5.12 ab	0.8 ± 0.02 bc	63.7 ± 8.56	407 ± 46.7 b	17.7 ± 1.82 a
p value	0.058	0.202	0.014	0.037	0.002	0.495	<0.001	0.018
significance	n.s.	n.s.	*	*	**	n.s.	***	*

Applied treatments: control (C) with water only; water suspension with 300 mg biochar/L (B300); water suspension with 600 mg biochar/L (B600); water suspension with 1200 mg biochar/L (B1200). The results are presented as the mean value of five replicates per treatment ± standard error. The data were subjected to one-way ANOVA. Significance markers indicate statistically significant differences as follows: * at p < 0.05; ** at p < 0.01; *** at p < 0.001; n.s.—not significant. For statistically significant results, Tukey’s post-hoc test was performed, and different letters within a column next to the mean values indicate statistically significant differences between treatments.

presents the micronutrient content in grapevine leaves measured at different phenological stages during the growing season.

At the flowering stage, boron content was highest in the B1200 treatment. Copper content was elevated across all biochar treatments. Iron and molybdenum levels showed no significant differences, while manganese content peaked in B300 and B1200. Sodium content was highest in B300 and comparable with B600 and B1200. Silicon content was also highest in B300, and zinc content was elevated in B300 and B1200.

At the setting stage, boron remained highest in B1200. Copper content peaked in B1200 and was comparable to the control. Iron content was highest in the control and comparable with B300 and B600. Manganese was highest in B600 and B1200, while molybdenum levels showed no significant variation. Molybdenum content showed no significant differences among treatments. Sodium content was highest in B1200. Silicon content was elevated in both B600 and B1200. Zinc content was high in all treatments except B300.

At the veraison stage, boron content was, again, highest in B1200 and comparable with B600. Copper content was elevated in both B600 and B1200. Iron was highest in the control treatment and comparable with B300 and B600. Manganese and molybdenum were highest in B1200 and comparable with B600. Sodium content peaked in B300 and B600 and was comparable to B1200. No significant differences were observed for silicon content. Zinc content was highest in B600.

At the harvest stage, no significant differences were found regarding boron, copper, and sodium content. Iron content was highest in the control and B300 treatments and comparable with B1200. Manganese was highest in B600 and comparable with other biochar treatments. Molybdenum peaked in B300 and was comparable to the control. Silicon content was highest in the control and B300 treatments. Zinc content was highest in B1200 and comparable with B600.

3.4. Grape Yield

Grape yield per vine was measured at the harvest stage and is presented in Figure 2. The highest yield was recorded for the B600 treatment, while the lowest was for the B300 treatment. Yields in the B1200 and control treatments were comparable and intermediate between the two extremes.

4. Discussion

The biochar used to prepare the foliar suspensions exhibited an alkaline pH, a high total carbon content, and notable quantities of macro- and microelements. These characteristics are consistent with results reported by Egri et al. [24]. The resulting foliar suspensions prepared with biochar also showed alkaline pH values, ranging from 9.21 to 9.42, while electrical conductivity (EC) values varied between 36 and 92 µS/cm. Optimal pH and EC values in foliar applications are critical to maximizing nutrient uptake efficiency and minimizing the risk of phytotoxicity in grapevines. The physicochemical properties of foliar spray mixtures can significantly influence the performance of active ingredients by affecting both formulation stability and interactions with the leaf surface, as noted by Assunção et al. [25]. The literature suggests that formulations with a near-neutral pH are often preferable, as they are less likely to disrupt the natural pH balance of the leaf surface and may enhance nutrient uptake and overall vine health [26]. Similarly, the EC value of a suspension is a crucial parameter in foliar treatment optimization. EC reflects the total ionic concentration in the spray suspension, and excessive EC values may cause osmotic stress or foliar burn, while excessively low values may indicate insufficient nutrient or additive content needed for effective uptake [25]. The pH and EC values of the applied biochar suspensions align with the average values reported for commonly used grapevine fungicides by Assunção et al. [25], which may explain why none of the applied biochar suspensions caused any visible damage to grapevine leaves.

Throughout the growing season, measurements of net photosynthetic assimilation (A) and intercellular carbon dioxide concentration (Ci) were conducted. No statistically significant differences in A or Ci were observed among the different foliar treatments. However, some trends emerged—specifically, treatment B600 exhibited higher net photosynthetic assimilation rates at the flowering, veraison, and harvest stages. Additionally, intercellular carbon dioxide concentration values at the veraison stage were higher in the B600 treatment, suggesting a potentially positive effect on photosynthesis. The results reported by Kumar et al. [17] showed that foliar treatments using biochar extracts from pine wood and wheat straw stimulated increases in chlorophyll a, chlorophyll b, and carotenoid contents, indicating enhanced photosynthetic capability. Similarly, Shahzadi et al. [18] observed that the application of nano-biochar solution increased plant pigments such as chlorophyll and carotenoids, as well as the leaf-relative water content of tomato plants under both stressed and non-stressed conditions. These findings contrast with our results, likely due to differences in the form of biochar used. Although those studies utilized biochar extracts of nano-biochar, which possess smaller particle sizes and greater solubility—which are factors that favor better foliar uptake—our research used biochar suspensions. Comparable results to ours were reported by Daler and Özkol [27], where the foliar application of another biostimulant, 5-aminolevulinic acid (5-ALA), on grapevine led to improvements in certain physiological parameters (e.g., plant growth), but it did not significantly affect net CO₂ assimilation or intercellular CO₂ concentrations. However, due to different chemical compositions and mechanisms of action between biochar and 5-ALA, a direct comparison is not appropriate. It is important to note that our results did not support the hypothesis that applying higher doses of biochar-based water suspensions could cause leaf shading and negatively affect photosynthesis. No adverse effects on photosynthetic activity were observed, even at the highest concentration used (B1200: 1200 mg/L).

Previous studies have reported that foliar application of nano-biochar suspension alleviated drought stress in tomato [18,28] and salinity stress in quinoa and wheat [29]. These effects were not observed in our study. Leaf water potential values during the grapevine growing season ranged from −0.47 MPa at flowering to −0.98 MPa at the veraison stage. These values fall within the ‘no water deficit’ (<−0.6 MPa) and ‘mild to moderate water deficit’ (−0.7 to −1.1 MPa) categories [30]. The absence of pronounced water deficit conditions can likely contribute to favorable meteorological conditions, the water-retentive properties of terra rossa soil with its high clay content, and well-timed soil and canopy management practices. The precipitation data indicated that the minimum monthly requirement (50 mm) was met in all months except June, which received slightly less (45 mm). Significant differences among treatments were found only at the harvest stage, where treatments B300 and B1200 showed the highest water deficit values but were comparable with B600. All three biochar treatments exhibited higher water deficit values compared to the control, but all remained within the ‘no water deficit’ category [30]. At flowering, the B600 treatment recorded the highest value, but as this was the initial stage of the experiment, it can be disregarded. At the setting and veraison stages, B1200 showed the highest values, suggesting a possible negative impact of the highest biochar concentration on leaf water potential. The substantial variation observed in leaf water potential across the flowering, setting, and veraison stages can be attributed to grapevines’ natural variability in water status. This variability can occur not only among different plants but also among leaves and shoots of the same plant. Contributing factors include leaf position (sun-exposed vs. shaded), leaf age, and local microclimatic conditions such as airflow and solar radiation [31]. For example, shaded leaves often have lower water potential due to reduced photosynthetic activity and transpiration, while sunlit leaves experience higher evaporative demand [32]. Although water deficit is known to influence photosynthesis, yield, grape composition, and wine sensory attributes [33], such effects were not observed in this study, likely due to the relatively small differences in leaf water potential among treatments.

Foliar application of biochar suspensions, particularly in nano-sized forms (nano-biochar or NBS), has been investigated for its potential to enhance plant growth and nutrient uptake. Nutrients from these suspensions can enter the leaf via stomatal uptake and cuticular penetration [34]. In our study, foliar treatments B600 and B1200 resulted in higher leaf concentrations of several elements (nitrogen, potassium, sulfur, boron, copper, and manganese) compared to the control and B300 treatments across most grapevine phenological stages, following the increasing biochar concentration gradient. These findings align with those of Rizwan et al. [35], who reported a linear increase in nutrient content in rice plants following foliar application of increasing concentrations of biochar mixed with nutrient solutions. Increased potassium levels in our study are also consistent with results reported by Mehmood et al. [36], where both soil and foliar applications of biochar-based fertilizers improved potassium uptake in wheat plants. The nutrients identified above play essential roles in grapevine physiology. Efficient nitrogen assimilation and partitioning are closely linked to vine performance and fruit quality [37]. Potassium is crucial for osmotic regulation and enzyme activation, contributing to stomatal function and carbohydrate translocation [38]. Sulfur, as another macronutrient, is indispensable for synthesizing sulfur-containing amino acids and various secondary metabolites essential in defense and stress responses. Verdenal et al. [37] emphasized sulfur’s involvement in grapevine metabolic processes and nutrient uptake dynamics. The same authors reported that boron is essential for cell wall formation and membrane stability, thereby influencing reproductive development, fruit set, and overall plant integrity. Leng et al. [39] specifically addressed mechanisms that control copper uptake and compartmentalization in grapevines, underscoring its importance in maintaining cellular redox balance and defense responses. Manganese is critical as a component of the oxygen-evolving complex in photosystem II and acts as a coenzyme in several redox reactions [37]. On the other hand, the present research noted that magnesium grapevine leaf content was often higher in B300 treatment compared to B600 and B1200, not following the biochar concentration gradient. This phenomenon may be attributed to nutrient antagonisms, particularly the interaction between potassium and magnesium. Peršurić Palčić et al. [40] reported that higher potassium content can suppress magnesium uptake in the leaves of Malvazija istarska grapevines. It is plausible that biochar foliar applications could similarly alter nutrient dynamics on the leaf surface, potentially leading to lower Mg content if the biochar formulation influences the balance between K and Mg. Across the studied phenological stages, calcium, boron, copper, and manganese content generally increased from flowering to harvest, regardless of the foliar treatment applied. The elemental composition of grapevine leaves, particularly concerning micronutrients such as Mn, Cu, and Zn, is strongly associated with grape quality. Manganese supports various enzymatic processes critical for plant health and development, while copper and zinc are vital for key metabolic functions and photosynthesis [41,42]. Deficiencies in these elements can impair physiological function, reduce vine vigor, and lower fruit quality [42]. Our results regarding calcium leaf content confirmed the results reported by Conradie et al. [43], who examined the seasonal uptake and partitioning of mineral nutrients in ‘Sultanina’ grapevines grown on different soil types. They found that calcium levels in leaves typically increase as the growing season progresses, with leaves absorbing a significant proportion of total Ca uptake during the fruit-set to harvest period, with 56.6% on sandy soil and 76.9% on alluvial soil. Similarly, Pradubsuk and Davenport [44] reported that boron, copper, and manganese concentrations in mature ‘Concord’ grapevine leaves typically rise from flowering to harvest, further corroborating our observations.

Yield, often the primary objective in the cultivation of many crops, is a crucial factor in agricultural production [45]. Foliar application of biochar has the potential to improve the microenvironment of the leaf by enhancing nutrient absorption, alleviating oxidative stress, and possibly modulating stomatal conductance [46]. These physiological changes can contribute to increased photosynthetic efficiency and improved water use efficiency, ultimately leading to enhanced crop yield [47]. Compared to soil amendments, foliar sprays offer more immediate nutrient uptake, thereby facilitating faster physiological responses that are essential for improved fruit set and berry development. In the present study, the B600 foliar treatment (600 mg of biochar per liter) resulted in a significantly higher grape yield, suggesting that this concentration may be optimal for maximizing grapevine productivity. This increase may be attributed to the concentrations of key nutrients observed in the leaves under B600 treatment, which likely influenced grapevine physiology and contributed to improved yield outcomes. Grape yield per vine serves as the foundational metric for calculating total yield per unit area. By multiplying the average yield per vine by the vineyard density, in this case, 5000 vines per hectare, an accurate estimation of yield per hectare can be determined. The B600 treatment led to a 37% increase in yield compared to the control, indicating that foliar biochar application can have a tangible, positive impact on grape productivity at the field scale. The significantly higher yield observed with B600 treatment highlights the potential of foliar biochar application as a promising and innovative agronomic strategy in grapevine cultivation. These findings underscore the importance of dosage optimization and suggest that targeted foliar biochar treatments could be an effective tool for enhancing both nutrient efficiency and overall crop performance. While increased grape yields can offer clear economic advantages through higher revenue and improved production efficiency, they may also introduce challenges related to market dynamics and potential impacts on product quality [48]. To ensure that such yield gains translate into sustainable economic benefits, careful planning and the adoption of responsible, sustainable viticultural practices are essential.

Moreover, to validate the consistency and generalizability of the observed benefits, long-term field trials should be conducted across different soil types and grapevine cultivars. These trials are crucial to confirm whether the positive effects of foliar biochar applications persist under varying environmental and agronomic conditions. Future research should focus on elucidating the underlying physiological mechanisms influenced by foliar biochar applications, as well as their long-term effects on grapevine health, yield stability, and fruit quality. In addition, practical considerations for the effective implementation and broader adoption of this technology should be addressed. These include optimizing biochar particle size to prevent nozzle clogging, determining the ideal volume of foliar suspension to apply per hectare, evaluating the use of surfactants to enhance leaf adherence and absorption, and assessing potential interactions with commonly used agrochemicals such as pesticides.

5. Conclusions

The application of biochar-based foliar treatments at varying concentrations during different grapevine phenological stages showed no significant effect on net photosynthetic assimilation or intercellular carbon dioxide concentration. Importantly, these findings confirmed that the potential leaf shading caused by biochar deposition did not negatively impact photosynthetic activity. Some positive trends in enhanced photosynthetic performance, particularly at the veraison and harvest stages, were observed in the B600 treatment, though these differences were not statistically significant. Regarding water status, the applied treatments resulted in significant differences in leaf water potential only at the harvest stage. Nevertheless, all treatments were categorized as exhibiting ‘no water deficit’, indicating that the biochar applications did not induce water stress under the prevailing environmental conditions. Treatments B600 and B1200, which involved higher concentrations of biochar (600 mg/L and 1200 mg/L, respectively), led to increased grapevine leaf content of nitrogen, potassium, sulfur, boron, copper, and manganese. Conversely, these treatments were associated with reduced magnesium content, likely due to strong antagonism between potassium and magnesium. Among all treatments, B600 produced the highest grape yield, achieving an increase of up to 37% compared to the control, indicating its potential as an optimal dose for improving grapevine productivity. These promising outcomes suggest that the foliar suspensions derived from grapevine-pruning-residue biochar could serve as a versatile and sustainable agronomic tool. Utilizing this often-underused biomass not only enhances grapevine physiology but also reduces reliance on commercial fertilizers. Future research should focus on long-term field applications of biochar foliar suspensions across various soil types and grapevine cultivars. In addition, the use of nano-biochar suspensions and biochar water extracts warrants further exploration. Investigating the enrichment of biochar during the quenching process with macro- and micronutrients could yield foliar formulations with enhanced nutritional profiles. Such strategies hold promise for maintaining grape and wine quality in the face of increasing environmental stress due to climate change.