The Effects of Biostimulants on the Physiological Processes of Yield Formation and Resistance of Apples to Spring Frosts

Ozherelieva, Zoya Evgen’evna; Prudnikov, Pavel Sergeevich; Stupina, Anna Yur’evna; Bolgova, Anzhelika Olegovna

doi:10.3390/horticulturae11091075

Open AccessArticle

The Effects of Biostimulants on the Physiological Processes of Yield Formation and Resistance of Apples to Spring Frosts

by

Zoya Evgen’evna Ozherelieva

^*

,

Pavel Sergeevich Prudnikov

,

Anna Yur’evna Stupina

and

Anzhelika Olegovna Bolgova

Russian Research Institute of Fruit Crop Breeding (VNIISPK), 302530 Zhilina, Russia

^*

Author to whom correspondence should be addressed.

Horticulturae 2025, 11(9), 1075; https://doi.org/10.3390/horticulturae11091075

Submission received: 19 July 2025 / Revised: 21 August 2025 / Accepted: 27 August 2025 / Published: 5 September 2025

(This article belongs to the Special Issue Effects of Biostimulants on the Growth and Development of Horticultural Crops)

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Abstract

The present research aimed to evaluate the effectiveness of new organo-mineral biostimulants in an apple orchard, including their relevance to spring frosts and to enhancing yield. The study evaluated the effects of foliar sprays with organo-mineral fertilizers on apple yield, comparing three treatments: 1—control (no treatment); 2—foliar spray with a 1% blend of “WPU” Antifreeze and 1% “WP Drip Ca + Mg”; 3—foliar application using a 3% solution of both “WPU” Antifreeze and “WP Drip Ca + Mg”. The NPC “White Pearl” foliar sprays exhibited cryoprotective properties to spring frosts through multiple mechanisms, i.e., prevention of cellular dehydration via elevated bound water content and accumulation of osmoprotective compounds including proline and soluble sugars. This research shows that the applied treatments improved carbohydrate metabolism by enhancing the biosynthesis of glucose and starch, as well as changing the donor–acceptor relationships between the leaf apparatus and the fruit toward the forming apple, promoting a better outflow of assimilates into ripening fruits. The 1% solution treatment enhanced apple yield by 70% (1.7-fold) relative to the untreated control. These findings indicate that the “White Pearl” organo-mineral fertilizer NPC (especially at 1% concentration) could serve as an effective supplement to conventional apple farming practices, boosting overall productivity.

Keywords:

biostimulants; foliar sprays; tolerance of spring frosts; yield; fruit quality

1. Introduction

The apple tree holds a leading position among fruit crops in Russia [1]. The main task of its cultivation is to obtain consistently high yields with good commercial and taste characteristics. To achieve these indicators, agrarians actively use mineral fertilizers and chemical protection means. However, extensive research indicates that these practices contribute to environmental contamination (affecting soil, water, and air), result in the accumulation of hazardous compounds in agricultural products, and promote the development of resistant pest and pathogen strains [2,3,4,5,6,7]. Traditional agrochemicals can be considered as an alternative to biological preparations, as they help reduce the environmental load [8,9]. Their effectiveness in some cases is comparable to complex agrotechnical measures [10,11]. Crucial nutrients such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg), which are part of many biostimulants, engage with soil contaminants via various adsorption methods. For example, certain nutrients that have a positive charge, such as potassium (K⁺) and calcium (Ca²⁺), may compete with heavy metal cations like lead and cadmium for spots on soil particles. This competition may result in a decrease in the ability to move and access these harmful substances [12]. Among the available and economically justified methods of optimizing the nutrition of fruit plantations, a special place is occupied by foliar sprays [13]. Although it cannot completely replace root fertilization, the use of biostimulants by spraying increases the resistance of plants to adverse external factors, enhances productivity, and improves the quality of fruits due to a more efficient assimilation of mineral elements. In modern agriculture, biostimulants are considered as a promising means of stimulating plant growth and increasing productivity, both in stressful and normal conditions. This is a significant step in the development of sustainable horticulture [14]. Despite encouraging research results demonstrating the potential of biostimulants in terms of mitigating abiotic stresses and improving product quality and yield [15,16], their mechanisms of action at the cellular, metabolic, and molecular levels remain understudied [17].

Biostimulants improve crop productivity by regulating metabolic and physiological processes, resulting in enhanced resilience to environmental stresses (both abiotic and biotic) as well as increased yield potential in terms of both size and quality. To lessen the effects of drought, researchers have looked into a number of strategies, including both conventional and cutting-edge methods. Conventional techniques, like the application of plant growth-promoting bacteria (PGPB) and morphological modifications, remain essential for improving drought resilience. Recent breakthroughs have provided innovative alternatives such as nanoparticle (NP) treatments and biochar, which enhance plant resilience. Biochar enhances soil moisture retention and nutrient accessibility, whereas nanoparticles augment water absorption and bolster molecular resilience under stress [18]. However, there is conflicting evidence about the effectiveness of biostimulants containing seaweed extracts. For example, in experiments conducted in Italy, foliar sprays with seaweed extracts (brown algae, Fucus spp.) did not lead to an increase in yield. Meanwhile, foliar sprays improved red color intensity and distribution in the Mondial Gala cultivar but not in Fuji [19]. At the same time, other studies have shown that similar preparations (Actiwave^®, a metabolic enhancer derived from the alga Ascophillum nodosum) can reduce manifestations of periodic fruiting on Fuji apple trees. Treated trees also showed higher leaf chlorophyll contents (by 12%), with a consequent increase in the rates of photosynthesis and respiration [20]. The liquid polycomponent fertilizer “Aminovit”, containing amino acids, nitrogen, trace elements (B—130 g/L; Mo, Cu, Mn, Fe, Zn—0.02 g/L), and humic acids, ensured a decrease in the shedding of ovaries and fruits due to the optimization of nutrition conditions. An analysis of the economic efficiency of the use of foliar sprays in apple cultivation which aimed to increase the yield and resistance to adverse environmental factors showed that in the foothill zone of the Krasnodar Territory, the profitability of fruit apple production of the Idared cultivar increased by an average of 1.4 times, that of the Renet Simirenko cultivar by 1.6 times, and that of the Golden Delicious cultivar by 1.9 times [21]. Regarding the blue honeysuckle cultivar, the use of a humic preparation and mineral fertilizer in the initial period of growth has been shown to have a positive effect on the average annual growth, which helps to increase the potential yield. As a result of that research, in the present study the application of a humic preparation of 150 mL/m² + Azofoska was selected, as these parameters led to the best outcomes: the mass fraction of nitrogen in the leaves was 2.64%, the mass fraction of mobile phosphorus compounds was 0.65%, the mass fraction of mobile potassium compounds was 1.51%, and average annual growth was 35 cm [22].

In this regard, studying the impact of new bio-preparations on resistance to abiotic stresses and productivity of apples is of significant scientific and practical interest.

The goal of the present research was to study the effect of biostimulants on the yield and resistance of apple trees to spring frosts, taking into account the modification of water and protein–carbohydrate metabolism.

2. Materials and Methods

2.1. Research Conditions

From 2021 and 2024, field trials were conducted in Central Russia to evaluate the novel organo-mineral fertilizer “White Pearl” NPC. This investigation, undertaken simultaneously in experimental orchards and the Laboratory of Physiology of Fruit Plant Resistance at the Russian Research Institute of Fruit Crop Breeding, represents a significant agricultural investigation targeting crop yield enhancement.

Trials were implemented on locally prevalent gray forest soil types (Table 1).

2.2. Analysis Hydrothermal Conditions 30 Days Before the Harvest of the Apple

Long-term studies have revealed significant variability in the hydrothermal regime during the critical 30-day period before harvesting. The pre-harvest period in 2024 exhibited exceptional climatic conditions characterized by elevated temperatures and severe drought compared to preceding years. Thermal accumulation analysis revealed that the sum of active temperatures (≥10 °C) surpassed previous years’ values by substantial margins: +113 °C versus 2021, +98.4 °C versus 2022, and +64.9 °C versus 2023. Hydrological data confirmed 2024 as the driest year, with precipitation deficits of 21.0 mm relative to 2021, 6.6 mm below 2022 levels, and 15.4 mm lower than 2023 records (Table 2).

2.3. Research Objects

The “White Pearl” natural plant complex (NPC) was evaluated on the Sinap Orlovsky (Malus domestica Borkh.) apple cultivar, a triploid late-winter maturity cultivar approved for cultivation in the Central and Central Chernozem regions of the Russian Federation, as well as in six regions of Belarus. This cultivar demonstrates particular susceptibility to physiological disorders, showing high vulnerability to both bitter pit manifestation during orchard production and extended storage periods [23,24] and superficial scald development during postharvest storage. The fruits are above average or large size (155 g) with a rounded-conical shape. The flesh is greenish-cream colored and juicy. Chemical composition of the fruit is as follows: sugars—9.9%, titrated acids—0.56%, ascorbic acid—13.4 mg/100 g, P-active substances—205 mg/100 g, and pectin substances—10.1%.

This study was conducted on the Sinap Orlovsky apple trees grafted onto semi-dwarfing rootstock (54–118), planted in 2013. The 6 m between rows and 3 m between trees spacing is a common semi-intensive system for apple orchards in temperate climates. Natural thinning was performed in-between tree rows, and herbicides were applied onto trunk strips (under-tree areas). Agrotechnics for apple trees in Central Russia involve a set of scientifically grounded practices to ensure healthy growth, high yields, and longevity of the trees.

In this study, there were three replicates in each variant. There were five trees in each group. The two factors used in the study were Factor A—Options and Factor B—Years.

Sinap Orlovsky is a popular late-winter apple cultivar known for its excellent storage qualities and adaptability to Central Russia. Below are its key phenological phases:

Budbreak onset—late April (II–III decade);
Flowering initiation—early to mid-May (I–II decade);
Flowering completion—late May (III decade);
Fruit harvest period—early to mid-September (I–II decade);
Leaf fall duration—October through November.

The “White Pearl” NPC is an organo-mineral fertilizer formulation engineered to improve fruit quality through optimized nutritional supplementation. This compound is classified as a non-hazardous agricultural input.

The NPC “White Pearl Universal (WPU)” Antifreeze is a mineral-based suspension derived from natural sources, incorporating concentrated needle extracts from spruce (Picea spp.), pine (Pinus spp.), and Siberian fir (Abies sibirica). The formulation contains the following mineral composition. The fertilizer composition comprised: SiO₂—5.6%, N total—6%, MgO—7000 ppm, CaO—5000 ppm, K₂O—0.2%, Al₂O₃—1600 ppm, Zn—150 ppm, Mo—200 ppm, and B—130 ppm.

NPC “White Pearl (WP) Drip Ca + Mg” is an organo-mineral formulation derived through aqueous extraction of oceanic biomass. The product combines marine-derived bioactive compounds with balanced calcium and magnesium mineral supplementation. The composition includes the following bio-elements: Ca—3490 ppm, Mg—2829 ppm, K—38.8 ppm, P—42.9 ppm, S—0.3 ppm, Fe—68.7 ppm, B—3.37 ppm, Mn—3.65 ppm, Zn—0.05 ppm, Cu—0.85 ppm, J—2.1 ppm, Se—0.003 ppm, Si—0.1 ppm, Mo—0.01 ppm, SiO₂—5.6%, CaO—0.4%, MgO—0.4%, Al₂O₃—0.16%, K₂O—0.2%, and Fe₂O₃—0.4%.

The organo-mineral fertilizers also include vitamins (fat-soluble)—A, D, E, and K; (water-soluble)—B₁, B₂, B₆, PP, and H; and phytochemicals—chlorophyll, flavonoids, sugars, proteins, and amino, sulfonic, and humic acids.

2.4. Procedures for the Use of “White Pearl” NPC in the Experiment

The experiment was conducted with three options:

Control (no treatment applied);
Plants receiving foliar spray of 1% solutions (NPC “WPU” Antifreeze + NPC “WP Drip Ca + Mg”);
Plants receiving foliar spray of 3% solutions (NPC “WPU” Antifreeze + NPC “WP Drip Ca + Mg”).

To protect the generative organs of Sinap Orlovsky trees from late spring frosts in early spring, they were treated twice with a 1% and 3% solution of NPC “WPU” Antifreeze. The first application was carried out in early April (during the “dormant bud–silver cone” phenophase). The second treatment was administered 20 days later (coinciding with the “mouse ear” phenophase). The third treatment with 1% and 3% solutions of NPC “White Pearl Universal” Antifreeze and NPC “White Pearl Drip Ca + Mg” was carried out during the “inflorescence emergence” phenological phase.

This investigation evaluated a summer foliar spray regimen using NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” at 1% and 3% concentrations to mitigate premature fruit abscission and improve fruit characteristics in apple trees. The treatment schedule targeted four crucial developmental stages, 14 days after flowering; “fruit-hazel” growth phase; “fruit-walnut” growth phase; and 25 days before apple harvesting, to increase yield. Solution preparation was as follows: 1% working solution: 100 mL of each formulation diluted in 10 L water; and 3% working solution: 300 mL of each compound mixed in 10 L water.

2.5. Analysis of the Fractional Composition of Water

Determination of Bound and Free Water Fractions in Apple Tree Tissues (Okuntsov–Marinchik Method) [25]: This analytical approach quantifies the ratio of bound to free water in plant tissues (specifically, the tissues of annual shoots and fruit buds) by assessing shifts in sucrose solution concentration following sample immersion. Tissue samples (0.4 g each) were weighed and immersed in a 30% sucrose solution (triplicate replicates per sample). The bound water fraction was analytically determined through differential measurement between total tissue hydration and free water content. Solution concentration was measured using a PAL-1 digital refractometer (Atago, Tokyo, Japan) with ±0.2% Brix accuracy. The total hydration of annual shoot tissues and reproductive buds was computed using the formula:

W = (m₁ − m₂)/m₁ × 100%,

(1)

where

W—Total hydration relative to wet mass, %;

m₁—Initial mass of fresh annual shoot and fruit bud tissue, g;

m₂—Mass of the same tissue after complete drying, g.

To determine the absolute dry weight, samples of annual shoot and fruit bud tissues were placed in pre-weighed aluminum containers and dried in an oven at 105 °C until a constant mass was achieved. The drying process continued until successive weight measurements remained unchanged, confirming the attainment of a constant mass.

2.6. Determination of Glucose, Starch and Proline

The concentrations of sucrose and glucose in annual shoot bark and fruit buds were analyzed spectrophotometrically using a modified resorcinol assay (λ = 520 nm) with triplicate measurements. We homogenized 0.5 g plant material in 10 mL of 80 °C ethanol and heated samples in a UT-4301 E water bath (Ulab, Shanghai, China) at 100 °C for 10 min to optimize extraction. We centrifuged extracts at 7000 rpm for 10 min (B4i centrifuge, Jouan, France). We combined 0.5 mL supernatant with 50 µL 5 N NaOH. We repeated heating (100 °C, 10 min) in a water bath. We added 0.5 mL ice-cold resorcinol reagent (100 mg resorcinol + 250 mg thiourea in 100 mL glacial acetic acid). We mixed it with 3.5 mL 30% HCl. We then heated the reaction mixture (100 °C, 10 min). We measured absorbance at 520 nm using a BioRad SmartSpecPlus spectrophotometer (Hercules, CA, USA). Finally, we quantified carbohydrate content against sucrose/glucose standard curves [26].

To determine starch content, finely chopped vegetable material (2 g) was ground in 5 mL of 80% calcium nitrate and 5 mL of water. The mixture was boiled at a low heat for 3 min to dissolve the starch. Ten mL of water was added to the flask. Then it was centrifuged at 2000–3000 rpm for 5 min. The liquid in the beaker was adjusted to the 50 mL mark using distilled water. Next, 2 mL of a 0.5% iodine solution was introduced into 5 mL of the prepared mixture. The solution was then centrifuged at 4000 rpm for 10 min. After centrifugation, 5 mL of a 5% calcium nitrate solution (containing 0.01% iodine) was added to the sediment. The mixture underwent another centrifugation cycle at 4000 rpm for 10 min. Ten mL of 0.1 N NaOH solution was added to the washed precipitate of the iodine-starch compound. The test tube was placed in a boiling water bath for 5 min. The solution was transferred to a 50 mL volumetric flask, to which there was added 0.3 mL of 0.5% iodine solution, 2 mL of 1 N HCl solution, and 20 mL of water. The optical density of the solution was measured at 590 nm [27].

The proline concentration was measured spectrophotometrically using ninhydrin-based analysis with triplicate experimental replicates. The assay was performed according to the following optimized methodology [23]. To do this, 500 mg of bark from annual shoots and fruit buds was ground in distilled water, boiled for 10 min at 100 °C in a UT-4301E, Ulab (Ulab, Shanghai, China) water bath. Following homogenization, the sample processing continued with these precise steps. We centrifuged the homogenate at 7000× g for 10 min using a B4i centrifuge (Jouan, Morlaàs, France). Samples were boiled at 100 °C for 1 h using a UT-4301E water bath (Ulab, Shanghai, China). Absorbance was read at 520 nm with a BioRad SmartSpecPlus spectrophotometer. The proline concentration was determined against a standard calibration curve prepared using analytical-grade L-proline (≥99% purity, Sigma-Aldrich, St. Louis, MI, USA). Results were expressed as milligrams of proline per kilogram of fresh plant tissue (mg/kg) [28,29].

2.7. Artificial Freezing of Apple Buds and Flowers

The experiment was conducted in a PSL-2KPH climate chamber (Espec, Osaka, Japan) to simulate spring frost conditions at −3 °C and −4 °C for 3 h. The temperature was reduced at a rate of 1 °C per hour. Branches from the test cultivar were collected, each containing 100 flowers and 100 buds. After freezing, samples were held at +22 °C until visible damage symptoms appeared. Visual inspection was used to evaluate browning in buds and flowers. The percentage of damage was calculated as: (number of damaged structures/total structures) × 100%.

2.8. Yield and Average Weight of Apple Fruits

The yield per tree (in kg) was determined through direct weighing of fruits at removable harvest maturity, following the established protocol [30]. For each experimental option, the mean yield per sample tree was computed as the ratio between the total fruit mass (harvested fruit crop + economically usable wind fallen fruits) and the number of trees in the option. The total orchard yield (in centners per hectare, c/ha) was computed using the following formula:

Y = A/B × 100,

(2)

where

Y—Yield, c/ha;

A—Average yield per 1 tree, kg;

B—Nutrition area of 1 tree, m²;

100—The coefficient of conversion of weight in kilograms to weight in hundredweight and m² area to hectares.

To evaluate fruit size characteristics, a random sample of 100 apples was selected from each experimental replicate. The average fruit weight was calculated by dividing the total weight of these 100 fruits by their quantity (100).

2.9. Statistical Analysis

The experimental data were analyzed using statistical methods, including one-way and two-way ANOVA (SPSS Version 22). Mean values were compared using Fisher’s least significant difference (LSD) test. A 95% confidence level (p < 0.05) was applied for all statistical evaluations.

3. Results

3.1. The Effect of “White Pearl” NPC on the Water Regime of the Sinap Orlovsky Apple Cultivar During the Spring Period

The use of 1% and 3% solutions of NPC “WPU” Antifreeze in the “dormant bud–silver cone” phenophase increased the hydration level of the annual shoots of the Sinap Orlovsky apple cultivar by 9.1% and 7.8%, respectively, compared to the control. In the subsequent “mouse ear” phenophase, the greatest increase in the shoot tissue hydration (by 18.0% relative to the previous stage) was observed in the control. At the same time, treatment with a 1% solution of the biostimulator provided only a 2.2% increase in this indicator. By the “inflorescence emergence” phenophase, a decrease in the hydration of shoot tissues by 8.5% was recorded in the control. In contrast to this, options with foliar sprays with 1% and 3% solutions of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” maintained the level of tissue hydration at a stable level (Table 3). This indicates a more effective preparation of plants for flowering due to the regulation of water exchange under the influence of the applied preparations.

In the initial development phases (“dormant bud–silver cone” and “mouse ear”), no significant differences in the degree of hydration of flower buds between the experiment options were found. However, significant deviations were detected at the “inflorescence emergence” phenophase. Thus, treatment with a 1% solution ensured the optimization of the water regime of flower buds compared to the control and the option with a 3% solution. Presumably, the minimum concentration of the organo-mineral composition contributed to the stabilization of the water balance and the activation of metabolic processes (Table 3). This assumption was confirmed by the results of biochemical analyses presented in the following sections of the study.

The highest amount of bound water in annual shoots of the Sinap Orlovsky cultivar was recorded in the “dormant bud–silver cone” phenophase in all experimental options. This was due to the reduced metabolic activity of plant tissues in the early spring period. As physiological processes were activated in the spring, a significant decrease in the proportion of bound water in the shoots was observed in all studied options. The most significant changes were noted in the “inflorescence emergence” phenophase. In the control option, the content of bound water decreased by 6.4%; when using a 1% solution of biostimulant it decreased by 4.5%; and when using a 3% solution it decreased by 9.5% (Table 4).

A different dynamic was identified in the flower buds. The maximum accumulation of bound water was recorded in the “mouse ear” phenophase, with the most pronounced effect observed in the options with treatments. In the control we registered an increase of 12.1%; in the case of 1% solution the content of bound water increased by 75.8%; and in the case of 3% solution the increase was 36.8%. Such a physiological response likely performs a protective function, safeguarding cellular structures from damage during spring frosts. In the subsequent phenophase “inflorescence emergence”, we registered the following decreases in the content of bound water: in the control it decreased by 7.8%; in the 1% solution option it decreased by 14.5%; and in the 3% solution option it decreased by 14.0%. However, the absolute indicators of the content of bound water in the treated options exceeded the control values by 12.3% and 14.0%, respectively (Table 4). Such a physiological adaptation is of significant practical importance as it reduces the risk of cellular dehydration, minimizes the likelihood of tissue damage, and increases plant resistance to extreme temperature effects during the critical development period. The obtained data indicate a pronounced physiological effect of the studied biostimulants, manifested in the modification of water exchange in plant tissues at different stages of organogenesis.

The highest concentration of free water in annual shoots was observed in the phenophase “inflorescence emergence” in all studied options, which was due to the activation of metabolic processes during this period of vegetation. Quantitative changes in the content of free water in annual shoots showed an increase of 6.4% in the control, and in the treatments with the 1% and 3% solutions the increase in free water content was 4.5% and 9.5%, respectively (Table 5).

The maximum free water contents in flower buds were also recorded in the phenophase “inflorescence emergence”. The analysis of the dynamics of free water content in the flower buds of the Sinap Orlovsky apple cultivar during the “inflorescence emergence” phenophase revealed the following increase in the content of free water in the flower buds: control by 9.7%; in the option with treatment with a 1% solution we registered a decrease of 22.2%; and in the option of treatment with a 3% solution it was 16.9% (Table 5). In the control option it was 6.4% higher relative to the 1% solution and 3.2% higher relative to the 3% solution of biostimulants. Thus, the lower amounts of free water in the treated options may indicate the activation of adaptation mechanisms. The experimental results demonstrate that adaptogenic treatments effectively regulate water metabolism in plants. These treatments alter the distribution of water fractions within plant tissues, helping to maintain optimal hydration levels and enhancing the plant’s ability to withstand environmental stressors.

Presumably, the “White Pearl” NPC preparations contributed to the optimization of the water regime of the Sinap Orlovsky apple cultivar plants, and as a result, the intensity of metabolic processes increased. This conclusion is confirmed by the results of biochemical studies, which are presented below.

3.2. The Effect of “White Pearl” NPC on the Level of Low-Molecular Osmoprotectors in the Bark of Annual Shoots and Flower Buds of Apple Trees in Spring

The first foliar sprays of the Sinap Orlovsky apple cultivar with a 1% solution of NPC “WPU” Antifreeze in the “dormant bud–silver cone” phase contributed to an increase of 15.6% in the bark and 17.2% in the flower buds of free proline content compared to the control (Table 6). However, treatment with the 3% solution did not have a significant effect on the level of the studied amino acid in the tissue under consideration. After the second foliar sprays, in the “mouse ear” phase, the content of free proline in the bark tissues decreased in all options. At the same time, the greatest decrease in the amino acid was observed in the option with treatment with the 1% solution of the preparation (by 90.4% compared to the date of the first treatment), whereas in the control the decrease in the amount of proline was 66.5%, and in the option with treatment with the 3% solution of the preparation it was 61.4% (Table 6). Against the background of a sharp decrease in the levels of free proline in the bark tissues in the option with 1% foliar sprays, it increased by 5.5 times in the generative organs compared to the “dormant bud–silver cone” phase; whereas, in the control it increased by 5.3 times, and in the option with the 3% solution it increased by 5.4 times. Apparently, treatment with solutions of the preparation contributed to the modification of protein metabolism, in particular, it intensified the biosynthesis of the amino acid in the buds, as well as the outflow of proline to the unfolding buds for the construction of protein molecules.

Foliar sprays analyses during the “inflorescence emergence” phase demonstrated that applying a 1% solution of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” significantly influenced proline metabolism. The 1% treatment reduced proline content by 20.9%, exceeding the decreases observed in the control (17.1%) and 3% treatment (16.7%). At the same time, in the tissues of flower buds, the level of proline under the influence of the 3% solution of the NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” decreased 5.4 times, while in the control it decreased 2.2 times, and under the influence of the 1% solution it decreased 3 times compared to the “mouse ear” phase (Table 6). The pronounced reduction in proline concentrations—particularly in bark and flower bud formation—suggests that both 1% and 3% solutions accelerate protein metabolism. This likely occurs through enhanced incorporation of proline into protein synthesis pathways.

By determining the sugar content, we established that treatment with a 1% solution of NPC “WPU” Antifreeze increased the carbohydrate content in the bark tissues. Thus, at the moment after the first treatment in the “dormant bud–silver cone” phase, the sugar level in the bark tissues increased by 15.3% compared to the control (Table 7). In flower buds, foliar sprays with the 1% solution of the preparation contributed to an 18.7% increase in sugar levels. At the same time, foliar sprays with the 3% solution in the bark tissues did not significantly change the content of disaccharides, but in flower buds it reduced it by half compared to the control.

During the “mouse ear” phase, after the second foliar sprays a decrease in the level of sugars was noted both in the bark tissues and in the tissues of the flower buds. Thus, the 1% and 3% solutions of the preparation reduced the amount of sugars in the bark tissues by 2.1 times and 1.9 times compared to the “dormant bud–silver cone” phase, and in the control the decrease was 1.8 times. In the flower buds in the options with the 1% and 3% solutions the amount of disaccharides decreased by 2.71 and 1.50 times, while in the control it was 1.4 times compared to the “dormant bud–silver cone” phase. The overall decrease in sugars, apparently, was associated with the beginning of active growth processes and their outflow due to the respiration process. Thus, the plants can obtain energy and plastic equivalents. A more intensive decrease in the amount of sugars in the tested options indicates, accordingly, a more active metabolism.

Against the background of the third foliar sprays in the “inflorescence emergence” phase, the amount of sugars in the bark tissues both in the control and in the option with the 1% solution of the preparation continued to decrease, but with less intensity, and in the option with the 3% solution the level of disaccharides did not significantly differ from the “mouse ear” phase. Ultimately, in the “inflorescence emergence” phase the options did not significantly differ from the control in terms of sugar content. At the same time, the greatest decrease in sugars, by 2.4 times, was noted in the control in the flower buds tissues. Furthermore, in the option with the 1% solution it decreased by 1.2 times, and in the case with the 3% solution we registered an upward trend of disaccharides compared to the “mouse ear” phase. As a result, in the “closed inflorescence” phase the amount of sugars in the experimental options exceeded the control by 28.6%.

Thus, the foliar sprays used accelerated carbohydrate metabolism in the example of sugars by increasing their formation, transport to flower buds, and expenditure on metabolic processes, acting as a substrate for respiration. At the same time, in such an important development phase as “inflorescence emergence” the levels of sugars in flower buds in the studied options were higher than control values.

3.3. Evaluating the Impact of “White Pearl” NPC Biostimulants on Frost Resistance in Apple Buds and Flowers During Spring

The results of assessing the frost resistance of the Sinap Orlovsky apple cultivar using “White Pearl” NPC preparations showed that treated plants demonstrated a significant increase in flower resistance after exposure to a temperature of −3 °C. Meanwhile, we registered a decrease in flower mortality by 19.7% for the 1% solution and 27.0% for the 3% solution of biostimulants. At the same time, a decrease in bud damage by 13.9–15.1% was identified for both treatment options. Foliar sprays with the 1% and 3% solutions of biostimulants reduced the degree of freezing of flowers and apple buds by 9.8% and 9.0%, respectively, and also reduced bud damage by 9.1% and 10.5% compared to the control option after exposure to a temperature of −4 °C (Figure 1a,b).

The extreme weather conditions of spring 2024 had a critical impact on fruit crops. In May, during the mass flowering of apple trees, they were subjected to an abnormal prolonged cooling with recurring frosts. According to the data of the weather station under the control of the Russian Research Institute of Fruit Crops Selection, the air temperature reached −6 °C (the minimum value for the last 50 years). This led to a loss of 70–80% of the harvest in most apple varieties. Under these conditions, the protective effect of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” on the Sinap Orlovsky apple cultivar was manifested. The results confirmed the data of laboratory tests during artificial freezing as in the control, 78.2% of flowers died; foliar sprays with the 1% solution reduced damage by 15.1%; and the 3% solution reduced the effect of freezing by 11.4%. The obtained data fully agree with the results of previous studies [31,32], demonstrating the stable effectiveness of the preparation under both controlled experimental conditions and extreme natural conditions. Thus, the use of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” increased the resistance of apple generative organs to spring frosts. This effect was likely associated with an increase in the proportion of bound water and the activation of carbohydrate–protein metabolism in plant tissues.

The “White Pearl” NPC foliar sprays exhibited cryoprotective properties through multiple mechanisms, including the prevention of cellular dehydration via elevated bound water content, and the accumulation of osmoprotective compounds including free proline (a known effective osmolyte) and soluble sugars. These mechanisms collectively enhanced frost resistance in orchard crops, significantly reducing spring frost damage to reproductive structures (buds and flowers).

3.4. The Impact of Foliar Sprays with “White Pearl” NPC on the Water Regime of the Sinap Orlovsky Apple Cultivar During the Summer Period

The conducted research showed that the use of the studied biostimulants during the summer period did not have a statistically significant impact on the indicators of hydration in the vegetative and generative organs of the Sinap Orlovsky apple cultivar. Thus, hydration in the leaf apparatus in all experiment options was in the average range (from 50.0% to 70.0%). In the fruits of the experimental variety, we observed a consistently high hydration (from 70.0% and above) (Table 8).

Foliar sprays with biostimulants during the summer period did not have a significant impact on the overall content of bound water in leaves and fruits. However, differences are observed in individual phenophases. Thus, in the “fruit-walnut” phase and 25 days before harvesting in the leaves of the plants treated with the 3% solution of biostimulants the content of bound water was higher by 10.0% and 7.8% (compared to the control), and 23.4% and 8.3% (compared to the 1% solution) (Table 9).

The minimum content of bound water in fruits was noted in the “fruit-walnut” phase, which is associated with active growth and the transition to a more mobile form of water. The decrease in bound water during the phase of active growth (“fruit-walnut”) was explained by the intensification of metabolism, i.e., water transitions into a free form, which facilitated the transport of nutrients. Meanwhile, 25 days before harvesting the level of bound water increased as follows: in the control it grew by 15.7%; in the 1% solution option it grew by 24.0%; and in the 3% solution option it grew by 24.7% (Table 9). The increase in bound water before harvesting was associated with the completion of fruit growth, a decrease in free water, and a slowdown in metabolic processes, which contributed to the accumulation of organic substances.

In the 25-day period before harvesting, we noted a 21.4–29.2% decrease in free water in the old apple leaves in all options compared to the “fruit-walnut” phase (Table 10). This was due to the reduction in metabolic activity of the leaves and the outflow of plastic substances (carbohydrates, amino acids) into the fruits and shoots, which ensured their growth and maturation.

In the fruits of the experimental apple variety, the minimum content of free water was noted 25 days before harvesting, which may be associated with a decrease in metabolic processes in maturing apples, an increase in the proportion of bound water, and intensive accumulation of organic substances in them. Thus, in the control fruits the level of free water decreased to a lesser extent, i.e., by 23.3%, and in the options treated with the 1% and 3% solutions of “White Pearl” NPC it decreased by 33.7 and 43.7%, respectively, relative to the “fruit-walnut” phenophase (Table 10).

In leaves, the reduction in free water was associated with natural aging and the redistribution of resources in favor of fruits. In fruits, the decrease in free water indicated a transition to ripening (reduction in metabolic activity, increase in the proportion of bound water). More intensive accumulation of dry substances was noted in the treated options (especially in the 3% concentration). Biostimulants (especially the 3% solution) enhanced the outflow of water and nutrients to the fruits, which improved their quality.

Thus, the obtained data showed that the use of “White Pearl” NPC optimized the water regime and metabolic processes in trees of the Sinap Orlovsky apple cultivar by improving the ratio of free and bound water in plant tissues of the leaf apparatus and ripening fruits.

3.5. The Influence of Foliar Sprays with “White Pearl” NPC on the Carbohydrate Metabolism of the Sinap Orlovsky Apple Cultivar During the Summer Period

In order to determine the effects of the preparation foliar sprays of the assimilation apparatus during the vegetation period, we determined the content of glucose and starch in leaf tissues, as well as in forming fruits. It was shown that foliar sprays with the preparations of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” at solution concentrations of 1% and 3% increased the glucose content in leaf tissues by 29% and 14%, respectively, and the starch content by 41% and 46% in the “fruit-walnut” phase (Table 11). At the same time, in the foliar sprays under study an increase in starch content in the developing ovary was observed, especially in the option with the 3% solution of the preparation (2.5 times higher than in the control), despite the treatments having no significant effect on the glucose content.

In foliar sprays of the 1% and 3% solution treatments during the “fruit-walnut” phase, leaf tissue glucose levels showed reductions of 47% and 35%, respectively, relative to the “fruit-hazel” phase measurements, whereas the control group exhibited only an 11% decrease in monosaccharide content. Starch content in the photosynthetic tissues demonstrated treatment-dependent declines of 38% and 25% for the 1% and 3% applications, contrasting with a 29% increase observed in control plants compared to the “fruit-hazel” phase. Conversely, fruit glucose content during the “fruit-walnut” phase showed pronounced increases of 43% and 80% for the 1% and 3% treatments, respectively, while control fruits displayed no statistically significant elevation. It is important to note that the starch content in the fruits which had been applied treatments also significantly increased by 2.9 and 1.7 times, whereas polysaccharide content increased by 1.4 times in the control. Apparently, foliar sprays changed the donor–acceptor relationships between the assimilation apparatus and the forming fruit, namely, they contributed to a better outflow of assimilates into maturing apples. This is evidenced by the more intensive decrease in the amount of monosaccharides and polysaccharides in the leaf apparatus of experimental plants and their increase in the forming fruit.

After the third treatment 25 days before harvesting, the maximum increase in glucose content and decrease in starch in the leaf apparatus was noted in the option with foliar sprays with the 3% solution of the studied preparations. Thus, the amount of glucose in the leaf in this option increased 2 times compared to the data obtained after the second foliar sprays in the “fruit-walnut” phase, and the starch content decreased 2.8 times. In the option with the 1% solution, the monosaccharide level in the leaf apparatus increased 1.6 times compared to the “fruit-walnut” phase and the starch content decreased 1.7 times. In the control, the glucose content in the leaf increased 1.4 times and the starch content decreased 1.3 times. Such a rise in glucose in the assimilation apparatus was apparently not so much associated with its de nova synthesis, but with the hydrolysis of starch for the purpose of carbohydrate translocation into ripening fruits. Indeed, when analyzing the content of the studied carbohydrates in fruits 25 days before harvesting, it was shown that foliar sprays with the preparations significantly changed the donor–acceptor leaf–fruit relationships towards increasing the outflow of assimilates into apples. Thus, the glucose content in fruits 25 days before harvesting in options with the 1% and 3% solutions increased 2.6 and 1.6 times, respectively, while starch content increased 4.3 and 3.8 times compared to the “fruit-walnut” phase. At the same time, in the control the level of glucose in the fruit increased 1.5 times, while starch content increased 2.6 times (Table 11).

Thus, the research has shown that the preparations used improved carbohydrate metabolism by enhancing the biosynthesis of glucose and starch, as well as changing the donor–acceptor relationships between the leaf apparatus and the fruit towards the forming apples, promoting a better outflow of assimilates into ripening fruits.

3.6. The Impact of Foliar Sprays with “White Pearl” NPC on the Yield of the Sinap Orlovsky Apple Cultivar

The most significant yield enhancement was observed with the combined application of 1% NPC “WPU” Antifreeze and 1% NPC “WP Drip Ca + Mg”, which increased yield by 1.7 times compared to untreated controls and exceeded other treatments by a factor of 1.6. In contrast, when applied at 3% concentration, the same biostimulant combination (NPC “WPU” Antifreeze + NPC “WP Drip Ca + Mg”) showed limited efficacy, providing only a 9.6% yield improvement relative to control plants. The optimal concentration for increasing apple yield turned out to be the 1% solution of adaptogenic preparations. A high concentration of the tested preparations (3%) was less effective, possibly due to excessive impact (Table 12).

Over the years of research, the highest yields were determined in 2022 and 2023 (favorable conditions). The lowest yield of the Sinap Orlovsky cultivar was in 2024 due to spring frosts, which severely damaged the flowers and ovaries and disrupted normal fruit formation. In 2022, compared to 2023, the yield of the studied cultivar was 1.6 times higher under control conditions. In the variants where foliar sprays were applied using 1% and 3% solutions of NPC “White Pearl”, the increase was 1.1 and 1.7 times, respectively. In 2023, the yield of this cultivar was significantly higher than in 2024. In the control variant the yield was 2.8 times higher, and after applying the 1% and 3% solutions of NPC “White Pearl” to the apple trees the yield increased by 3.8 and 2.7 times, respectively. In 2024, when the weather conditions were unfavorable, the yield of the Sinap Orlovsky cultivar was 4.5 times lower in the control samples. When using foliar sprays with the 1% and 3% solutions of “White Pearl” NPC, the yield decreased by 4.1 and 4.6 times, respectively (Table 12).

Thus, foliar sprays with the 1% solution of “White Pearl” NPC biostimulants significantly increased yield, probably due to improved absorption of calcium and magnesium (important for the strength of fruit cell walls and photosynthesis) and increased resistance to spring frosts.

3.7. The Impact of Foliar Sprays with “White Pearl” NPC on the Weight of the Sinap Orlovsky Apple Cultivar Fruits

Foliar sprays with “White Pearl” NPC fertilizers significantly enhanced fruit weight in Sinap Orlovsky apples. Relative to untreated controls, mean fruit weight increased by 24.2 g when treated with the 1% “WPU” Antifreeze + 1% “WP Drip Ca + Mg” formulation, compared to a 17.6 g increase observed with the 3% concentration of the same biostimulants (Table 13). This represents a 37.5% greater weight enhancement at the lower concentration. The superior performance of the 1% formulation in both yield and fruit weight parameters suggests optimized nutrient uptake and assimilation efficiency. The reduced efficacy at higher concentrations may reflect nutrient oversaturation in plant tissues, potentially leading to metabolic imbalance or feedback inhibition of critical physiological processes.

4. Discussion

Weather factors play a key role in gardening. Even highly effective agricultural techniques cannot compensate for the impact of spring frosts, which can lead to a 100% loss of fruit crops. In this regard, the development of resilient technologies aimed at reducing the impact of abiotic stresses on plants is a priority task. Among various technologies aimed at mitigating the negative impact of abiotic stresses on plants, the most promising is the use of environmentally friendly compounds, such as biostimulants [33,34].

In this regard, for the first time in the conditions of Central Russia, tests of the new biostimulant “White Pearl” NPC were conducted using the example of an apple tree. As a result of the tests, it was found that in plants of the Sinap Orlovsky apple cultivar treated with “White Pearl” NPC the intensity of protein–carbohydrate and water exchange increased, which is of great importance in the conditions of recurrent spring frosts. It is known that metabolites such as carbohydrates and amino acids play a vital role in protecting plants from abiotic stresses, acting as osmolytes and scavengers of active forms of oxygen [35]. Our research demonstrated that foliar applications of 1% and 3% concentrations of NPC “WPU” Antifreeze and NPC “WP Drip Ca + Mg” biostimulants effectively mitigated low-temperature stress in apple trees. These treatments appeared to safeguard cellular integrity by preventing dehydration, potentially through increased accumulation of osmoprotectants, particularly free proline and soluble sugars. The cryoprotective efficacy was particularly evident in reproductive tissues, where NPC “White Pearl Universal” Antifreeze and NPC “White Pearl Drip Ca + Mg” applications substantially reduced frost damage. Specifically, the 1% solution lowered flower freezing rates by 19.7% at −3.0 °C and 9.8% at −4 °C, while the 3% formulation showed greater protection at −3.0 °C (27.0% reduction) but comparable effects at −4 °C (9.0% reduction). Additionally, both treatment concentrations reduced bud damage in Sinap Orlovsky by 9.1% to 15.1% across both treatment options. These results confirm the prospects of using “White Pearl” NPC in the system of protecting fruit crops from spring frosts. It is especially important that the preparations showed effectiveness for both flowers and buds, which significantly extends the period of their protective action. Foliar sprays of plants with “Humate+7” and “Energin” in combination with mineral fertilizers increased the winter hardiness of raspberries. The shoot frost damage on these variants was 1.9–2.0 points, and the bud damage was 24.0–26.1%. Due to the activation of regeneration processes, the overall condition of the plants at the end of the growing season was rated at 3.8–3.9 points, which was considered good [36]. Previous research by V.P. Popova and colleagues [37] demonstrated the efficacy of organo-mineral foliar fertilizers containing macro- and microelements combined with amino acid complexes in enhancing apple trees’ adaptive capacity. In grapevines, foliar application of SWE facilitated photosynthetic recovery following drought stress, whereas soil drenching had no significant physiological impact [38]. The efficacy of biostimulants in enhancing abiotic stress tolerance in agricultural and horticultural crops is linked to several physiological and biochemical mechanisms. These mechanisms include strengthening the root system to enhance nutrient uptake and assimilation; improving photosynthetic efficiency and leaf-water relations; increasing the accumulation of osmolytes such as sorbitol, proline, betaine, and glycine; reducing oxidative stress by lowering hydrogen peroxide and malondialdehyde levels, and enhancing antioxidant defenses through increasing activities of enzymes like catalase and superoxide dismutase; and optimizing water use efficiency by reducing transpiration and stomatal resistance [39,40,41].

In our current study, summer applications of the 1% NPC “WPU” Antifreeze + 1% NPC “WP Drip Ca + Mg” solution promoted more vigorous fruit growth and maturation through multiple physiological mechanisms. The treatment positively influenced carbohydrate metabolism, water regulation, and source–sink relationships between leaves and fruits. These physiological improvements translated into significant yield increases of 1.7-fold and 1.6-fold (compared to control and other treatments, respectively), along with substantial enhancements in average fruit weight (24.2 g and 17.6 g for the respective comparisons). The obtained data suggest that the application of the new biostimulant “White Pearl” NPC had a complex effect on the water regime of apple trees of the Sinap Orlovsky apple cultivar. This effect was manifested in the improvement of the water-holding capacity of apple leaves and fruits, potentially increasing resistance to stresses. Before harvesting, the proportion of free water in apple fruits decreased and the content of bound water increased, especially when treated with biostimulants, indicating active apple maturation. Changes in apple leaves were due to natural aging and the redistribution of organic substances. Other researchers also report an increase in apple yield when using the “Mival-Agro” biostimulator. This preparation is a silicon–organic biostimulator and exhibits cryoprotector and adaptogen properties [42]. Three-fold foliar top dressing with the organo-mineral fertilizer “EvricorForte+7” (at a rate of 1.5 L/ha) contributed to an increase in the content of ascorbic acid in strawberries; specifically, the levels of ascorbic acid were 61.8 mg/100 g in Anastasiya (an increase of 3.5% compared to the control) and 60.2 mg/100 g in Orletz (an increase of 6.1%) strawberries [43]. The yield of raspberry berries increased by 14.4% in the variant with four foliar sprays of “Humate+7” [36]. The organo-mineral fertilizer “Naliv”, based on BioHumate from horse manure and vegetable raw materials (humic and fulvic acids and amino acids, including proline), increased the content of monosaccharides in ripening apples by 1.8 times and the sucrose content by 10% [44]. Research has demonstrated that biostimulants derived from alfalfa protein hydrolysate, marine algae extracts, and B-complex vitamins significantly enhance both the visual and qualitative characteristics of apple fruits. Furthermore, zinc-containing biostimulants have proven particularly effective in mitigating physiological storage disorders [45], while simultaneously improving fruit coloration and flavor profiles. Foliar sprays with preparations “Calcium 44 LG”, “Alga Ca”, and “Algical” have been able to reduce the loss of Renet Kubansky apples from bitter pitting during storage. The composition of “Calcium 44 LG” includes humic acids and calcium in chelated forms. “Alga Ca” and “Algical” are organo-mineral fertilizers prepared from the extract of Laminaria digitate seaweed and have a high calcium content [46].

These findings collectively suggest that incorporating biostimulants into horticulture holds considerable promise, particularly when cultivating in marginal or challenging agricultural conditions.

5. Conclusions

The obtained data indicate the ability of bio-preparations to induce protective mechanisms and preserve the reproductive organs of apple varieties under temperature stress, as well as to alter donor–acceptor relationships between the leaf apparatus and the fruit towards the forming apple, promoting a better outflow of assimilates into maturing fruits.

Based on our experimental findings, we propose the following optimized application schedule using 1% solutions of both NPC “White Pearl Universal” Antifreeze and “White Pearl Drip Ca + Mg” to enhance frost resistance and yield in apple cultivation:

“Dormant bud–silver cone”;
“Mouse ear”;
“Inflorescence emergence”;
14 days post-bloom;
“Fruit-hazel”;
“Fruit-walnut”;
25–30 days before harvesting.

Author Contributions

Z.E.O.—formal analysis, investigation, resources, project administration, writing—original draft, and writing—review and editing; P.S.P.—resources, data curation, and visualization; A.Y.S.—resources; A.O.B.—resources. All authors have read and agreed to the published version of the manuscript.

Funding

The research was carried out at the expense of a grant from the Russian Science Foundation No. 24-26-00041, https://rscf.ru/project/24-26-00041/ (accessed on 30 March 2025).

Data Availability Statement

The original data presented in the study are openly available in database of apple cultivar—VNIISPK, https://vniispk.ru/varieties/sinap-orlovskii (accessed on 3 September 2025).

Acknowledgments

The authors express their gratitude to AgroPlus Group of Companies LLC for providing the adaptogenic preparations used in this research at no cost.

Conflicts of Interest

The authors declare no conflicts of interest.

References

Sedov, E.N. Apple breeding history, goals, methods and results. Agric. Biol. 2007, 1, 3–15. [Google Scholar]
Levitan, L.; Merwin, I.; Kovach, J. Assessing the relative environmental impacts of agricultural pesticides: The quest for a holistic method. Agric. Ecosyst. Environ. 1995, 55, 153–168. [Google Scholar] [CrossRef]
Ju, X.T.; Kou, C.L.; Zhang, F.S.; Christie, P. Nitrogen balance and groundwater nitrate contamination: Comparison among three intensive cropping systems on the North China Plain. Environ. Pollut. 2006, 143, 117–125. [Google Scholar] [CrossRef]
Poulsen, M.E.; Naef, A.; Gasser, S.; Christen, D.; Rasmussen, P.H. Influence of different disease control pesticide strategies on multiple pesticide residue levels in apple. J. Hortic. Sci. Biotechnol. 2009, 84, 58–61. [Google Scholar] [CrossRef][Green Version]
Pathak, H.; Nedwell, D.B. Nitrous oxide emission from soil with different fertilizer, water levels and nitrification inhibitors. Water Air Soil Pollut. 2011, 129, 217–228. [Google Scholar] [CrossRef]
Damos, P.; Colomar, L.A.; Ioriatti, C. Integrated fruit production and pest management in Europe: The apple case study and how far are from the original concept? Insects 2015, 6, 626–657. [Google Scholar] [CrossRef]
Khan, A.A.; Wang, Y.F.; Zeb, A.; Hayat, K.; Alhoqail, W.A.; Soliman, M.H. Beneficial elements and their roles against soil pollution. In Beneficial Elements for Remediation of Heavy Metals in Polluted Soil; Elsevier: Amsterdam, The Netherlands, 2025; pp. 1–32. [Google Scholar]
Ryabchinskaya, T.A.; Kharchenko, G.L. Ecologized strategy of fruit and berry crop protection. Plant Prot. Quar. 2008, 7, 10–12. [Google Scholar]
Kochkina, A.M.; Kashirskaya, N.Y. Efficiency of apple tree’s protection systems against scab. Pomic. Small Fruit Cult. Russ. 2015, 43, 286–289. [Google Scholar]
Holb, I.J.; Dremák, P.; Bitskey, K.; Gonda, I. Yield response pest damages and fruit quality parameters of scab-resistance an scab-susceptible apple cultivars in integrated and organic production systems. Sci. Hortic. 2012, 145, 109–117. [Google Scholar]
Valiušaitė, A.; Uselis, N.; Kviklis, D.; Lanauskas, J.; Rasiekevičiūtė, N. The effect of sustainable plant protection and apple tree management on fruit quality and yield. Zemdirbiste-Agric. 2017, 104, 353–358. [Google Scholar] [CrossRef]
Palansooriya, K.N.; Shaheen, S.M.; Chen, S.S.; Tsang, D.C.; Hashimoto, Y.; Hou, D.; Bolan, S.; Rinklebe, J.; Ok, Y.S. Soil amendments for immobilization of potentially toxic elements in contaminated soils: A critical review. Environ. Int. 2020, 134, 105046. [Google Scholar] [CrossRef]
Sergeeva, N.N. Efficiency of special fertilizers in the compositions of tank mixtures with the means of protection of plants in fruit orchard. Fruit Grow. Vitic. South Russ. 2018, 49, 65–75. [Google Scholar]
Rai, N.; Rai, S.P.; Sarma, B.K. Prospects for abiotic stress tolerance in crops utilizing phyto- and bio-stimulants. Front. Sustain. Food Syst. 2021, 5, 754853. [Google Scholar] [CrossRef]
Van Oosten, M.J.; Pepe, O.; De Pascale, S.; Silletti, S.; Maggio, A. The role of biostimulants and bioeffectors as alleviators of abiotic stress in crop plants. Chem. Biol. Technol. Agric. 2017, 4, 5. [Google Scholar] [CrossRef]
Zaid, A.; Mohammad, F.; Fariduddin, Q. Plant growth regulators improve growth, photosynthesis, mineral nutrient and antioxidant system under cadmium stress in menthol mint (Mentha arvensis L.). Physiol. Mol. Biol. Plants 2020, 26, 25–39. [Google Scholar] [CrossRef]
Nephali, L.; Piater, L.A.; Dubery, I.A.; Patterson, V.; Huyser, J.; Burgess, K.; Tugizimana, F. Biostimulants for plant growth and mitigation of abiotic stresses: A metabolomics perspective. Metabolites 2020, 10, 505. [Google Scholar] [CrossRef] [PubMed]
Khan, A.A.; Wang, Y.-F.; Akbar, R.; Alhoqail, W.A. Mechanistic insights and future perspectives of drought stress management in staple crops. Front. Plant Sci. 2025, 16, 1547452. [Google Scholar] [CrossRef] [PubMed]
Malaguti, D.; Rombolà, A.D.; Gerin, M.; Simoni, G.; Tagliavini, M.; Marangoni, B. Effect of Seaweed Extracts-Based Leaf Sprays on the Mineral Status, Yield and Fruit Quality of Apple. Acta Hortic. 2002, 594, 357–359. [Google Scholar] [CrossRef]
Spinelli, F.; Fiori, G.; Noferiny, M.; Sprocatty, M.; Costa, G. Perspectives on the use of a seaweed extract to moderate the negative effects of alternate bearing in apple trees. J. Hortic. Sci. Biotechnol. 2009, 84, 131–137. [Google Scholar] [CrossRef]
Yaroshenko, O.V.; Sergeyeva, N.N.; Gaponenko, A.V. Organo-mineral foliar feeding in the technology of growing apple trees in the south of Russia. Subtrop. Ornam. Hortic. 2019, 70, 133–142. [Google Scholar] [CrossRef]
Golovunin, V.P. Influence of humic growth stimulator and mineral fertilizer on the vegetative development of blue honeksuckle. Contemp. Hortic. 2023, 4, 138–144. Available online: https://old.journal-vniispk.ru/pdf/2023/4/37.pdf (accessed on 18 August 2025).
Gudkovskii, V.A.; Kozhina, L.V.; Balakirev, A.E.; Nazarov, Y.B.; Kuzin, A.I. Promising technology to control bitter pit and other postharvest pathologic diseases. Acta Hortic. 2021, 1325, 151–158. [Google Scholar] [CrossRef]
Sedov, E.N.; Sedysheva, G.A.; Krasova, N.G.; Serova, Z.M.; Gorbacheva, N.G.; Galasheva, A.M.; Yanchuk, T.V.; Pikunova, A.V.; Van de Veg, E. Origin, economical and cytoembryological characteristics of triploid apple cultivar ‘Sinap Orlovsky’. Russ. Agric. Sci. 2017, 1, 14–18. [Google Scholar] [CrossRef]
Ozherelieva, Z.Е.; Prudnikov, P.S.; Zubkova, М.I.; Krivushinaа, D.А.; Knyazev, S.D. Determination of Frost Resistance of Straw-berries in Controlled Conditions; VNIISPK: Orel, Russia, 2019. [Google Scholar]
Turkina, М.V.; Sokolova, S.V. Study of membrane transport of sucrose in plant tissue. Plant Physiol. 1972, 1, 912–919. [Google Scholar]
Kabashnikova, L.F.; Kalitukho, L.N.; Derevensky, A.V. Quantitative Analysis of Free and Bound Carbohydrates in a Single Sample of Plant Tissue; BSPU: Minsk, Belarus, 2003. [Google Scholar]
Bates, L.S.; Waldren, R.P.; Teare, I.D. Rapid determination of free proline for water-stress studies. Plant Soil 1973, 39, 205–207. [Google Scholar] [CrossRef]
Prudnikov, P.S.; Ozherelieva, Z.Е. Physiological and Biochemical Methods for Diagnosing the Resistance of Fruit Crops to Drought and Hyperthermia; VNIISPK: Orel, Russia, 2019. [Google Scholar]
Sedov, Е.N.; Ogoltsova, T.P. (Eds.) Program and Methodology of Fruit, Berry and Nut Variety Study; VNIISPK: Orel, Russia, 1999. [Google Scholar]
Ozherelieva, Z.E.; Prudnikov, P.S. Impact of the B-Plus White Pearl (Belyi Zhemchug) reparation on the spring frost tolerance, yield and quality of apple cross. Hortic. Vitic. 2022, 6, 24–32. [Google Scholar] [CrossRef]
Ozherelieva, Z.; Prudnikov, P.; Nikitin, A.; Androsova, A.; Bolgova, A.; Stupina, A.; Vetrova, O. Adaptogenic Preparations Enhance the Tolerance to Spring Frosts, Yield and Quality of Apple Fruits. Horticulturae 2023, 9, 591. [Google Scholar] [CrossRef]
Ali, O.; Ramsubhag, A.; Jayaraman, J. Biostimulant properties of seaweed extracts in plants: Implications towards sustainable crop production. Plants 2021, 10, 531. [Google Scholar] [CrossRef] [PubMed]
Rouphael, Y.; Colla, G. Editorial: Biostimulants in Agriculture. Front. Plant Sci. 2020, 11, 40. [Google Scholar] [CrossRef]
Sarma, R.K.; Saikia, R. Alleviation of drought stress in mung bean by strain Pseudomonas aeruginosa GGRJ21. Plant Soil 2014, 377, 111–126. [Google Scholar] [CrossRef]
Rezvyakova, S.V.; Rezvyakova, E.S. Evaluation of the effects of growth stimulants for improving winter hardiness and yield of raspberry. Bull. Agrar. Sci. 2017, 5, 3–11. [Google Scholar] [CrossRef]
Popova, V.P.; Sergeeva, N.N.; Fomenko, Т.G.; Yaroshenko, О.V.; Nenko, N.I. Principles of increasing the resistance of garden cenoses to stress factors and changes in the level of soil fertility. Sci. Work. North Cauc. Fed. Sci. Cent. Hortic. Vitic. Winemak. 2019, 23, 89–99. [Google Scholar] [CrossRef]
Frioni, T.; VanderWeide, J.; Palliotti, A.; Tombesi, S.; Poni, S.; Sabbatini, P. Foliar vs. soil application of Ascophyllum nodosum extracts to improve grapevine water stress tolerance. Sci. Hortic. 2021, 277, 109807. [Google Scholar] [CrossRef]
Souri, M.K.; Hatamian, M. Aminochelates in plant nutrition: A review. J. Plant Nutr. 2019, 42, 67–78. [Google Scholar] [CrossRef]
Fiorentino, N.; Ventorino, V.; Woo, S.L.; Pepe, O.; De Rosa, A.; Gioia, L.; Romano, I.; Lombardi, N.; Napolitano, M.; Colla, G.; et al. Trichoderma-based biostimulants modulate rhizosphere microbial populations and improve N uptake efficiency, yield, and nutritional quality of leafy vegetables. Front. Plant Sci. 2018, 9, 743. [Google Scholar] [CrossRef]
Luziatelli, F.; Ficca, A.G.; Colla, G.; Baldassarre Švecová, E.; Ruzzi, M. Foliar application of vegetal-derived bioactive compounds stimulates the growth of beneficial bacteria and enhances microbiome biodiversity in lettuce. Front. Plant Sci. 2019, 10, 60. [Google Scholar] [CrossRef]
Kalmykova, О.V. The effectiveness of using biological products in an apple orchard in the conditions of the Lower Volga region. Bull. Altai State Agrar. Univ. 2014, 4, 20–23. [Google Scholar]
Mushinsky, A.A.; Aminova, E.V.; Avdeeva, Z.A.; Borisova, A.A.; Tumaeva, T.A. The effect of organic fertilizer on the productivity and quality of strawberries. Pomic. Small Fruits Cult. Russ. 2019, 59, 335–342. [Google Scholar] [CrossRef]
Doroshenko, T.; Ryazanova, L.; Petrik, G.; Gorbunov, I.; Chumakov, S. Features of the economical yield formation of apple plants under non-root nutrition in the Southern Russia organic plantings. In Proceedings of the BIO Web of Conferences BIOLOGIZATION, Krasnodar, Russia, 21–23 September 2021; Volume 34, p. 05004. [Google Scholar] [CrossRef]
Soppelsa, S.; Kelderer, M.; Casera, C.; Bassi, M.; Robatscher, P.; Andreotti, C. Use of Biostimulants for Organic Apple Produc-tion: Effects on Tree Growth, Yield, and Fruit Quality at Harvest and During Storage. Front. Plant Sci. 2018, 9, 1342. [Google Scholar] [CrossRef]
Prichko, T.G.; Smelik, T.L. Assessment of efficiency of new preparations contained calcium in the fight against of apple bitter pits. Sci. Publ. FSBSO NCRRIHV 2015, 7, 143–146. [Google Scholar]

Figure 1. Degree of frost damage to flowers (a) and buds (b) of the Sinap Orlovsky apple cultivar under controlled conditions, %. Distinct lowercase letters indicate statistically significant differences (p ≤ 0.05, LSD test).

Table 1. The soil profile characteristics across experimental plots.

Depth, cm	pH_KCl	Humus, %	H_a, cmol(+) kg⁻¹	Content
				P₂O₅	K₂O	Ca²⁺	Mg²⁺
				mg/kg		mmol/100 g
0…20	5.06	4.61	3.46	177.12	75.68	14.98	4.39
20…40	5.04	3.81	3.37	129.18	58.00	15.59	4.58
40…60	5.13	2.78	2.76	128.48	56.12	14.76	4.77

Table 2. Meteorological conditions during the 30-day pre-harvest period for Sinap Orlovsky apple cultivars (2021–2024 growing seasons).

Year	Average Daily Temperatures Sum ≥ 10 s °C	Precipitation Amount, mm	HTC
Year	30 Days Before Harvest
2021	458.5	33.7	0.74
2022	473.1	19.3	0.41
2023	506.6	28.1	0.55
2024	571.5	12.7	0.22

Table 3. Tissue hydration of annual shoots and flower buds of the Sinap Orlovsky apple cultivar in the spring period, %.

Option	Development Phases
Option	“Dormant Bud–Silver Cone”	“Mouse Ear”	“Inflorescence Emergence”
Annual shoots
Control (without treatment)	45.02 ± 0.47 ^a	53.12 ± 0.11 ^c	48.98 ± 0.38 ^a
1% NPC “WPU” Antifreeze + 1% NPC “WP Drip Ca + Mg”	49.10 ± 1.39 ^c	50.18 ± 1.11 ^b	50.02 ± 1.45 ^b
3% NPC “WPU” Antifreeze + 3% NPC “WP Drip Ca + Mg”	48.55 ± 1.30 ^b	48.55 ± 1.09 ^a	48.33 ± 0.48 ^a
LSD₀₅	2.00	2.74	F_f < F_t
Flower buds
Control (without treatment)	76.07 ± 0.35 ^ab	81.47 ± 0.37 ^ab	83.13 ± 0.12 ^a
1% NPC “WPU” Antifreeze + 1% NPC “WP Drip Ca + Mg”	75.43 ± 0.23 ^a	81.05 ± 1.09 ^ab	84.82 ± 0.59 ^b
3% NPC “WPU” Antifreeze + 3% NPC “WP Drip Ca + Mg”	77.30 ± 2.52 ^b	80.83 ± 0.96 ^a	83.15 ± 0.59 ^a
LSD₀₅	F_f < F_t	F_f < F_t	1.08