Introduction

Canopy management plays a key role in the modulation of berry composition, as it contributes to controlling the decoupling of sugar and anthocyanins accumulation, which is particularly evident in some red varieties, such as Sangiovese, Cabernet Sauvignon, Merlot, Nero di Troia and Uva Longanesi (Rombolà et al., 2011; Palliotti et al., 2013; Poni et al., 2013; Tessarin et al., 2014; Baiano et al., 2015; Filippetti et al., 2015; Bondada et al., 2016; Tessarin et al., 2016; Pastore et al., 2017; Tessarin et al., 2018), and its effects depends on the terroir and climate conditions.

In the context of climate change, grape growers, especially those who cannot practice irrigation, need to carry out summer pruning practices (e.g., shoot topping/trimming, leaf removal and shoot positioning) in order to be able to preserve grape quality until harvest and to deliver healthy grapes to the winery (Palliotti et al., 2014; Poni et al., 2018). Defoliation is a common intervention whose effects in vine physiology and berry composition largely depend on treatment timing and intensity (Palliotti et al., 2014; Poni et al., 2018). Defoliation in the fruit zone is one of the most important and commonly applied canopy management interventions in viticulture (Ivanišević et al., 2020). This technique is performed on grapevines to improve light penetration and air circulation around the clusters (Ivanišević et al., 2020). The right moment for defoliation depends on the region, variety and type of wine produced (Ivanišević et al., 2020).

Performed on different genotypes and in different growing conditions, early defoliation usually involves the removal of about 6–7 main basal leaves before flowering, resulting in a significant decrease in fruit-set, which in turn increases the loosening of clusters and tolerance to rot (Poni et al., 2018). Moreover, this technique, irrespective of genotype, markedly improves grape composition (total soluble solids, anthocyanins, phenols and aromatic compounds) and wine sensory properties (Poni et al., 2018). However, defoliation just before anthesis should be carefully applied, because it can decrease must titratable acidity, increase must pH (Risco et al., 2014) enhancing malic acid degradation (Gatti et al., 2015), increase berry sunburn (Lopes et al., 2019) with negative consequences in terms of wine quality. Risco et al. (2014) and Lopes et al. (2019) reported a cumulative negative effect of early defoliation on vine bud fertility.

Late defoliation (carried out at the onset of veraison or later) is a frequently adopted practice that curtails the development of fungal diseases. It allows healthy bunches to be maintained for longer on the vine, resulting in the attainment of adequate levels of phenolic and aromatic compounds (Kalua and Boss, 2009).

Controlling the phytosanitary status of bunches during ripening is crucial, particularly in organic and biodynamic viticultural systems (Botelho et al., 2016); this is mainly due to there being less effective active ingredients for controlling bunch rot, which has led to research on agroecological strategies, such as using essential oil vapour (Burggraf and Rienth, 2020) and carrying out post-veraison trimming (Bondada et al., 2016; Tessarin et al., 2018). In addition, the EC Regulation (EC, 2012) for the production of organic wines provides a series of restrictions on determinate oenological practices (e.g., dealcoholisation) and limits the amount of sulphites that can be added to the must during winemaking (Parpinello et al., 2015). Due to such restrictions, organic wine producers face difficulties in managing grapes affected by rot in the cellar.

Basal leaf removal at veraison can induce changes in berry composition, especially a decrease in total soluble solids (Pastore et al., 2013; Pastore et al., 2017; Tessarin et al., 2014). The main drawback of basal leaf removal at veraison is the possible decrease in berry anthocyanins (Pastore et al., 2013; Tessarin et al., 2014), which is also mirrored in wines. However, basal leaf removal at the end of veraison does not seem to result in any significant changes in berries (Tessarin et al., 2014).

Late bunch-zone defoliation carried out at post-veraison on cv. Aglianico did not change berry soluble solids or the alcohol concentrations of young wines without decreasing berry anthocyanin levels and the wine colour (Caccavello et al., 2017). The post-veraison defoliation of leaves above the bunch zone of cv. Sangiovese potted vines (Poni et al., 2013) and of those grown in field conditions (Palliotti et al., 2013) without exposing the bunches to direct solar radiation caused a decline in photosynthetic capacity of the vines, resulting in a decrease in berry soluble solids and a reduction in wine alcohol levels without any changes in anthocyanins and polyphenols levels in the berries and wines (Palliotti et al., 2013; Poni et al., 2013). Physiologically, leaf removal is based on the fact that, around veraison, the leaves above the bunch on the apical two-thirds of the canopy are the most photosynthetically functional (Poni et al., 1994).

In the context of climate change high temperatures are frequent in the first phases of ripening; therefore, protecting bunches from solar radiation is essential for preserving berry composition and wine colour (Tessarin et al., 2014). Grapevines trained to vertical shoot positioning (VSP) systems were found to undergo more heat damage compared to those with sprawling, non-positioned canopies (Dry, 2011). Dry (2011) proposed creating a “semi-ballerina” effect on VSP canopies in warm to hot sunny climates; this consists in positioning long shoots downwards, thereby shading the bunches (Supplementary Figure 1) in the warmest hours of the day and reducing the risk of bunch damage. Specific research is required to determine the possible implications of this technique in terms of the effects on grapes and wine quality.

Sangiovese is one of the most widespread grape varieties in Italy, covering around 54000 ha, which constitutes 10.8 % of the national grape growing area (Focus OIV, 2017). Sangiovese has a relevant agronomic and economic role, since it is used for producing hundreds of different wines, such as Chianti Classico, Brunello di Montalcino, Romagna Sangiovese, which must undergo an aging period of a minimum of 24 months before commercialisation (Rinaldi et al., 2021). This cultivar displays a sensitive anthocyanin profile (Mattivi et al., 2006; Castellarin et al., 2012) due to its high percentage of unstable dihydroxy pigments, which is mirrored in wine quality parameters (Arapitsas et al., 2012).

Sangiovese is rich in skin flavonols, in particular quercetin. Quercetin increases with cluster exposure to light and thus after defoliation, especially early (pre-flowering) defoliation (Lanati et al., 2021), which is performed to regulate yield and reduce cluster compactness and susceptibility to fungal pathogens (Poni et al., 2006). Quercetin is beneficial for wine quality (Boulton, 2001) and human health (Derosa et al., 2021). However, its precipitates in wines are detrimental to wine quality; wines from Sangiovese are among those that are very sensitive to quercetin haze (Gambuti et al., 2020; Lanati et al., 2021).

For the above-mentioned reasons, the achievement of multiple goals, especially the attainment of Sangiovese wines suitable for aging, constitutes a considerable challenge for organic grape growers.

Therefore, our research on cv. Sangiovese aimed to achieve the following main goals: 1) improve the grape sanitary status, 2) preserve the berry skin anthocyanins and technological parameters, 3) enhance the berry flavonols, 4) improve the quality of the wines undergoing aging, and 5) achieve the former goals without altering the yield, budburst and fruitfulness in the season being studied and the following ones.

We assessed the effects of post-veraison and pre-harvest defoliation and post-veraison shoot positioning on vine physiology and grape quality and sanitary status, as well as wine composition throughout storage, in an organically-managed and rainfed Sangiovese vineyard.

The experimental hypothesis was that post-veraison and pre-harvest defoliation and post-veraison shoot positioning are all able to trigger changes in berry composition, leading to the formation of compounds beneficial for the quality of wine undergoing aging without altering vine yield, budburst and fruitfulness.

Materials and methods

1. Plant material and experimental layout

The experiment was conducted in 2013 and 2014, in a mature vineyard planted in 2003 with Vitis vinifera L., cv. Sangiovese (clone FEDIT 30 ESAVE) grafted onto Kober 5BB and trained to a cordon de Royat training system with an 80 cm high cordon (vertical shoot positioning, VSP). The vineyard was located in Tebano (Faenza, RA), Italy (44°17ʹ7ʹʹ N, 11°52ʹ59ʹʹE, 117 m a.s.l), on a medium slope (< 8°), with southeast/northwest- and downhill-oriented rows. The climate of this area is subcontinental temperate, with hot, humid summers and cold winters. Rainfall averages 600 to 700 mm per year. Vines were spaced 1.0 m apart within the row and 2.8 m between rows, for 3571 vines/ha. From 2007 onwards, the vineyard was managed as organic in accordance with Reg. EC 834/2007 (EC, 2007), with neither irrigation nor fertilisation.

The loamy clayey and alkaline soils of the vineyard comprise 2.2 % organic matter, 1.5 ‰ nitrogen concentration, 14.7 % total carbonates, 6.7 % active lime, 10 μg/g assimilable phosphorus and 188 μg/g assimilable potassium. Spontaneous vegetation was present in the alternate planting rows. Annually, at the end of each growing season, herbaceous species were sown in alternate planting rows, such as fava bean (Vicia faba), barley (Hordeum vulgare) or subterranean clover (Trifolium subterraneum). The ground cover was managed by mowing the vegetation in late spring, which maintained the biomass on the soil surface.

The vineyard was treated against diseases and pests using products conforming to EC Regulations (EC, 2002). Copper (an average of 6 kg/ha/year) and sulphur (an average of 70 kg/ha/year) broad-spectrum fungicides were applied. Vines were spur-pruned to two count nodes, with 12-14 nodes per vine being retained at the end of February. The non-count shoots (shoots arising from the base buds of the spur) were removed at the beginning of the season, with 12 uniformly distributed shoots per meter of cordon being kept. The bunch number was adjusted to 16 per vine by bunch thinning at veraison. The experimental design included four treatments: an untreated control (CK); two different defoliation times: post-veraison defoliation (DEF I) and pre-harvest defoliation (DEF II) (Figure 1); and shoot positioning through the “semi-ballerina” effect (SB) (Supplementary Figure 1). The DEF I treatment was imposed at post-veraison, when the berries had reached an average of 15 °Brix (86 and 92 days after flowering, DAF, in 2013 and 2014 respectively), which was measured by refractometer (Digital Refractometer HI 96811, Hanna instruments, Milan, Italy). The DEF II treatment was imposed at pre-harvest (101 DAF, 19.8 °Brix in 2013 and 106 DAF, 17.4 °Brix in 2014; Supplementary Table 2). The SB treatment was performed at 86 and 92 DAF in 2013 and 2014 respectively. The control treatment (CK) comprised long canes possessing around 24 nodes trailing over both sides of the canopy. Defoliation treatments were performed by manually removing all main leaves and laterals up to the eighth node. The “semi-ballerina” treatment (Dry, 2011) consisted in positioning long canes downwards (around 24 nodes) on one side of the canopy exposed to the south-west. The treatments were applied to the same vines in each season. Each treatment was replicated three times in a randomised-block design. The treatments were applied on vines within three parallel rows that were divided into three consecutive blocks with 30 vines per repetition. For each repetition, the measurements were made on 10 vines positioned in the central row of the three parallel rows. All vines included in the experiment were subjected to the same soil management with alternate cover crops.

Photosynthetic Active Radiation (PAR) at bunch level was measured in the afternoon at post-veraison using a Skye Quantum Sensor (Llandrindod Wells, Powys, UK).

Figure 1. Vines subjected to late defoliations in the organically-managed and rainfed Sangiovese vineyard.

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Figure 2. Seasonal trends of berry skin glucosylated flavonols (mg/g of skin), measured in 2013 and 2014 on cv. Sangiovese.

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Results

Discussion

In previous studies, a reduction in total soluble solids (TSS) at harvest was obtained only when the leaf area (LA)/yield ratio was lower than 0.8–1.2 m²/kg (Kliewer and Dokoozlian, 2005; Poni et al., 2013). In our work, the decrease in total leaf area per vine (TLA) (-30-35%) that occurred in the defoliated (DEF I and DEF II) vines did not result in enhanced photosynthetic activity in the remaining leaves compared with the control (CK) vines. These different results may be explained by the limited influence of removing old leaves in our experiment (134 days in 2013 and 147 days in 2014 for DEF I leaves; 149 days in 2013 and 161 days in 2014 for DEF II leaves), which are characterised by low photosynthetic activity compared to that of intermediate and apical leaves (Kriedemann et al., 1970).

Moreover, in our study, the plant nutritional status (Supplementary Table 1) and the physiological status were similar for all treatments, and the photosynthesis values were within the standards of Sangiovese vines (Penazzi et al., 2011; Tessarin et al., 2018). This suggests that the reiteration of late defoliations or shoot positioning did not have any adverse effects on these parameters.

Our data suggest that in a season characterised by high rainfall during the ripening period it is important to improve the bunch microclimate by avoiding over-shading and by removing leaves and laterals from the bunch zone, preferentially at post-veraison. The possibility of reducing the attack of Botrytis on cv. Sangiovese bunches is of paramount importance for organic growers, lacking effective products against this fungus. Therefore, the implementation of these agricultural practices, while taking into account the meteorological conditions of each season, is of great importance in organic viticulture.

Berry weight and technological parameters (TSS, pH and titratable acidity (TA)) at harvest were not affected by the treatments; this result is consistent with other studies on late defoliation (Caccavello et al., 2017; Pastore et al., 2017). However, some studies have produced different results: post-veraison defoliation of leaves above the bunch zone in cv. Sangiovese potted vines (Poni et al., 2013) or in field conditions (Palliotti et al., 2013) have been found to induce a decrease in soluble solids of the berry (Palliotti et al., 2013; Poni et al., 2013) and a reduction in alcohol in the wines (Palliotti et al., 2013), without changing anthocyanins and polyphenols in berries (Palliotti et al., 2013; Poni et al., 2013) or wines (Palliotti et al., 2013). As discussed earlier, the removal of the basal leaves did not induce any compensation phenomena in the photosynthetic activity of the remaining younger leaves. Presumably, the sugars stored as plant reserves were translocated into the berries (Candolfi-Vasconcelos et al., 1994; Rossouw et al., 2017). The exposure of bunches to direct solar radiation and the consequent higher berry temperature did not result into any reduction in TA (or pH increase) in the berries, possibly because the removal of the leaves resulted in a decrease in the organic acid-neutralising cations (particularly K) that were translocated via the phloem to the berries (Villette et al., 2020).

Flavonols may play a crucial role as co-pigments in young red wine by stabilising the anthocyanins and creating a stable association to form polymeric pigments, whose importance for the colour of older red wines is well-known. When changes in the abundance of individual flavonols were observed (2014), a higher percentage of the sum of quercetin-glucoside and glucuronide was recorded in berry skins from the vines that underwent late defoliation and shoot positioning treatments; moreover, DEF II vines showed a higher percentage of kaempferol-glucoside and a lower percentage of myricetin-glucuronide and myricetin-glucoside compared with the CK vines (Table 3). In a study by Pastore et al. (2013), an increase in the concentration of cv. Sangiovese berry skin flavonols was also observed following the removal of leaves and laterals at pre-bloom and veraison, and similar to our study, higher quercetin and kaempferol percentages were detected compared to the control vines. This phenomenon occurs, because the synthesis of flavonols is promoted by the exposure of clusters to direct sunlight (Downey et al., 2004).

The concentration of flavonols in berries have been shown to strongly correlate with degree of fruit exposure (Price et al., 1995; Haselgrove et al., 2000) and bunch shading to significantly reduce flavonols levels (Downey et al., 2004; Matus et al., 2009). However, flavonol concentrations have also been reported to decrease following leaf removal treatments (Matus et al., 2009; Baiano et al., 2015); it is likely that the high temperatures within the berry skin after exposure to direct sunlight adversely affected flavonol accumulation, thus overriding the positive effects of the light.

The effects of late DEFs on total flavonol concentration at harvest may be partially due to the increased light condition in the bunch zone. In the measurements performed in the afternoon, the Photosynthetic Active Radiation (PAR) (Skye Quantum Sensor, Llandrindod Wells, Powys, UK) at bunch level varied in the range 30-250 μmol/(m²·s) in vines subjected to the SB effect, 150-250 μmol/(m²·s) in CK vines and 1200-2200 μmol/(m²·s) in DEF vines. In these hottest hours of the day, the clusters of DEF vines showed higher temperatures than those of CK and SB vines (data not shown). However, previous research has shown that temperature may have little or no impact on berry flavonol biosynthesis (Price et al., 1995; Haselgrove et al., 2000, Spayd et al., 2002; Mori et al., 2005).

In this study, the higher exposure to light of the bunches in both of the defoliated (DEF I and II) vines did not affect total anthocyanin concentration at harvest (Table 3). It has been reported that depriving bunches from light may have no effect on colour or, conversely may influence anthocyanin composition and/or concentration, depending, for instance, on the variety (Downey et al., 2004; Cortell and Kennedy, 2006) or the duration of the treatment (Li et al., 2013). The effects of light on anthocyanin concentration are also closely dependent on the increase in berry temperature as a consequence of increased sunlight exposure, since high berry temperature can inhibit colour development (Bergqvist et al., 2001; Mori et al., 2005; Mori et al., 2007). Temperature has more effect on anthocyanin accumulation than light (Spayd et al., 2002, Mori et al., 2005, Tarara et al., 2008). Although the DEF bunches experienced higher temperatures, total glucosylated anthocyanin concentrations were similar for all treatments, and changes in anthocyanin composition were observed (Table 3). While the profile and structure of skin anthocyanins are known to influence intensity and stability of wine colour, there is little information regarding the influence of agronomic practices (e.g., irrigation, defoliation) on single anthocyanins (Tessarin et al., 2014; Theodorou et al., 2019). The data obtained in this experiment seem to suggest that bunch exposition to higher temperatures may change the proportion of total glucosylated anthocyanins (Table 3). Concerning the relative abundance of the single anthocyanin glucosylated forms, some changes were observed among the treatments; in particular, the 3ʹ4ʹ-OH/3ʹ4ʹ5ʹ-OH anthocyanin ratio at harvest was always higher in DEF I berries, as well as in the second season in DEF II, compared to the CK and SB ones. This increase in the di-substituted to tri-substituted ratio was in accordance with the effect of veraison defoliation in the study by Pastore et al. (2017). This could be explained by a metabolic shift leading to higher accumulation of cyanidin and a lower accumulation of malvidin-3-glucoside in vines subjected to late leaf and lateral removal in the bunch zone (Table 3). Similarly, in cv. Sangiovese vines subjected to veraison-leaf removal in the bunch zone, Pastore et al. (2013) found a higher percentage of cyanidin-3-glucoside and a lower percentage of malvidin-3-glucoside compared to CK at harvest, despite there being no changes in the concentration of total anthocyanins. The modifications to anthocyanin percentage may be, in part, explained by the increased light interception by bunches in the canopies that underwent late defoliation treatments, with an increase in di-substituted anthocyanins forms, particularly cyanidin-3-glucoside, and a decrease in the tri-substituted ones (Table 3). Such changes exhibit similarities to changes in the proportion of flavonols (Table 3) and may be partly associated with the latter; indeed, the synthesis of anthocyanins from flavonols occurs through specific metabolic pathways (Castellarin et al., 2012). The di-substituted to tri-substituted anthocyanin ratio is a potential indicator of the mode of the actions triggered by late defoliation treatments; its changes indicate a possible effect of light on the metabolic pathways involved in the synthesis of anthocyanins. The changes induced by DEF I and DEF II on the anthocyanin profile likely contribute to explaining the observed changes in the wines (Table 5).

The additional shading produced by SB shoot positioning on one side of the canopy did not have any affect on anthocyanins and flavonols levels. This indicates that the berries were sufficiently protected by leaves.

To our knowledge this is the first investigation on the multiple effects of the described practices on berries (Table 3) and on wine throughout storage (Table 4, Table 5). Moreover, the implications of the “semi-ballerina effect” through shoot positioning on wine quality were assessed for the first time (Table 4, Table 5).

Late defoliations (post-veraison or pre-harvest) and the “semi-ballerina” effect through shoot positioning significantly influenced some of the colour components studied in the wines, while maintaining the basic properties of the wines (e.g., alcohol strength, titratable acidity, volatile acidity, dry matter and pH) in the 2013 and 2014 vintages (Table 3). In particular, the SB treatment had higher small polymeric pigments than CK in wines after 16 months of storage, and DEF I wines showed higher total polymeric pigments than CK in wines after 4 months of storage (Table 5). Likewise, Tessarin et al. (2018) found that late trimming in Sangiovese resulted in a higher concentration of small and large polymeric pigments in stored wines. Data suggest that bending downwards lignified canes at post-veraison (Supplementary Figure 1), which involved some fractures, mimicked the action of trimming performed in the same phenological stage. Although the statistical analysis did not show any significant differences among treatments for the other parameters under investigation (Table 5), the data indicate that DEF I and SB treatments resulted in a colour enhancement in the wines of the 2013 vintage, the season being characterised by higher temperatures during ripening. This fact also shows the importance of the timing of the treatment, since the treatments that showed the highest values were DEF I and SB, both applied at post-veraison. Colour components, tannins and iron-reactive phenols are of great importance for the evolution of aging wines. Our results are consistent with those obtained for cv. Nero di Troia in a study by Baiano et al. (2015); however, in this variety, the late defoliations influenced the chemical composition of the wines.

In the 2014 wines, evaluating the effects of the treatments was not easy due to low alcohol and polyphenol concentrations; nevertheless, overall, a similar trend was observed. Collectively, these components might have contributed to an effect on the final colour intensity of the wine with a sensory impact on the wine. The loss of anthocyanins during storage may be due to the degradation of the anthocyanins or their incorporation into oligomeric and polymeric pigments - most likely into pigmented tannin-anthocyanin polymers (LPP) via anthocyanin-acetaldehyde cross-linked oligomers and pyranoanthocyanins (SPP) (Harbertson et al., 2003), as evident from the data on polymeric pigments. In fact, after one year of storage, the total polymeric pigments, fractioned as LPP and SPP, increased significantly (Table 5).

It is worth nothing in this study that the positive effects of the late defoliation treatments on the colour of the wine are not due to an enhanced concentration of berry skin anthocyanins (Table 5), but rather due to LPP and SPP pigments (Table 5). This increase may also be related to variations in the concentration of polyphenols in the seed due to late defoliation and shoot positioning treatments. Most studies have focused on early defoliation treatments, with results showing contrasting effects on seed polyphenols at harvest. Talaverano et al. (2016) reported a decrease in the concentration of seed polyphenols after the pre-flowering defoliation of Tempranillo vines. Kotseridis et al. (2012) recorded different results for the amount of seeds phenols in vines defoliated after flowering compared to the control vines, depending on the cultivar and the severity of the defoliation. The ability to drive the polymeric pigment reaction forward depends on many factors, including the levels of anthocyanins, tannins and acetaldehyde, the pH, the levels of SO₂ and the temperature (Harbertson et al., 2003). Our results suggest that late defoliation, particularly DEF I, and SB can substantially improve the quality of Sangiovese wines produced organically, increasing the parameters correlated with colour in a warm season as 2013.

Conclusions

The late defoliations (DEF I and DEF II) applied in the two consecutive seasons on the same cv. Sangiovese vines did not alter budburst, fruitfulness, leaf photosynthetic activity, vine water status, yield, berry technological parameters or berry skin anthocyanins. However, there was a marked increase in the concentrations of total flavonols in the two contrasting seasons as a result of enhanced light conditions in the bunch zone after the removal of leaves and laterals. Furthermore, late defoliations, especially DEF I, and “semi-ballerina” shoot positioning (SB) to a minor extent, limited the severity of Botrytis bunch rot. The results clearly indicate that the defoliations at post-veraison or pre-harvest have a beneficial effect on berry composition and grape sanitary status.

Oenological benefits induced by late leaf removal at post-veraison and pre-harvest and by the “semi-ballerina” effect through shoot positioning were observed in cv. Sangiovese wines throughout storage. In particular, these canopy management practices positively influenced wine components related to the final colour intensity without altering the basic chemical characteristics of wine.

The positive implications of late defoliations on wine colour are not related to enhanced concentrations in berry skin anthocyanins, but to increased flavonol levels and polymeric pigments. The increase in flavonols after the imposition of late defoliations indicates this practice can have positive implications for berry and wine composition, even when applied at pre-harvest.

“Semi-ballerina” shoot positioning and post-veraison defoliation improved the wine colour, particularly in the first season, which was characterised by higher temperatures and lower rainfall; this technique therefore shows high potential in the context of climate change. A more detailed metabolic analysis is required to better understand which compounds contributed to the observed effects, and thus be able to improve wine properties related to the aging of Sangiovese.

The choice of the proper timing of late defoliation (post-veraison vs. pre-harvest), should consider meteorological conditions in order to successfully both improve grape quality and contain and cluster rot. Technological advances mean that mechanical defoliation is also possible at pre-harvest, without damaging the bunches. Our results may further contribute to providing useful recommendations on canopy management for grape growers who produce organic Sangiovese wines subject to aging.

Acknowledgements

The Authors would like to thank Dr. Anna Maria Bregoli and Dr. Esther Hernández Montes for providing essential comments on the manuscript.

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