Introduction
Grapevine (Vitis vinifera L.) is one of the major crops in the world, which is especially used for the production of grape berries to produce wines (OIV, 2022). Nonetheless, grapevine cultivation is subject to a variety of problems, including diseases, which diminish both grape berry yield and quality. Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases in vineyards, particularly affecting V. vinifera cultivars, which can cause losses of up to several millions of euros (Gouveia et al., 2024; Koledenkova et al., 2022). P. viticola is an oomycete and an obligate biotrophic agent, which infects all the green organs of grapevine, particularly the leaves and young berries, triggering a cascade of discoloration, necrosis and ultimately defoliation. P. viticola is a polycyclic pathogen: mature oospores (sexual spores) germinate and produce macrosporangia which release zoospores (asexual spores) under wet conditions during the spring (Rossi et al., 2008; Rossi & Caffi, 2007). These zoospores swim to stomata and encyst. Then, a germ tube is produced and forms an appressorium to penetrate the intercellular space and uptake nutrients with haustoria (Allègre et al., 2007; Unger et al., 2007). After that, mycelium growth is latent and symptoms of primary infection appear 5 to 18 days later on the adaxial leaf surface (yellow area called “oilspot”) (Rossi et al., 2013). With moist conditions and warm temperature (> 25 °C), sporangia emerge from stomata onto the abaxial surface of leaves (“white downy”) (Fröbel and Zyprian, 2019; Unger et al., 2007). The cycle of secondary infections starts with the release of zoospores from these sporangia by the action of wind or raindrops (Rossi et al., 2013). At the end of the growing season, oospores are formed in the infected leaves, shoots and berries as a result of the union of male (antheridia) and female (oogonia) structures which have differentiated from mycelial hyphae (Rossi et al., 2013).
Currently, to control downy mildew, the most economical and effective strategies rely on chemicals (Clippinger et al., 2024; Peng et al., 2024). Copper-based (e.g., Bordeaux mixture) and synthetic fungicides are the main chemical products used in viticulture (Koledenkova et al., 2022), and they are subject to regulation by the European Union (Dir. 2009/128/EC and Reg. 1107/2009/EC). However, in addition to the potential harm of these chemicals on humans and the environment, their extensive use has led to soil contamination and the development of fungicide resistance, with the emergence of resistant strains of P. viticola (Massi et al., 2021).
It is worth noting that winegrowers often opt for an Integrated Pest Management (IPM) approach, in which control solutions are used that minimise risks towards health and the environment (Pertot et al., 2017). These include specific cultural practices (Clippinger et al., 2024; de Bem et al., 2016), resistant grapevine cultivars (Divilov et al., 2018) and biological control products that comprise biotic agents (e.g., fungi, yeast, bacteria (El-Sharkawy et al., 2018; Zhang et al., 2017)) or natural antimicrobial substances (e.g., plant extracts and essential oils (Dagostin et al., 2010; Gabaston et al., 2017)).
While stilbenes, the main phytoalexins found in grapevine, are well-known for their antimicrobial, and particularly anti-oomycete activities (Gabaston et al., 2017; Pébarthé-Courrouilh et al., 2024; Taillis et al., 2022), other polyphenols such as tannins are also of interest for their antimicrobial properties. Tannins are the fourth most abundant components to be extracted from biomass (Arbenz & Avérous, 2016). They are considered as specialised metabolites in plants, have a molecular weight ranging from 500 to 30000 Da, and are characterised by the presence of one or more aromatic rings. They are grouped into four main classes: hydrolysable tannins, complex tannins, phlorotannins and condensed tannins (Serrano et al., 2009). Hydrolysable tannins are composed of polyesters of a sugar moiety and organic acids. After acid hydrolysis, gallotannins and ellagitannins release gallic acid and ellagic acid, respectively. Phlorotannins are polymers or oligomers of phloroglucinol-derived units. Complex tannins are compounds containing structural elements of hydrolysable tannins and condensed tannins. Condensed tannins (also known as proanthocyanidins) are oligomers or polymers of flavanol units linked through covalent B-type inter-flavanol bonds (de Hoyos-Martínez et al., 2019; Serrano et al., 2009). They are widely distributed throughout the plant kingdom and are synthesised in almost all the different parts of plants (e.g., seeds, roots, twigs, bark, wood, leaves and fruit) (Barbehenn & Constabel, 2011), with different monomeric compositions depending on the part and the plant studied (de Hoyos-Martínez et al., 2019). Grape seed tannins belong to the procyanidin group and are mainly composed of catechin, epicatechin and epicatechin-3-O-gallate units, such as flavanol, while skin tannins are mainly prodelphinidins composed of gallocatechin and epigallocatechin flavanol units (Mattivi et al., 2009).
Tannins are also known to act on plant defences and can be either constitutive (phytoanticipins) or inducible (phytoalexins) in response to pathogens and herbivores (insects and animals) (Barbehenn & Constabel, 2011; Sharma, 2019; Treutter, 2006; War et al., 2012). In grapevine, they have been found to accumulate after bacterial (Wallis & Chen, 2012), viral (Gutha et al., 2010) and fungal attacks (Del Río et al., 2004), making them good candidates for grapevine protection. Their role in plant defence is due in large part to their ability to complex and precipitate proteins, such as pectinases, cellulases, amylases, β-galactosidases, lipases, proteases and laccases (Khanbabaee & Ree, 2001; Pizzi, 2019; Sharma, 2019; Vignault et al., 2020). In addition, some tannin extracts (bay laurel, black oak and white oak extracts) have shown antimicrobial activities against oomycetes that have infected plants such as Phytophthora ramorum (Stong et al., 2013) and Aphanomyces cochlioides (Islam et al., 2002). In light of these characteristics, we can hypothesise that tannins are good biopesticide candidates for protecting vineyards from downy mildew. The tannins present in the co-products (e.g., chestnut wood, nut galls, grape skins and seeds) of many cultivated plants could be easily recovered and valorised, thereby enhancing their value and promoting a circular economy. In the oenology sector, tannins (gallotannins, ellagitannins and condensed tannins) have largely been used to ensure wine stability, their main sources being nut galls and tara (gallotannins), oak and chestnut (ellagitannins), grape, acacia, quebracho and mimosa (condensed tannins) (Vignault et al., 2018).
The aim of this study was to evaluate the antimicrobial effects of tannins on Plasmopara viticola. To this end, we first characterised the chemical composition of five tannin extracts (one from nut gall, one from chestnut wood, one from grape skin and two from grape seeds). Then, we evaluated their capacity to inhibit downy mildew sporulation. The concentrations inhibiting 50 % of downy mildew sporulation, sporangia release and zoospore motility were determined for the two most active extracts. Finally, the homemade crude grape seed tannin extract was fractionated according to the polymerisation degree of condensed tannins. The resulting fractions were tested for their anti-mildew activity.
Materials and methods
1. Chemical and reagents
All solvents were HPLC grade. Acetic acid (HPLC grade) and hydrochloric acid (HPLC grade) were obtained from Fisher Scientific (Illkirch, France). Folin-Ciocalteu reagent, sodium hydroxide, sodium carbonate, ascorbic acid, phloroglucinol, as well as catechin, epicatechin, epigallocatechin, epicatechin-3-O-gallate (ECG), vescalagin and ellagic acid were purchased from Sigma-Aldrich (St. Quentin, Fallavier, France). Procyanidins B1-B4 were obtained from Extrasynthèse (Genay, France). Ethanol, used for the biological assays, was purchased from VWR® (Radnor, USA) and Milli-Q water was obtained using a Milli-Q® reference water purification system from Merck Millipore (Burlington, USA).
2. Tannin extracts and chemical characterisation
Five tannin extracts were used in this study: one nut gall commercial extract (Tanin Galalcool® SP), one chestnut wood commercial extract (Kingbrown®), one grape skin commercial extract (Tanin VR Skin®) and two grape seed extracts (one homemade and one commercial (Tanin VR Grape®)). All commercial extracts were provided by Laffort® (Bordeaux, France) and King Tree (Arras, France). The homemade grape seed tannin extract was prepared applying the procedures described in Ćurko et al. (2014). Grape seeds were manually separated from Cabernet-Sauvignon grapes collected at maturity from a Bordeaux vineyard. The seeds were frozen under liquid nitrogen and freeze-dried for two days, then ground in a ball grinder. A 6 g portion of the obtained powder was extracted using 55 mL of acetone/water (80:20, v/v) for 8 h, by repeating three extraction cycles on the powder. After centrifugation, the supernatants were combined and evaporated under reduced pressure at 30 °C to remove the organic solvents. Then, the obtained crude tannin extract was dissolved in 200 mL of water/ethanol (95:5, v/v) and extracted three times with 200 mL of chloroform to remove the lipophilic material. The obtained aqueous phase was then evaporated under reduced pressure at 30 °C to remove any traces of organic solvents, and then freeze-dried to obtain a crude grape seed tannin extract.
Each tannin extract was characterised in terms of richness and composition using the analytical methods described below. All the analyses were carried out in triplicate (at a minimum).
The total phenolic content of the tannin extracts was estimated using the Folin-Ciocalteu assay according to the method proposed by Lorrain et al. (2012) using 96-well microplates. The tannin richness of the grape skin and seed extracts were determined using a modified Bate-Smith method according to Lagarde et al. (2023) and Ribéreau-Gayon and Stonestreet (1966). The monomeric and dimeric flavanol content of the skin and seed tannins extract were determined by HPLC-UV-fluo according to Lagarde et al. (2023) and Ćurko et al. (2014), while the proanthocyanidins’ mean degree of polymerisation (mDP), as well as the monomeric composition, were determined by HPLC-UV after phloroglucinolysis depolymerisation, following the procedure described by Drinkine et al. (2007). The content and composition of ellagitannins in the chestnut wood tannin extract were determined by HPLC-UV-MS according to Gancel et al. (2023).
3. <italic>Plasmopara viticola</italic> assays
Isolate ANN-01 of the oomycete Plasmopara viticola (originating from a vineyard in Charente (France)), was maintained in the laboratory by performing weekly subcultures according to the procedure used by Corio-Costet et al. (2011). Briefly, V. vinifera cv. Cabernet-Sauvignon leaves collected from two-month-old cuttings (grown in greenhouses located in Villenave-d’Ornon, France) were inoculated with P. viticola. After seven-days, sporangia were collected and suspended in cold water. Microdroplets (15 µL) of this suspension (10.103 sporangia/mL) were deposited on the abaxial surface of the Cabernet-Sauvignon leaves, which were then placed on Whatman paper in Petri dishes. One-day latter, droplets were removed, and dishes were incubated for six more days. Incubation was carried out in controlled conditions (24 ± 2 °C and 16/8 h light/dark).
3.1. Sporulation assays
Fresh leaves (the third and fourth leaves on the apex) of two-month-old foliar cuttings of Vitis vinifera cv. Cabernet-Sauvignon were collected and washed with tap water. Foliar discs that were 22 mm in diameter were deposited on a moistened Whatman paper in 90 mm diameter Petri dishes (3 mL of water, 6 discs per dish).
Tannin extract solutions were prepared extemporaneously in EtOH 0.2 %, and 400 mg/L was sprayed onto the foliar discs until microdroplets formed (32 ± 4 µL per disc, 50-100 µm droplet size). An ethanol concentration up to 1 % did not affect oomycete sporulation (Pezet et al., 2004). After drying the extract solutions in a laminar flux cabinet, the foliar discs were inoculated with 15 µL droplets (3 per disc) of P. viticola in a sporangia solution of 10.103 sporangia/mL. The inoculated discs were incubated in Petri dishes for one day in darkness before the sporangia droplets were removed. After that, the dishes were placed in a culture room with a photoperiod of 16/8 h day/night at 24 ± 2 °C. The downy mildew sporulation density was evaluated 7 days after inoculation. It was estimated as the percentage of mildew sporulation. The control condition (EtOH 0.2 %) was set as 0 % sporulation inhibition, corresponding to the maximal sporulation level of the oomycete, and sporulation inhibition values for all other conditions were compared to this control (Corio-Costet et al., 2011). Each condition corresponded to the mean of three biological replicates and the experiments were carried out four times each.
The concentrations that inhibited 50 % of mildew sporulation (IC50) were calculated for the most active extracts (i.e., the homemade and commercial grape seed extracts). To this end, the followed final concentrations were used: 0, 300, 350, 400, 450, 500 and, 550 (for the homemade grape seed extract) and 700 mg/L (for the commercial grape seed extract). Each condition was the mean of three Petri dishes (biological replicates) containing six foliar discs each (technical replicates). Experiments were conducted five and seven times on the homemade and the commercial grape seed extracts, respectively.
3.2. Zoospore motility assays
Fresh sporangia of P. viticola were collected in Milli-Q water and the suspension was adjusted to a final concentration of 15.104 sporangia/mL. The sporangia solution was kept for 2 h in the dark at ambient temperature to ensure zoospore release (Hong & Scherm, 2020; Krzyzaniak et al., 2018). After that, the zoospore solution was mixed with grape seed extracts (homemade or commercial) or ethanol (0.2 %, considered as control). In the literature, a final concentration of less than 1 % ethanol has been reported to not affect the zoospore motility of P. viticola (Pezet et al., 2004). The solutions were then incubated for 5 min in darkness before carrying out the optical microscopic observations (DM750, Leica®, 400-fold magnification), for which 15 µL of solution was deposited on a glass slide and a reading of the motile and immobile zoospores at a defined point was taken for 30 s, according to the procedure described by Pezet et al. (2004) and Taillis et al. (2022).
The following concentrations of the two grape seed extracts (homemade and commercial) were tested: 0, 25, 50, 75, 100, 125 and 150, as well as 175 mg/L in the case of the commercial grape seed extract. In addition to the IC50, we determined the IC100 ; i.e., the lowest tested concentration that showed total inhibitory activity.
At each extract concentration, the mean of three biological replicates (reaction mixtures) was calculated and each biological replicate was analysed in three different microscopic observations (technical replicates). All the experiments were carried out in triplicate.
3.3. Zoospore release assays
Fresh sporangia of P. viticola were collected in Milli-Q water and the suspension was adjusted to reach a final concentration of 15.104 sporangia/mL. The grape seed extract solutions or EtOH 0.2 % (control condition) were added to the sporangia suspension. The samples were immediately placed in the dark at ambient temperature for 2 h, to enable zoospore release from the sporangia. After that, for each sample, three readings of 30 sporangia (full or empty) were performed under an optical microscope (ML100, VWR®, 400-fold magnification) according to the procedure described by Raveau et al. (2024).
The following concentrations of the two grape seed extracts (homemade and commercial) were tested: 0, 5, 10, 20, 40 and 80 mg/L. EtOH 0.2 % did not affect zoospore release from the sporangia (data not shown). The percentage inhibition of zoospore release in the control conditions (EtOH 0.2 %) was set to 0 %, and the zoospore release inhibition percentage in all the other conditions was calculated relative to the control. It was thereby possible to determine the IC50 of the two tannin extracts.
At each extract concentration, the mean of three biological replicates (reaction mixtures) was calculated, and each biological replicate was analysed in three different microscopic observations (technical replicates). All the experiments were carried out in triplicate.
4. Homemade grape seed extract fractionation, chemical characterisation and sporulation assays
The homemade grape seed extract was fractionated using the method described by Lorrain et al. (2012), that was slightly modified. Briefly, the crude extract was first dissolved in 250 mL of water/ethanol (95:5, v/v) and extracted three times using ether (250 mL) to obtain the monomeric-small oligomeric fraction (Fraction 1) after removing the organic solvent under reduced pressure. Then, the aqueous phase underwent an extraction process three times using ethyl acetate (250 mL) to obtain the oligomeric fraction (Fraction 2) after removing the organic solvent under reduced pressure. The remaining aqueous phase was frozen and freeze-dried to obtain the polymeric fraction (Fraction 3).
Each tannin fraction was characterised in terms of richness and composition using the analytical methods described in Section 2. All the analyses were carried out in, at the minimum, triplicate.
The antimicrobial action on downy mildew sporulation was assessed (at 400 mg/L) as described in Section 3.1. Each condition corresponded to the mean of three biological replicates and each experiment was carried out four times.
5. Statistical analyses
Statistical analyses were performed using R Studio software (2022.07.1). After assessing residue normality (Shapiro test) and homoscedasticity (Levene test), multiple comparison analyses were performed using a one-way ANOVA followed by a post-hoc test (Tukey), or with a non-parametric ANOVA (Kruskal Wallis) followed by a non-parametric post-hoc test (Pairwise Wilcoxon). For comparison analyses of two groups, we used Student tests (t-tests). Significant differences were set at p < 0.05.
Dose response curves for grape seed extracts in all biological assays on P. viticola were generated by plotting the relative inhibition values against log10 of the extract concentration tested. The IC50 and R2 were determined from the regression equation generated through the linear part of the sigmoid curve on Excel software (2019 MSO 64 bits), according to Corio-Costet et al. (2011). The R2 is a measure of the correlation between the concentration of the tannin extracts and the inhibition effects.