Sakuranetin, a Natural Flavonoid, Promising to Manage Grapevine Diseases

Abstract

Botrytis cinerea and Plasmopara viticola, the causal agents of grey mold and downy mildew, respectively, are two major grapevine pathogens whose control largely relies on synthetic fungicides, raising environmental and health concerns. Plant-derived secondary metabolites, particularly flavonoids involved in plant defense, represent promising sustainable alternatives. Among them, sakuranetin, a flavanone aglycone known for its antifungal activity in rice, remains poorly explored for grapevine protection. In this study, sakuranetin was purified from cherry branches (48 mg) and structurally characterized using UHPLC-ESI-QTOF-MS and NMR analyses. Its antifungal activity against B. cinerea and P. viticola was evaluated through in vitro, in vivo and in planta assays. For B. cinerea, our results showed a significant in vitro inhibition of mycelium growth, with EC₅₀ values of 16.43 mg·L⁻¹, while no protection of detached berries was observed. Against P. viticola, sakuranetin has no effect on the release of zoospores, but there is a total inhibition of spore germination at 1 mg·L⁻¹ in vitro, confirmed in vivo on a foliar disc. In planta, no significant protection is observed at 25 mg·L⁻¹, even if some targeted defense genes are induced. Further studies are needed to determine the best concentration of sakuranetin to use to manage B. cinerea and P. viticola in planta.

1. Introduction

Grapevines (Vitis vinifera L.) are a crop of major economic importance, cultivated for the production of table grapes, raisins and, mainly, wine grapes [1]. Each year, global grape production reaches approximately 78 million tons over 7.1 million hectares [2]. Grapevines host a wide range of pathogens, among which fungal and oomycete species are the most damaging, causing major economic losses [3,4]. In particular, Plasmopara viticola, responsible for downy mildew, and Botrytis cinerea, the causal agent of grey mold [5], can cause yield losses of up to 75% [6] and 50% [7], respectively. The control of these diseases still relies heavily on synthetic chemicals [8], despite their well-documented adverse effects on human health and the environment [9,10,11]. In addition, the repeated use of fungicides with a single mode of action promotes the emergence of resistant pathogen strains [12]. Copper-based products remain among the main control strategies for grey mold and downy mildew [13,14], particularly in organic viticulture. However, copper is no longer considered a sustainable solution due to its accumulation in soils, toxicity to non-target organisms, and persistence, which leads to residues in grapes and wine [15]. Regulatory restrictions have further reduced the number of available active substances, increasing risks regarding sustainable disease management [16,17,18]. In this context, the development of environmentally friendly alternatives is urgently needed. Biocontrol strategies, based on natural mechanisms such as competition, antimicrobial metabolite production or induction of plant defense, represent promising solutions [19,20]. Among these approaches, the use of plant-derived natural compounds is particularly attractive (19). Medicinal and aromatic plants are rich sources of secondary metabolites, including flavonoids, which play key roles in plant defense and stress adaptation [21,22,23,24,25]. Flavonoids are one of the most widespread classes of plant secondary metabolites, with more than 9000 structures described to date [26,27]. Structural modifications such as O-methylation enhance their physicochemical properties, including stability and lipophilicity, thereby increasing their antimicrobial activity [28,29,30]. Sakuranetin (4′,5-dihydroxy-7-methoxyflavanone), a methylated flavonoid first described in 1908 in the cortex of the cherry tree bark (Prunus spp.) [31], is a well-known phytoalexin involved in resistance to several fungal and bacterial pathogens [32,33,34,35]. It also plays an important role in the biotic stress responses of woody species and exhibits strong antifungal activity against wood-decaying fungi [36,37,38]. Despite its promising properties, no studies have yet investigated the effects of sakuranetin against B. cinerea and P. viticola in grapevine. This study therefore aims to evaluate the protective potential of sakuranetin against these two major grapevine pathogens. Sakuranetin was first purified from Prunus avium branch extracts, an important co-product in arboriculture and widely available. Then, the protection in vivo and in planta is evaluated against both P. viticola and B. cinerea, and, finally, its mode of action is investigated.

2. Results

2.1. Purification of Sakuranetin from Cherry Branch Extract

From the crude extract of cherry branches (Prunus avium var. burlat), ~50 mg of sakuranetin was purified. The compound was identified by mass spectrometry, showing a retention time identical to the analytical standard (Figure 1A), with an estimated concentration of 15 ± 2 mg·g⁻¹ dry matter. Flash chromatography localized sakuranetin in fraction F, yielding 12 ± 2 mg·g⁻¹ of dry extract. This step was repeated five times before preparative HPLC purification, resulting in 48 mg of sakuranetin (Figure 1B). In negative ESI-MS mode, the [M–H]⁻ ion at m/z 285.1 matched the theoretical mass (C₁₆H₁₄O₅; 286.28 g·mol⁻¹). Retention time and chromatographic profile were identical to the commercial standard. Structural identity was confirmed by NMR spectroscopy. ¹H NMR (300 MHz, acetone-d₆) showed characteristic signals at δ 2.75 and 3.22 (CH₂), 3.85 (OCH₃), 5.47 (H-2), aromatic protons between 6.05–7.41, and phenolic protons at 8.50 and 12.14. ¹³C NMR (75 MHz, acetone-d₆) confirmed the flavanone skeleton. Signal assignments were validated by 2D NMR (COSY, HSQC, HMBC) for both the standard and purified compound, while the DEPT-135 experiment was performed only on the purified sakuranetin. Complete overlap with the standard spectra confirmed purity and identity (Supporting Information, Figures S1–S6).

2.2. In Vitro Inhibition of Sakuranetin Against P. viticola and B. cinerea

For P. viticola, non-linear regression showed a clear dose–response relationship, with an IC₅₀ of 6.59 mg·L⁻¹ (Figure 2A). Significant inhibition occurred from 5 mg·L⁻¹ (p < 0.05), with concentrations of up to 100 mg·L⁻¹ progressively reducing sporulation (Figure 2B), reaching ~90% inhibition at 25 mg·L⁻¹. Visual observations confirmed strong inhibition at 25 mg·L⁻¹ and little to no sporulation at 75 mg·L⁻¹ (Figure 2C). For B. cinerea, the IC₅₀ was 16.43 mg·L⁻¹ (Figure 3A). Mycelial growth was significantly inhibited from 5 mg·L⁻¹, with a dose-dependent decrease up to 100 mg·L⁻¹ (Figure 3A,B). A plateau was reached above 25 mg·L⁻¹, indicating no further increase in activity, as confirmed visually (Figure 3C). At all concentrations, sakuranetin exhibited a fungistatic rather than a fungicidal effect.

2.3. No Antifungal and Anti-Oomycete Activity of Sakuranetin in Planta and In Vivo

The concentration of 25 mg·L⁻¹ sakuranetin was selected based on the in vitro plateau (~80% inhibition), but this efficacy was not reproduced in planta against P. viticola. Both control and treated plants showed high infection levels, with up to 11 infected leaves out of 12 (Figure 4A), and similar infection intensity, unlike the positive control (Figure 4B). However, infection values under sakuranetin were more homogeneous and slightly shifted toward lower intensities, as confirmed by the Kolmogorov–Smirnov test (p = 0.0013). For B. cinerea, in vivo assays on detached berries also failed to confirm in vitro activity. Both control and treated berries showed high proportions of severe infection (class 4), whereas those treated with Géoxe were mostly uninfected (class 0) (Figure 5A,C). Visual assessments confirmed the absence of a protective effect at 25 mg·L⁻¹ (Figure 5B).

2.4. Anti-Germinative Action and Mycelial Growth Inhibitor of Sakuranetin

No significant effect of sakuranetin on P. viticola zoospore release was observed at 3 or 6 h (Figure 6A). However, zoospore germination was completely inhibited at 1 mg·L⁻¹, without affecting motility (Figure 6B,C). In planta, epifluorescence microscopy showed extensive mycelial development and sporulation in controls (Figure 7A), whereas sakuranetin (25 mg·L⁻¹) moderately reduced mycelial growth, with lower fluorescence intensity and smaller sporulation areas (Figure 7C). The presence of sporangiophores and sporangia remained similar in treated and control tissues, indicating no effect on their formation (Figure 7B,D).

2.5. Sakuranetin Induces the Expression of Some Defence Genes of Vitis vinifera

In uninfected plantlets, sakuranetin (25 mg·L⁻¹) significantly induced VvPOX4 expression compared with both the water control and Bion^® (4.11-fold, p ≤ 0.05; Figure 8A), while VvCHI, VvCHS, VvWRKY1 and VvSTS1 were unchanged. In infected plantlets, VvPOX4 was also significantly upregulated (3.85-fold, p ≤ 0.05), reaching levels comparable to Bion^® (Figure 8B). VvPR1 showed a slight increase under sakuranetin (1.20-fold, p ≤ 0.05) and Bion^®, whereas VvCHI, VvCHS and VvSTS1 remained unaffected. Overall, VvPOX4 displayed the strongest and most consistent induction, while other genes showed limited or no response.

3. Discussion

Sakuranetin is widely recognized as a key component of plant defense against diverse pathogens. In rice, it accumulates in infected tissues and reaches higher levels in resistant cultivars than in susceptible cultivars [39,40,41]. It exhibits strong antifungal activity, inhibiting Magnaporthe oryzae growth, spore germination, and germ tube elongation at 100 µM [39], and interferes with clathrin-mediated endocytosis, disrupting effector delivery and enhancing resistance [42]. Its activity extends to other pathogens, improving resistance to Ustilaginoidea virens [43] and affecting bacterial species such as Burkholderia glumae and Xanthomonas oryzae [34]. In other species, UV-induced accumulation enhances resistance to B. cinerea [44], and it has been identified in potato cultivars resistant to Phytophthora infestans [45]. Transcriptomic analyses further revealed extensive gene reprogramming in M. oryzae, suggesting multiple modes of action [46]. Together, these findings highlight sakuranetin as a multifunctional defense molecule combining antimicrobial activity, modulation of defense signaling, and interference with pathogen infection, supporting its evaluation against P. viticola and B. cinerea.

3.1. Variability and Technical Constraints in Sakuranetin Extraction

Sakuranetin and its glycosylated form, sakuranin, are widespread flavonoids found in various plant extracts and derived products such as honey [31]. They are particularly abundant in the genus Prunus, especially in cherry species [47]. In this study, sakuranetin was purified from Prunus avium branches at 15 ± 2 mg·g⁻¹ dry weight, markedly higher than previously reported values (~0.7 mg·g⁻¹), from stems [48]. This difference may reflect the predominance of sakuranin and methodological factors [49], but it is more likely due to natural variability in polyphenol content linked to environmental and cultivation conditions [50,51]. An alternative approach could exploit the abundance of sakuranin, which can be converted into sakuranetin by mild acid or enzymatic hydrolysis using β-glycosidases, potentially improving yield.

3.2. High Direct Activity of Sakuranetin Against Plasmopara viticola and Botrytis cinerea

This study provides the first evidence of sakuranetin activity against P. viticola. In vitro, it showed a low IC₅₀ of 6.59 mg·L⁻¹ (21 µM; Figure 2), placing it among the most active natural compounds against grapevine downy mildew. Its activity is comparable to stilbene phytoalexins such as ε- and δ-viniferin (IC₅₀ = 12 and 18 µM) [52,53,54], whereas flavones from Glechoma hederacea showed no activity at 500 mg·L⁻¹ [55]. Mechanistically, sakuranetin did not affect zoospore release but completely inhibited germination at 1 mg·L⁻¹ (≈3.49 µM), as confirmed by epifluorescence microscopy. Similar effects have been reported for dehydroeffusol, larixyl acetate [56] and larixol [57], indicating comparable or higher activity. Against B. cinerea, sakuranetin also showed antifungal activity (IC₅₀ = 16.43 mg·L⁻¹; 57 µM), without complete inhibition of mycelial growth, suggesting a fungistatic effect. Its activity exceeds that of several terpenes [58] and phenylpropenes [59], but remains slightly lower than δ-viniferin [60]. Compared with most flavonoids, including gnaphaliin A [61], sakuranetin ranks among the most potent. Overall, these results identify sakuranetin as a promising natural antifungal compound active against both P. viticola and B. cinerea.

3.3. Sakuranetin, a Promising Molecule for Protecting Plants, Needs to Be Improved

Although in vitro results were promising, they were not confirmed in vivo or in planta, as sakuranetin showed no significant protective effect. Against P. viticola, a slight inhibitory trend with more homogeneous infection levels was observed (Figure 4), as indicated by the Kolmogorov–Smirnov test, whereas no protection occurred against B. cinerea (Figure 5). This discrepancy likely reflects limited bioavailability at the infection site. Antifungal efficacy depends not only on intrinsic activity but also on maintaining sufficient in planta concentrations. Several physico-chemical constraints may reduce availability, including limited solubilization, adsorption to cuticular waxes due to lipophilicity [62,63], photodegradation [64], and wash-off under high humidity [65]. Biological and temporal factors may also contribute. Longer infection dynamics in planta may reduce efficacy if the compound degrades before key stages such as spore germination. Tissue complexity may further enhance pathogen development compared to simplified in vitro systems. In B. cinerea, wounding-induced nutrient leakage may have increased infection pressure, masking moderate effects. Consistently, sakuranetin only partially inhibits conidial germination under low nutrient conditions [66]. Detoxification mechanisms may also play a role, as reported for M. oryzae and R. solani [67,68]. Although not demonstrated in B. cinerea, its ability to degrade compounds such as resveratrol [69] and methylated isoflavones [70] suggests that similar processes could occur. Such discrepancies between in vitro and in planta activity are common and often reflect limits in stability, diffusion, or bioavailability, as reported for Warburgia ugandensis [71] and Larix decidua [57]. Thus, the lack of in planta efficacy does not question sakuranetin intrinsic activity but highlights constraints related to its delivery and persistence. These aspects will be addressed in future studies to optimize its efficacy and further investigate its role in grapevine defense.

3.4. Activation of the Targeted Defense Gene of Grapevine by Sakuranetin

Sakuranetin is the main flavonoid phytoalexin in rice, induced by stress and involved in resistance, notably through modulation of pathogen effector endocytosis. Although not identified as a phytoalexin in grapevine, it may act as a signaling or priming compound. We therefore tested whether exogenous sakuranetin could activate grapevine defenses. At 25 mg·L⁻¹, sakuranetin induced a response similar to Bion^®, with significant upregulation of VvPOX4 and VvPR1, while VvCHI, VvCHS, and VvSTS1 were unchanged. However, the VvPR1 increase (~1.2-fold) is likely of limited biological relevance. In non-infected plants, sakuranetin specifically induced VvPOX4, suggesting activation of an oxidative response involving class III peroxidases, potentially supported by ROS production, as reported for other flavonoids such as naringenin [72]. The lack of VvPR1 induction indicates weak activation of the salicylic acid pathway. After P. viticola infection, VvPOX4 remained induced, while VvPR1 showed only a slight increase, preventing firm conclusions about SA pathway activation. Overall, sakuranetin triggers a partial defense response mainly via oxidative pathways, without conferring effective protection under the tested conditions.

4. Materials and Methods

To improve readability while maintaining reproducibility, only the essential methodological details are presented in the main text, whereas full experimental procedures and technical specifications are provided in the Supporting Information (pp. 8–15). Readers are strongly encouraged to consult the Supporting Information for detailed Materials and Methods.

4.1. Obtaining Sakuranetin from Plant Material

4.1.1. Extraction and Preparation of the Crude Extract from Cherry Tree Branches

Cherry branches (Prunus avium var. burlat) were collected in south-eastern France (Drôme department, Chestnut site, Montélimar; 44°33′31″ N, 04°45′03″ E) and processed as described in [73] with minor modifications. Ground material was extracted by reflux maceration in ethanol/water (70/30, v/v) at 70 °C for 30 min. The extract was filtered, centrifuged, concentrated under reduced pressure and freeze-dried to obtain the crude extract.

4.1.2. Detection and Identification of Sakuranetin in Crude Cherry Branch Extract

LC–MS analyses were performed using an Agilent 1290 UPLC system coupled to a 6545 Q-ToF mass spectrometer with UV-DAD detection (Agilent Technologies, Santa Clara, CA, USA). Separation was achieved on a C18 column at 40 °C using a water/acetonitrile gradient (0.1% formic acid). MS analyses were conducted in negative ESI mode (m/z 50–1050). Data were processed with MassHunter (version 12.0, Agilent Technologies, Santa Clara, CA, USA) and compared with internal and public databases (NIST, Metlin). Sakuranetin was identified based on its exact mass ([M–H]⁻ = 285.0763 m/z) and confirmed using a commercial standard by retention time and MS data comparison.

4.1.3. Two-Step Purifications of Sakuranetin from Crude Cherry Branch Extract

Step 1—Fractionation by Flash Chromatography and Identification of the Fraction Containing Sakuranetin:

Preliminary fractionation of the crude extract was performed using a Puriflash PF-5.250-UV800 system (Interchim, Montulçon, France) equipped with a C18 reverse-phase column. For each run, 1 g of crude extract was adsorbed onto celite, dried, and loaded onto a dry-load cartridge. Elution was carried out using water and isopropanol (both containing 0.1% acetic acid) under a stepwise gradient at 50 mL·min⁻¹. Fractions were collected based on UV detection (254 and 280 nm) and pooled according to their spectral profiles. The fraction containing sakuranetin was identified by UPLC-ESI-QTOF-MS as described in Section 4.1.2.

Step 2—Purification of Sakuranetin by Preparative HPLC from the Fraction of Interest:

Sakuranetin was purified by preparative HPLC using a Waters system equipped with PDA and MS detection (Waters Corporation, Milford, MA, USA). Samples were injected in partial loop mode and separated on a C18 column (250 × 21.2 mm, 5 µm) at 20 mL·min⁻¹. The mobile phase consisted of water and methanol (both with 0.1% formic acid) using a linear gradient from 100% A to 100% B. UV detection was monitored between 190 and 400 nm, and MS analyses were performed in negative ESI mode (m/z 50–500). Sakuranetin was collected based on UV signals and its characteristic [M–H]⁻ ion at m/z 285.1.

4.1.4. Structural Verification of Purified Sakuranetin

Structural confirmation of purified sakuranetin was performed by NMR spectroscopy to exclude isomeric compounds. ¹H, ¹³C and 2D NMR experiments (COSY, HSQC, HMBC, DEPT-135) were acquired on a Bruker 300 MHz spectrometer equipped with a cryoprobe (Bruker, Billerica, MA, USA). Samples were dissolved in acetone-d₆ and analyzed in 3 mm tubes. Data acquisition was performed using TopSpin software version 3.2 (Bruker, Billerica, MA, USA), and spectra were processed and interpreted using MestReNova version 14.0.0-23239 (Mestrelab Research, Santiago de Compostela, Spain). The resulting spectra were compared with those of a commercial sakuranetin standard analyzed under identical conditions, confirming structural identity.

4.1.5. Solvent and Reference Substance

The sakuranetin analytical standard was purchased from Extrasynthese (Lyon, France) and its identity and purity (≥90%) were confirmed by UV–visible diode array spectroscopy, LC–MS and NMR analyses. Acetonitrile used for UPLC analyses was obtained from VWR (VWR international, Radnor, PA, USA), while formic and acetic acids were supplied by Fisher Scientific (Thermo Fishcer Scientific, Waltham, Massachusetts, USA). Ethanol, methanol and isopropanol used for extraction, fractionation and purification were purchased from VWR. Ultrapure water was produced using a Milli-Q Integral 5 system (Merck Millipore, Burlington, MA, USA).

4.2. Evaluation of the Protective Activity of Purified Sakuranetin Against Two Major Grapevine Pathogens

4.2.1. Biological Material

For P. viticola, both in vitro and in planta assays were conducted using Vitis vinifera cv. Chardonnay plantlets derived from in vitro culture and acclimatized in greenhouse conditions. In vitro experiments were performed on leaf discs, while in planta assays used plantlets at the 12-leaf stage. Inoculation was carried out by spraying a spore suspension (1 × 10⁴ spores·mL⁻¹) onto the abaxial leaf surface, followed by incubation at 24 °C (24 h dark, then 16 h/8 h light/dark). P. viticola was maintained on sterilized grapevine leaves on agar. Botrytis cinerea (strain BC630) was cultured on Potato Dextrose Agar (PDA) from glycerol stocks and incubated at 20 °C in the dark.

4.2.2. In Vitro Evaluation of Purified Sakuranetin

For both pathogens, Sakuranetin was prepared at concentrations of 0, 1, 5, 10, 25, 50, 75, and 100 mg·L⁻¹ in water containing 1% ethanol. The solutions were kept in an ultrasonic bath until use to maintain the solubility of sakuranetin.

Evaluation of Anti-Oomycete Activity Against Plasmopara viticola

Leaf discs (15 mm) from acclimatized Vitis vinifera cv. Chardonnay plantlets were placed on agar (12 discs per treatment). Sakuranetin was applied 24 h prior to inoculation, with controls receiving solvent only. Discs were inoculated with a P. viticola spore suspension (1 × 10⁴ spores·mL⁻¹) and incubated (24 h dark, then 7 days at 22 °C, 16 h/8 h light/dark). Experiments were performed in triplicate. Disease development was quantified by image analysis using Fiji version 20250529-2217 (ImageJ distribution, National Institutes of Health, Bethesda, MD, USA) as described in [74] and expressed as percentage inhibition relative to the control.

Evaluation of Antifungal Activity Against Botrytis cinerea

PDA medium was prepared and supplemented with sakuranetin (1% ethanol final). Mycelial plugs (3 mm) of B. cinerea from one-week-old cultures were placed at the center of Petri dishes (6 replicates per treatment) and incubated at 20 °C in the dark for 3 days. Mycelial growth was quantified by image analysis using Fiji and expressed as percentage inhibition relative to the control. Experiments were performed in triplicate.

4.2.3. Confirmation of the Anti-Plasmopara viticola Activity in Planta of Purified Sakuranetin on Acclimatised Plantlets

Greenhouse Experiment and Inoculation with Plasmopara viticola

The experiment was conducted in a greenhouse (20–28 °C, natural light) using acclimatized grapevine plantlets. Treatments were divided into two blocks, a protection block (disease assessment) and an elicitor-effect block (leaf sampling), to avoid interference between sampling and disease development. Each treatment included six plants. Preventive applications were performed 24 h before inoculation. P. viticola inoculation was carried out by spraying a sporangial suspension (2 × 10⁴ sporangia·mL⁻¹), followed by incubation (24 h dark, then 10 days under day/night conditions with high humidity). In the protection block, three treatments were tested: a negative control (water + 1% ethanol), a positive control (Bordeaux mixture), and sakuranetin (25 mg·L⁻¹). This concentration corresponded to the onset of the dose–response plateau (~80% efficacy in vitro), supporting its relevance for in planta evaluation.

Evaluation of the Protection, Image Processing and Data Acquisition

After incubation, leaves were collected, placed in moist Petri dishes, and stored at 4 °C prior to imaging. The abaxial surface was photographed, and disease development was quantified using Fiji version 20250529-2217 (ImageJ distribution, National Institutes of Health, Bethesda, MD, USA) with thresholding based on Trainable Weka Segmentation (TWS). Images were pre-processed (contrast enhancement, gradient filtering with MorphoLibJ) before analysis. The model, trained on representative leaves, included four classes (background, leaf tissue, sporulation, veins) (Supporting Information, Figure S8). Infection was quantified in the protection block as the proportion of leaf area covered by sporulation relative to total leaf surface.

4.2.4. Confirmation of the In Vivo Anti-Botrytis cinerea Activity of Purified Sakuranetin on Detached Berries

Plant Material/Detached Berries

Grape bunches (white Muscat table variety) were commercially sourced. Berries were detached with pedicels, surface-sterilized in 70% ethanol, rinsed with sterile water, and placed on racks with pedicels inserted into water to maintain physiological conditions. Each treatment included 48 berries.

Treatments and Inoculation of Botrytis cinerea

Treatments were applied preventively 24 h before inoculation by spraying the berries, followed by drying and a 24 h incubation period. Solutions were prepared in water containing 1% ethanol, and sakuranetin was applied at 25 mg·L⁻¹. A commercial fungicide (Géoxe WG^®, fludioxonil) was used as a positive control. Berries were artificially wounded (two punctures per berry) to facilitate infection [75], and each wound was inoculated with a spore suspension (1 × 10⁶ spores·mL⁻¹). Samples were incubated for 7 days under high humidity (16 h/8 h light/dark).

Quantification and Evaluation of the Protection

To quantify the protection, a rating scale was established to classify each berry according to its degree of infection, thereby enabling an objective comparison of the different modalities (Supporting information; Figure S7).

4.3. Elucidation of Protective Activity Through Mechanistic Study of Purified Sakuranetin

4.3.1. Microscopic Observation at Different Stages of the Plasmopara viticola Infection Cycle

Impact on Sporangia Release, Motility and Spore Germination:

A spore suspension was mixed with sakuranetin (1, 10 and 100 mg·L⁻¹; final spore concentration 1 × 10⁴ spores·mL⁻¹, 1% ethanol) in 96-well plates. Each condition was tested in six replicates. Observations were performed by optic microscopy EVOS M7000 (Thermo Fishcer Scientific, Waltham, Massachusetts, USA) at ×20 at 0, 3 and 6 h, and images were analyzed using Fiji version 20250529-2217 (ImageJ distribution, National Institutes of Health, Bethesda, MD, USA). Zoospore release was expressed as the percentage of empty sporangia, while germination was assessed qualitatively based on germ tube formation.

Impact on Sporangiophore Development:

Sporulation of P. viticola was observed on leaf discs using a 3D digital microscope Keyence VHX-7000, (Keyence Corporation, Osaka, Japan) at ×100–×400 magnification. Images were acquired with VHX software version 1.3 (Keyence Corporation, Osaka, Japan) and adjusted for brightness and contrast only. Observations were performed on at least six leaf discs per condition and were representative of the results.

Impact on Mycelial Network Development:

Leaf discs were observed by epifluorescence microscopy Olympus BX43, equipped with U/B/G filters (Olympus Corporation, Tokyo, Japan). Images were acquired using the Infinity Analyze software version 7.1 (Lumenera Corporation, Ottawa, ON, Canada). Observations focused on discs treated with 100 mg·L⁻¹ sakuranetin. Infected discs were cleared (ethanol, NaOH, NaClO), stained with aniline blue, and examined after mounting in water.

4.3.2. Evaluation of the Elicitor Potential of Sakuranetin on Acclimatised Plantlet

Treatment and Conditions:

This experiment was conducted under the same conditions as those described in Section 4.2.3 using the same plant material. Bion^® (acibenzolar-S-methyl, 2 g·L⁻¹) was used as a positive control. Leaves (positions 3 and 4 below the apex) were sampled 4 days after treatment in non-inoculated plants, or 3 days post-inoculation following infection 24 h after treatment. Only plants from the elicitor-effect block were used, and samples were immediately frozen in liquid nitrogen. The sampling time was selected to assess the persistence of the elicitor effect several days after treatment [76].

Gene Expression Monitoring by RT-qPCR

Total RNA was extracted from leaf powder using PureLink™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA. Expression of five defense-related genes (VvSTS1, VvPR1, VvCHS, VvCHI and VvPOX4) was quantified by RT-qPCR using VvEF1a, VvUBE2 and VvACT7 as reference genes. Each biological replicate was analyzed in triplicate. RT-qPCR was performed using SYBR Green chemistry, and relative expression levels were calculated using the ΔΔCq method after normalization.

4.4. Statistics

Statistical analyses were performed using GraphPad Prism 10 version 10.6.0 (GraphPad Software, San Diego, CA, USA). Data were tested for normality, and appropriate parametric or non-parametric tests were applied. P. viticola in vitro data were analyzed using the Kruskal–Wallis test, whereas B. cinerea data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Zoospore release data were analyzed using the Šidák multiple-comparison test, P. viticola infection in planta using the Kruskal–Wallis test, and gene expression data by one-way ANOVA followed by Dunnett’s test versus the control. Differences were considered statistically significant at p < 0.05.

IC50 values were estimated using a four-parameter logistic (4PL) regression model fitted to log10-transformed concentrations:

$$Y=Bottom+{\displaystyle \frac{Top-Bottom}{1+{10}^{\left(lo{g}_{10}\right(I{C}_{50})-X)\times HillSlope}}}$$

where Y is the response and X is the log10 inhibitor concentration. IC50 values and their 95% confidence intervals were derived from the fitted model. The standard deviation of IC50 was estimated by error propagation from the log-transformed scale:

$$S{D}_{lo{g}_{10}\left(I{C}_{50}\right)}={\displaystyle \frac{C{I}_{95\%}^{upper}-C{I}_{95\%}^{lower}}{2\times 1.96}}$$

This approach ensures consistency with the non-linear regression model while expressing variability on the linear IC50 scale. Zoospore release data were analyzed using the Šidák multiple-comparison test, P. viticola infection in planta using the Kruskal–Wallis test, and gene expression data by ANOVA followed by Dunnett’s test versus the control. Differences were considered significant at p < 0.05.

5. Conclusions

Overall, this study highlights the biological potential of sakuranetin purified from cherry branches, a widely available arboricultural by-product, for the management of B. cinerea and P. viticola in grapevine. Sakuranetin exhibited strong in vitro activity against both pathogens, with IC₅₀ values among the lowest reported for natural flavonoids. Its pronounced anti-germinative effect, particularly on P. viticola zoospores, suggests that it targets early stages of the pathogen infection cycle. However, this efficacy was not confirmed in planta under the tested conditions, as no significant protective effect was observed at the applied concentration. This discrepancy may have resulted from several factors, including limited compound availability at the site of action or the absence of an appropriate formulation. In addition, sakuranetin treatment was associated with the modulation of specific defense-related responses in grapevine. While these observations suggest a potential role in defense activation, our results do not allow us to conclusively demonstrate a biologically effective elicitor activity under the conditions tested. Future studies will aim to elucidate the reasons underlying the lack of in planta efficacy. Several hypotheses will be explored, including the determination of optimal application concentrations and the development of improved formulations to enhance sakuranetin stability, solubility, and bioavailability, with the objective of increasing its effectiveness under in planta conditions.