Effect of vegetal ground cover crops on wine anthocyanin content

Scientia Horticulturae 211 (2016) 384–390

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Effect of vegetal ground cover crops on wine anthocyanin content Y. Bouzas-Cid a , J. Portu b , E.P. Pérez-Álvarez b , A. Gonzalo-Diago b , T. Garde-Cerdán b,∗ a b

Viticulture and Enology Research Station of Galicia (EVEGA-INGACAL), Ponte San Clodio s/n, 32428 Leiro, Ourense, Spain Instituto de Ciencias de la Vid y del Vino (CSIC-CAR-UR), Ctra. de Burgos Km. 6, 26007 Logro˜ no, Spain

a r t i c l e

i n f o

Article history: Received 17 June 2016 Received in revised form 15 September 2016 Accepted 16 September 2016 Keywords: Soil management Phenolic compounds Wine colour Mencía Grape

a b s t r a c t Wine colour is a quality index that can provide information about conservation state, age or the presence of defects. Anthocyanin compounds are colour-related molecules and their concentration is affected by several factors such as grape variety, berry maturity degree or cultural practices. The aim of this work was to determine the anthocyanin composition of Mencía wines and how this is affected by the establishment of different cover crops (native vegetation, ryegrass and subterranean clover) respect to soil tillage treatment. This study was carried out during two consecutive seasons in the same vineyard. The use of cover crops significantly affected wine anthocyanin content; even though, their basic attributes were not altered by the treatments. In 2013, the wines from the ryegrass treatment had a significantly greater total anthocyanins concentration and, in 2014, the wines under the native cover had the highest concentration of these compounds. In both years, wines coming from the treatment under subterranean clover had a lower concentration of total anthocyanins when compared with those from the rest of the treatments. Cover crops increased wine anthocyanin concentrations when compared to the tilled treatment. However, the type of cover crop causing the highest increases differed from year to year. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Phenolic compounds play an important role as constituents of wine colour, thus contributing to the wine sensory quality. Within this family of compounds, anthocyanins are the most important molecules that are found in red grape skins. They are extracted during the winemaking process, providing the bluish-red colour characteristic of red wines. Although anthocyanins are the main compounds responsible for red-wine colour, other phenolic compounds indirectly contribute to it. For instance, flavanols contribute to wine astringency, but they also take part in wine colour stability and in wine ageing capacity (Gómez-Mínguez et al., 2006). Moreover, flavonols and phenolic acids influence wine colour by means of copigmentation reactions (Escribano-Bailón and Santos-Buelga, 2012). One of the most important factors that influence wine phenolic composition is grape variety, namely genetics (González-Neves et al., 2007). Other factors to consider are the climatic conditions of each growing season, cluster exposure to sunlight (Song et al., 2014), berry maturity degree and cultural practices, such as soil

∗ Corresponding author. E-mail addresses: [email protected], [email protected], [email protected] (T. Garde-Cerdán). http://dx.doi.org/10.1016/j.scienta.2016.09.026 0304-4238/© 2016 Elsevier B.V. All rights reserved.

management with cover crops (Pérez-Álvarez et al., 2015) or the foliar application of nitrogen compounds (Portu et al., 2015a). In the Ribeiro Designation of Origin (DO), Galicia (NW Spain), different traditional grapevine varieties are cultivated. Among the red cultivars, ‘Mencía’ (Vitis vinifera L.) is the most relevant one. This variety has high fertility and productivity, being appropriate for the production of young red wines (Consello Regulador del Ribeiro, 2015), which are traditionally considered as fruity and with high alcoholic degree (12–14% vol.). Several authors (Calleja and Falqué, 2005) described the aromatic profile of this red variety, which is characterised by its high content in different families of varietal aromatic compounds such as linalool and citronellol. The Galician wine sector is concerned about the excessive vegetative growth of Mencía, which can cause imbalances between vegetative growth and yield, affecting grape quality (Jackson and Lombard, 1993). A cultural practice that may be used to control vine growth is soil management with cover crops, since they compete with vines for water and nutrients, thus limiting vine vegetative growth (Giese et al., 2014; Yuste, 2005). This reduction in vegetative growth caused by cover crops might provide a higher sunlight exposure on the cluster zone through a decrease in shoot secondary growth. This fact may reduce green pruning operations, and of cluster thinning thus improving the polyphenol metabolism in the berries. Therefore, wines would retain more of these compounds, improving their colour properties and structure, hence,

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having a higher quality. Previous studies reported that the use of cover crops as a soil management strategy in vineyards increased total anthocyanins content in red grapes (Pérez-Álvarez et al., 2013) and, therefore, in wine (Trigo-Córdoba et al., 2015). However, the influence that the use of cover crops may have on wine anthocyanin detailed composition has not been studied yet. In this context, the aim of this work was to assess the effect of three different cover crops (native vegetation, ryegrass and subterranean clover) respect to a control treatment (tillage) on the anthocyanin composition of Mencía wines. As cover crops increased wine anthocyanins total concentration (Trigo-Córdoba et al., 2015), in the current study, we hypothesised the individual anthocyanins would also be increased by the use of this soil management strategy. 2. Materials and methods 2.1. Experimental design The experiment was carried out during two consecutive vintages (2013 and 2014) in a Mencía (Vitis vinifera L.) vineyard located within the experimental farm of the Viticulture and Enology Research Station of Galicia (EVEGA) in Leiro, Ourense, NW Spain (latitude 42◦ 21.6 N, longitude 8◦ 7.02 W, altitude 115 m above sea level). The vineyard was planted in 2007 on 196-17C rootstock at a spacing of 2.3 × 1.25 m (3478 vines/ha) and vines were trained to a vertical trellis on a single cordon system spur-pruned in winter (10–12 buds/vine) oriented in the East-West direction. Treatments consisted of four different soil management systems in the inter-rows: i) soil tillage (ST); ii) native vegetation (NV, included grass species such as genus Bromus and Lolium, and broadleaf species such as Bellis perennis L., Senecio vulgaris L., Vicia sativa L., and genus Trifolium and Conyza); iii) English ryegrass (Lolium perenne L.) sown at 40 kg/ha (ER); and iv) subterranean clover (Trifolium subterraneum L.) sown at 30 kg/ha (SC). Treatments with cover crops were mowed three times per year, when vegetation reached 20 cm height. Soil tillage alleys were disked during the growing season, in order to eliminate weeds. The treatments were replicated three times in a complete randomized block design. Each replicate consisted of three rows with seven vines per row. The five vines in the centre of the middle row were used for measurements and the rest acted as buffers (Trigo-Córdoba et al., 2015). The soil at the site is an Inceptisol (Soil Survey Staff, 2010) of sandy-loam texture (68% sand, 19.4% slit and 12.6% clay), with pH (H2O) of 5.8 and organic matter content of 4%. The climate in the region is characterised as temperate, humid with cool nights (Fraga et al., 2014). In 2013 and 2014, the annual rainfall was 1283 and 1301 mm and the mean temperature was 13.5 and 14.2 ◦ C for 2013 and 2014, respectively. Data on vegetative growth, yield and must quality from this experiment have been reported elsewhere (Trigo-Córdoba et al., 2015). 2.2. Winemaking and storage Grapes from the different treatments were manually harvested on the same day, when berries achieved an appropriate balance between probable alcoholic grade and titratable acidity, and were transported to the experimental winery in 20-kg field boxes. Winemaking was performed separately at EVEGA on samples of about 35 kg from each treatment. Grapes were mechanically destemmed and transferred to 35L stainless steel containers. During grape processing, 50 mg/L of SO2 were added to the mass. Fermentations were carried out at room temperature (22–24 ◦ C). Grand rouge XG (LamotheAbiet, Bordeaux, France) yeast was added following manufacturer’s instructions. The wine lots were punched down once a day until the

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end of alcoholic fermentation (8 days). Then, they were pressed, racked into new tanks and kept at room temperature for a couple of days. Then, wines were kept at 4 ◦ C in a chamber for a period of approximately one month for cold stabilization. After this period, wines were filtered, bottled and stored at 10 ◦ C. 2.3. Analytical methods 2.3.1. General attributes of wines Basic attributes of wines (alcohol content, titratable acidity, pH, malic and tartaric acids) were determined by Fourier transform infrared spectrometry (FTIR) using a WineScan FT120 analyser (FOSS Electric, Barcelona, Spain), calibrated according to the official methods (OIV, 2009). Determinations of these attributes were performed in duplicate. 2.3.2. Colour attributes An ultraviolet-visible spectrophotometer model Thermo Helios Zeta (Thermo Scientific Ltd, Leicester, UK) was used to determine wine colour attributes, using the methodology described by Zamora (2003), and summarized here. Prior to the measurements, wines were kept at room temperature (20 ◦ C) during a period of 24 h, and then they were filtered through a pore size of 0.22 ␮m. Colour intensity and tonality were determined through direct reading of wines at three different wavelengths: 420, 520 and 620 nm. Total polyphenol index (TPI) was determined by reading at a wavelength of 280 nm, after diluting the sample to a factor of 100. The concentration of total anthocyanins in wines was quantified according to the discolouration experimented by the addition of metabisulphite to the samples and reading in the spectrophotometer at 520 nm (Zamora, 2003). Results were expressed in mg/L. Total tannins of Mencía wines were determined after an acid hydrolysis of the samples. Then, readings in the spectrophotometer were made at 550 nm (Zamora, 2003). The concentration of these compounds was expressed in g/L. All colour determinations were performed in duplicate. 2.3.3. Determination of anthocyanins compounds in wines Anthocyanins in Mencía wines were determined in duplicate by high-performance liquid chromatography (HPLC) with direct injection of 10 ␮L of sample, previously centrifuged (4000 rpm, 10 ◦ C, 10 min) and filtered through a pore size of 0.22 ␮m (Easyprep, Quebec, Canada). The anthocyanins identified were 3-O-glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin and their corresponding acetyl and trans-p-coumaroyl derivatives. In addition, four pyranoanthocyanins were determined: vitisins A and B, 10-hydroxyphenyl-pyranomalvidin-3-glucoside (10-HP-pymv-3-glc) and 10-dihydroxyphenyl-pyranomalvidin-3glucoside (10-DHP-pymv-3-glc). A HPLC 1260 Infinity (Agilent Technologies, Palo Alto, CA, USA) equipped with a photodiode array detector was used. The separation was carried out in a column Licrosphere® 100 RP18 in reverse phase (4.0 × 250 mm, with particle size of 5 ␮m, Agilent), with precolumn Licrosphere® 100 RP18 (4 × 4 mm, with particle size of 5 ␮m, Agilent). ˜ The methodology developed by Castillo-Munoz et al. (2007) was followed. The analysis temperature was 40 ◦ C with a flow rate of 0.630 mL/min. Mobile phases used in the separation of compounds were: (A) acetonitrile/water/formic acid in proportion 3/88.5/8.5 (v/v/v) and (B) acetonitrile/water/formic acid in proportion 50/41.5/8.5 (v/v/v). The elution gradient was: 0 min, 6% B; 15 min, 30% B; 30 min, 50% B; 35 min, 60% B; 38 min, 60% B; 46 min, 6% B. Detection was performed at the maximum absorption wavelength of anthocyanins in the ultraviolet-visible region, 520 nm. The compounds were identified according to their retention time

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and absorption spectrum in the ultraviolet-visible region, which were obtained from standards or previous publications (Castillo˜ et al., 2007; Gómez-Alonso et al., 2007; Lago-Vanzela et al., Munoz 2011). The quantification of each compound was made according to the most abundant anthocyanin found in wines, malvidin-3-Oglucoside. Therefore, the anthocyanins were expressed as mg/L of malvidin-3-O-glucoside. 2.4. Statistical analysis The effect of the studied cover crops on the general attributes of wines and concentrations of anthocyanins was assessed using ANOVA. Means were separated using the Tukey´ıs Honest significant Difference test. The software used was Statistical Product and Service Solutions version 20 (SPSS Statistics for Windows, Armonk, NY, USA). Principal component analysis (PCA) was used to separate wine samples from the different treatments according to their anthocyanin profiles, using InfoStat Professional 2012 version software (www.infostat.com.ar). 3. Results and discussion 3.1. General attributes of wines Table 1 shows the basic attributes of the Mencía wines according to the treatments established in the vineyard inter-rows for the two years studied (2013 and 2014). No significant differences among treatments were observed for any of the attributes considered (alcohol, titratable acidity, pH, tartaric acid and malic acid) (Table 1). These results are in agreement with previous works that have not found any significant difference for the oenological parameters in Tempranillo (Pérez-Álvarez et al., 2015) and Sauvignon blanc wines (Fourie et al., 2007). Colour attributes are determining factors for ageing a wine in barrels. The recommended minimum value for anthocyanins is 400 mg/L, for tannins is 2 g/L and for total polyphenol index (TPI) is 40 (OIV, 2009). Taking into account the results obtained (Table 1), the red wines produced with the Mencía variety met the minimum requirements for wine ageing in oak barrels. In 2013, there were no significant differences on colour intensity between wines from the cover crops and control treatments (Table 1). However, wines from NV and SC had significantly greater colour intensity values than wines from ER. No significant differences among treatments were detected in 2014. The colour intensity values found in the current study are in agreement with those found in previous works (Soto-Vázquez et al., 2010). However, winemaking procedures have a great influence on the value of this attribute. This result confirms that no loss of colour has occurred in our wines despite the fact of being bottled one or two years prior to analysis. Therefore, as will be seen in the next section, the changes in the concentration of anthocyanins may be due to copigmentation and polymerisation reactions among these compounds (García-Falcón et al., 2005). The total polyphenol index (TPI) did not show significant differences among treatments in the studied years (Table 1). In 2013, the total anthocyanins concentration was significantly different between treatments. Wines from the ST and ER treatments showed higher anthocyanins concentrations than those from NV and SC (Table 1). However, in 2014, the wines from the cover crop treatments had higher total anthocyanins concentrations respect to those from the ST treatment. These results were in accordance with other varieties such as Tempranillo (Pérez-Álvarez et al., 2013) and the agronomic performance of this vineyard (Trigo-Córdoba et al., 2015), because berries from the cover crop treatments were smaller than those from the ST treatment.

Irrigation (Casassa et al., 2015; Bucchetti et al., 2011) or defoliation (Hunter et al., 1995) carried out in the vineyard can be other agronomic techniques, besides the cover crops, able to increase wine anthocyanins concentrations. Finally, in 2013, the wines from the ST, ER and SC treatments presented greater tannins concentrations than the wine obtained under NV. However, no significant differences among treatments were observed in 2014 (Table 1). 3.2. Anthocyanin composition of the wines Tables 2 and 3 show the concentrations of the seventeen individual anthocyanins quantified in Mencía wines for the two years, 2013 and 2014, respectively. In addition, the concentration of the four pyranoanthocyanins quantified (vitisin A, vitisin B, 10-dihydroxyphenyl-pyranomalvidin-3-glucoside and 10-hydroxyphenyl-pyranomalvidin-3-glucoside) are shown. The most abundant anthocyanin in all the studied wines for the two years was malvidin-3-O-glucoside, in spite of the significant differences observed among soil management treatments. Malvidin derivatives were clearly the most abundant type of anthocyanins, accounting for around 80% of total anthocyanins, followed by petunidin derivatives, which accounted for 8% of total anthocyanins. This finding is in agreement with previous works on Mencía (Soto-Vázquez et al., 2010) and it could suggest that trihydroxylated anthocyanins (i.e. delphinidin-, petunidin- and malvidin-types) are preferentially synthesized over dihydroxylated anthocyanins (i.e. cyanidin- and peonidin-types) in this grape variety. In addition, wines rich in trihydroxylated anthocyanins tend to display a more purple/blue colour (Castellarin et al., 2006). Moreover, pyranoanthocyanins concentrations in the current work are in agreement with those reported for other grape varieties (Blanco-Vega et al., 2014). In 2013, the wines from ER presented a significantly greater concentration of total anthocyanins (Table 2), when compared with the rest of the treatments. In addition, these wines showed a higher concentration in all the individual anthocyanins. The fraction of 3-O-glucosides represented 75–85% of the total wine anthocyanin content. The concentrations of delphinidin-3-O-glucoside, malvidin3-O-glucoside, peonidin-3-O-acetylglucoside and pyranoanthocyanin vitisin B presented significant differences among the four treatments in the year 2013 (Table 2). Wines from the ER treatment showed higher concentrations of these compounds, whereas SC and NV wines presented the lowest concentrations. The pyranoanthocyanins formation is intimately linked to wine ageing and colour stability during this process. These compounds give lower colour intensity to wines because they provide orange instead of red tones (Rentzsch et al., 2007). However, these molecules are of great interest in aged wines since, contrary to flavanol-anthocyanin adducts, their small size keep them from precipitating (Rentzsch et al., 2007). Wines from ER showed a high concentration in pyranoanthocyanins and, thus, lower colour intensity when compared with the rest of the wines from 2013 (Table 1). Principal component analysis (PCA) showed that the anthocyanins concentrations are able to explain 90.4% of the variance found in the wines from the four treatments (Fig. 1). The individual anthocyanins that presented high loads in principal component 1 (PC1) were delphinidin-3-Oglucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, petunidin-3-O-acetylglucoside, peonidin-3-O-acetylglucoside, malvidin-3-O-acetylglucoside, petunidin-3-O-coumarylglucoside, malvidin-3-trans-Opeonidin-3-O-coumarylglucoside, coumaroylglucoside, malvidin-3-O-caffeoylglucoside and 10-dihydroxyphenyl-pyranomalvidin. PC1 explained 59% of

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Table 1 General attributes of Mencía wines from the different soil management treatments (ST, soil tillage; NV, native vegetation; ER, English ryegrass; SC, subterranean clover) in 2013 and 2014.

Alcohol (% vol) Titratable acidity (g/L tartaric acid) pH Tartaric acid (g/L) Malic acid (g/L) Colour intensity Total polyphenol index (TPI) Total anthocyanins (mg/L) Total tannins (g/L)

Year

ST

NV

ER

SC

2013 2014 2013 2014 2013 2014 2013 2014 2013 2014 2013 2014 2013 2014 2013 2014 2013 2014

13.5 a 13.7 a 3.4 a 4.7 a 4.46 a 4.47 a 2.9 a 2.0 a 1.5 a 1.2 a 10.1 ab 10.6 a 65 a 58 a 674.6 b 664.1 a 2.0 b 2.0 a

13.2 a 13.7 a 3.4 a 4.9 a 4.41 a 4.23 a 3.0 a 1.7 a 1.8 a 2.0 a 11.3 b 12.2 a 62 a 65 a 588.0 a 781.4 b 1.8 a 2.1 a

13.3 a 12.8 a 3.7 a 4.7 a 4.34 a 4.13 a 3.0 a 1.7 a 1.8 a 1.3 a 8.3 a 10.4 a 69 a 59 a 721.0 b 714.9 b 2.2 b 2.1 a

13.0 a 11.6 a 3.9 a 3.8 a 4.32 a 4.21 a 2.8 a 1.9 a 1.6 a 0.2 a 13.3 b 10.3 a 68 a 60 a 551.3 a 758.6 b 2.0 b 2.0 a

For each attribute and year, values with different letters are significantly different between treatments (p ≤ 0.05). Table 2 Anthocyanin compounds (mg/L of malvidin-3-O-glucoside) of Mencía wines (mean ± standard deviation) from the different soil management treatments (ST, soil tillage; NV, native vegetation; ER, English ryegrass; SC, subterranean clover) in 2013. 2013

Delphinidin-3-O-glc Cyanidin-3-O-glc Petunidin-3-O-glc Peonidin-3-O-glc Malvidin-3-O-glc Delphinidin-3-O-acglc Cyanidin-3-O-acglc Petunidin-3-O-acglc Peonidin-3-O-acglc Malvidin-3-O-acglc Delphinidin-3-O-cmglc Cyanidin-3-O-cmglc Petunidin-3-O-cmglc Peonidin-3-O-cmglc Malvidin-3-O-cmglc-cis-cmglc Malvidin-3-O-cmglc-trans-cmglc Malvidin-3-O-cfglc Total anthocyanins Vitisin A Vitisin B 10-DHP-pymv-3-glc 10-HP-pymv-3-glc Total pyranoanthocyanins

ST

NV

ER

SC

5.82 ± 0.09c 1.86 ± 0.04a 12.19 ± 0.08b 6.61 ± 0.05b 140.09 ± 1.17c 2.59 ± 0.06a 1.84 ± 0.04a 2.38 ± 0.14b 2.56 ± 0.06b 18.37 ± 0.21b 1.45 ± 0.07a nd 1.30 ± 0.03c 1.88 ± 0.02c nd 7.81 ± 0.02c 1.08 ± 0.01a 207.82 ± 1.10b 1.46 ± 0.02a 1.85 ± 0.01d 1.74 ± 0.05a 1.93 ± 0.05c 6.98 ± 0.07a

4.60 ± 0.02b 1.99 ± 0.06a 9.20 ± 0.19a 5.82 ± 0.21a 129.78 ± 2.42b 2.17 ± 0.08a 1.92 ± 0.01ab 1.66 ± 0.01a 1.89 ± 0.03a 12.07 ±0.23a 1.44 ± 0.01a nd 1.09 ± 0.00a 1.36 ± 0.02a nd 4.54 ± 0.04a 1.22 ± 0.00b 180.76 ± 2.42a 2.66 ± 0.11b 1.34 ± 0.01a 1.93 ± 0.04b 1.85 ± 0.03bc 7.78 ± 0.17b

6.94 ± 0.08d 2.23 ± 0.05b 14.12 ± 0.42c 9.10 ± 0.09c 151.07 ± 0.50d 3.62 ± 0.34b 2.18 ± 0.07b 2.79 ± 0.09b 3.70 ± 0.09d 20.29 ± 0.30c 1.52 ± 0.05a nd 1.32 ± 0.01c 2.38 ± 0.00d nd 8.06 ± 0.01c 1.05 ± 0.01a 230.37 ± 0.09c 2.87 ± 0.04b 1.74 ± 0.02c 1.76 ± 0.02a 1.75 ± 0.00ab 8.13 ± 0.05b

4.10 ± 0.15a 1.98 ± 0.00a 9.25 ± 0.09a 5.34 ±0.16a 111.03 ± 1.76a 3.54 ± 0.31b 2.50 ± 0.12c 2.62 ± 0.17b 3.20 ± 0.10c 19.65 ± 0.27c 1.51 ± 0.01a nd 1.18 ± 0.01b 1.74 ± 0.03b nd 5.49 ± 0.21b 1.19 ± 0.02b 174.33 ± 3.21a 4.35 ± 0.14c 1.53 ± 0.00b 1.83 ± 0.05ab 1.67 ± 0.04a 9.38 ± 0.23c

For each compound, values with different letters are significantly different between treatments (p ≤ 0.05). nd = not detected. Abbreviations: glc (glucoside), acglc (acetylglucoside), cmglc (coumaroylglucoside), cfglc (caffeoylglucoside), 10-DPH-pymv-3-glc (10-dihydroxyphenyl-pyranomalvidin-3-glucoside), 10-HP-pymv-3-glc (10hydroxyphenyl-pyranomalvidin-3-glucoside).

the variance in our samples and clearly separated wines from ER (positive side of PC1) than those from NV (negative side of PC1). The individual anthocyanins that presented high loads in PC2 were delphinidin-3-O-acetylglucoside, cyanidin-3-Oacetylglucoside, delphinidin-3-O-coumaroylglucoside, vitisin A and 10-hydroxyphenyl-pyranomalvidin-3-glucoside. PC2 explained 31.4% of the variance in our samples and clearly separated wines from SC (positive side of PC2) than those from ST (negative side of PC2). Moreover, the wines from NV and SC had a lower total anthocyanins concentration than those from ST and ER. In a previous study (Trigo-Córdoba et al., 2015), the use of cover crops increased wine anthocyanins concentrations with respect to the control treatment under soil tillage. This fact can be explained by the smaller berries in the cover crop treatments, which increased their skin-pulp relative proportion. Therefore, under

the same maceration conditions, the anthocyanins concentrations were higher in the cover crops. In 2014, the wines from NV presented the highest total anthocyanin concentration, while those from SC showed the lowest one (Table 3). This result could be associated with the spontaneous malolactic fermentation suffered by SC wines. In this year, 75–78% of total anthocyanins were from the 3-O-glucosides family. In 2014, the wines from ST presented a significantly higher pyranoanthocyanins concentration with respect to NV, ER and SC; the wines from this latter treatment had the lowest concentration of pyranoanthocyanins (Table 3). The fact that ST wines showed a high concentration of pyranoanthocyanins implies a more orange tonality when compared to the wines from the rest of the treatments. However, a higher concentration of these compounds may play a key role in colour stability of long-aged wines.

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Table 3 Anthocyanin compounds (mg/L of malvidin-3-O-glucoside) of Mencía wines (mean ± standard deviation) from the different soil management treatments (ST, soil tillage; NV, native vegetation; ER, English ryegrass; SC, subterranean clover) in 2014. 2014

Delphinidin-3-O-glc Cyanidin-3-O-glc Petunidin-3-O-glc Peonidin-3-O-glc Malvidin-3-O-glc Delphinidin-3-O-acglc Cyanidin-3-O-acglc Petunidin-3-O-acglc Peonidin-3-O-acglc Malvidin-3-O-acglc Delphinidin-3-O-cmglc Cyanidin-3-O-cmglc Petunidin-3-O-cmglc Peonidin-3-O-cmglc Malvidin-3-O-cmglc-cis-cmglc Malvidin-3-O-cmglc-trans-cmglc Malvidin-3-O-cfglc Total anthocyanins Vitisin A Vitisin B 10-DHP-pymv-3-glc 10-HP-pymv-3-glc Total pyranoanthocyanins

ST

NV

ER

SC

12.66 ± 0.17c 3.03 ± 0.07c 24.23 ± 0.66c 15.50 ± 0.41b 222.03 ± 5.22bc 6.54 ± 0.14b 2.23 ± 0.01c 4.57 ± 0.11b 7.75 ± 0.26b 32.80 ± 0.26b 1.95 ± 0.12b nd 1.85 ± 0.03b 4.65 ± 0.15b nd 13.20 ± 0.27b 1.14 ± 0.01b 354.13 ± 7.74b 2.89 ± 0.02d 2.99 ± 0.15b 1.42 ± 0.01c 2.84 ± 0.03d 10.14 ± 0.15c

12.88 ± 0.14c 3.30 ± 0.10c 23.94 ± 0.12c 16.89 ± 0.52b 229.57 ± 0.64c 6.51 ± 0.16b 2.07 ± 0.03bc 4.96 ± 0.03b 8.80 ± 0.04c 36.45 ± 0.54c 2.10 ± 0.10b nd 2.07 ± 0.00c 6.00 ± 0.04c nd 17.46 ± 0.11c 1.20 ± 0.00c 374.19 ± 0.62c 2.46 ± 0.01c 3.01 ± 0.00b 1.29 ± 0.01b 2.35 ± 0.01b 9.10 ± 0.01b

11.56 ± 0.15b 2.58 ± 0.16b 21.71 ± 0.20b 16.34 ± 0.61b 210.63 ± 2.32b 6.41 ± 0.20b 1.98 ± 0.00b 4.81 ± 0.12b 9.09 ± 0.35c 36.69 ± 0.60c 2.04 ± 0.02b nd 2.12 ± 0.01c 6.39 ± 0.10d nd 17.23 ± 0.44c 1.13 ± 0.02b 350.72 ± 4.24b 2.32 ± 0.02b 2.94 ± 0.08b 1.27 ± 0.02b 2.44 ± 0.00c 8.96 ± 0.08b

8.12 ± 0.06a 2.00 ± 0.04a 15.00 ± 0.19a 9.40 ± 0.08a 151.77 ± 0.58a 4.60 ± 0.19a 1.69 ± 0.09a 3.40 ± 0.12a 5.55 ± 0.00a 23.77 ± 0.32a 1.52 ± 0.04a nd 1.56 ± 0.01a 3.80 ± 0.08a nd 10.66 ± 0.09a 1.05 ± 0.00a 243.89 ± 0.63a 1.85 ± 0.04a 2.21 ± 0.06a 1.16 ± 0.02a 1.96 ± 0.01a 7.19 ± 0.02a

For each compound, values with different letters are significantly different between treatments (p ≤ 0.05). nd = not detected. Abbreviations: glc (glucoside), acglc (acetylglucoside), cmglc (coumaroylglucoside), cfglc (caffeoylglucoside), 10-DPH-pymv-3-glc (10-dihydroxyphenyl-pyranomalvidin-3-glucoside), 10-HP-pymv-3-glc (10hydroxyphenyl-pyranomalvidin-3-glucoside).

2013 5.00 Vitisin A

Cin-3-acglc

SC

Df-3-cmglc Df-3-acglc

2.50

Pn-3-acglc Pt-3-acglc Cn-3-glc Mv-3-acglc

PC 2 (31.4%)

Mv-3-cfglc 10-DHP-pymv-3-glc

Pn-3-cmglc

0.00

Pn-3-glc NV Df-3-glc

-2.50

ST

ER

Pt-3-cmglc Pt-3-glc Mv-3-trans-cmglc Vitisin B

Mv-3-glc

10-HP-pymv-3glc -5.00 -5.00

-2.50

0.00

2.50

5.00

PC 1 (59.0%) Fig. 1. Principal component analysis (PCA) of Mencía wines anthocyanins concentration from different soil management treatments (ST, soil tillage; NV, native vegetation; ER, English ryegrass; SC, subterranean clover) in 2013.

The principal component analysis (Fig. 2) showed a clear separation of SC wine from the rest of the wines of 2014 season and is able to explain 97.6% of the variation found in the wines from the four treatments. In this case, PC1 accounted for more than 80% of the variance in our dataset; hence it had high loadings from all of the individual anthocyanins. The clear separation of the SC wine from the rest of the wines indicate that SC wine showed lower concentrations of all individual antho-

cyanins when compared with the wines from the rest of the treatments. In contrast, PC2 only accounted for 13.6% of the variance in our samples. Only three compounds contributed significantly to this PC: peonidin-3-O-coumaroylglucoside, vitisin A and 10-dihydroxyphenyl-pyranomalvidin-3-glucoside. No clear separation of wines was provided by PC2. In general, the HPLC results indicated that total anthocyanin compounds were increased by ER in 2013 and NV in 2014 when

Y. Bouzas-Cid et al. / Scientia Horticulturae 211 (2016) 384–390

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2014 7.00

10-DHP-pymv-3-glc 10-HP-pymv-3glc Vitisin A Cin-3-acglc Cn-3-glc Pt-3-glc Df-3-glc ST Df-3-acglc Mv-3-glc Vitisin B Mv-3-cfglc Df-3-cmglc Pn-3-glc NV ER Pt-3-acglc Mv-3-acglc Pn-3-acglc Pt-3-cmglc Mv-3-trans-cmglc Pn-3-cmglc

PC 2 (13.6%)

3.50

0.00

SC

-3.50

-7.00 -7.00

-3.50

0.00

3.50

7.00

PC 1 (84.0%) Fig. 2. Principal component analysis (PCA) of Mencía wines anthocyanins concentration from different soil management treatments (ST, soil tillage; NV, native vegetation; ER, English ryegrass; SC, subterranean clover) in 2014.

compared to control wines. This finding is of oenological interest since anthocyanins are the key compounds in red wine colour. The increase of wine total anthocyanins by these cover crops could be explained by water and/or nutrients deficit in control vines. Moderate water deficit is known to increase grape anthocyanins. This fact has been explained by changes in the expression of the genes responsible for anthocyanin synthesis (Castellarin et al., 2007) as well as by reducing fruit growth (Bucchetti et al., 2011). In this respect, it has been widely reported that cover crops reduce water availability (Celette et al., 2008; Monteiro and Lopes, 2007). Moreover, it has been suggested that the establishment of cover crops decreases nutrient availability (Celette et al., 2009; Pérez-Álvarez et al., 2015). In this respect, excessive nitrogen availability is considered to result in low coloured wines while moderate supply could result in greater anthocyanin content (Bell and Henschke, 2005; Portu et al., 2015b). However, this study has also shown significant differences among the different types of cover crops and the influence of the vintage in its effect. Differences between the vintages could be due to weather conditions which also influence vine water status. As for the different behaviour of the cover crops, it is known that the species also influences vine response, due to a different development over the vine growing season. In our particular case, native vegetation and ryegrass grew more rapidly and covered a higher proportion of the surface between vine rows than subterranean clover (Trigo-Córdoba et al., 2015). Moreover, after veraison, vines under the NV treatment showed more negative values of stem water potential with respect to the other treatments indicating that these vines suffered a mild to moderate water stress. However, in 2014 vines under ST presented a greater foliar surface that caused that their stem water potential values were similar to those fo the vines under NV. These observations could partially explain the differences detected in wine anthocyanin concentration. Nevertheless, the absence of significant differences in stomatal conductance and yield suggested that Mencía vinesshowed an adequate physiological status of the vines independently of the treatment (Trigo-Córdoba et al., 2015).

4. Conclusions Cover crops did not significantly affect the general attributes of the studied wines, although significant differences were found in their anthocyanin composition. In our case, wine colour attributes were similar to those previously reported for Mencía wines and storage conditions were the same for all treatments; therefore, we can conclude that differences among wines were caused mainly by the treatments imposed in the vineyard and not by other factors. Wines from ER and NV showed significantly higher total anthocyanin concentrations in 2013 and 2014, respectively. The main group of anthocyanins found in the studied wines was that of 3-O-glucosides, and the main compound was the anthocyanin malvidin-3-O-glucoside. Although the anthocyanin profile of Mencía wines was not altered by the cover crop treatments, the concentrations of these compounds were greater in wines from the ryegrass treatment, especially in 2013. Acknowledgements This research was funded by Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) through project RTA201100041-C02-01, with 80% European Social Fund (FEDER) funds. Y. Bouzas-Cid thanks INIA for the Ph. D. scholarships. E.P. PérezÁlvarez and A. Gonzalo-Diago thank INIA and Gobierno de La Rioja for their contracts and J. Portu and T. Garde-Cerdán thanks INIAGobierno de La Rioja for their contracts. References Bell, S.J., Henschke, P.A., 2005. Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res. 11, 242–295. Blanco-Vega, D., Gómez-Alonso, S., Hermosín-Gutiérrez, I., 2014. Identification, content and distribution of anthocyanins and low molecular weight anthocyanin derived pigments in Spanish commercial red wines. Food Chem. 158, 449–458. Bucchetti, B., Matthews, M.A., Falginella, L., Peterlunger, E., Castellarin, S.D., 2011. Effect of water deficit on Merlot grape tannins and anthocyanins across four seasons. Sci. Hortic. 128, 297–305.

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