Foliar Applications of Calcium, Silicon and Their Combination: A Tool to Improve Grape Composition and Quality

Citation: Garde-Cerdán, T.; González-Lázaro, M.; Alonso-Ortiz de Urbina, D.; Sáenz de Urturi, I.; Marín-San Román, S.; Murillo-Peña, R.; Torres-Díaz, L.L.; Pérez-Álvarez, E.P.; Fernández, V. Foliar Applications of Calcium, Silicon and Their Combination: A Tool to Improve Grape Composition and Quality. Appl. Sci. 2023,13, 7217. https://doi.org/10.3390/ app13127217 Academic Editors: Joanna Kapusta-Duch, Teresa Leszczy´nska and Ewa Pi ˛atkowska Received: 30 May 2023 Revised: 9 June 2023 Accepted: 12 June 2023 Published: 16 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). applied sciences Article Foliar Applications of Calcium, Silicon and Their Combination: A Tool to Improve Grape Composition and Quality Teresa Garde-Cerdán1,* , Miriam González-Lázaro 1, David Alonso-Ortiz de Urbina 1, Itziar Sáenz de Urturi 1, Sandra Marín-San Román1, Rebeca Murillo-Peña 1, Lesly L. Torres-Díaz 1, Eva P. Pérez-Álvarez 1,* and Victoria Fernández 2 1 Grupo VIENAP, Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Ctra. de Burgos Km. 6, 26007 Logroño, Spain; [email protected] (M.G.-L.); [email protected] (D.A.-O.d.U.); [email protected] (I.S.d.U.); [email protected] (S.M.-S.R.); r[email protected] (R.M.-P.); [email protected] (L.L.T.-D.) 2Departamento de Sistemas y Recursos Naturales, Universidad Politécnica de Madrid, Ciudad Universitaria, s/n., 28040 Madrid, Spain; v[email protected] *Correspondence: teresa.garde.cer[email protected] (T.G.-C.); [email protected] (E.P.P.-Á.) Abstract: Foliar nutrient sprays are broadly used in commercial agriculture. To date, the application of Ca and Si has been little explored in vineyard production but may lead to positive responses at various levels. Therefore, the aim of this work was to evaluate the effect of supplying single or combined calcium (Ca, 120 mM) and silicon (Si, 120 mM) sprays in Tempranillo grape composition. Foliar treatment with Ca + Si foliar enhanced all families of aromatic compounds, whereas single Ca and Si sprays induced lower effects. Regarding phenolic compounds, all foliar treatments led to minor effects. However, all three foliar Ca and Si treatments increased the total grape amino acid content. Consequently, the application of combined Ca and Si sprays to a vineyard is recommended as a tool for improving grape quality. Keywords: volatile composition; aroma; phenolic compounds; nitrogen composition; Tempranillo; foliar application; must; grape 1. Introduction Grapevine is a major agricultural crop that is often exposed to variable stress conditions during its lifespan and growing cycle [ 1 , 2 ]. Among other husbandry techniques, fertilization has been shown to play a key role in grapevine cultivation, since it may affect berry and vine quality [ 3 , 4 ], in addition to improving disease resistance [ 5 , 6 ] and tolerance to other stress factors [7]. Calcium is an essential nutrient for plants that plays key signaling and structural roles [ 8 ]. Roots absorb this element from the soil solution, and Ca is subsequently transported to the shoots in the xylem [ 8 ]. Calcium mobility in plants is generally low, this element being chiefly accumulated in mature organs despite the high Ca requirements of actively growing plant parts such as expanding leaves, flowers or fruits [9,10]. The delivery of Ca to developing fruits depends on xylem transport and transpiration [ 11 , 12 ], but a contribution of pedicel phloem transport has also been reported [ 9 ]. The high stomatal densities of leaves compared to fruits lead to higher transpiration and Ca accumulation rates in foliar tissues compared to fruiting organs [12]. Although Ca is normally available in soils in sufficient amounts, the occurrence of localized Ca deficiencies (i.e., Ca-related disorders) in fruits and vegetables is a common agricultural production problem, which can lead to major economic losses at harvest and postharvest [ 13 ]. Calcium-related disorders may involve an array of alterations including the deterioration of cell walls and membranes [ 13 ], with many physiological and anatomical Appl. Sci. 2023,13, 7217. https://doi.org/10.3390/app13127217 https://www.mdpi.com/journal/applsci Appl. Sci. 2023,13, 7217 2 of 15 symptoms in plant organs as recently described for grapevine [ 14 ]. Given the limited Ca translocation from leaves to fruits during fruit growth and development [ 10 , 15 ], the supply of Ca as sprays onto fruit surfaces is recommended [ 16 , 17 ], as also evaluated in some studies performed with grapevine [3]. On the other hand, Si is a nonessential element that has been found to be beneficial for improving crop yield and quality and also plant tolerance to various abiotic and biotic stress factors [ 18 ]. The effect of a Si supply on grapevine has been assessed in a few investigations [ 19 – 21 ], which reported positive changes in grape quality and must composition. Silicon is often provided as foliar sprays that have been shown to induce positive plant responses in terms of improved quality, yield and pest and disease tolerance [22,23]. Foliar nutrient sprays are nowadays widely used in plant production, chiefly as a complementary strategy to root treatments [ 24 , 25 ]. Foliar-applied nutrient solutions may be absorbed by plant surfaces via stomata, the cuticle and cuticular irregularities, trichomes, veins and other modified epidermal structures, such as lenticels [ 26 ]. Research in the last 50 years largely focused on the mechanisms of cuticular penetration [ 24 ], with limited focus on the permeability of other epidermal structures such as stomata or trichomes, which may have a major contribution to the foliar absorption process [ 26 , 27 ]. Recent studies are providing evidence for their major physical and chemical heterogeneity of plant surfaces, which are generally covered with a cuticle [ 26 ]. Indeed, the occurrence of cell wall polysaccharides in [ 28 – 30 ] and even at the very surface of the cuticle [ 31 ] has been shown for few species and plant organs, and this may have major implications for understanding the mechanisms of agrochemical spray foliar permeability or pest and pathogen attack, among others [26]. Aware of the positive results reported after the application of Ca and Si sprays to crop plants in the few studies available as reported above, the aim of this investigation was to evaluate the effect of Ca and Si supplied as either single elements or in combination, in terms of Tempranillo berry yield, quality and subsequent must chemical composition under the prevailing growing conditions in La Rioja (Spain). 2. Materials and Methods 2.1. Vineyard, Treatments and Grape Samples Tempranillo grape (Vitis vinifera L.) variety plants grown in Finca La Grajera (Spain) were used for trial development. The vine was planted in 1995 (2.80 m × 1.20 m) and the experiment was performed during the 2020 season. Foliar sprays included Ca nitrate (CaNO 3 4H 2 O ≥ 99.0%, Sigma-Aldrich-Merck, Darmstadt, Germany) or/and 120 mM silicic acid potassium solution (Tradecorp, Madrid, Spain), which were applied separately or in combination. The commercial products were used to prepare solutions with concentrations of 120 mM Ca, 120 mM Si and 120 mM Ca + 120 mM Si. Foliar sprays contained 0.1% (v/v) Genapol X-80 surfactant (Sigma-Aldrich-Merck). Control plants were foliar treated with 0.1% Genapol solutions. All treatments were applied twice, at veraison and one week later, and approximately delivered 200 mL of foliar spray solution per plant. Foliar applications were carried out in triplicate and were performed following a completely randomized block design, with 3 vines for each treatment and repetition. For harvesting, grapes from all treatments were picked at the optimal technological maturity, i.e., when the weight of 100 berries remained constant and the probable alcohol reached 13 (% v/v). A random set of 150 grape berries was picked, per treatment and replicate, and frozen at − 20 ◦ C until the determination of volatile (50 berries), phenolic (50 berries) and nitrogen (50 berries) composition. Another set of 100 berries was used to obtain the average berry weight. Then, the berries were crushed, and the physico-chemical parameters were determined in the must. Appl. Sci. 2023,13, 7217 3 of 15 2.2. Must Physico-Chemical Parameters Must general parameters were analyzed by the OIV [ 32 ] official methods by measuring total acidity, pH, ◦ Brix and probable alcohol. Glucose, glucose + fructose (calculating fructose concentration by the difference), tartaric and malic acids, total phenols, ammonium and amino nitrogen (N) (yeast assimilable N (YAN) was the sum of the two N fractions) determined using Miura One equipment (TDI, Barcelona, Spain). As treatments included 3 repetitions, the results of enological parameters also presented as the mean of 3 determinations (n = 3). 2.3. Volatile Compound Determination The volatile composition was determined in the musts by headspace solid-phase microextraction (HS-SPME) and subsequent analysis by GC-MS (Agilent, Palo Alto, CA, USA), according to the method described by Garde-Cerdán et al. [ 33 ]. Briefly, aliquots of 9 mL must plus 2.5 g of NaCl and 10 µ L of 2-octanol (internal standard) were put in 20 mL vials. Solutions were then stirred, and the vials were closed for subsequent GC-MS analysis. After completion of the extraction process, the fiber was inserted into the GC injection port ( 250 ◦C ) for 15 min. The capillary column used was SPB ™ -20 ( 30 m ×0.25 mm I.D. ×0.25 µm of film thickness) (Supelco, Bellenfonte, PA, USA). Identification of volatile compounds was carried out using the NIST library, by comparing with the mass spectra and retention time of the chromatographic standards. Since foliar applications involved 3 repetitions, the results of grape volatile compounds are expressed as an average of 3 replicates (n = 3). 2.4. Analysis of Phenolic Compounds 2.4.1. Grape Phenolic Compound Extraction Grape berry phenolic compounds were extracted according to Garde-Cerdán et al. [ 34 ]. Briefly, 50 g grapes were added to a mixture of methanol/water/formic acid (50:48.5:1.5, v/v/v) (50 mL). Then, this mixture was homogenized (18,000 rpm, during 1 min). After that, samples were macerated (ultrasonic bath) for 10 min and, then, were centrifuged (3640 × g, 10 ◦ C, 10 min). The supernatant was separated, and the resulting pellet was extracted once again. Then, the supernatants collected were mixed, and samples were stored at −20 ◦C for further analysis. 2.4.2. Extract of Non-Anthocyanin Phenolic Compounds The extraction of non-anthocyanin phenolic compounds was performed following the procedures described by Garde-Cerdán et al. [ 34 ], by using PCX SPE cartridges ( 500 mg , 6 mL ; Bond Elut Plexa, Agilent, Santa Clara, CA, USA). Cartridges were put in the extraction system (Visiprep TM SPE Vacuum Manifold, Sigma-Aldrich-Merck). Then, 3 mL of grape phenolic extracts were diluted with 0.1 N HCl (9 mL). The cartridges were conditioned with methanol (5 mL) and water (5 mL). After that, the diluted samples were passed through the cartridges and washing was undertaken (5 mL of 0.1 N HCl + 5 mL of water); 2 × 3 mL of ethanol were used in order to elute the non-anthocyanin phenolic compounds fraction. Then, this fraction was dried in an evaporator (35 ◦ C) and redissolved in a methanol ( 1.5 mL ) aqueous solution (20% v/v). The anthocyanin-free fraction was employed in order to determine flavonols, hydroxycinnamic and hydroxybenzoic acids, stilbenes and flavanols. 2.4.3. Phenolic Compound Determination Phenolic compounds were analyzed as described by Garde-Cerdán et al. [ 34 ] using an Agilent 1260 Infinity II chromatograph equipped with a diode array detector (DAD). Samples were filtered and injected into a Licrospher ® 100 RP-18 column (Agilent) with pre-column Licrospher ® 100 RP-18 (Agilent) (40 ◦ C). For the analysis of anthocyanins, grape extracts were injected (10 µ L). For the analysis of non-anthocyanin phenolic compounds (flavonols, hydroxycinnamic and hydroxybenzoic acids, stilbenes and flavanols), 20 µ L was injected. For quantification, DAD chromatograms were extracted: anthocyanins (520 nm), Appl. Sci. 2023,13, 7217 4 of 15 flavonols (360 nm), hydroxycinnamic acids and stilbenes (320 nm) and gallic acid and flavanols (280 nm). Since field treatments were performed in triplicate, the results for phenolic compounds are the average of the analyses of three samples (n = 3). 2.5. Nitrogen Compound Determination The analyses of amino acids were carried out by HPLC using an Agilent 1260 Infinity Series coupled to a DAD and a fluorescence detector (FLD) according to Garde-Cerdán et al. [ 35 ]; 100 µ L of sarcosine and 100 µ L de norvaline (internal standards) were added to 5 mL of must. The mixture was submitted to a derivatization with o-phthaldialdehyde (Agilent) and with 9-fluorenylmethylchloroformate (Agilent). The nitrogen compound separations were undertaken on a Hypersil ODS (250 × 4.0 mm, I.D. 5 µ m, Agilent) column, the injected volume being 10 µ L. The identification of the nitrogen compounds was carried out according to the retention times of the standards (Sigma-Aldrich, St. Louis, MO, USA). Their quantification was undertaken using the calibration graphs (R2> 0.96). Since the foliar applications were carried out in triplicate, the results of amino acids are expressed as the average of the 3 replicates (n = 3). 2.6. Statistical Analyses The statistical elaboration of the data was conducted using SPSS Version 21.0 (SPSS, Chicago, IL, USA). Physico-chemical parameters and volatile, phenolic and nitrogen compounds data were managed using the variance analysis (ANOVA) (p≤0.05). 3. Results and Discussion In this study, we assessed the potential of Ca and Si sprays supplied two times during the growing season for improving grape quality by measuring several physico-chemical parameters. Positive results were obtained, especially when treating vines with combined Ca plus Si sprays as described in the paragraphs below for the different variables measured. 3.1. Must Enological Parameters Table 1shows general grape parameters for control and foliar sprayed vines, which were supplied Ca, Si and Ca + Si. Foliar Ca application decreased the weight of 100 berries, as previously observed by Martins et al. [ 36 ]. This effect may be associated with a delay in berry maturation. However, ◦ Brix was unaffected by the three foliar treatments (Table 1). On the other hand, Ca spraying increased total phenols, amino N and YAN contents in must compared to the control must; except for total phenols, the effect of Ca application was significant in comparison with Si and Ca + Si foliar treatments (Table 1). Foliar Si sprays increased the fructose must content in comparison to the values recorded for control, Ca and Ca + Si musts. Furthermore, Ca + Si foliar application led to musts with a higher ammonium N content than control and Si musts (Table 1). Therefore, the overall effect of foliar treatments on general must parameters was slight. Gomes et al. [ 37 ] described an increase in some acids in the grapes after spraying vines with Si and Ca, in contrast with the general parameters shown in Table 1. However, Losada et al. [ 21 ] observed an absence of effect on the ◦ Brix, pH and total acidity of must after foliar application of monosilicic acid to grapevines, which is in agreement with our results (Table 1). Appl. Sci. 2023,13, 7217 5 of 15 Table 1. General parameters in musts from control and Ca, Si and Ca + Si foliar treatments. Control Ca Si Ca + Si Weight of 100 berries (g) 235.30 ±25.45 b 183.03 ±33.68 a 222.00 ±19.29 ab 239.63 ±0.60 b ◦Brix 22.80 ±1.73 a 22.53 ±1.10 a 24.83 ±1.76 a 24.00 ±1.11 a Probable alcohol (% v/v) 13.33 ±1.18 a 13.13 ±0.76 a 14.72 ±1.22 a 14.14 ±0.77 a Glucose + Fructose (g/L) 231.95 ±19.45 a 232.52 ±11.04 a 265.52 ±23.85 a 249.23 ±19.62 a Glucose (g/L) 116.27 ±10.39 a 115.63 ±5.28 a 130.83 ±11.32 a 124.31 ±1048 a Fructose (g/L) 115.68 ±9.09 a 111.68 ±9.22 a 133.02 ±31.74 b 118.19 ±16.90 a pH 3.53 ±0.14 a 3.73 ±0.14 a 3.61 ±0.02 a 3.68 ±0.24 a Total acidity (g/L) * 5.23 ±0.89 a 4.72 ±0.16 a 4.95 ±0.10 a 5.40 ±0.89 a Tartaric acid (g/L) 5.02 ±0.37 a 5.03 ±0.59 a 4.42 ±0.13 a 4.48 ±0.86 a Malic acid (g/L) 2.64 ±0.05 a 2.21 ±0.26 a 2.23 ±0.18 a 2.63 ±0.46 a Total phenols (mg/L) 472.47 ±42.30 a 722 ±172.75 b 625.57 ±6.58 ab 592.33 ±50.60 ab Ammonium nitrogen (mg N/L) 91.78 ±15.13 a 123.50 ±5.07 ab 98.02 ±13.29 a 138.84 ±27.30 b Amino nitrogen (mg N/L) 108.24 ±7.92 a 205.92 ±30.80 b 119.97 ±17.16 a 97.97 ±9.24 a YAN (mg N/L) ** 200.02 ±22.12 a 329.42 ±35.87 b 217.99 ±26.04 a 236.81 ±23.80 a * As g/L of tartaric acid. ** YAN: yeast assimilable nitrogen. All parameters are listed with the standard deviation (n = 3). Different letters indicate significant differences between the treatments (p≤0.05). 3.2. Effect of the Ca, Si and Ca + Si Foliar Treatments on Must Volatile Composition The must volatile compounds content of control and Ca, Si and Ca + Si foliar treatments is shown in Figures 1and 2and Table 2. Terpenoids are one of the most important families of varietal compounds for aromatic typicity, since these compounds contribute to floral and citrus aroma [ 38 ]. The application of Ca + Si sprays to vines strongly increased the total must terpenoids content (Figure 1l) because of the increase in the concentration of limonene, p-cymene, linalool, α -terpineol, nerol, geraniol and neral (Figure 1a–d,f,g,k), although the content of geranyl acetone showed a decrease (Figure 1j). Foliar Ca application also showed positive effects in terms of varietal aromatic quality, as it increased the synthesis of p-cymene, geraniol and neral (Figure 1b,g,k), consequently raising the total terpene concentration (Figure 1l). However, foliar Si treatment did not affect the total concentration of this family of aromatic compounds (Figure 1l), despite it increased p-cymene, linalool and nerol concentration (Figure 1b,c,f), whereas it decreased the content of isogeraniol and geranic acid (Figure 1h,i). Regarding C 13 norisoprenoids, which together with terpenoids is the other major family of varietal compounds [ 39 – 41 ], the only treatment that significantly favored their synthesis was the foliar application of Ca + Si (Figure 2), with the single exception of β -ionone, whose concentration decreased in the musts after this application (Figure 2c). Calcium spray application to grapevines had no effect on the amount of these aromatic compounds (Figure 2), while the application of Si only affected the concentration of β -ionone in the musts (Figure 2c), decreasing it, so that the concentration of total C 13 norisoprenoids also decreased (Figure 2g). As for benzenoid compounds, all spray treatments had an influence on the concentration of these compounds, which generally increased, especially in the Ca + Si combined treatment (Table 2). The application of Ca and Si favored the synthesis of 2-phenylethanol and 2-phenylethanal, while the Ca + Si treatment also increased the concentration of eugenol in the musts (Table 2). Benzenoid compounds also belong to the varietal aroma, conferring pleasant, fruity aromas, and 2-phenyethanol is characterized by the aroma of rose [ 42 ]. These compounds come from phenolic acids or from aromatic amino acids [ 41 , 43 ]. Appl. Sci. 2023,13, 7217 6 of 15 Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 of 16 Figure 1. Terpenoids concentration (µg/L) in musts of control (C) and Ca, Si and Ca + Si foliar treatments. The mean volatile compound contents are shown the standard deviations SD (n = 3). Different letters indicate significant differences between foliar applications (p ≤ 0.05). 0.00 0.05 0.10 0.15 0.20 0.25 Concentration (mg/L) (a) Limonene 0.00 0.10 0.20 0.30 0.40 0.50 0.60 Concentration (mg/L) (c) Linalool aab b c 0.00 0.02 0.04 0.06 0.08 Concentration (mg/L) (e) Citronellol aa a a 0.00 0.10 0.20 0.30 0.40 Concentration (mg/L) (d) a-Terpineol aaa b 0.00 0.05 0.10 0.15 0.20 0.25 Concentration (mg/L) (b) p-Cymene a bb c 0.00 0.20 0.40 0.60 0.80 Concentration (mg/L) (f) Nerol a ab b c 0.00 0.50 1.00 1.50 2.00 2.50 Concentration (mg/L) (g) Geraniol a b ab c 0.00 0.20 0.40 0.60 0.80 Concentration (mg/L) (i) Geranic acid a bc b c 0.00 0.05 0.10 0.15 Concentration (mg/L) (k) Neral aa bb 0.00 0.05 0.10 0.15 0.20 0.25 Concentration (mg/L) (j) Geranyl acetone a b bb 0.00 1.00 2.00 3.00 4.00 5.00 Concentration (mg/L) (l) Total terpenoids C Ca Si Ca+Si aa b c 0.00 0.05 0.10 0.15 0.20 Concentration (mg/L) (h) Isogeraniol a bab b Figure 1. Terpenoids concentration ( µ g/L) in musts of control (C) and Ca, Si and Ca + Si foliar treatments. The mean volatile compound contents are shown the standard deviations SD (n = 3). Different letters indicate significant differences between foliar applications (p≤0.05). Appl. Sci. 2023,13, 7217 7 of 15 Appl. Sci. 2023, 13, x FOR PEER REVIEW 7 of 16 Figure 2. C13 norisoprenoids concentration (µg/L) in musts from control (C) and Ca, Si and Ca + Si foliar treatments. The volatile compound concentrations are means ± SD (n = 3). Different letters indicate significant differences between foliar applications (p ≤ 0.05). As for benzenoid compounds, all spray treatments had an influence on the concentration of these compounds, which generally increased, especially in the Ca + Si combined treatment (Table 2). The application of Ca and Si favored the synthesis of 2-phenylethanol and 2-phenylethanal, while the Ca + Si treatment also increased the concentration of eugenol in the musts (Table 2). Benzenoid compounds also belong to the varietal aroma, conferring pleasant, fruity aromas, and 2-phenyethanol is characterized by the aroma of rose [42]. These compounds come from phenolic acids or from aromatic amino acids [41,43]. 0.00 2.00 4.00 6.00 8.00 10.00 Concentration (mg/L) (a) (E)-b-Damascenone aa b a 0.00 0.50 1.00 1.50 2.00 Concentration (mg/L) (c) b-Ionone a a b b 0.00 0.05 0.10 0.15 0.20 Concentration (mg/L) (e) TDN aa b a 0.00 0.20 0.40 0.60 0.80 Concentration (mg/L) (d) b-Cyclocitral aa b a 0.00 0.10 0.20 0.30 0.40 0.50 Concentration (mg/L) (b) (Z)-b-Damascenone aa b a 0.00 0.20 0.40 0.60 0.80 1.00 Concentration (mg/L) (f) Methyl jasmonate a a b a 0.00 2.00 4.00 6.00 8.00 10.00 12.00 Concentration (mg/L) (g) Total C13 norisoprenoids C Ca Si Ca+Si bab c a Figure 2. C 13 norisoprenoids concentration ( µ g/L) in musts from control (C) and Ca, Si and Ca + Si foliar treatments. The volatile compound concentrations are means ± SD (n = 3). Different letters indicate significant differences between foliar applications (p≤0.05). Of the two esters found in the musts, only the concentration of methyl salicylate was influenced by foliar applications, decreasing in the Si treatment and increasing in association with Ca + Si spray application, the total content following a similar trend (Table 2). Nonetheless, esters are present in grapes in small quantities. These compounds are related to fruity aromas and, hence, play an important role in wine aroma because they are formed in high amounts during the alcoholic fermentation [44]. Appl. Sci. 2023,13, 7217 8 of 15 Table 2. Benzenoid compounds, esters, alcohols, carbonyl compounds and C6 compounds concentrations (µg/L) in musts from control and Ca, Si and Ca + Si foliar treatments. Control Ca Si Ca + Si Benzenoid compounds 2-Phenylethanol 6.68 ±1.23 a 11.32 ±1.07 b 14.86 ±2.54 b 14.84 ±3.53 b 2-Phenylethanal 8.25 ±1.35 a 18.58 ±4.08 b 16.69 ±2.10 b 26.84 ±5.05 c Benzyl alcohol 2.40 ±0.48 ab 1.85 ±0.47 a 2.46 ±0.49 ab 2.78 ±0.12 b Eugenol 0.02 ±0.00 a 0.03 ±0.01 a 0.04 ±0.01 a 0.06 ±0.01 b Total 17.35 ±3.05 a 31.78 ±2.62 b 34.04 ±3.53 b 44.52 ±8.47 c Esters Hexyl acetate 0.07 ±0.01 ab 0.06 ±0.01 a 0.06 ±0.01 a 0.09 ±0.02 b Methyl salicylate 0.12 ±0.01 b 0.11±0.02 b 0.06 ±0.01 a 0.33 ±0.02 c Total 0.20 ±0.02 b 0.18 ±0.01 b 0.12 ±0.01 a 0.42 ±0.01 c Alcohols 1-Heptanol 0.05 ±0.01 a 0.13 ±0.02 b 0.18 ±0.02 b 0.30 ±0.06 c 1-Octanol 0.48 ±0.08 a 0.76 ±0.12 a 0.70 ±0.10 a 1.83 ±0.34 b 1-Nonanol 0.24 ±0.04 a 0.27 ±0.05 a 0.51 ±0.09 b 0.96 ±0.14 c 1-Octen-3-ol 0.80 ±0.08 a 0.70 ±0.14 a 0.67 ±0.03 a 1.19 ±0.12 b 2-Ethyl-1-hexanol 0.89 ±0.15 b 0.57 ±0.12 a 0.56 ±0.12 a 2.38 ±0.11 c Total 2.46 ±0.34 a 2.44 ±0.24 a 2.62 ±0.12 a 6.66 ±0.42 b Carbonyl compounds Heptanal 0.06 ±0.00 a 0.08 ±0.02 a 0.05 ±0.01 a 0.11 ±0.02 b Octanal 0.07 ±0.01 a 0.08 ±0.01 a 0.07 ±0.01 a 0.06 ±0.01 a (E)-2-Octenal 0.15 ±0.03 a 0.16 ±0.03 a 0.14 ±0.01 a 0.17 ±0.03 a Nonanal 0.94 ±0.08 a 1.37 ±0.32 b 0.79 ±0.14 a 1.63 ±0.19 b (E)-2-Nonenal 0.14 ±0.03 a 0.23 ±0.05 b 0.12 ±0.02 a 0.21 ±0.02 b Decanal 0.22 ±0.04 c 0.15 ±0.02 b 0.12 ±0.02 ab 0.09 ±0.02 a (E,E)-2,4-Hexadienal 2.83 ±0.17 b 1.79 ±0.41 a 1.12 ±0.20 a 2.86 ±0.55 b (E,E)-2,4-Heptadienal 2.29 ±0.42 c 1.16 ±0.20 b 0.56 ±0.12 a 2.57 ±0.34 c (E,E)-2,4-Nonadienal 0.16 ±0.02 b 0.15 ±0.02 ab 0.11 ±0.02 a 0.26 ±0.03 c (E,E)-2,4-Decadienal 0.04 ±0.01 a 0.04 ±0.01 a 0.03 ±0.01 a 0.06 ±0.01 b γ-Decalactone 0.31 ±0.06 a 0.42 ±0.07 ab 0.43 ±0.10 ab 0.63 ±0.17 b 6-Methyl-3,5-heptadien-2-one 0.05 ±0.01 b 0.02 ±0.00 a 0.03 ±0.00 a 0.07 ±0.01 c Total 4.95 ±0.19 b 4.49 ±0.13 b 2.99 ±0.36 a 6.15 ±0.77 c C6 compounds Hexanal 80.75 ±18.07 a 100.09 ±17.52 a 86.51 ±13.31 a 111.05 ±20.55 a n-Hexanol 46.85 ±5.20 a 45.33 ±10.17 a 66.65 ±2.15 b 65.55 ±11.66 b (E)-2-Hexenal 36.31 ±7.23 a 43.23 ±4.70 a 35.66 ±6.12 a 46.02 ±9.12 a (Z)-3-Hexen-1-ol+(E)-2-Hexen-1-ol 2.95 ±0.57 ab 2.09 ±0.39 ab 1.95 ±0.40 a 3.01 ±0.69 b Total 166.86 ±22.47 a 190.74 ±22.85 ab 190.78 ±19.23 ab 225.63 ±41.71 b The volatile compounds contents are listed with the standard deviation (n = 3). Different letters indicate significant differences between foliar applications (p≤0.05). Regarding alcohols, only the Ca + Si foliar treatment increased their total concentration (Table 2). The application of Ca had an inverse effect on the concentration of two alcohols, 1-heptanol and 2-ethyl-1-hexanol, the first one increasing and the latter decreasing (Table 2). The same result was observed for Si, and, moreover, after this foliar application, 1-nonanol concentration increased in the musts. These compounds are related to the green and herbaceous aromas of grapes and wines [45]. The largest family of volatiles found in the musts was carbonyl compounds, whose total content increased when Ca + Si was applied to the vines and decreased when Si was applied, with no effect associated with Ca sprays (Table 2). Calcium treatments actually favored the synthesis of nonanal, (E)-2-nonenal, decanal and (E,E)-2,4-heptadienal, whereas it decreased the concentration in the musts of (E,E)-2,4-hexadienal and 6-methyl3,5-heptadien-2-one. Foliar application of Si decreased the must concentrations of decanal, (E,E)-2,4-hexadienal, (E,E)-2,4-heptadienal and 6-methyl-3,5-heptadien-2-one. However, Appl. Sci. 2023,13, 7217 9 of 15 the combined Ca + Si application favored the synthesis of most of these compounds, i.e., heptanal, nonanal, (E)-2-nonenal, (E,E)-2,4-nonadienal, (E,E)-2,4-decadienal, γ -decalactone and 6-methyl-3,5-heptadien-2-one, and only decreased the content of decanal (Table 2). Carbonyl compounds are presented in low quantities in grapes. These compounds mostly are formed during alcoholic fermentation and present one or more aldehyde and ketone functions [46]. Finally, the C6 compounds, which are pre-fermentative aromas related to green and herbaceous notes [ 45 ], were hardly modified by the foliar treatments supplied to the vineyard, since only the concentration of n-hexanol was affected, increasing with the applications of Si and Ca + Si (Table 2) and only showing a total concentration increase with Ca + Si spray. We failed to find any study in which the effect of Ca and Si application to the vineyard was evaluated in terms of the aromatic composition of the grapes, as assessed in our investigation. 3.3. Influence of Foliar Ca, Si and Ca + Si Treatments on Grape Phenolic Compounds Table 3shows the results of grape phenolic composition in control and foliar-treated grapevines. Table 3. Phenolic compound concentration (mg/kg) in grapes from control and Ca, Si and Ca + Si foliar treatments. Control Ca Si Ca + Si Anthocyanins Delphinidin-3-glc 78.63 ±7.06 a 73.38 ±16.52 a 117.93 ±13.51 b 93.43 ±8.72 ab Cyanidin-3-glc 13.36 ±0.27 a 11.78 ±3.35 a 25.50 ±7.91 b 16.47 ±4.16 ab Petunidin-3-glc 61.52 ±3.98 a 59.88 ±11.44 a 50.19 ±56.05 a 72.35 ±3.09 a Peonidin-3-glc 26.46 ±1.77 a 27.53 ±7.04 a 44.99 ±13.29 a 35.04 ±0.83 a Malvidin-3-glc 188.29 ±8.85 a 217.85 ±27.95 a 216.19 ±4.13 a 225.16 ±47.53 a Delphinidin-3-acglc 7.59 ±0.56 a 7.56 ±0.80 a 8.55 ±0.29 a 8.04 ±0.55 a Cyanidin-3-acglc 3.86 ±0.04 a 3.83 ±0.14 a 3.79 ±0.17 a 3.84 ±0.04 a Petunidin-3-acglc 5.89 ±0.34 a 5.82 ±0.44 a 6.05 ±0.08 a 6.05 ±0.36 a Peonidin-3-acglc 16.72 ±0.57 a 16.07 ±1.86 a 18.33 ±1.12 a 17.62 ±1.24 a Malvidin-3-acglc 3.68 ±0.11 a 3.64 ±0.08 a 3.54 ±0.11 a 3.61 ±0.03 a Delphinidin-3-cmglc 11.23 ±1.01 a 12.89 ±0.81 a 10.92 ±1.15 a 12.33 ±1.02 a Cyanidin-3-cmglc 5.30 ±0.13 ab 4.82 ±0.38 a 6.22 ±0.63 b 5.61 ±0.81 ab Petunidin-3-cmglc 4.14 ±0.09 a 4.33 ±0.22 a 3.71 ±0.06 a 4.23 ±0.48 a Peonidin-3-cmglc 13.96 ±0.17 a 14.21 ±2.08 a 14.57 ±1.19 a 14.56 ±0.53 a Malvidin-3-cis-cmglc 4.92 ±0.31 ab 5.18 ±0.16 b 4.38 ±0.17 a 4.98 ±0.48 ab Malvidin-3-trans-cmglc 9.03 ±0.42 a 9.14 ±1.01 a 10.44 ±0.73 a 10.41 ±0.24 a Malvidin-3-cfglc 55.04 ±3.91 a 62.37 ±8.52 a 49.38 ±11.05 a 58.57 ±11.17 a Total 509.62 ±15.49 a 540.28 ±68.71 a 594.69 ±82.25 a 592.32 ±40.16 a Flavonols Myricetin-3-glcU+3-gal 23.61 ±1.16 a 32.53 ±7.83 a 29.08 ±1.15 a 31.82 ±3.84 a Myricetin-3-glc 78.09 ±2.09 a 111.54 ±32.97 a 110.22 ±8.88 a 121.79 ±16.72 a Quercetin-3-glcU 27.17 ±2.55 a 42.39 ±17.18 a 36.57 ±4.26 a 35.06 ±3.43 a Quercetin-3-glc 31.39 ±5.88 a 50.16 ±23.86 a 49.84 ±4.98 a 61.51 ±12.84 a Laricitrin-3-glc 13.31 ±0.59 a 21.15 ±5.21 ab 20.06 ±1.54 ab 23.91 ±4.28 b Kaempferol-3-gal 8.30 ±0.02 a 8.64 ±0.17 a 8.99 ±0.55 a 9.30 ±0.74 a Kaempferol-3-glcU+3-glc 1.92 ±0.20 a 3.74 ±2.55 a 2.70 ±0.25 a 4.13 ±1.65 a Isorhamnetin-3-glc 3.05 ±0.92 a 4.91 ±2.12 a 9.29 ±0.56 b 11.78 ±1.66 b Syringetin-3-glc 7.71 ±0.35 a 12.75 ±3.74 a 12.11 ±1.81 a 15.74 ±4.55 a Total 195.55 ±9.54 a 287.80 ±94.12 a 278.85 ±17.29 a 315.04 ±48.23 a