Impact of Ozonated Water on the Fungal Colonies, Diversity and Fruit Quality of Grapevine in Northern Europe

Abstract

Due to the frequent use of fungicides in viticulture, resistant plant pathogens have emerged, necessitating environmentally friendly alternatives. This research aimed to determine the effect of ozonated water (OW) spraying on fungal colonies present on grapevine leaves and berries, as well as on the biochemical composition of the berries. ‘Regent’ grapevines were grown in a high plastic tunnel and sprayed with OW from post-flowering to harvest. The fungal population on the phyllosphere of grapevine leaves and berries was evaluated using the serial dilution plating method. The taxonomic composition of the predominant fungal colonies was characterized using internal transcribed spacer amplicon sequencing. OW treatment significantly decreased fungal colonies on grapes but had no significant effect on grapevine leaves. The fungal colonies were dominated by Botrytis cinerea, Penicillium brevicompactum, and Fusarium sp. OW treatment significantly reduced the total sugar content in grapes (from 160 to 154 g L⁻¹) and increased the total acid content (from 7.2 to 8.6 g L⁻¹). The fruit polyphenol content increased from 431 to 508 mg 100 g⁻¹, and antioxidant activity was significantly enhanced. It can be concluded that OW treatment is effective in reducing fungal colony forming units on grapes in vineyards. OW treatment affected the sugar, acid, and polyphenol content in grapes, but not to a degree that would present specific challenges for winemakers.

1. Introduction

Viticulture is an important part of the agricultural sector. According to the International Organization of Vine and Wine, the world’s vineyard surface area in 2023 was 7.2 million hectares [1], the largest producers being China, Italy and France [2]. Climate change impacts grape yield and quality through rising temperatures and changes in pathogen geographical distribution [3]. Addressing these challenges may include alternative cultivars or changing the geographic location.

Estonia lies in the northern part of the wine industry world. Estonia and Lithuania were added to the coolest northern viticulture zone A of the European Union in December 2021, thus officially acknowledged as wine-producing countries [4]. Estonian viticulture and the wine industry are in the initial development phase, and grape growers are looking for sustainable growing technologies. Grapevines are highly vulnerable to pathogens, which can lead to a significant reduction in yield as well as product quality [5]. Traditionally, viticulture has relied heavily on fungicides to combat Botrytis cinerea. This approach has unintentionally fuelled the rise of resistant plant pathogens, diminishing the efficacy of chemical interventions [6]. Fungicides can also affect wine quality: Song et al. [7] showed that at the maximum residue limit, hexaconazole, difenoconazole, flutriafol, tebuconazole, and propiconazole significantly inhibited the growth of Saccharomyces cerevisiae during winemaking, changing the fermentation profile and metabolites in comparison to the control. Zhao et al. [8] reported that tebuconazole had a negative impact on the fruity and floral characteristics of wines and also altered wine color.

Pesticides used in viticulture have a significant impact on the environment. Copper-based fungicides (such as the Bordeaux mixture, (CuSO₄+Ca(OH)₂) have been intensively used in Europe since the end of the 19th century to control fungal diseases in vines [9]. Copper applied to eroded vineyard soils can easily reach ground and surface waters [10], where it can be toxic to aquatic organisms [11]. Besides Cu, residues of other fungicides have been found in vineyard soils. Bermúdez-Couso et al. [12] found concentrations of fludioxonil and cyprodinil (used for Botrytis rot control) in vineyard soils reaching 349 and 462 μg kg⁻¹, respectively. To mitigate further environmental contamination and minimize harmful effects on ecosystems, it is essential to focus on environmentally friendly alternatives to synthetic pesticides.

Ozone is a natural substance in the atmosphere and one of the most potent sanitizers against various microorganisms [13,14]. The product of ozone degradation is oxygen, so it leaves no residues on treated commodities. One of the key attributes of ozone is its potency in both air and water. The postharvest fumigation of grapes with ozone has been effective against gray mold [15,16]. There are some reports demonstrating that ozonated water (OW) can be used in Vitis vinifera plants. Pierron et al. [17] reported that OW completely suppressed spore germination of the esca-associated fungus Phaeoacremonium aleophilum in vitro, and at 9 weeks post-inoculation, fungal development was significantly reduced in planta. To our knowledge, the effect of OW during growth on fungal diversity and grape quality in northern countries in high polytunnels has not been studied. Colony forming units (CFUs) of fungi are a critical metric in microbiology, representing the number of viable fungal cells capable of forming colonies. The CFU assay has remained the gold standard for measuring viability across disciplines [18,19].

The hypothesis of the present work was that frequent OW treatment reduces the amount of CFUs in grapevines, and since ozone is a powerful oxidizer, it has an enhancing effect on the antioxidant activity of berries. The aim of this research was to determine the effect of OW spraying on the fungal CFUs on grapevine cultivar ‘Regent’ leaves and grapes, as well as on the biochemical compound content in the berries.

2. Materials and Methods

2.1. Experimental Site and Treatments

Vines of the cultivar ‘Regent’ were grown in a commercial vineyard in a high plastic tunnel in South Estonia (57°58′58.6″ N 26°34′04.2″ E). Vitis vinifera ‘Regent’ is a noir grapevine cultivar developed through the crossbreeding of the cultivar ‘Diana’ and ‘Chambourcin’ in Germany [20]. The cultivar exhibits resistance to powdery mildew (Erysiphe necator) and downy mildew (Plasmopara viticola). The tunnel, which had no foundation, was 28 m in length, 7.6 m in width, and 4.6 m in height, and was covered with 0.18 mm thick UV-stable low-density polyethylene. The vineyard was established in 2013 using vines grafted onto SO4 rootstock. Vines were planted with a 1.5 m × 2 m spacing and trained on low double-trunk trellises. The vine rows were oriented from north to south, which is a common practice in viticulture to ensure uniform sunlight exposure throughout the day as the sun moves from east to west. The experimental site was characterized by a high-productivity sandy loam soil classified as Haplic Luvisol, with adequate drainage. The 2022 growing season (April–October) was slightly cooler and markedly drier compared to the 1991–2020 climatic baseline. The mean temperature during this period was 11.7 °C, which is 0.5 °C lower than the long-term average of 12.2 °C. Total precipitation amounted to 333 mm, representing a 27% decrease from the long-term seasonal mean of 456 mm. April (4.4 °C vs. the average 5.9 °C) and May (10.3 °C vs. 11.5 °C) were colder than average, while June was unusually warm (17.3 °C vs. the average 15.5 °C) but dry (50 mm vs. 88 mm). September was particularly cool (9.3 °C vs. 11.8 °C). The driest months were June, August (52 mm vs. 79 mm), and September (34 mm vs. 55 mm). The ground was covered with woven ground cover fabric, and no fertilizers or additional irrigation systems were used. Lateral shoots were consistently pruned back to two leaves throughout the growing season. At the beginning of berry coloration (BBCH81), leaves were removed from the cluster zone.

The trial was conducted with two treatments: a control (20 vines), where no spraying was performed (including water, as it could have promoted disease development in the tunnel), and a treatment with OW (20 vines). The experiment was conducted using a block design. Randomization was not applied to prevent the transfer of ozonated water to control plants. Spraying with OW started after grapevine flowering (BBCH71), during the berry formation stage. The entire plants were treated weekly from 6 July 2022, until harvest, for a total of 12 applications. Both leaves and grapes were sprayed. An ozone generator, model EOD Medi (EOD OY, Finland), was used. The submersible ozonator produces OW from tap water at a concentration of up to 2 ppm, and the concentration was monitored during each spraying.

2.2. Assessment of Fungal Colony Forming Units and Species Identification

Grapevine leaves were collected twice: randomly and aseptically at the onset of berry coloration (BBCH 81) on 19 July and again at harvest time (BBCH 89) on September 29, from the middle part of the shoots. At each sampling date, we randomly selected only mature leaves, avoiding very young apical leaves and the oldest basal leaves. Undamaged grapes were collected only once, at harvest. Samples of grapes (20 berries randomly selected from approximately 10 clusters) and leaves (40 discs of 10 mm diameter, cut with a sterile cork borer from approximately 15 leaves per treatment) were processed under sterile conditions. The samples were diluted in a sterile saline solution (0.9% w/v NaCl), and serial dilutions ranging from 10⁻¹ to 10⁻⁴ were prepared for each sample to ensure accurate colony counting, as described in Perazzolli et al. [21].

Aliquots (0.1 mL) of each dilution were then enumerated using the gold-standard spread plate method by spreading with an automatic pipette onto PDA (potato dextrose agar) (Biolife, Milan, Italy) plates supplemented with ampicillin (25 μg mL⁻¹) to inhibit bacterial growth. Each treatment was performed in triplicate. The plates were incubated at 24 °C for 72 h, after which colonies were counted and expressed as CFU mL⁻¹. For statistical analyses, only plates with ≤100 standardized colonies were used.

After the initial incubation of leaf and berry samples collected at harvest time, the most prevalent fungal colonies—those exhibiting distinct morphology and the highest frequency—were selected for species detection. These colonies were carefully re-cultured onto new, sterile PDA plates. The subcultured colonies were then incubated under the same conditions (24 °C for 72 h) to obtain pure cultures. Following incubation, the mycelia from the pure cultures were subjected to molecular analysis for species identification.

DNA from 0.2 g of fungal cultures was extracted using the GeneJET genomic DNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. Fungal samples were amplified using the ITS1F+ITS4 primer pair for fungi [22,23]. PCR was performed in a 25 μL volume comprising 0.5 μL of each primer (20 μM), 5 μL HOT FIREPol blend master mix Ready to Load (Solis Biodyne), 1 μL DNA extract, and 18 μL double-distilled H₂O. Thermocycling conditions included the initial 15 min denaturation at 95 °C, followed by 35 cycles of 15 s of denaturation, 30 s of annealing at 55 °C, and 60 s of elongation at 72 °C, with a final 10 min elongation before a hold at 4 °C.

The electrophoresis of PCR products was carried out at 75 V for 55 min using 1% agarose (SeaKem^® LE Agarose; Lonza, Rockland, ME, USA) gels. A Solis Biodyne 100 bp DNA Ladder (Solis Biodyne, Tartu, Estonia) was loaded in the first and last lanes as molecular-size markers. PCR products were visualized under UV light using Uvidoc gel documentation system (Cambridge, UK). PCR products were sequenced at the Estonian Biocentre in Tartu using the ITS5 primer [22]. The sequences were edited using BioEdit version 7.2.5 [24]. BLAST searches for fungal taxon confirmation were performed at GenBank (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 20 March 2023).

All analyzed isolates are deposited in the Tartu Fungal Collection subcollection (TFC-FP) at the Estonian University of Life Sciences, Estonia. Sanger sequences of subcultured colonies were deposited in the UNITE database (https://unite.ut.ee/; accessed on 10 February 2025).

2.3. Chemical Analyses of Grapes

For the measurement of total sugars, fructose and glucose, and total acids, malic and tartaric acids, the frozen (−20 °C) grape samples were thawed at room temperature until manual juice pressing was possible. The obtained juice was transferred to a 5 mL syringe. Juice samples were injected into the flow system and scanned using a horizontal platinum diamond attenuated total reflectance (ATR) single-reflection sampling module cell, mounted in a Bruker Alpha FT-IR Wine analyzer (Bruker Optics GmbH, Ettlingen, Germany). Before scanning, juice samples were automatically stabilized in the cell at 40 °C, as recommended by the manufacturer, with a reference background spectrum recorded between different samples using deionized water. The ATR-NIR spectra were recorded by OPUS software version 7.5 (Bruker Optics GmbH, Ettlingen, Germany). The spectrum of each sample was obtained by taking an average of 120 scans. The accuracy of prediction for malic acid was ±0.6 g L⁻¹ and for tartaric acid ±0.3 g L⁻¹. The accuracy of prediction for glucose was ±0.9 g L⁻¹, for fructose ±0.8 g L⁻¹, and for total sugars ±1.7 g L⁻¹. Spectra were recorded over the 4000–400 cm⁻¹ region, and diffuse reflectance measurements were obtained. This analytical setup has previously demonstrated reliability and effectiveness in the analysis of grape and pomegranate juice quality parameters [25,26].

The total polyphenol content of the berry samples was determined in three replicates by the Folin–Ciocalteu phenol reagent method [27]. The results are expressed as mg of gallic acid equivalent (GAE) per 100 g of fresh skins weight (mg 100 mg⁻¹ GAE). The absorbance was measured after 2 h at 765 nm. The antioxidant activity was determined by applying the DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging method [28] and expressed as percentage (%) DPPH radical scavenging activity. The absorbance was measured after 60 min at 515 nm. Spectrophotometric measurements were made with a Cary 60 UV-Vic spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA, USA).

2.4. Statistical Analyses

CFU values were log-transformed before statistical analyses to scale the data more evenly. For CFU analyses, the Bonferroni correction was applied (Statistica 12.0, Palo Alto, CA, USA). A one-way analysis of variance (ANOVA) was applied to evaluate the effect of ozonated water treatment (independent variable) on the chemical composition of grapes, with sugars, acids, polyphenol content, and antioxidant activity as dependent variables (Statistica 12.0, Palo Alto, CA, USA). Mean comparisons were conducted using the post hoc Fisher’s least-significant difference (LSD) test to confirm statistically significant differences between treatments. The results in the figures are presented as means of three replications and standard deviation. Different letters and asterisks in the figures indicate statistically significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results

3.1. Fungal Colonies on the Grapes and Grape Leaf Surface

The analysis of CFU under different treatment conditions revealed significant variations. For leaf samples at the fruit set stage, the mean log CFU value was significantly higher in the control treatment (3.688 ± 0.075) compared to the OW treatment (3.343 ± 0.075) (Bonferroni correction, p = 0.03; Figure 1). However, no significant differences were observed among pairwise comparisons during the harvest (Figure 1).

OW treatment significantly affected the total number of CFUs on grapes. For berry samples, the mean log CFU value was significantly lower in the OW treatment (3.111 ± 0.069) compared to the control treatment (3.729 ± 0.069) (Bonferroni correction, p = 0.003; Figure 1).

The analysis of CFU plates revealed that the most prevalent re-cultured isolates were identified as Fusarium sp., Penicillium brevicompactum, and Botrytis cinerea (

Table 1. Fungal species detected from re-cultured isolates.

Sample Code	Fungal Collection Code in TFC-FP 1	BLAST Best Hit	Sample Origin
FP285-1	101374	Botrytis cinerea	Grape
FP284-1	101375	Penicillium brevicompactum	Grape
FP278-1	101376	Fusarium sp.	Grape
FP221-2	101377	Cladosporium cladosporioides	Grape
FP222-1	101378	Botrytis cinerea	Leaf
FP226-1	101379	Botrytis cinerea	Leaf
FP277-1	101380	Penicillium brevicompactum	Grape
FP316-1	101381	Botrytis cinerea	Leaf
FP319-1	101382	Gibellulopsis nigrescens	Leaf
FP324-1	101383	Cladosporium cladosporioides	Grape

¹ All analyzed isolates are deposited in the Tartu Fungal Collection subcollection (TFC-FP) at the Estonian University of Life Sciences, Estonia. Sanger sequences of sub-cultured colonies were deposited in the UNITE database (https://unite.ut.ee/).

). These fungal species were consistently identified across multiple samples (both leaves and grapes), indicating their dominance in the studied environment. Notably, their prevalence was observed independently of whether the samples were subjected to OW treatment or maintained as controls, suggesting an adaptability to the conditions present in both treatments.

3.2. Grape Chemical Parameters

The results indicated that OW treatment influenced the sugar profile of grapes (Figure 2). In particular, a statistically significant reduction was observed in sugar concentrations, with fructose levels decreasing from 84 to 80 g L⁻¹ (p < 0.01), glucose levels from 75 to 72 g L⁻¹ (p < 0.05), and total sugar content from 160 to 154 g L⁻¹ (p < 0.01).

However, the effect of OW on acid content followed the opposite direction: OW treatment significantly increased the acid content in grapes (Figure 3). Malic acid levels rose from 2.8 to 4.3 g L⁻¹ (p < 0.01), while tartaric acid increased from 3.2 to 4.0 g L⁻¹ (p < 0.05). Consequently, the total acid content increased from 7.2 to 8.6 g L⁻¹ (p < 0.01), with all observed differences being statistically significant.

The total polyphenol content of control grapes was 431 mg 100 g⁻¹ GAE, and of OW-treated grapes, 508 mg 100 g⁻¹ GAE, with the effect being statistically significant (p < 0.01) (Figure 4a). A significant increase was also observed in antioxidant activity, which increased from 91 to 95% (p < 0.05) (Figure 4b).

4. Discussion

4.1. Fungal Colonies on the Grapes and Grapevine Leaf Surface

The present study demonstrated that OW treatment significantly influenced fungal CFUs on both grapevine leaves and berries, though its effects varied across different growth stages. At the onset of berry coloration (BBCH 81), OW treatment reduced fungal CFUs on leaves compared to the control. This suggests that OW had an inhibitory effect on early fungal colonization, potentially by disrupting fungal spores or altering the phyllosphere microbiota. Roy et al. [29] indicated that the primary mode of fungal spore inactivation by ozone appears to be nucleic acid damage. However, by the harvest stage, no significant differences were observed between treatments, suggesting that when fungal loads are larger, OW may not be sufficient to significantly reduce colony numbers, as demonstrated by Gao et al. [30], and Tanuwidjaja and Fuka [19]. One of the reasons could be the difference in the density of the plant canopy: during the initial stages of berry coloration, the canopy is sparser; later, the denser canopy influences the moisture conditions within the canopy, thereby facilitating the spread of fungi. Sinfort et al. [31] proved that the level of pesticide losses during spraying is among other factors, dependent on the stage of the development of the vegetation.

The absence of significant differences in leaf CFU counts at harvest suggests that additional factors, such as environmental conditions may influence the antimicrobial effect of ozone. The half-life of ozone is known to vary from seconds to up to almost an hour, depending on environmental conditions and dissolved organic compounds [32]. A more pronounced effect of OW was observed on the berries, where a significant reduction in fungal CFUs was recorded at harvest. This can be explained by the fact that leaves surrounding the fruit had been removed before berry coloration, which allowed for better contact between the berries and the OW. This finding is consistent with previous studies highlighting the antifungal properties of ozone, particularly its effectiveness in reducing postharvest fungal contamination [16]. The observed decrease in fungal CFUs on berries suggests that OW may help reduce fungal infections and microbial loads that negatively impact fruit and wine quality. The underlying mechanism for this reduction likely involves the oxidative properties of ozone, which can damage fungal cell membranes and inhibit spore germination.

The persistence of Botrytis cinerea, Fusarium sp., Penicillium brevicompactum, and Cladosporium cladosporioides identified across leaf and berry samples, irrespective of treatment conditions, underscores their ecological resilience and adaptability. Notably, their ability to persist under the challenging climatic conditions at the northern edge of viticulture has not been previously reported. The Fusarium species are widely recognized for their pathogenicity in plants, causing devastating diseases such as wilts and rots, which can significantly impact agricultural productivity. In viticulture, several Fusarium species have been identified as pathogens affecting grapevines. For instance, Fusarium oxysporum has been associated with root rot in grapevines, leading to symptoms like leaf yellowing and plant decline [33]. Similarly, Penicillium sp. encompasses a diverse group of fungi known for its saprotrophic nature and possible mycotoxin synthesis that contribute to food spoilage and potential health risks [34,35]. Botrytis cinerea, a notorious phytopathogen, is responsible for gray mold disease, a major concern in agriculture due to its ability to infect a broad range of crops and cause severe postharvest losses [36]. One of the examples of the adaptability of Botrytis cinerea to chemical stressors is its ability to develop fungicide resistance. The resistance of B. cinerea to succinate dehydrogenase inhibitors (SDHIs, e.g., boscalid) and quinone outside inhibitors (QoIs, e.g., pyraclostrobin) has been reported in several countries [37,38,39]. In the future, it will be important to specifically investigate the effects of OW treatment on the prevalent fungal contaminants, including pathogenic fungi, to better understand its potential as a targeted disease management tool.

‘Regent’ is a wine grape cultivar, making the impact of berry-associated fungal species particularly relevant for wine quality. Botrytis cinerea infection has been shown to retard alcoholic fermentation and impart earthy or musty odors to wines [40,41]. The Fusarium species may inhibit the synthesis of volatile compounds important for wine aroma [42]. Common grape-associated fungi such as Cladosporium, Aspergillus, and Penicillium contribute to grape rots and mycotoxin production. Although they do not grow in wine, their impact results primarily from grape damage [43]. In this study, Aspergillus spp. was not detected on ‘Regent’ berries, aligning with their preference for warmer climates, whereas Penicillium spp., more common in cooler regions like northern Europe, was found. Higher fungal counts, particularly of Penicillium, have been linked to wine spoilage [44]. However, crushing grapes for must preparation significantly reduces mold diversity [45]. Notably, regional fungal communities may also contribute to fine-scale terroir, influencing the distinctiveness of single-vineyard wines.

4.2. Grape Chemical Parameters

The results of the current research indicated that OW treatment influences grape metabolism, leading to lower sugar accumulation and higher acid retention. This shift in the sugar-to-acid balance could have implications for fruit ripening, taste, and winemaking properties. These results contrast with those reported by Campayo et al. [46], who observed an increase in sugar content following single and multiple applications of OW, suggesting an improvement in technological maturity. The impact of OW on grape acids is in line with previous studies indicating that treatment frequency and application technique play a crucial role in shaping the chemical composition of grapes. For instance, a study by Campayo et al. [47] demonstrated that a combination of endotherapy (trunk injection) and spraying resulted in higher acidity levels in wines, which implies an increased acid concentration in grapes. This supports our observation that OW treatment can shift the balance between sugars and acids, an essential factor affecting grape and wine quality.

The sugar content of wine grapes is an important parameter, since it can be used to calculate the potential alcohol content of wine. The expected ethanol production can be estimated based on the measured sugar concentration, applying the official European conversion ratio of 16.83 g L⁻¹ of sugar to produce 1% ethanol. The sugar content of the tested grapes was reduced from 160 to 154 g L⁻¹, from which it can be concluded that the potential ethanol content would have been reduced from 9.5 to 9.1%. The obtained results are important for increasing the ethanol content by adding sugar during winemaking. In viticulture zone A, it is permitted to add sugar to increase the ethanol content up to 3%. This means that it is possible to produce dry wine.

A significant increasing effect was observed on the polyphenol content and antioxidant activity of grapes in the current experiment. These findings are consistent with previous studies that suggest that ozone-induced oxidative stress may trigger specific metabolic pathways in plants [48]. One proposed mechanism involves the stimulation of the phenylpropanoid biosynthetic pathway, where ozone exposure increases the expression of key enzymes such as phenylalanine ammonia-lyase and flavonol synthase. This leads to the enhanced biosynthesis and accumulation of polyphenolic compounds, particularly flavonols like quercetin-7-O-glucoside and kaempferol-3-O-glucoside, which contribute to the antioxidant potential of grapes. A significant impact on the berries was also associated with cultivation technology. At the onset of berry coloration, leaves surrounding the berries were removed, exposing the berries directly to OW. The total polyphenol content in grapes is influenced by several factors, including environmental conditions (e.g., sunlight, soil type), vine growth conditions, fruit ripeness at harvest, and grape cultivar [49]. García-Martínez et al. [50] demonstrated that spraying ‘Cabernet Sauvignon’ grapevines with OW increased the content of phenolic compounds and the potential of aromatic compounds in grapes. Similarly, Campayo et al. [51] found that OW spraying influenced the biochemical composition of ‘Bobal’ grapes, including phenolic and aromatic compounds. Due to its oxidative effect, OW also had a positive impact on the antioxidant activity of the experimental grapes. Modesti et al. [52] found that treatment with OW significantly increased the antioxidant activity. A similar result was also obtained by Christopher et al. [53], who found that this effect is significantly related to the magnitude of cultivar characteristics.

Overall, the findings suggest that OW may serve as an effective intervention for modulating fungal populations within high-polytunnel vineyard environments, with consequential effects on vinification processes. Nonetheless, longitudinal investigations are essential to fully elucidate its long-term impacts on vine health, microbial community structure, and fruit compositional quality.

5. Conclusions

The results of this study demonstrated that OW at a concentration of 2 ppm was effective in reducing fungal CFU counts on grapevine leaves at the onset of berry coloration (BBCH 81), but not at harvest (BBCH 89), when the foliage had become significantly denser. However, OW treatment led to a significant reduction in fungal CFUs on berries at harvest, a favorable outcome for wine grape production as it supports better microbial control during the winemaking process.

Additionally, OW treatment influenced grape berry metabolism by increasing polyphenol content and antioxidant capacity, while simultaneously reducing sugar levels and increasing acid concentrations. As a result, the potential ethanol yield would be expected to decrease slightly, from 9.5% to 9.1%. Given that in Nordic viticultural regions it is common practice—and legally permitted—to add sugar during winemaking to raise ethanol content by up to 3%, OW treatment did not negatively impact grape quality to a degree that would present specific challenges for winemakers.

Furthermore, the persistence of Botrytis cinerea, Fusarium sp., Penicillium brevicompactum, and Cladosporium cladosporioides across samples, irrespective of treatment conditions, was reported for the first time in a vineyard in Estonia, highlighting their adaptability to northern viticultural environments.