Introduction

Viticulture is a sector with great socio economic importance worldwide, the largest cultivated area being in Europe and the top wine producing countries being Italy, France and Spain (OIV, 2023, https://www.oiv.int/what-we-do/global-report?oiv). However, grapevine development and production are increasingly subject to a biotic (drought, salt, mineral nutrition disturbances, light and temperature) and biotic (wounding, pathogens and herbivores) stress, all of which contribute to the overuse of chemical fertilisers and synthetic pesticides (Samuels et al., 2022). Therefore, there is a need and an increasing demand for more ecologically-sustainable agricultural products, as outlined by the United Nations Sustainable Development Goals (SDGs). Within this framework, the “From Farm to Fork” strategy aims to reduce chemical usage and promote sustainability, one of its objectives being to achieve 25 % of agricultural land under organic production by 2030.

Grapevine plants are among the most frequently treated crops due to their high susceptibility to a number of pathogens, particularly fungi and oomycete. Plasmopara viticola (P. viticola) (Berk. & M.A. Curtis; Berl. & De Toni) a biotrophic oomycete, the causal agent of grapevine downy mildew, is one of the most devastating diseases in vineyards worldwide. The incidence of downy mildew has increased notably in recent years in Europe (Koledenkova et al., 2022). While most commercially important European grapevine cultivars (Vitis vinifera) are highly susceptible to this pathogen, differences in level of sensitivity to it have been documented (Boso et al., 2014). P. viticola severely attacks flower clusters, leaves and young berries, and is therefore controlled with frequent applications of chemical fungicides and copper-based formulations to avoid decreases in yield and quality. Depending on the meteorological conditions, up to twelve fungicide applications per growing season may be necessary to control it. The use of copper can cause environmental problems owing to its accumulation in the soil, with negative side effects, such as decreases in yield and soil microbial diversity (Gessler et al., 2011; Lamichhane et al., 2018). Despite its unfavourable ecotoxicological profile, the use of copper is still tolerated for its unique properties as a wide-spectrum fungicide and bactericide. However, restrictions have been applied by the European Commission on the number of pesticide treatments (Directive 2009/128/EC, http://data.europa.eu/eli/dir/2009/128/oj) and on the maximum allowed quantity per year of copper fungicides (Regulation 2002/473/EC, http://data.europa.eu/eli/reg/2002/473/oj). Moreover, copper, which is still considered the most important antifungal product in organic viticulture, has been added to the list of candidates for substitution (European Commission Implementing Regulation 2018/84, http://data.europa.eu/eli/reg_impl/2018/84/oj), and has been further limited to 4 kg per hectare/year spread over 7 years from February 2019 (European Commission Implementing Regulation 2018/1981, http://data.europa.eu/eli/reg_impl/2018/1981/oj).

The application of environmentally friendly products has become increasingly important in the last few years. Biofungicides, biostimulants and resistance inducers are sustainable biocontrol products that are being currently studied to counteract the overuse of chemical fungicides (Jindo et al., 2022). Among biostimulants, the use of seaweed extracts is increasing. The application of algal extracts has been shown to stimulate defense mechanisms and plants growth (Cappelletti et al., 2016; García-Sánchez et al., 2022; Gutiérrez‐Gamboa et al., 2019). In viticulture in particular, foliar applications have been proven to improve grape and wine composition (i.e., nitrogen, phenolic and volatile contents (Gutiérrez‐Gamboa et al., 2019; Jindo et al., 2022; Rantsiou et al., 2020). However, information regarding the effects of using seaweed extract for the prevention of grapevine fungal diseases is scarce. When used in several foliar treatments on grapevine, a crude extract of the green seaweed Ulva armoricana, rich in the polysaccharide ulvan, showed anti-powdery mildew activity (90 % reduction of symptoms) (Jaulneau et al., 2011). In fact, defense mechanism activation related to the jasmonic acid pathway has been related to ulvan (Jaulneau et al., 2010; Jaulneau et al., 2011). The treatment of grapevine plants with laminarin, a polysaccharide from the brown algae Laminaria digidata, reduced the infection of Botrytis cinerea and P. viticola by 55 % and 75 %, respectively (Aziz et al., 2003). Gauthier et al. (2014) and Pagliarani et al. (2020) have provided insights into the modifications that occur within molecular pathways related to resistance following seaweed extract applications in studies that applied “omics'' technologies, microarray analysis and high throughput sequencing, respectively. The defense mechanisms that are activated under pathogen stress vary, depending on the sensitivity of the grapevine genotypes to the disease (Molitor et al., 2018; Nogueira Júnior et al., 2020; Adrian et al., 2024), and thus the elicitation of plant response by plant protection products or biostimulants can vary depending on the plant’s genetic background (Godínez-Mendoza et al., 2023).

Recently, an aqueous extract from Rugulopteryx okamurae (R. okamurae) - an invasive brown alga that has had a huge negative environmental, social and economic impact on the Andalusian coast (southern Spain) - was found to be a grapevine biostimulant under greenhouse conditions (Zarraonaindia et al., 2023). The extract induced grapevine defense by the expression of immunity genes and secondary metabolites, such as stilbenes, phytohormones and antioxidant enzymes, which are involved in protecting Tempranillo plants from stress and increasing their resistance to it. Additionally, the fungal community composition of the leaves was altered following the treatment, and an increase in the abundance of several beneficial genera known for their antifungal or antagonistic properties occurred in the treated grapevines. However, it remains to be studied whether the elicitation by the extract could indeed provide protection for grapevines against fungi and oomycetes. In addition, while variations to downy mildew resistance have been previously evidenced, even between clones of the same grapevine variety (Boso et al., 2006), a very limited number of studies have addressed these differences and unraveled the underlying mechanisms. To address these gaps in knowledge, the objective of the present study was to assess the efficacy of a R. okamurae aqueous extract in inducing resistance to P. viticola in two clones of Vitis vinifera L. cv. Tempranillo. The molecular and metabolic responses of the clones together with the changes to plant physiology were investigated after i) R. okamurae extract applications, and ii) P. viticola inoculation. In addition, the efficiency of the treatments for protecting the grapevines from downy mildew disease was assessed. The differential response of each of the clones to the treatments is discussed here, shedding more light on the influence of the interaction between genotype and elicitor on level of plant protection.

Materials and methods

1. Rugulopteryx okamurae extract elaboration and biochemical characterization

The brown macroalgae R. okamurae (Div. Phaeophyta, O. Dictyotales) was collected from Algeciras (Cadiz, Spain, 5° 25′ 34.75′′ W, 36° 4′ 37.56′′ N) in April 2022. After harvesting, the seaweed biomass was washed with desalinised water to remove epiphytes and salt, then drained and frozen at −80 °C, and freeze-dried (Cryodos, Telstar, Spain). To produce the extracts, batches of 50 g of freeze-dried R. okamurae were combined with 500 mL of milliQ water and subjected to vigorous shaking in a thermoshaker at 70 ºC for 2 h. The product was centrifuged, the supernatant stored and the solid phase mixed once more with 500 miliQ water and then shaken for 2 h at 70 ºC and centrifuged again. The two liquid phases resulting from the centrifugations were combined, frozen and then lyophilised. The composition of the aqueous extract was characterised. Briefly, ash content, CNS content, macronutrients (Ca, K, Mg, P), micronutrients (Si, Cl, Fe, Mn, Br, Cu, Zn), ashes, total proteins, total lipids and total carbohydrate were analysed according to Zarraonaindia et al. (2023) (Table S1).

2. Experimental design

2.1. Biostimulation and infection

Two clones of Vitis vinifera L. cv. Tempranillo plants (VN40 and RJ43) grafted on Richter-110 rootstock were provided by two commercial nurseries (Vitis Navarra and Viveros Villanueva, Navarra, Spain, respectively). The plants (n = 30 per clone) were grown in a containment level 2 greenhouse (NCB2), in 5 L pots containing an enriched nutrient substrate of organic matter (90 %), sphagnum peat (160 g/L), calcium carbonate (7 g/L), NPK fertiliser (1.5 g/L) and trace elements (PotgrondH, Klasmann-Deilmann GmbH, Germany). The plants were grown for 2 months, with a 16 h day/8 h night photoperiod at an average room temperature of 18 ºC and were irrigated to field capacity when necessary.

When the plants had 10 to 12 fully expanded leaves, they were treated with either R. okamurae extract (RO, 6 g/L), water (W) or copper (BB, cuprous oxide 4.2 g/L, Nordox 75 WG, NordoxIndustrier, Oslo, Norway) spraying 3 times on leaves once every 48 hours (Figure 1). The soil in the pots was protected from the rinse with filter paper during each application. Ten plants were used per treatment type and clone type. To all the treatments (W, BB, RO) 0,1 % Retenol (Daymsa, Zaragoza, Spain) was added as a coadjutant.

Figure 1. Experimental design.

Forty-eight hours after the last treatment, all the plants (n = 60) were inoculated with a suspension of P. viticola sporangia by pulverisation on the abaxial side of the leaf with a manual hand sprayer (Figure 1). P. viticola isolate was collected in 2022 from a commercial vineyard (Etxano, Biscay, Spain), isolated and regularly multiplicated in the laboratory on detached leaves of V. vinifera cv Tempranillo until use. Sporangia concentration was quantified using a Thomahaemato cytometer (GMBH + CO KG, Germany) as described by Llamazares De Miguel et al. (2022). The sporangia was dissolved in commercial mineral water, and its concentration was adjusted to 5 x 10⁴ sporangia/ml.

2.2. Sample collection and analysis

Three crucial time-points were studied: S₀ sampling, the initial sampling prior to the application of the treatments (Day 0); S₁ sampling, 24 h after foliar application of the last treatment, referred to as the biostimulation time-point (1 day post-biostimulation, 1dPB); S₂ sampling, 24 h after P. viticola inoculation, referred to as the post-inoculation time-point (1dPI) (Figure 1).

To assess the initial physiological state of the plants, the photochemical parameters of the leaves and plant height were determined in S₀ (n = 60 plants). At the biostimulation time-point (S₁, 1dPB), the 5th leaf (starting from the apex) was sampled, and a leaf from two plants was pooled to create five replicate samples per treatment type/clone. Leaves were milled in liquid nitrogen and were then used for gene expression and secondary metabolites analysis (including enzyme activity and polyphenols). The 7th leaf was used to study photochemical parameters in n=10 plants/treatment. In this case, while the same sampling approach was used 1 day after inoculation (S₂, 1dPI), the 4th and 6th leaf of two plants were pooled together for gene, polyphenol and enzyme analysis (n = 5 biological replicates per treatment/clone). In addition, disease development was evaluated by visual observation in all the treated plants (W, BB and RO, n = 60) at three different monitoring times: 3, 10 and 17 days after observing the first symptoms. Incidence and severity rates were measured as a percentage and then transformed into a 0–7 scale similar to the EPPO evaluations for the efficacy evaluation of fungicides: a sporulation of 0 % was given a “0” value, a sporulation lower than 5 % a “1” value, a sporulation lower than 10 % a “2” value, a sporulation lower than 25 % a “3” value, a sporulation lower than 50 % a “4” value, a sporulation lower than 75 % a “5” value, and a sporulation higher than 75 % a “6” value. Those values were transformed into a disease index (DI) using the Townsend-Heuberger formula (1943) to obtain the Incidence Disease Index and the Severity Disease Index, respectively.

3. Gene expression analysis

RNA was extracted with Spectrum^TM Plant Total RNA kit (Sigma-Aldrich Co., MI, USA), following the manufacturer’s instructions, from water-treated and RO-treated plants leaves at the S₁ and S₂ timepoints. For each biological replicate (n = 5 per treatment/clone), DNA was removed by On-Column DNase I Digestion Set (Sigma-Aldrich Co., MI, USA) during the extraction protocol. First-strand cDNAs were synthesised, starting from around 750 ng of total RNA, using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific Waltham, MA, USA) and following the manufacturer’s instructions. cDNA samples were analysed for gene expression using the INRAE BC2 Grape platform (https://bc2grape-inrae.fr/nos_services/neovigen). The expression of genes was monitored by quantitative polymerase chain reaction (qPCR) using an implemented version of the NeoViGen microarray and a Fluidigm® de BioMark HD, following the protocol described by Dufour et al. (2016). The array contained five reference genes and a total of 89 targeted genes: 74 from Dufour et al. (2016) and 15 from Bodin et al. (2020). NADH and RNAse P were the negative and positive control, respectively. A list of the genes is provided in Table S2.

The comparative Ct (2−ΔΔCt) method was used to calculate transcript expression levels, as all the genes showed similar amplification efficiencies (between 90 and 110 %). The gene expression profiles of RO-treated and water-treated (W) plants were compared 24 h after treatment application (S1) and 24 h after P. viticola inoculation (S2), respectively. To determine the significance of a differential gene expression, the fold change threshold was fixed at 1.5 or -1.5 (log transformed) and a one-way analysis of variance (ANOVA) was carried out in SPSS 15.0 (SPSS Corporation, Chicago, IL, USA) per gene; the ANOVA values were considered statistically significant when p < 0.05.

4. Polyphenol analysis

Polyphenols were extracted from W, BB and RO leaves which had been collected in S1 and S2 following the method described by Krzyzaniak et al. (2018) with modifications. Briefly, 50 mg of freeze-dried powdered leaves was extracted using 10 mL of methanol and by shaking overnight at 4ºC. The supernatants were evaporated, redissolved in methanol:water (30:70), and then passed through a cartridge C₁₈ (Scharlab, Spain) to clean up the samples. Polyphenols were eluted in methanol:water (90:10), concentrated under vacuum, redissolved in methanol:water (50:50), and finally filtered through a 0.22 µm filter (PTFE Teknokroma, Barcelona, Spain). For compound identification, 20 μL of the samples were analysed using a Waters HPLC system (Milford, MA, USA) equipped with a model 1525 pump, a W2707 injector and a Waters 2996 photodiode detector, and a Mediterranean Sea C18 column (Tecknokroma, Barcelona, Spain) (RP-18, 25 × 0.46 cm; 5 μm particle size), with a precolumn of the same material. Caftaric acids, coutaric acid, cis- and trans-piceid, and viniferins (e and d) were quantified at 320 nm with their respective standards; trans-Resveratrol was quantified at 306 nm as trans-resveratrol; cis-Piceid was quantified as cis-piceid with its respective standard generated by UVC treatment on trans-piceid. Quercetin 3-glucoside and quercetin-3-glucuronide were quantified at 360 nm as their respective standard. Concentrations were expressed in mg/L.

A one-way analysis of variance (ANOVA) of the mean values was used to test for differences between time-points and treatments. Significant results (p ≤ 0.05) were then evaluated via the LSD test, using Statistix version 9.0 (Analytical Software, Tallahassee, FL, USA).

5. Analysis of the antioxidant enzymes

The activity of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) was determined as described in Pérez-López et al. (2009). In summary, ground leaf tissue (40 mg fresh weight) of 5 replicates per treatment/clone was extracted using enzyme-specific buffers (0.6 mL per sample). The resulting homogenates were subjected to centrifugation at 16,100 g for 25 min. Superoxide dismutase (SOD) activity was determined via a spectrophotometric assay based on the reduction of ferricytochrome-c, utilising xanthine and xanthine oxidase as sources of superoxide radicals. One unit of SOD was defined as the amount of enzyme required to achieve a 50 % decrease in the ferricytochrome-c reduction rate. For the glutathione reductase (GR) assay, the activity was measured by monitoring the decrease in absorbance at 340 nm, corresponding to the NADPH oxidation dependent on oxidized glutathione (GSSG), at 25 °C. Ascorbate peroxidase (APX) activity was evaluated by tracking the ascorbate (ASA) oxidation at 290 nm. Statistical analysis was carried out using SPSS version 28.0 (IBM Corp, Armonk, NY, USA). The differences between the treatments were assessed via a one-way analysis of variance (ANOVA) on the mean values. When ANOVA indicated significant differences (p ≤ 0.05), Tukey’s post-hoc test was employed to compare the tested treatments.

6. Photochemical parameters

Measurements of the photochemical efficiency of photosystem II (φ_PSII or Fv/Fm) and the leaf chlorophyll level (SPAD index) were performed in situ at each sampling point (on the 5th leave in S₀ and S_1, and on the 7th in S₂) using a FluorPen FP 100 fluorometer (Photon Systems Instruments, Brno, Czech Republic) and a Konica-Minolta SPAD-502 (n = 60 plants, n = 10 per treatment/clone). Chlorophyll a, chlorophyll b, carotenoids, xantophylls and phaeophytinization quotient chlorophyll a were determined in 0.7 cm diameter leaf discs from the 7th leaf in S1 and S2. Statistical analysis was conducted in SPSS28.0 0 (IBM Corp. Armonk, NY: IBM Corp, USA) by means of an ANOVA and a Tukey´s test.

Results and discussion

1. Initial state (S₀): basal differences between VN40 and RJ43 Tempranillo clones

Being the most widespread red variety in Spain, Tempranillo is well known throughout the world. In the present work, the two clones VN40 and RJ43 were assessed in order to gain a broader perspective of our findings. According to the commercial nurseries, the VN40 clone plants exhibit higher production but lower vigour and compactness than the RJ43 clone, which may be key aspects regarding infection development.

During the two-month growing period in the greenhouse prior to biostimulation and pathogen inoculation, a delayed growth of the RJ43 clone was observed. Accordingly, in S₀, the measured development parameters (stem height, number of leaves, number of stems) showed that the RJ43 clone plants (n = 30) were less developed than the VN40 clone plants (n = 30) (Table S2), thereby already indicating differences in vegetative and phenological stages between these clones at this initial time-point.

2. Biostimulation state (S₁, 1dPB): responses to Rugulopteryx okamurae extract (RO) application

Grapevine leaves were sampled after three applications of water (W), copper (BB) and seaweed (RO) (S₁, 1dPB, Figure 1) to assess the metabolic response of the plants. At this sampling point, our results are compared with a previous study conducted on Tempranillo VN40 clone, in which two foliar treatments with the same seaweed extract were conducted (Zarraonaindia et al., 2023).

2.1. Gene expression modulation by RO

Differential expression analysis was carried out on the targeted 89 genes (Table S3) following three applications of R. okamurae (RO) and Water treatment (W). The results revealed that the extract significantly enhanced the expression of three genes belonging to the PR protein group (PR3, PR4bis and PIN) and four other genes, each involved in a different functional group: CHI2 (only in RJ43), HSR-203J, GST1 and CAD2 genes related to the flavonoid pathway, lipoxygenase pathway, redox status regulation and cell wall reinforcement, respectively (Figure 2A). In addition, the applied extended gene expression analysis using NeoViGen microarray strategy (compared to the 15 targeted genes in Zarraonaindia et al. (2023)) allowed down-regulated genes to be identified after seaweed applications, among which PR8, PR12, CHORS2, CAGT, EDS1b and SAPB2a showed the highest inhibition values, with log values (RO versus Water plants) close to -2 (Figure 2A).

The GST1 gene, commonly associated with ascorbate peroxidase (APX) levels (Gullner et al., 2018), exhibited the highest induction values following RO treatment, with a 4.62-fold change in VN40 and a 4.83-fold change in RJ43, consistent with previous findings in Zarraonaindia et al. (2023), which showed that RO extract preferentially induced jasmonic acid (JA) related genes, while inhibiting salicylic acid (SA) responsive genes. In particular, PR3, PR4bis and PIN (gene function of PR6), whose expressions are known to be strongly dependent on JA signalling (Ali et al., 2018; Van Loon et al., 2006), were significantly higher in RO-treated leaves. Moreover, the expression of HSR-203J, known to be enhanced by JA signalling (Dufour et al., 2016), was also increased by RO treatments. On the other hand, RO was found to down-regulate PR-8, PR-12, PR-14, EDS1b, SAMPT1 and SAPB2a genes, which are known to be related to SA signalling (Adrian et al., 2012; Bodin et al., 2020; Dufour et al., 2016); this thus corroborates the antagonistic regulation of both JA and SA pathways suggested by other authors (Paolacci et al., 2017).

Both clones showed similar upregulated and downregulated gene profiles after R. okamurae treatments. However, overall, VN40 plants showed a more consistent downregulation of five genes, three of which were related to SA pathway ones (PR8, PR12 and SAPB2a), a nitrate transporter (NrT2) and a glycosyltransferase (CAGT). By contrast, in five cases (PR3, PIN, CHI2, GST1 and CAD2) upregulation was more pronounced in the RJ43 clone (Figure 2A). CAD2 expression, involved in the biosynthesis of monolignols (main component of lignin), was significantly upregulated by RO, especially in RJ43 plants. Lignans play an important role in plant defense and, indeed, one of the characteristic responses of plants to injury and disease is the reinforcement of cell walls (Ninkuu et al., 2022). Therefore, RO treated plants, and particularly the RJ43 clone, might have higher protection responses than untreated plants in the event of a pathogen infection.

2.2. Induction of phenolic compounds by RO

The concentrations of polyphenols in the leaves of Tempranillo VN40 plants increased after treatment with R. okamurae (Figure 3A). The concentrations of caftaric acid and coutaric acids were significantly higher in RO- and BB-treated plants than in the control (water). Consistent with this, at this sampling point, significant increases in stilbenes (total piceid and E-resveratrol) were also observed in RJ43 plants relative to the control ones (Figure 3B). Viniferins, on the other hand, were detected under the detection limits. The same stilbenes as those found in Zarraonaindia et al. (2023) were identified, albeit in higher concentrations, which is probably due to the additional third RO treatment applied in the present experiment.

In general, polyphenols reached higher concentrations in VN40 leaves than in RJ43 without any significant differences. However, stilbenes, key compounds for responding to biotic stresses, reached similar concentrations in RO leaves for both clones, which suggests that biostimulation with regards to these compounds is similar in these clones. Flavonols (quercetin 3-glucoside + glucuronide and myricetin 3-glucoside) were not affected by the treatments, which corraborates our previous results in Zarraonaindia et al. (2023). An increase in flavonoids has previously been observed after seaweed treatment in the berries of Tempranillo blanco plants (Cataldo et al., 2022), but, to our knowledge, such an increase has not been associated with leaves. In general, the results regarding phenolic compounds indicate that both clones are equally prepared for a possible infection.

2.3. Activity modulation of the antioxidant enzyme system by RO

SOD, APX and GR enzymes have been described as being related to defense mechanisms in plants. Generally, biostimulants derived from algae are known to induce the antioxidant system (García-Sánchez et al., 2022). However, in VN40 plants, SOD and GR activities did not show any significant differences between the three different treatments (W, BB and RO). By contrast, APX activity significantly increased for RO- and BB-treated plants (1.6 and 2.9 times higher than that of water respectively; Figure 4A), indicating that both treatments were able to reduce H₂O₂. This could be related to a H₂O₂ signaling mediated by the biostimulation, preparing VN40 for any potential stress. At the end of signalling, H₂O₂ must be metabolised, for which APX is needed. This is an interesting strategy, since H₂O₂ is a reactive oxygen species which can damage plant cells (Hasanuzzaman & Fujita, 2022). Importantly, the already high intrinsic SOD values found in the VN40 plants indicate that this clone relies heavily on H₂O₂ signalling and regulation in its basal state (under no biotic or abiotic stress). According to Pérez-López et al. (2015), high SOD activity could be considered an advantage as it helps plants protect themselves against potential oxidative damage. Therefore, overall, this high basal SOD could be the reason why the antioxidant system response to biostimulation was less pronounced in VN40 plants. Unfortunately, no reference was found in the literature to the effect of biostimulants on Vitis vinifera antioxidant enzymes. However, by comparing our results with those obtained under non-stress conditions, it can be noted that the SOD values observed in our study are higher than those reported for SOD in non-stressed Tempranillo (Salazar-Parra et al., 2012) and Öküzgözü (Özden et al., 2009).

By contrast, all the enzymes studied in RJ43 plants were affected by the RO treatment. SOD activity decreased 2.2- and 1.8-fold for RO and BB treatments relative to the control plants, respectively. Meanwhile, for the RO and BB treatments, there was an approximate 1.9-fold increase in APX and 1.7- and 1.3-fold increase in GR (Figure 4B). The increase in GR in RJ43 plants allows them to regenerate the important antioxidant molecule glutathione and increase the ability of plants to prepare for an oncoming stress (Noctor & Foyer, 1998). It is important to note that RJ43 seems to rely on APX rather than SOD when reducing the accumulation of H₂O₂.

Thus, our results indicate that each clone modulates antioxidant enzymes differently after RO biostimulation, with the RJ43 clone modulation benefiting more, since GR is activated to a greater extent than in the VN40 clone. However, it should be noted that elicitation may have been lower in the VN40 plants due to their already high basal SOD activity.

2.4. Modifications to photosynthetic pigments and photochemical parameters by RO

While the composition of photosynthetic pigments in RJ43 clone plants were not affected by treatments, VN40 clone plants decreased both chlorophyll a and b, as well as carotenes and xanthophylls, which is in line with the SPAD results (Table 1). Conversely, maximum photochemical efficiency (φ_PSII) decreased in the RJ43 plants, while the VN40 clone maintained it at levels similar to those of the control samples (W), despite significantly decreasing the greenness (SPAD) (Table 1). Previous studies have shown that the application of seaweeds in general, and the brown macroalgae Ascophyllum nodosum in particular, results in increased chlorophyll content in the leaves of different plants (e.g., tomato, dwarf French bean, wheat, barley and maize); this is likely due to the inhibition of chlorophyll degradation, which is caused partly by compounds, such as betaines, present in the seaweed extracts (Sangha et al., 2014). The R. okamurae extract used here does not contain betaine lipids, as was revealed by biochemical characterisation conducted by Córdoba-Granados et al. (2024), and as would be expected for an aqueous extract, which might justify the differences found with the aforementioned publication.

In general, our results revealed no effect, or just slight negative effects (depending on the clone) of the RO treatment on photosynthetic pigments and the photochemical parameters associated with them. However, it is worth mentioning that the VN40 plants were at a more advanced phenological stage than the RJ43 plants, which may also have contributed to the reduction in photosynthetic pigments observed in VN40. Nonetheless, the reduction was slight, and the values were still within the established range representing good photosynthesis activity in grapevine leaves (Casanova-Gascon et al., 2018).

3. Post inoculation state (S₂, 1dPI): responses to Plasmopara viticola

Grapevine resistance to downy mildew has a large genotypic component and defense reactions occur with variable promptness and magnitude, timing and intensity. Generally, it is assumed that highly susceptible grapevines show a weak defense response to P. viticola infection (Perazzolli et al., 2012), and therefore they rely heavily on external compounds to help withstand disease. Copper-based products directly target the pathogen, making them the most common control measure. By contrast, it is less common for seaweed to be directly toxic to fungi, their protective efficiency coming rather from their ability to elicit defense mechanisms in the plant. In this section, the effect of P. viticola at the molecular and metabolic levels is evaluated in Tempranillo clones (RJ43 and VN40) in order to assess the effectiveness of R. okamurae as an elicitor against downy mildew.

3.1. Gene expression modulation after Plasmopara viticola inoculation

The expression levels of most RO-modulated genes either remained stable or decreased at S₂ (1dPI) compared to S₁ (1dPB), a pattern that could be due to Tempranillo's inherent sensitivity to P. viticola. Tempranillo’s response to infection can usually be expected to be moderate, although P. viticola is known to be able to modulate JA pathway-related genes in certain grapevine varieties (Figueiredo et al., 2015; Guerreiro et al., 2016). In this study, in the RJ43 clone, six out of the seven RO-induced genes, particularly the PR4bis gene in the JA pathway, showed lower expression values after inoculation (Figure 2B; Table 2). Interestingly, while the expression levels also decreased in RO-treated VN40 plants, CHI2 expression markedly increased (332 %-fold change from S₂ to S₁), suggesting that this gene was primed by the seaweed application.

Remarkably, while the inhibition of SA-related genes was not maintained after infection in the VN40 plants, their downregulation was strengthened in the RJ43 clone (Figure 2B). Gene expression downregulation was particularly marked in PR12 and NrT2, as well as in PR8 and CALS, in RJ43 after P. viticola inoculation; this suggests that the extract was able to prime the plants, and therefore the inhibition of the expression of these genes became stronger when the grapevines were further subjected to biotic stress. Several stimuli have been shown to prime plants for an improved response to future stress, thereby triggering earlier, faster and/or stronger responses (Hilker et al., 2016; Mauch-Mani et al., 2017), among which seaweed extracts can be highlighted (Islam et al., 2021). It should be noted that elicitors may also be potentially used in defense priming strategies by downregulating certain genes (Landi et al., 2017), as is the case of susceptibility genes (S-genes) (Liu et al., 2022). However, to our knowledge, no previous evidence of downregulation associated with the priming of plant defense response genes has been described in the literature to date; therefore, the downregulation observed for PR12, NrT2, PR8 and CALS in the previously biostimulated plants requires further validation.

3.2. Concentrations of phenolic compounds after Plasmopara viticola inoculation

Total polyphenol concentration increased in all plants after infection; however, no clear trend was observed between treatments or clones (data not shown). Only the RJ43 clone plants exhibited an increase in total piceid, this enrichment being more acute in the plants previously treated with seaweed (1.15-fold for W, 1.31-fold for BB and 1.84-fold for RO). However, piceids, in their glucoside form, are known to be inactive against P. viticola (Gabaston et al., 2017). Other stilbenes have been directly correlated with resistance to fungal disease, and differences in the content and accumulation of stilbenes between susceptible and resistant genotypes is known to be key to overcoming infections (Burdziej et al., 2021; Paolocci et al., 2014; Vrhovsek et al., 2012). The biosynthesis of complex stilbenes in grapevine leaves is typically observed during infection around six days after inoculation (Vrhovsek et al., 2012; Burdziej et al., 2021), which may explain the low concentrations and differences observed in our early sampling; i.e., just 24 hours after infection. Despite this early sampling, ε-viniferin and ω-viniferin were detected in both clone plants in all the treatments (W, BB, RO), ε-viniferin concentration being significantly higher in the RO samples of RJ43 plants (Table S4). Susceptible genotypes are thought to accumulate resveratrol and piceid during infection, while other stilbenes, such as ε- and δ-viniferins, have been found to be mostly produced in resistant genotypes (Boso et al., 2012; Paolocci et al., 2014). Therefore, the increase in viniferins observed in the present study suggests that, despite Tempranillo being considered a susceptible variety, the clones studied here were exhibiting a certain level of resistance to infection. Further samplings at later time-points would provide valuable insights into the extent of this resistance.

3.3. Antioxidant system activity modulation after Plasmopara viticola infection

In both clones, SOD activity decreased with respect to the S₁ sampling point, especially in plants that had not been previously biostimulated, indicating that the oomycete was likely downregulating SOD activity in order to inhibit the plants’ defense mechanisms (Nascimento et al., 2019). Interestingly, in the VN40 clone, plants previously elicited by RO showed an increase of 138 %, 93 % and 73 % for SOD, APX and GR respectively in their leaves, relative to the water-treated plants. In the RJ43 clone, however, RO-biostimulated plants did not alter SOD in response to P. viticola inoculation, but APX and GR activities increased by 112 % and 57 % relative to the water samples. The significant induction of APX correlated with the pronounced overexpression of the GST1 gene (Figure 2A) in the RJ43 clone, suggesting that the ROS scavenging and detoxification mechanisms triggered by RO application in S₁ were further enhanced following P. viticola infection, thus helping to protect the cells from the combined signal induced by both the seaweed and the pathogen.

Overall, the results indicate that RO biostimulated the plant´s antioxidant metabolism, which was activated after pathogen inoculation, maintaining most of the enzymes at high values in both clones. This indicates that both clones potentially reduce the development capacity of the oomycete. The high SOD values in VN40 could promote some resistance, as high H₂O₂ levels are toxic for fungi and prevent the development of the pathogen. In fact, reactive oxygen species play an important role in pathogen resistance by directly strengthening host cell walls via the cross-linking of glycoproteins, lipid peroxidation and the activation of ROS-signalling networks, leading to the establishment of a resistance response (Figueiredo et al., 2017). However, as mentioned previously, H₂0₂ could also be toxic for plants (Hasanuzzaman & Fujita, 2022; Mittler et al., 2004); therefore, we hypothesise that to counteract it, VN40 regulated the high H₂0₂ levels with a higher activation of APX. These strategies seem to allow the delicate equilibrium between ROS production and scavenging to be maintained (Mittler et al., 2004).

Thus, we can conclude that R. okamurae seaweed treatment was able to prime both clones of the Vitis vinifera plants, allowing the plants to respond more efficiently when the infection occurred than the plants that had not been previously biostimulated by R. okamurae.

3.4. Modifications to photosynthetic pigments and photochemical parameters by Plasmopara viticola

Few significant differences were found in terms of pigments and photochemical parameters between treatments after infection (S₂). The VN40 clone plants treated with RO did not show any significant differences when compared to the water treated plants (Table S5). With regards to the RJ43 clone plants, no differences in terms of pigments, SPAD or photosynthetic efficiency were found between treatments; this demonstrates that any negative effect observed at the biostimulation sampling point (S₁) (Table 3) was transitory and quickly reverted.

P. viticola has been shown to affect the photosynthesis of susceptible grapevine cultivars by reducing the diffusion of CO₂ in leaf mesophyll (Jermini et al., 2020) and by inhibiting the enzymes involved in chlorophyll synthesis (Nogueira Júnior et al., 2020). However, in the present study, the sampling was performed one day after pathogen inoculation, and therefore the above effects had not yet been observed.

4. Evaluation of the protection conferred by Rugulopteryx okamurae extract against Plasmopara viticola

The protective efficiency of the copper-based treatment (BB) was higher than that of RO and occurred earlier in the period of infection: by days 3 and 10 for the incidence of clone VN40 and RJ43, respectively, and by day 10 for the severity of both clones (Figure 5). Moreover, a different level of protection was observed depending on the clone, which was more evident by day 17. While in VN40 the incidence and severity index were not significantly different between RO and W samples, in RO-treated RJ43 plants a disease reduction was observed, with 20 % incidence reduction and 25% severity reduction, both of which were significant. All these results align with the findings observed for this clone during both the biostimulation (S₁) and post-inoculation stages (S₂), which had already indicated that RO treatment elicited a higher response in RJ43 clone plants. It is widely known that clones show different susceptibility to fungal diseases (Molitor et al., 2018), as is the case for varieties (Boso et al., 2023). In fact, targeted clone selection represents an integral tool in the control strategy in Integrated Pest Management contributing to pesticide reduction in viticulture.

Because most research has been conducted in-vitro (e.g., on leaf discs), few scientific studies report the efficacy of agricultural products at the whole-plant level under controlled conditions (reviewed by: Adrian et al., 2024; Taillis et al., 2022). Furthermore, while various commercial plant protection products are known for their ability to inhibit fungal infections in grapevine (Llamazares De Miguel et al., 2022), little is known about such ability in seaweed-based products. The R. okamurae aqueous extract applied here contained alginate, fucoidan and laminarin (Córdoba-Granados et al., 2024) that could in part be responsible for the elicitation and disease suppression observed, as polysaccharides have been previously demonstrated to have such activity. For instance, a crude extract of Ulva armoricana green seaweed, rich in the polysaccharide ulvan, showed anti-powdery mildew activity in the leaf disc and in the plant after several grapevine foliar treatments (Jaulneau et al., 2011). The treatment of grapevine plants with laminarin, a polysaccharide from the brown algae Laminaria digidata, was found to reduce the infection of Botrytis cinerea and P. viticola by 55 % and 75 %, respectively (Aziz et al., 2003). Therefore, the present work represents a stepping-stone towards understanding the capacity of seaweed to protect plants from disease in agriculture, and natural defense elicitation by R. okamurae extract was demonstrated. However, further studies are needed to determine the relationship between the conferred rate of protection by R. okamurae and P. viticola disease progression, as the protection induced by natural elicitors could indeed be affected by the time interval between elicitation and pathogen attack, or the number of elicitation treatments given, among other factors. Comment by EMMA CANTOS VILLAR: I propose has been demonstrated (in the current paper)

Conclusion

Rugulopteryx okamurae extract (RO) was able to induce defense reactions in grapevine plants at both molecular and metabolic levels. Importantly, plants that had been previously treated with the seaweed responded more efficiently to Plasmopara viticola infection, leading to a reduction in the disease incidence and severity indexes. This suggests that Rugulopteryx okamurae elicitation was effective in priming plants, ultimately protecting them from the disease. The defense elicitation and extent of protection were however clone-dependent, being more effective in the tempranillo RJ43 clone plants.

Overall, the results of this study contribute to the future development of sustainable products based on seaweed formulations, which could be used in environmentally friendly protection strategies in viticulture. Despite RO-mediated disease suppression being less effective than with the copper-based treatment, the seaweed aqueous extract still showed promise as a sustainable biocontrol candidate. Its use in combination with other conventional products would be advisable for more effective disease control, and it could contribute to reducing the reliance on and dosage of copper in viticulture, a growing necessity and a hot topic in the agriculture sector. Furthermore, the results of this study present an opportunity to valorise the invasive R. okamurae seaweed by integrating it into a circular system. Further studies are required to characterise the bioactive compounds in the extract and to elucidate the precise mechanisms behind elicitation and disease suppression, given the complexity of the involved factors. Importantly, the differences in efficacy observed, even between clones, underscore the need to explore grapevine genomics and epigenomics further, extending research beyond grapevine variety.

Acknowledgments

This research is supported by SEAWINES Project (PID2020-112644RR-C21 and -C22) financed by MCIN/AEI/10.13039/501100011033. Juan José Córdoba-Granados thanks the support from PRE2021-100476 MICIU/AEI /10.13039/501100011033 and FSE+. Asier Cámara thanks the support from Ikertalent 2022 predoctoral programme financed by the Basque Government. Grant EQC2021-007261-P funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR.