Sustainable Viticulture: E ﬀ ects of Soil Management in Vitis vinifera

: Soil management in vineyards is of fundamental importance not only for the productivity and quality of grapes, both in biological and conventional management, but also for greater sustainability of the production. Conservative soil management techniques play an important role, compared to conventional tillage, in order to preserve biodiversity, to save soil fertility, and to keep vegetative-productive balance. Thus, it is necessary to evaluate long-term adaptation strategies to create a balance between the vine and the surrounding environment. This work sought to assess the e ﬀ ects of following di ﬀ erent management practices on Vitis vinifera L. cv. Cabernet Sauvignon during 2017 and 2018 seasons: soil tillage (T), temporary cover cropping over all inter-rows (C), and mulching with plant residues every other row (M). The main physiological parameters of vines (leaf gas exchange, stem water potential, chlorophyll ﬂuorescence, and indirect chlorophyll content) as well as qualitative and quantitative grape parameters (technological and phenolic analyses) were measured. Signiﬁcant di ﬀ erences in gas exchanges related to the di ﬀ erent season and inter-row management were observed. C showed more negative values of water potential, due to the grass–vine competition, especially when water availability was lower. The competition exerted by C led to di ﬀ erences in fruit setting with impact on yield; therefrom, signiﬁcant di ﬀ erences also in sugar and anthocyanic content were observed.


Introduction
The European Community's focus on environmental protection issues and increased sensitivity on the part of agricultural entrepreneurs drive scientific research to sustainable production techniques for the environment [1]. To achieve sustainable viticulture, innovative techniques with low environmental impact must be identified and used, ensuring that productivity and quality are maintained over time [2,3].
Agriculture must be the natural balance between plant and atmosphere, to obtain healthier products and ward off environmental degradation [4][5][6][7].
It has been shown that the vineyard inter-row soil management affects grapes; nonchemical weed control (harrowing, mulching), tillage and nutrient application, and other interventions affect vine functioning to varying extents [24][25][26].
Soil management methods have an effect on photosynthesis and stomatal conductance of the vines [27,28] and the nutrient uptake of the grapes is affected by soil temperature, soil compaction, and soil moisture. For example, a positive correlation between the high soil temperature and the uptake of nitrogen (N), potassium (K), calcium (Ca), and magnesium (Mg) exists [29].
Unfavorable soil conditions, such as low water content due to mishandling of soil, can cause plant stress, with negative effects both on growth and yield [30][31][32]. Water deficit also decrease photosynthetical activity [33,34] and affects the differentiation and abortion of flowers, fruit sets, and berry sizes [35,36].
Cover cropping, in territories with very low summer rainfall and with high evaporative demand, can be detrimental if the competition for water leads to severe vine water stress and consequently to negative effects on berry quality, yield, and growth [37]. Grapevines growing under heat stress experience a significant drop in photosynthesis due to stomatal limitations, leading to a reduction in water use efficiency [38]. In high vigor situations, the management of the green soil covers can lead to an improvement in the state of the vines because the increase in water consumption can lead to reduction of the vegetative growth of the vine and as a result an advantage in the microclimate of the fruit area and in the quality of the grapes can be obtained [39][40][41]. Many scientific experiments and tests have been carried out to better detect the influence of different floor covers in grapevine vegetative growth, yield, berry, and wine quality [9,[41][42][43]. In the Mediterranean basin vineyard cover cropping is still debated and the few results of the scientific experiments are often contradictory and conflicting [44].
Cover cropping is considered as the practical application of ecological principles such as diversity, crop interaction, and other natural regulation mechanisms [45]. Available resources, such as light, water, and nutrients are more efficiently used by the intercrop than by the main crop. Management of these differences in competitive capacities between crops and intercropping species could lead to yield advantages and produce crop quality improvements with consequent changes in grape quality, and decrease the herbicide use and associated risks, such as vine damage by spray drift, evolution of weed resistance [46,47].
Cover cropping in vineyards was a common practice in Europe [48]. Nowadays, vineyard cover cropping is widely used in areas with frequent summer rainfall to remove excess water and nitrogen, but the benefits of cover crops also include soil erosion control [49,50], nitrogen and organic matter management. For example, a reduction in the soil nitrate can induce a low level of must nitrogen content [51,52], improve soil structure, increase water penetration [53] and retention, decrease direct soil water evaporative losses, reduce grapevine vegetative vigor, thus enhancing grape and must quality [54,55].
According to Guerra and Steenwerth [9], cover crops can improve soil and vine health, and may influence vine vigor by adjusting parameters such as canopy efficiency and shoot growth period. Moreover, cover crops increased juice anthocyanins, soluble solids, and other phenolic components and decreased titratable acidity.
It is necessary to have a balanced vineyard system/intercropping, picking out crop species accurately and applying a suitable management to obtain benefits; the same studies [56][57][58] have shown that the use of cover crops in vineyards has detrimental effects, such as yield reductions due to water and nutrients competition.
Several studies found that pH were reduced by cover cropping relative to tillage due to an increase in the ratio of tartaric to malic acids [8,59]; another observed an increase in juice anthocyanins and tannins as affected by permanent cover crop [12,60]; this is probably due to a yield reduction and subsequent must concentration.
Mulch is any material, other than soil, placed or left on the soil surface for soil and water management purposes [61] and mulching involves leaving crop residues or other materials on the soil surface for soil and water conservation and keeping favorable and stable environments for vine growth [62]. The positive effects of mulching (both organic and/or synthetic) can be summarized as follows: (i) soil protection against the impact of rain and consequent erosion reduction [63], (ii) major water storage [64], (iii) improved infiltration capacity [65], (iv) decrease in evaporation [66], (v) improved soil structure and organic content [67], (vi) best root development [68], (vii) growing activity of some species of earthworms and crop performance [69].
Use of mulches in orchards has been found to increase plant growth and yields [70]; the yield increase as a result of mulch treatment was attributed to the improved soil environmental conditions of reduced diurnal temperature fluctuations and increased soil water availability for vine production [71]. Moreover, mulched compost can reduce water loss by evaporation and drainage into deeper soil horizons [72] and increased photosynthesis per vine (µmol CO 2 plant −1 s −1 ) at flowering, when the berries were pea sized and at maturity [73].
Mulch composed of fresh plant residues increased grape juice titratable acidity (TA) and, in dry years, increased juice soluble solids [74] and increased the K content with a resulting slight increase in grape juice pH [75]. According to Coventry [76] mulch increased Brix, total phenolics, flavonols, and anthocyanins of "Cabernet Franc" berries at harvest compared to tillage.
Vineyard soil management practices with sustainable objectives can be considered as a first protection strategy to improve grapes quality and reduce the effects of climate change [77].
Considering the above, a research project to evaluate the effect of soil management practices on Vitis vinifera, cv. Cabernet Sauvignon was carried out in a Tuscan vineyard.

Experimental Design and Settings
The experiment was set up in the Chianti Classico area (Lat 43 • 39 35" N-Long 11 • 11 5" E), Tuscany, Italy, located at an elevation of 270 m a.s.l. facing South-West exposure. The measurements were performed from 2017 to 2018. The 10-year-old vineyard of the red cv. Cabernet Sauvignon (Vitis vinifera L.), clonal selection R 5, grafted on 420 A rootstock, was planted with a spacing of 1.2 m (between vines) × 2 m (between rows) (≈4166 vines/ha). Vines were trained on a vertical shoot positioning and spur-pruned with a single cordon system, at 70 cm above ground with six spurs (12 buds per vine).
The vines did not have irrigation and were grown using standard cultural practices. Soil horizons presented a clay texture with the following characteristics: clay 39.8%; silt 34.8%; sand 25.4%; organic matter 2.0%; pH (H 2 O) 7.6. Soil analyses were performed according to the official method by the Italian Ministry of Agriculture.
The following three soil management methods were tested: mulching with straw and vegetable residues (M), cover crop (33% Trifolium incarnatum, 33% Lolium multiflorum, 33% Festuca arundinacea), (C) and mechanical cultivation tillage (T). Every treatment was located in five contiguous rows, with five replications per treatment. There was a total of 25 rows per management practice.
With mechanical cultivation, the soil was cultivated three times after the autumn ploughing. The straw and vegetable mulch were applied in the year 2017 (7 kg/m 2 ), and it was renewed in May 2018. The thickness of straw mulch layer was 3 cm based on the study by Sharma et al. [78]. The length of the trimmed vegetable mulch was about 5 cm.
Mean, maximum, minimum air temperatures ( • C), and global radiation (W m −2 ) of the growing season were collected daily from a weather station nearby (Ecotech, Germany) as well as rainfall (mm) and air relative humidity (%).

Leaf Gas Exchanges, Stem Water Potential, Leaf Chlorophyll a Fluorescence and Content
Net photosynthesis (P n ), stomatal conductance (g s ), and transpiration rate (E) were measured between 10 and 12 a.m. on 10 fully developed and intact leaves per treatment (one each vine, 10 replicates) using a portable infrared gas analyzer (model Ciras 3, PP Systems, Amesbury, MA, USA). Water use efficiency (eWUE) was calculated as the ratio of photosynthesis to transpiration. Measurements were performed, setting the leaf chamber flow at the prevailing environmental condition (ambient temperature, ambient CO 2 concentration ≈400 ppm, saturating photosynthetic photon flux of 1300 µmol m −2 s −1 ) at the three phenological stages as mentioned above.
Stem midday water potential (MPa, Ψ m ) was determined between 12:30 a.m. and 13:30 p.m. with a pressure chamber (model 600, PMS Instrument Co., Albany, OR, USA) on 10 fully expanded leaves for soil management covered with aluminum foil at least 60 before measurement [80].
Chlorophyll a (Chl-a) fluorescence transients of 30 dark-adapted leaves were recorded using a chlorophyll fluorometer (Handy-PEA ® , Hansatech Instruments, Norfolk, UK). F v (the variable) and F m (the maximal) Chl-a fluorescences were collected by applying a saturating flash of actinic light at 3000 µmol photons m −2 s −1 for 1" and used to calculate Fv/Fm: the maximum quantum yield of photosystem PSII following Maxwell and Johnson methodology [81]. Chl-a content in leaves was estimated by 502 SPAD device (Konica Minolta Inc., Tokyo, Japan).
Chl-a content, Chl-a fluorescence, and midday stem water potential were taken on the same leaves used for leaf gas exchange measurements at the same three stages.

Berry Composition
At harvest 100 berries were randomly harvested from each replication of every soil treatment (M, C, T) choosing 10 sample vines (10 berry samples per plant) to perform technological maturity assessments (total of 500 berries per treatment). The samples were individually weighed with a digital scale (PCE Italia Ltd., Capannori, Italy) and the berries were squeezed to analyze the content of sugar, pH, and titratable acidity (TA). The Brix (sugar content) was evaluated using a refractometer (ATAGO, Bellevue, WA, USA); pH was evaluated using a portable pH meter (Hanna instrument, Woonsocket, RI, USA), and g L −1 tartaric acid (TA) was calculated by manual glass burette on a 10 mL sample using 0.1 M NaOH to an end-point of pH 7.0. Another 100-berry sample/replication/treatment (10 berry samples per plant) was used to determine phenolic maturity parameters (i.e., phenolic contents and total and extractable anthocyanin) in berry [82].

Statistical Analysis
Soil treatments were investigated with one-way ANOVA (p ≤ 0.05). Mean values (calculated and expressed as mean ± SE) were separated by Fisher's least significant difference (LSD) post-hoc test (p ≤ 0.05). All statistical analyses were performed using SPSS Statistic 25 (IBM, Armonk, NY, USA).

Climate Parameters
The 2017 and 2018 microclimate conditions of the experimental area are reported in Figure 1. Average, maximum, and minimum air temperatures were measured in both seasons during the experiment (June-September). The 2017 season proved to be the warmest and the least rainy, on the contrary 2018 proved to be a rainy and cooler year. The warmest period in both years was August: in 2017 the average monthly temperature was 2 • C higher than in 2018.

Leaf Gas Exchanges, Stem Water Potential, Leaf Chlorophyll a Content, and Chlorophyll Fluorescence
The physiological parameters of V. vinifera in the three different soil managements (mulching, M; cover cropping, C and tillage T) are presented in Table 1 and Figure 2. Table 1. Physiological parameters. Net photosynthesis (P n ), stomatal conductance (g s ), water use efficiency (eWUE), and midday stem water potential (Ψ m ) of V. vinifera treated with three different soil management practices. Measurements were conducted at full veraison, mid maturation, and full maturation. Data (mean ± SE, n = 10) were subjected to one-way ANOVA. Different letters within the same parameter and row indicate significant differences among M, T, and C (Least Significant Difference (LSD) test, p ≤ 0.05).   Tables 2 and 3 show the composition of V. vinifera berries among three different soil management practices, under two years, in terms of technological and phenolic maturity.   No significant differences were found in physiological parameters (P n, g s ) of 28 July 2017 among the different soil management practices (Table 1).

Berry Composition
In vines with mulching in both vintage higher values of P n , eWUE, and Ψ m were generally found. No significant difference was found in Fv/Fm and chlorophyll content in leaves of V. vinifera (Figure 2A-D). Tables 2 and 3 show the composition of V. vinifera berries among three different soil management practices, under two years, in terms of technological and phenolic maturity.     To determine the optimum harvesting time, grapes from three different experimental plots were periodically tested with a digital hand refractometer and harvesting time was considered when the content of berries attained approximately more than 25 Brix. No significant difference in pH was found ( Table 2). On the contrary, at mid maturation and full maturation a significant difference in sugar content, berry weight, and acidity between mulching and cover cropping were observed in two seasons ( Table 2).

Berry Composition
The greatest differences were found under the composition of anthocyanins (Table 3). At mid and full maturation, C berries showed significantly higher extractable anthocyanin content than M and T berries in 2017. At full maturation, C berries showed significantly higher extractable content than T and it showed significantly higher total anthocyanin content than T and M for berries in 2018. No difference in both vintages in the extractable polyphenols at full maturation were observed.

Discussion
Current vineyard soil management practices derive from research conducted over the last 30-40 years with the aim of preserving organic matter. The recommended techniques are designed to balance the winemaker's conflicting objectives. The main objective is to maintain a soil environment that promotes proper vegetative growth of the vine taking into account both the needs of the vine and the characteristics of the site, ensuring that nutrients and water are not excessively consumed by competing vegetation [83].
The present study assessed, at a subregional level, the importance of soil management as a potential adaptation measure to ensure the future sustainability of grapevine yields in Tuscany.
In our study, at veraison 2017, full maturation 2017, and veraison, mid, and full maturation 2018, Pn, eWUE, were lower in T and C than in M plants. It is probable that, in vine C and T, photosynthesis, and consequently the eWUE, were almost exclusively depressed by water stress (i.e., decreased water potential) [84], as a consequence of the hottest period of the growing season (maximum temperature high above 40 • C). In full maturation (2017 and 2018) the values of photosynthesis, water use efficiency, and water potential in C were lower than the others, this may derive from a competition of the cover crop with the vines: cover crops can affect soil properties, including spatial and temporal modification of the water in the soil profile [85].
On the contrary, physiological parameters were positively influenced in the M practice, probably due to better water retention in the soil (i.e., less negative water potential).
Similarly, several authors reported that the application of mulching can enhance physiological and/or growth parameters in different horticultural species [86][87][88][89][90] and showed that mulching can indeed improve yields in regions under low water availability [28,91,92]. Results highlight the importance of water availability as a widely recognized main limiting factor for grapevine productivity under Mediterranean like environmental conditions [93,94]. The better water relation in M practice could be due to the reduction in soil water evaporation limited by the mulching. Some investigation on root system depth could help to clarify this aspect.
No differences were found in gs in the hottest period in both vintages: Cabernet Sauvignon reflects its conservative behavior (i.e., anisohydric) under water-stress condition without witnessing a drop in values [95,96].
The fluorescence of chlorophyll providing different parameters on photosynthetic flows (considered a reliable indicator of plant stress monitoring; [97][98][99]) leads us to the conclusion that in both years there were no excessively limiting situations for plants (no significant differences were found in the two vintages).
In terms of grape traits, in our study, technological maturity was influenced by soil management practices. At full maturation (5 September 2017 and 17 September 2018) C showed higher sugar content than M in accordance with Zang et al. [100]. Mulch composed of fresh plant residues and tillage increased grape juice TA while, in dry years, C increased juice soluble solids [72,74]. This is probably due to a reduction in soil water availability that leads to a greater concentration of the must in the berries. Unlike Carsoulle [101], in our study no differences in pH were found.
There were also increases in the berry weight on 5 September 2017 and in all three times in 2018 in favor of M and T. This is probable due to the enhancement of physiological performances promoted by mulch that may have positively affected berry metabolism and on the growth of berry resulting in improved grape quality (berries more hydrated). Like many studies [12,102,103], we have found that the titratable acidity was reduced by the cover crop compared to the other treatments probably due to an increase in the ratio of tartaric acid to malic acid. In berries with cover cropping, berry weight decreased in comparison with M and T: maybe severe heat stress and water scarcity, especially in 2017 with a stem water potential of 1.5 Mpa, promoted the dehydration of berries through water loss via apoplast path to rachis [104], with declines in productivity (i.e., lower berry weight in C). Cover cropping definitely competes with vines because of nutrients and water. As regards the total and extractable anthocyanins, the differences were found starting from 28 July in 2017 and from 15 August in 2018.
Both in 2017 and 2018 in full maturity, differences in the anthocyanin content were found: C turned out to be the one with the highest content expressed in mg/L, indeed it has been shown that sugar levels in the grape berry are closely associated with accumulation of pigment and total phenolics [105,106], (i.e., C on both 5 September 2017 and 17 September 2018 showed a higher sugar content than T and M).
The relationship between levels of anthocyanins, total sugars, and berry weight in the ripening grapes was studied in the fruit during the period from veraison to maturity from many authors [107][108][109]. The higher anthocyanin content in C is deduced to be also due to a greater water stress which led to a lower berry weight [35].
Vineyard floor management has multiple goals that encompass improving weed management and soil conservation, reducing soil resource availability to control vine vigor, and influencing desirable aspects in wine quality [9]. Compared to tillage and mulching, cover cropping showed a higher use of water during the season [41], which induced a lower leaf water potential up to harvest in both years. These differences in the state of the vine water influenced the weight of the berry, the accumulation of sugar in the fruits, and the anthocyanin content. Conversely even the literature suggests that mulch conserves water [71,[110][111][112]; mulch enhanced grapes quantity and quality, in general the sugar content was more balanced (Brix) while maintaining an excellent anthocyanin and total polyphenol content of the Cabernet Sauvignon berries in both harvests. Mulch also reduced the water stress and increased photosynthesis.

Conclusions
One of the objectives of modern viticulture is in fact to look for more balanced and less alcoholic wines while maintaining a correct content of polyphenols and anthocyanins [113].
Our results indicate that mulching can be a valuable tool for enhancing wine quality in this wine growing region for soils with low water availability; it therefore appears to be a valid soil management technique that is sustainable, conservative, and less impactful than tillage.
Our results allow to conclude that the choice for a cover crop is not the best one due to plant-cover crop water competition that leads to excessive stress on the plants affecting grape quantity and quality and leading to excessively alcoholic and unbalanced wines.
Although the cultivation of the vine compared to most other crops requires less water for its growth and maturation, the expected climate changes (i.e., the reduction of rain and rising temperatures; [114,115]) will intensify the water stress on the vines, particularly in regions with limited water availability.
The best practice for each vineyard site is determined in part by vineyard design, soil type, and climatic conditions of the vineyard site [116,117]. However, long-term trials are needed in order to verify these effects, particularly on water stress and its consequences for quality.
Author Contributions: G.B.M. and E.C. designed, conceptualized, and organized the experiment. E.C., L.S. and S.S. followed the execution of treatments, made measurements of gas exchanges, water potentials, and sampled berries. E.C. also processed data and carried out statistical analyses. E.C. wrote the original draft manuscript, which was edited and improved by G.B.M. and P.S. All authors have read and accepted the published version of the manuscript.
Funding: This research has not received any specific grant from funding agencies in private or public commercial sectors.