Table

titre du tableau
Year	Treatment	Sugar per berry(mg/berry)	Total Soluble Solids (g/L)	Titratable acidity(g/L)	pH	Tartaric acid(g/L)	Malic acid(g/L)	M/T
2015	CC	331 a	226 b	6.4	3.60	4.89 a	7.70 a	1.57
	PFD	383 b	239 a	6.2	3.64	4.55 b	7.46 b	1.63
2017	CC	353	212 b	6.4	3.32	n/d	n/d	n/d
	PFD	391	232 a	6.2	3.30	n/d	n/d	n/d
Treatment	**	***	ns	ns	**	**	ns
Year	ns	ns	ns	***	-	-	-
T x Y	ns	ns	ns	ns	-	-	-
2018	CC	372	234	7.7	3.27	4.18	8.54	2.04 a
	PFD	381	235	8.1	3.34	4.50	8.45	1.87 b
2019	CC	357 b	217 b	7.7	3.32	3.32 b	7.76 b	2.33 a
	PFD	416 a	228 a	7.6	3.41	4.01 a	8.44 a	2.10 b
Treatment	**	ns	ns	**	***	ns	**
Year	ns	**	ns	**	***	**	**
T x Y	ns	ns	ns	ns	ns	**	ns

Tritable acidity = g of tartaric acid per litre, M/T = malic to tartaric acid ratio. Different letters for a given year indicate significant differences between treatments post hoc test by Fisher; no letters indicate absence of significance (p-value ≤ 0.05). Factorial analyse: treatment, year, T x Y: treatment per year at p-values ≤ 0.10 (*), 0,05 (**), 0.01 (***). ns: no significance. Different factorial ANOVA correspond to different vineyard sites.

Table

titre du tableau
Year	Treatment	Anthocyanin Potential (mg EMG/L-1)	Anthocyanin Potential extractable (mg EMG/L)	Polyphenol Total Index (A280)	Extractability (%)
2016	CC	1339 b	661 b	44 b	50
	PFD	1638 a	830 a	54 a	52
2017	CC	1877 b	862 b	49 b	54 a
	PFD	1989 a	974 a	60 a	50 b
Treatment	***	***	**	ns
Year	***	***	**	ns
T x Y	ns	ns	ns	*
2018	CC	2059 a	948 a	62	53 b
	PFD	1792 b	681 b	62	59 a
2019	CC	1979 b	909 b	-	52
	PFD	3051 a	1361 a	-	55
Treatment	ns	ns	-	*
Year	***	**	-	**
T x Y	***	***	-	ns

EA (%) = extractability of anthocyanin. n/d = non data. Different letters for a given year indicate significant differences between treatments post hoc test by Fisher; no letters indicate absence of significance (p-value ≤ 0.05). Factorial analyses: treatment, year, T x Y: treatment per year at p-values ≤ 0.10 (*), 0,05 (**), 0.01 (***), ns: no significance.Different factorial ANOVA correspond to different vineyard sites.

The weather conditions varied highly from year to year, which is common in Uruguay (Tiscornia et al., 2016). The amount of rainfall during the cycle, and its distribution and thermal conditions during the different phenological periods, largely explain the yearly differences in yield, vegetative growth, sanitary status of harvest and grape composition (Ferrer et al., 2020).

The highest levels in cluster rot incidence occurred in 2017, due to weather conditions (high rainfall during the ripening period) being favourable for grey mould development. During 2019, the largest amount of rainfall occurred between the 31st and 34th stages (Coombe, 1995), when the efficiency of the defence mechanisms of the berries was high, preventing Botrytis cinerea infections (Nair and Hill, 1992).

The incidence of bunch rot was notably reduced by PFD, in agreement with previous reports (Tardaguila et al., 2010; Molitor et al., 2011; Sternad Lemut et al., 2015). The increased bunch exposure and decreased compactness in 2017 were negatively related to rot incidence levels, which is also in agreement with previous studies (Poni et al., 2006; Molitor et al., 2011). In general, decreasing bunch compactness caused by PFD was not consistent. According to Tello and Ibáñez (2018), the environmental conditions during the growth cycle are widely known to modulate this characteristic. A trend of increasing berry weight for the PFD treatment was observed, with differences only found in the last season; this could be due to an increase in bunch compactness and, consequently, in cluster rot susceptibility. Conversely, increased bunch exposure generated less favourable microclimatic conditions for the development of bunch rot (Table 2). Relative humidity levels were reduced during certain hours of the day; this was likely due to the higher incidence of solar radiation, resulting in an increase in temperature and in vapour pressure deficit within the bunch zone, thus decreasing the periods of wet tissue after a rain event (Figure 3). In addition, increased exposure improves airflow and the effectiveness of phytosanitary control (English et al., 1989; Molitor et al., 2015; Sternad Lemut et al., 2015).

Rainfall amount and distribution determined vine vegetative growth capacity and recovery of leaf area losses caused by PFD (Figure 2), which occurred in the 2016 and 2019, as previously reported by Poni et al. (2006). Despite the conditions in the first season being the driest, the PFD vines were able to recover their leaf area. 74 % of the rainfall occurred during the first stages of vine development, a critical period for leaf area establishment (Champagnol, 1984). Furthermore, the water storage capacity of the soil was high until January, and it probably fulfilled the vine water requirements during the first stages of the growth cycle (Figure 1). In the second season, the absence of regrowth can be mainly explained by the continuity of PFD, the consecutive application inducing the carryover effect of a larger decrease in yield and low vigour, as confirmed by a decrease in DMPC and PW values (Table 4). In a regime of continuous PFD, it is possible that reserves are mobilised from perennial parts to make up for deficiencies caused by early leaf losses, leading to a significant decrease in yield and vigour across the seasons, as suggested by Candolfi-Vasconcelos et al. (1994) and Risco et al. (2013). Regarding the new plants in 2018, the absence of vegetative regrowth on PDF vines and lower vegetative expression can be explained by October to February rainfall being below historic values, affecting secondary shoot development. In the fourth season, low yields per plant and high water availability were both recorded from October until February, likely causing a shift in physiological balance towards the observed vegetative expression. In our study, the capacity for Tannat grapevines exposed to severe leaf removal in the early stages of the growth cycle to recover leaf area was conditioned by the interaction of prevailing environmental conditions during critical periods for leaf area establishment (particularly rainfall during the spring months) and vine capacity as expressed by the DMPC.

The drop in yield of PFD plants varied between 11 and 39 % when compared to control vines, which agrees with previous studies by Poni et al. (2006), Tardaguila et al. (2010), Risco et al. (2013) and Verdenal et al. (2018). The yield was mainly affected in terms of number of berries per cluster, thus having an impact on bunch weight (R²= 0.88, p-value < 0.001); this result is in accordance with previous studies (Poni et al., 2006; Palliotti et al., 2011; Gatti et al., 2012). According to the results of the factorial analysis, PFD did not differ in berry weight from CC; this variable only differed statistically in the fourth season, when it was greater than CC. This result is different from studies carried out in a semiarid climate where a decrease in berry weight was observed (Poni et al., 2006; Risco et al., 2013; Gatti et al., 2012). The result observed in the fourth season could be due to a lower number of berries per bunch that promote cell division in the mesocarp during the herbaceous growth stage of berries, and when there is no water restriction (which was the case); this can result in compensatory growth of the berries during the ripening stage (Ojeda et al., 2002; Petrie and Clingeffeler, 2006). There was always a greater drop in yield from PFD vines after continuous PFD had been carried out for the second time; this can be explained by the carryover effect of removing leaves in the preceding season, which adds to the effect of PFD in the current season, as suggested by Risco et al. (2013). Reduction in leaf area due to PFD affects fruit set, yield and vine capacity expressed as DMPC. In such a situation, this could imply a re-translocation of carbohydrate reserves to other sink organs like berry during maturity, thus reducing available stocks for the next cycle (Candolfi-Vasconcelos et al., 1994; Gómez del Campo et al., 2002). PFD did not result in decreased bud fertility the following year (Table 3), which is consistent with Poni et al. (2006) and Verdenal et al. (2018), but contrasts with results of studies by Sabbatini and Howell (2010) and Risco et al. (2013). Slight increases in bud fruitfulness were recorded depending on the year, which can probably be explained by the impact of an increase in light intensity and temperature (known to be particularly important in induction and differentiation processes) on the first buds of the shoot (Figure 3), which could have resulted from the leaf removal in this variety at this stage (Buttrose, 1970; Collins et al., 2020). Our results confirm the potential to use PFD as a yield management practice as an alternative to traditional bunch thinning (Poni et al., 2006), the latter being a common practice in the production of quality wines in many viticultural regions, including in Uruguay. Manual cluster thinning is an expensive practice, which does not always have the desired results. In addition, pre-veraison cluster thinning in Tannat has been reported to result in yield compensation due to an increase in bunch weight, which also increases compactness and susceptibility to rot (Ferrer and González-Neves, 2002). PFD did not involve any yield compensation and it also improved the microclimate in the bunch zone, reducing humidity levels within the canopy (Table 2 and Figure 3) and improving the sanitary status of the harvest (Table 3). Regarding the interannual variability of the yield from both treatments, the most affected component was the number of bunches per vine, which is also influenced by the environmental conditions during floral induction in the previous season, mildew incidence in the current season, and level of vigour in vines (Vaillant-Gaveau et al., 2014; Ferrer et al., 2017).

The weather conditions contributed to modulating the physiological balance of the plants across the seasons, which is in agreement with Echeverría (2017). While the Ravaz index was different between years, similar values were obtained in the two first seasons, indicating equilibrium situations (Ferrer et al., 1997). The third and fourth seasons, however, were completely different. In 2018, the balance shifted towards grape production, while in the fourth^{season it shifted towards wood production, due to the low yield and high-water availability during the whole growth cycle (Table 4 and Figure 1). The ranges of leaf to fruit ratio values recorded in this study (Table 4) are in agreement with those reported by Echeverría (2017), who studied the same variety in the same conditions. Without any restrictions on vegetative growth, higher leaf to fruit ratio values were obtained for the PDF vines, which is in accordance with previous studies (Poni et al., 2006; Tardaguila et al., 2010). PB (SA/dry matter) was also higher in PFD vines, explained by the lower fruit set and, consequently, the lower yield; when plants regained leaf area after early leaf removal this translated into a higher dry matter production capacity per leaf area unit. This could at least partially explain the improvement of some oenological attributes, such as sugar concentration in must and grape anthocyanin potential. Furthermore, PDF vines are likely to develop a more efficient canopy, due to having less internal leaves (Table 2) and younger leaves from secondary shoots, which contribute to improving microclimatic conditions for photosynthesis (Smart and Robinson, 1991; Poni et al., 2006).}

In terms of grape composition, the concentration of sugars in the must was higher for PFD vines in three out of the four years of evaluation. The decrease in yield observed in the present study produced a greater capacity for the remaining berries to accumulate sugars, as was suggested by Kliewer (1970); this is supported by the fact that the number of berries per bunch correlated negatively (R² = -0.53, p-value < 0.001) with the amount of sugar per berry. Despite the absence of differences in LF ratio in 2017, the early defoliated plants achieved a higher concentration of total soluble solids. This may be due to a lower number of berries per bunch (< 29 % when compared to CC), a greater exposure of bunches to sunlight (> 104 % when compared to CC), and differences in the canopy structure (Table 2), improving microclimatic conditions for photosynthesis and ripening (Figure 3). PFD did not induce any consistent changes in tritable acidity or pH, in accordance with Poni et al. (2006), Tardaguila et al. (2010), Gatti et al. (2012) and Mirás-Avalos et al. (2019). Lower M/T ratios in PFD must were recorded during the 2018 and 2019 vintages, and can be mostly explained by higher tartaric contents. The higher amount of light intercepted by a PFD canopy could lead to a large amount of CO₂ being used in tartaric biosynthesis (Kliewer and Schultz, 1964). The fact that there were fewer shaded adult leaves on PDF plants meant that a higher amount of tartaric acid was produced and subsequently exported to the berry. This process could mitigate the presumed increase in malic acid catabolism caused by the high sun exposition of bunches, keeping acidity levels stable, as suggested by Gatti et al. (2012). In 2019, the high-water availability during pre-veraison and post-veraison promoted vegetative growth at the expense of sugar concentration. The quantity of sugar per berry was higher in PFD, this variable was related to berry weight (R² = 0.67, p-value = 0.001), in accordance with previous studies (Dai et al., 2011; Gil et al., 2015; Mirás-Avalos et al., 2019). The capacity for accumulating solutes was not restricted like in other studies under semi-arid conditions (Mirás-Avalos et al., 2019). The increase in sugar concentration observed in PFD must during the "unfavourable" seasons (2017 and 2019) could result in winegrowers receiving a higher price for the grape.

Values for anthocyanin potential, extractable anthocyanin, phenolic richness and extractability in Tannat grapes were in agreement with those reported by González-Neves et al. (2004). Seasonal environmental conditions had an effect on how PFD influenced grape anthocyanin potential (Table 6). Anthocyanin concentration was not improved by PFD when both rainfall was lacking during the critical period for regaining leaf area after leaf removal and yield per vine was high (which occurred in 2018). LF explained a high proportion of variability in anthocyanin content per berry (Figure 4). Consequently, vine yield also influenced anthocyanin content per berry. However, is widely known that other factors during ripening, such as bunch light exposure, water status and temperature conditions, have an influence on the synthesis and degradation of these compounds (Smart and Robinson, 1991). The data collected here support other studies on the influence of yield and leaf to fruit ratio on the accumulation of secondary metabolites in the grape berry (Howell, 2001; Kliewer and Dokoozlian, 2005; Tardaguila et al., 2010; Echeverría, 2017; Mirás-Avalos et al., 2019). Despite the fourth season being unfavourable for the accumulation of sugars in the berry, the concentration of anthocyanins was exceptionally high. A lower linear relation between sugar quantity and anthocyanin quantity in the berries was found (R² = 0,09, p-value = 0,03), which indicate a multifactorial control of anthocyanin biosynthesis in grape.

Carrying out PFD on the Tannat variety in a location with a humid climate had different effects depending on the seasonal weather conditions. PFD proved to be a suitable technique for yield control, where declines in yield were accumulative in a continuous application regime. Yield per vine was reduced by 11 to 39 % with respect to CC. In a year with favourable conditions for bunch rot, the technique showed a notable effectiveness for improving the sanitary status of the grapes and contributed to the improvement of some oenological attributes, such as sugar concentration, total polyphenols and anthocyanin content. However, these improvements were relative and appear to depend on the capacity for the recovery of vegetative growth, yield per vine, weather conditions and their partial interaction. PFD could be useful for correcting excess of vigour in grapevines. However, factors limiting vegetative growth, such as water supply during the critical stages of leaf area establishment, could lead to undesirable effects on grape composition and vine life. Despite these possible drawbacks, our study showed PFD to be a promising technique for vigorous grapevine varieties, like Tannat under humid climate conditions.

This work was supported by CSIC VUSP project “Study of the cluster rot complex and application of techniques for its control” and CSIC 330 “Study of the plant environmental interaction according to climatic regions to determine the yield, composition and typicity of Tannat grapes”. The authors are grateful to professor José Manuel Miras Ávalos for his contribution to improving this article, and to Establecimiento Juanico for allowing us to set up the trials in their commercial vineyards.