Introduction

Under current climate change pressures, obtaining grapes with adequate acidity at harvest is one of the main challenges for growers, especially if the goal is producing sparkling wines. This issue arises from two main occurrences: i) higher temperatures enhance the degradation of malic acid; ii) grape maturity may occur under suboptimal climatic conditions due to advanced phenology (Palliotti et al., 2014). For this reason, the introduction of new varieties or the reconsideration of minor and insofar neglected cultivars or clones are gaining enormous interest and popularity (Palliotti et al., 2014, Poni et al., 2020, Antolín et al., 2020). However, the criteria for the identification of performing genotypes in terms of ‘acidity at harvest' are complex. Late veraison and ripening can be limited traits since they do not necessarily mean that grapes maintain adequate acidity in relation to a satisfying sugar concentration (Poni et al., 2018, Frioni et al., 2020a). Considering the evolution and the destination of organic acids in grapes, the pre-veraison malic acid pool could represent a promising trait. In the same framework, an eventual genetic control over malic acid degradation rates could hint at additional information. The same can be said for the ratio between minimum acidity and pre-veraison malate abundance or for the sugars/acidity ratio. However, any of the above-mentioned parameters seems to underestimate some of the factors involved or, in any case, to provide an incomplete overview of grape ripening.

In this work, we compared the fruit, must, and wine composition of a local, widely grown white grape variety in the Colli Piacentini area (cv. Ortrugo, ORT) with those of a minor autochthonous variety, namely, Barbesino (BRB), over four consecutive seasons. Our goal was to clarify the relationships between the evolution of titratable acidity during ripening and a wide range of parameters concerning organic acids catabolism. We hypothesised that minimum grape malic acid concentration is a genetic trait deriving from malic acid degradation rates displayed at different TA concentrations. The general aim of this study was the identification of the proper parameters to describe the attitude of a genotype to retain satisfying acidity at harvest.

Materials and methods

Results

3. Grape ripening and malic acid degradation rates

ORT and BRB showed a very similar grapes TSS accumulation pattern in any of the four years of study (Figure 1).

Figure 1. Seasonal dynamic of grapes total soluble solids (TSS, A–D) and titratable acidity (TA, E–H) in grapevines cv. Ortrugo (ORT) and cv. Barbesino (BRB) in 2017 (A,E), 2018 (B,F), 2019 (C,G) and 2020 (D,H). DOY= Day Of the Year. Asterisks denote significant difference within date between the two treatments per p < 0.05.

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The onset of TSS accumulation differed according to the season, but both varietals started accumulating sugars concomitantly in all the years. Final TSS was higher in 2017 and 2020 than in 2018 and 2019. Conversely, TA was significantly higher in BRB from preveraison to the end of the season in all the years (Figure 1). Pre-veraison differences in TA were higher in 2017 (+ 9.5 g/L in BRB) and lower in 2020 (+ 3.2 g/L in BRB). Then BRB maintained higher TA until the end of the season with final differences scoring + 2.2 g/L in 2017, + 2.7 g/L in 2018, + 2.3 g/L in 2019 and + 4 g/L in 2020. As a result, data pooled over the four years demonstrate that BRB exhibits higher TA independently by the TSS concentration (Supplementary Figure 3). Differences in grape malic acid during ripening tracked those in grape malic acid concentration (Figure 2A–D). In any of the four years of study, the differences between BRB and ORT peaked right after veraison, and, despite a quite steady reduction of the gap later in the season, in all the years, the final grape malic acid concentration was higher in BRB. Both varietals exhibited slower sugar accumulation rates on the lower part of the parcel, while no difference in TA decrease was observed within the different sections of the rows, independently by the cultivar.

Figure 2. Seasonal dynamic of grapes malic acid concentration (A–D) and malic acid degradation rates (E–H) in grapevines cv. Ortrugo (ORT) and cv. Barbesino (BRB) in 2017 (A,E), 2018 (B,F), 2019 (C,G) and 2020 (D,H). DOY= Day Of the Year. Asterisks denote significant difference within date between the two treatments per p < 0.05.

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The analysis of malic acid degradation rates (Figure 2 E–H) reveals that in any of the four years, ORT starts losing malic acid earlier than BRB. Maximum malic acid degradation rates were achieved earlier by ORT in 2017, 2019 and 2020, while peaks coincided in 2018. However, maximum malic acid degradation rates were higher in BRB in 2017, 2018 and 2020. In these three years, BRB showed higher rates also later in the season until the last sampling dates, with a few exceptions. In 2019 only, maximum malic acid degradation rates were higher in ORT, but later in the season, BRB showed higher rates until the end of the season, such as in 2017, 2018 and 2020.

Polynomial third-grade correlations were fitted between grapes TSS and malic acid degradation rates in both varietals for all the years (Figure 3). Analysis revealed that the relationship between TSS and malic acid degradation rates were significantly affected by the season, with BRB showing: i. higher malic acid degradation rates at TSS ranging from 8 °Brix to 15 °Brix, except for 2019; ii. Higher malic acid degradation rates at the TSS interval 15–20 °Brix, except for 2018 when curves were coinciding for TSS > 18 °Brix.

Figure 3. Correlation (A–D) between grapes total soluble solids concentration (TSS) and malic acid degradation rates in grapevines cv. Ortrugo (ORT) and cv. Barbesino (BRB) in 2017 (A), 2018 (B), 2019 (C) and 2020 (D). Correlation (E–H) between grapes malic acid concentration and malic acid degradation rates in grapevines cv. Ortrugo (ORT) and cv. Barbesino (BRB) in 2017 (E), 2018 (F), 2019 (G) and 2020 (H). All correlations were significant per p < 0.05.

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Significant polynomial third-grade correlations were also built for the correlation between instantaneous grapes malic acid concentrations and malic acid degradation rates displayed in the period (Figure 3). BRB showed consistently higher malic acid degradation rates for malic acid concentration > 15 g/L, while the rates were consistently lower when malic acid was lower than 10 g/L.

4. Fruit composition at harvest and final wine characteristics

Picking grapes at TSS = 20 °Brix, no difference was found in TSS or tartaric acid concentration between treatments, whereas BRB showed significantly lower pH (–0.14) and higher TA and malic acid concentration (+ 4.09 g/L and + 2.43 g/L, respectively), independently by the year (Table 2). The tartaric acid on malic acid ratio was significantly higher in ORT due to the low malic acid final concentration.

Table 2. Grapes composition and organic acids concentration in grapevines cv. Ortrugo (ORT) and cv. Barbesino (BRB) over four seasons (2017–2020).

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	TSS (Brix)	pH	Titratable acidity (g/L)	Malic acid (g/L)	Tartaric acid (g/L)	HT/HM1
ORT1	21.2	3.15	5.02	0.52	6.52	12.5
BRB	20.9	3.01	9.11	2.95	7.81	2.65
V	ns2	**	***	***	ns	***
Y	*	***	**	***	ns	**
VxY	ns	ns	ns	ns	ns	ns

¹ ORT = Ortrugo, BRB = Barbesino, V = Variety, Y = Year, HT/HM = Tartaric acid/malic acid ratio.

²*,** and *** denote significant difference per p < 0.05. ns = no significant difference.

Table 3 shows the oenological parameters of ORT and BRB wines produced in 2020. All wines were regularly fermented to dryness (residual sugars < 0.80 g/L) within 10 days. No differences were found in ethanol content. ORT wines showed a higher pH (3.20) and a TA of 3.91 g/L, while BRB had a significantly higher TA (5.04 g/L) and lower pH (3.00). Tartaric acid concentrations ranged from 3.71 to 3.80 g/L, with no difference due to the variety. On the other hand, the differences in titratable acidity were mainly due to malic acid, which was significantly higher in BRB wines (2.76 g/L vs 1.02 g/L in ORT wines).

Table 3. Oenological parameters and organic acids profile of white wines obtained by cv. Ortrugo (ORT), cv. Barbesino (BRB) in 2020.

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	Attributes	ORT	BRB	V1
General parameters	Ethanol (% v/v)	10.17	12,50	ns
	Glucose (g/L)	0.06	0.06	ns
	Fructose (g/L)	0.71	0.70	ns
	pH	3.20	3.00	**
	Titratable Acidity (g/L of sulfuric acid)	3.91	5.04	***
	Volatile acidity (g/L of acetic acid)	0.31	0.40	**
	Free SO2 (mg/L)	17	12	**
	Total SO2 (mg/L)	82	87	ns
Organic acids	L-(+) Tartaric acid (g/L)	3.90	3.81	ns
	L-(-) Malic acid (g/L)	1.02	2.96	***
	Latic acid (g/L)	0.19	0.17	ns
	Acetic acid (g/L)	0.16	0.29	***

¹*,** and *** denote significant difference per p < 0.05, p < 0.01, p < 0.005, respectively. ns means no difference. V = Variety.

Discussion

Maintaining adequate acidity in grapes is one of the main current issues for producing white and sparkling wines in warm climates (Palliotti et al., 2014, Poni et al., 2018). While managing grape sugars is quite easier due to their dependence to leaf to fruit ratio and crop load (Kliewer and Dokoozlian, 2005), controlling organic acids is definitely more complicated since they respond mainly to air temperatures and genetic traits (Famiani et al., 2014; Ford, 2012). For this reason, the most efficient strategies to increase grape acidity consist of the so-defined long-term adaptation strategies, encompassing, for instance, site selection, altitude, and varietal choice (Palliotti et al., 2014). In this framework, many research groups and wine districts are re-considering old neglected cultivars and clones (Arrizabalaga-Arriazu et al., 2018; Arrizabalaga-Arriazu et al., 2020; Arrizabalaga-Arriazu et al., 2021, Frioni et al., 2020a, 2020b, Antolín et al., 2020, 2022, Torres et al., 2018, Medrano et al., 2018), or identifying quantitative trait locis codifying for acidity to breed new varieties coping with warming trends (Bigard et al., 2018; Bigard et al., 2020).

In this context, the definition of the best parameters to describe the attitude of a genotype in maintaining satisfying acidity at the final stages of ripening is a key factor. In our work, ORT had lower acidity at harvest than BRB in four consecutive years, with an average difference of 4.07 g/L (Table 2). Such a consistent and stable behaviour was accompanied by a lower pre-veraison TA and malic acid concentration (Figures 1 and 2A–D). However, no significant correlation was found between the seasonal pre-veraison malic acid concentration and its final minimum abundance when pooling the two varietals together, nor considering them separately (data not shown). Therefore, seasonal final malic acid concentration depended on other factors.

After veraison, malic acid is mainly lost due to berry respiration, which, in turn, is driven by temperatures and substrate abundance (Ford, 2012). In grapes, this process leads to a drastic reduction of the initial malate pool from 20–40 g/L to 0–4 g/L. In white/sparkling wines, optimal technological ripening consists of the balance between TA and TSS (Poni et al., 2018). However, Figure 2E–H demonstrates that time of achievement of maximum malic acid degradation rates changes according to the genotypes, even if those exhibit an identical TSS accumulation pattern (Figure 1). In detail, ORT started degradation of malic acid earlier in all four years, then rates peaked earlier than BRB in 2017, 2019 and 2020 (Figure 2E,G,H), whereas in 2018, the two cultivars achieved maximum malic acid degradation rates concomitantly (Figure 2F). Data suggest that a strong varietal factor drives timings in malic acid degradation rates, which can significantly vary between genotypes independently by the TSS accumulation onset time and later trend. This confirms that TSS dynamics and timings cannot be taken as a representative parameter to define varietal TA and malic acid loss trends.

Additionally, other factors leading to malic acid loss is dilution due to berry growth and volume increase. In our work, BRB and ORT started from similar pre-veraison berry weight levels and exhibited no difference at harvest (Supplementary Figure 2, Table 1). Therefore, higher final malic acid concentrations in BRB seem to be unrelated to a different berry volume expansion from veraison to harvest.

Surprisingly, in three years out of four, BRB had higher maximum post-veraison malic acid degradation rates. In 2019 only, ORT scored higher maximum rates (Figure 2G). The 2019 season was the coolest among the four examined, displaying delayed veraison and TA loss as compared to the others, and this can be one of the reasons for such occurrence. In detail, ORT maximum malic acid degradation rates ranged between 0.89 g/L (2020) and 1.24 g/L in 2017, whereas in BRB, rates ranged between 0.92 g/L (2019) and 1.93 g/L (2018). Overall, our data suggest that maximum malic acid degradation rates depend on the pre-veraison malic acid abundance, and that varieties exhibiting higher initial malate pool are also those more sensitive to weather evolution in terms of seasonal changes in maximum malic degradation rates.

After veraison, malic acid concentration and its degradation rates were consistently higher in BRB in all the years examined (Figure 2). Therefore, low malic acid degradation rates during ripening do not mean higher final acidity in grapes. Conversely, our data demonstrate that a simple analysis of malic acid loss rates can be misleading since the varietals displaying lower post-veraison malic acid degradation rates are those having the lower final TA and malate concentration.

As expected, correlations between TSS and TA or malic acid depict higher TA in BRB for any TSS value (data not shown), in agreement with Frioni (Frioni et al. 2020a, Frioni et al., 2020b). In Figure 3, a polynomial correlation between grapes TSS and malic acid degradation rates was built for the two genotypes. Interestingly, in any of the four years, BRB showed higher rates for TSS >15 °Brix, even if in 2018 (Figure 3B), correlations of the two varietals overlapped for TSS > 18 °Brix. Considering the above-mentioned link between malic acid degradation rates and its abundance, higher rates at TSS > 15 °Brix mean retaining higher TA and malic acid at TSS, approaching optimal values for harvest.

In Figure 3E–H, correlations between instantaneous malic acid concentration and malic acid concentration rates are reported. Data reveal that higher malic acid degradation rates reported for BRB in Figure 2E–H are essentially a smokescreen: even if rates are higher in BRB for malic acid concentrations > 15 g/L, ORT displays higher malic acid degradation rates for malate < 10 g/L in all the years examined. Additionally, despite a certain seasonal variability in terms of peaks and their occurrence, the transition from higher rates in BRB to higher rates in ORT was something consistent and steady in all four years. This transition occurred at 13 g/L in 2017, at 12.5 g/L in 2018, at 16 g/L in 2019 and at 10 g/L in 2020. Our data demonstrate that there is a strict genetic control over malic acid degradation rates at varying malic acid concentrations and that this control easily overcomes seasonal effects, which instead affect a simple analysis of TSS, TA, veraison times, and, particularly, malic acid degradation rates.

Notably, BRB exhibited an anticipated berry growth right after veraison. This could have contributed to the high malic acid degradation rates found in BRB at malic acid concentration rates >10 g/L. Therefore, we cannot exclude a possible role in malic acid degradation rates due to different timing in berry volume expansion.

Wine analytic data confirms that if malo-lactic fermentation is not undertaken, wines tend to preserve the relative differences in TA at harvest (Table 3). Although the pH and titratable acidity of wines is an essential parameter for ensuring wine quality, the organic acids (i.e., tartaric acid and malic acid) and low molecular weight play a crucial role in maintaining the chemical and microbiological stability of the wine, its colour, and taste (Mato et al., 2005).

Finally, when evaluating genotypes for their adaptability to climate change, productivity cannot be exchanged for better fruit composition. In our work, BRB demonstrated a comparable yield to ORT, with potentially lower susceptibility to rots, due to its lower cluster compactness (Table 1).

In conclusion, our work demonstrates that when evaluating genotypes of grape acidity, malic acid degradation rates itself can be a misleading parameter since rates mainly respond to malic acid abundancy, and varietals having higher malic acid pool pre-veraison or during ripening exhibit higher rates, no matter what the final TA or malic acid concentration is. Conversely, our work highlights the potentialities of the analysis of relationships between instantaneous malic acid concentration and malic acid degradation rates. The polynomial correlations built allow us to identify the genotypes that, in the given environment, can reduce the loss of acidity when most of the pre-veraison malic acid pool is already depleted and maintain a final high must acidity. The approach can be of relevance for the determination of varieties maintaining higher acidity at different scales: i. In farm, when establishing new vineyards; ii. In wine districts revising appellations according to local or international germplasm; iii. At the research level, for phenotyping or breeding new varieties or for selecting accessions within progenies.

Acknowledgements

The work was partially funded by Emilia-Romagna Rural Development 2014–2020 for Operational Groups (PSR Emilia-Romagna 2014–2020 Mis. 16.01.01) projects EIP-AGRI ValorInVitis (n. 5004320, call 2016) and EIP-AGRI SalViBio (n. 5150112, call 2019). Participation in GiESCO was funded by UCSC internal funds.

The authors thank ‘Azienda Vitivinicola Mossi 1558’, Roberto Miravalle and the partners of the Operational Groups ValorInVitis and SalViBio for the support and topic discussions. Special thanks to Maurizio Zamboni, Luigi Mossi and Mario Fregoni for preserving plant material over time and for establishing the varietal collection. Authors thank Maria Giulia Parisi, Silvia Pagani, Elia Romanini and Leonardo D’Intino for technical help.

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