Soil management in semi-arid vineyards: Combined effects of organic mulching and no-tillage under different water regimes

1 EUROPEAN JOURNAL OF AGRONOMY 1 2 Type of manuscript: Regular paper 3 4 Title: Soil management in semi-arid vineyards: combined effects of organic mulching 5 and no-tillage under different water regimes 6 7 Authors: Ignacio Buesa1,2,*, José M. Mirás-Avalos2,3, José M. De Paz1, Fernando Visconti1, 8 Felipe Sanz1,2, Antonio Yeves1, 2, Diego Guerra1,2, Diego S. Intrigliolo1,2 9 10 Affiliations: 11 1 Instituto Valenciano de Investigaciones Agrarias (IVIA). Centro Desarrollo Agricultura 12 Sostenible (CEDAS), Unidad asociada al CSIC “Riego en la agricultura mediterránea”, 13 Apartado Ofcial, 46113 Moncada, Valencia, Spain. 14 2 Dept. Riego. Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), 15 Campus Universitario de Espinardo, PO Box 164, 30100 Murcia, Spain. 16 3 Unidad de Suelos y Riegos (asociada a EEAD-CSIC). Centro de Investigación y Tecnología 17 Agroalimentaria de Aragón (CITA), 50059, Montañana, Zaragoza, Spain. 18 19 20 * Corresponding author: 21 Instituto Valenciano de Investigaciones Agrarias (IVIA): Unidad asociada al CSIC “Riego 22 en la agricultura mediterránea” 23 Carretera CV-315, Km 10,7, 46113 24 Moncada, Valencia, Spain 25 Phone: +34 650578152 26 E-mail: [email protected] 27 28 Word count: 9782 (from abstract to references, both included) 29 Number of tables: 6 30 Number of figures: 3 31 2 Abstract 32 Optimizing water use in vineyards is crucial for ensuring the sustainability of viticulture in 33 semi-arid regions, and this may be achieved by minimizing direct water evaporation from the 34 soil through the use of mulching. In this context, the current study aimed at assessing the 35 combined effects of the vine-row application of an organic mulch (vine prunings) and no-36 tillage under two water regimes on soil properties, plant water and nutritional status, yield 37 and must composition of grapevine (Vitis vinifera L.) cv. Bobal grown under semi-arid 38 conditions. For this purpose, a field experiment in a split-plot design was carried out for three 39 years (2016-2018) in a mature Bobal vineyard located in Eastern Spain. Two soil 40 management strategies (tillage and organic mulching with no-tillage) were assessed under 41 two water regimes (rainfed and deficit drip irrigation) with four replications per combination. 42 Vine responses were determined by measuring midday stem water potential, leaf nutrient 43 concentrations, pruning weight, yield components and grape composition. Soil properties 44 were assessed at the end of the experiment. Mulching and no-tillage positively affected vine 45 water status under both water regimes, resulting in reductions in grape phenolic composition. 46 Interactive effects of both water regime and soil management on water use efficiency were 47 found. Regardless of soil management practice, irrigation increased yield and pruning weight 48 when compared to rainfed conditions. Soil management had slight effects on vine nutritional 49 status. At the end of the experiment, soil compaction increased and infiltration decreased as 50 a consequence of mulching and no-tillage. Organic mulch and no-tillage improved vine water 51 status, however, considering the final soil surface compaction and low water infiltration rate, 52 longer-term studies are necessary to assess the sustainability of combining both practices. 53 54 3 Keywords: Drip irrigation; Soil management; Sustainable viticulture; Vitis vinifera L.; 55 Water relations. 56 57 Abbreviations: DO (Designation of Origin); ETo (Reference evapotranspiration); WR 58 (Water regime); SM (Soil Management); RT (Rainfed tilled); RM (Rainfed mulched and no-59 tilled); IT (Irrigated tilled); IM (Irrigated mulched and no-tilled); EU (experimental unit); 60 stem (midday stem water potential); CCE (Calcium carbonate equivalent); TSS (Total 61 soluble solids); TA (Total acidity); ANOVA (Analysis of variance). 62 63 1. Introduction 64 In the current scenario of global change, sustainability is becoming a serious concern 65 in viticulture due to the large extension of this crop in many different environmental 66 conditions. Especially in semi-arid regions, vine water requirements generally exceed the 67 average annual rainfall, making water the most important resource for the sustainability of 68 viticulture (Medrano et al. 2015). Grapevine (Vitis vinifera L.) water requirements range 69 between 300 and 700 mm to complete its growing cycle (López-Urrea et al. 2012; Medrano 70 et al. 2015), which, under the Mediterranean climate, coincides with the driest months of the 71 year, making irrigation scheduling and timing critical for vine performance and grape 72 composition (Intrigliolo et al. 2012). In dry regions, irrigation competes for water with other 73 uses and could result in an overexploitation of surface and groundwater resources, thus 74 compromising the sustainability of viticulture (Chaves et al. 2007). Furthermore, evaporative 75 demand is expected to rise due to the increased global air temperature and intensity of 76 climatic anomalies, such as droughts and heat waves (Fraga et al. 2016). In response to the 77 increase in temperature and evaporative demand, greater vine transpiration rates are 78 4 expected, leading to further depletion of soil water content and/or increased vine water stress 79 (Dayer et al. 2020; Flexas et al. 2010). In addition, to ensure viticulture sustainability, a 80 balance between inputs and outputs of nutrients within the farm system is crucial, as 81 grapevines strongly react to nutrient deficit in terms of vine yield and particularly grape 82 composition (Keller et al. 2005). In this regard, soil nutrient storage capacity and accessibility 83 are influenced by soil texture, rooting depth, and organic matter content, but the nutrient 84 availability is modified by soil moisture and pH. 85 Nowadays, in most of the semi-arid regions of grapevine production, as well as many 86 of the “new world” viticulture areas, minimum water and nutrition requirements are not met 87 (García-Escudero et al. 2013; Medrano et al. 2015). Therefore, optimizing water use in 88 vineyards and its interaction with vine nutrition is a subject of paramount importance to 89 secure sustainability in viticulture (Quemada and Gabriel 2016). As a consequence, a great 90 research effort has been made to determine the best strategies of irrigation (timing, schedule, 91 rates) and its relation with crop nutrition that allow reasonable yields with good 92 organoleptical quality (Buesa et al. 2017; Gaiotti et al. 2017; Intrigliolo et al. 2012; Jackson 93 and Lombard 1993; Keller et al. 2005; Romero et al. 2013; Schreiner et al. 2013; Vos et al. 94 2004; Pérez-Álvarez et al. 2017). However, other agricultural practices besides irrigation and 95 fertilization might improve water use efficiency (WUE) and increase soil nutrient availability 96 in vineyards by reducing soil water evaporation and runoff, thus maximizing green water use 97 (Medrano et al. 2015; Vos et al. 2004). 98 In this context, soil management (SM) practices allowing the control of weeds, the 99 alleviation of soil compaction, the reduction of soil erosion, the enhancement of nutrients and 100 water uptake, and the modulation of vine vigour and yield, amongst others (Celette et al. 101 2009; Guerra and Steenwerth 2012; Steenwerth and Belina 2008) are of special importance 102 5 for grapevine performance and, consequently, for wine quality (Lopes et al. 2011; Trigo-103 Córdoba et al. 2015). Several SM practices can be used in vineyards to achieve the 104 aforementioned goals, including tillage, application of herbicides, cover crops and 105 organic/inorganic mulches (Gaudin et al. 2010; Guerra and Steenwerth 2012; Salomé et al. 106 2016). Whatever the case, to choose the best practice for each location the following factors 107 have to be taken into account: vine age, vineyard plantation design, soil type, environmental 108 regulations, objectives of the winery, and climatic conditions (Ripoche et al. 2011; Steinmaus 109 et al. 2008). 110 In this regard, tillage is the most traditional soil management technique in vineyards 111 worldwide because it is an effective way of controlling weeds (Guerra and Steenwerth 2012) 112 and, at least initially, increasing water infiltration into the loosened soil and decreasing 113 capillary continuity (Triplett and Dick 2008). In spite of this benefit, tillage has also several 114 disadvantages including soil compaction and thus loss of structure, cumulative shrink of 115 fertility and soil organic matter, increased risk of soil erosion and damage to vine roots as 116 well as directional spread of soil pests and pathogens (Hamza and Anderson 2005; 117 Steenwerth and Belina 2008; Garcia et al. 2019; Bordoni et al. 2019). The use of herbicides 118 is another choice and though herbicides has been proven easy to use, cost-effective and more 119 efficient than tillage for controlling weeds, the risk of toxicity and the potential of herbicide 120 residues leaching into waterbodies (Tourte et al. 2008) limit their use for managing the soil 121 in the vineyard inter-rows. As a third alternative, in the last decades, the use of cover crops 122 has become a common vineyard SM practice because of its many benefits including soil 123 protection against erosion, regulation of vine growth, weed suppression, habitat for beneficial 124 predators and improved soil fertility and water-holding capacity (Gaudin et al. 2010; Fourie 125 2011; Linares-Torres et al. 2018; Morlat and Jacquet 2003; Pérez-Álvarez et al. 2015, Virto 126 6 et al. 2012). Despite these advantages, the adoption of cover crops as a SM strategy in 127 Mediterranean vineyards is limited by the concern of an excessive competition for nutrients 128 and water between these crops and the grapevines (Celette et al. 2008, 2009; Monteiro and 129 Lopes 2007). Finally, mulching may be an alternative for overcoming all these concerns and 130 provide additional benefits to the soil and grapevines (Morlat et al. 2008; Prosdocimi et al. 131 2016). Indeed, organic mulching is a sustainable agronomic practice that is widely used for 132 weed control, preventing soil erosion and improving general soil properties, including the 133 minimization of water loss through evaporation and runoff, thus improving infiltration of 134 water into the soil and increasing vineyard biodiversity (Morlat and Chaussod 2008; 135 Pinamonti 1998; Varga and Májer 2004; Medrano et al. 2015). Moreover, this organic 136 mulching has been reported to be positive not only for soil but also for grapevine yield and 137 must composition (Mundy and Agnew 2002; Pinamonti 1998). Furthermore, mulching could 138 contribute to a circular economy (recycling of pruning residues), increasing soil organic 139 matter content and nutrients, water-holding capacity and inhibiting the growth of weeds 140 (Ferrara et al. 2012; Montanaro et al. 2017). In addition, the use of pruning waste on soil 141 would avoid the presently used, more conventional practice of burning pruning waste, and 142 therefore, reduce emissions of CO2 and other greenhouse gases into the atmosphere by 143 increasing CO2 capture into the soil (Montanaro et al. 2017). Other alternatives for the 144 application of vine prunings are to compost them together with manure or winery wastes or 145 even to carbonize them to obtain biochar (Mundy and Agnew 2002; Baronti et al. 2014; 146 Gaiotti et al. 2017). In any case, increases in nutrient recirculation and release are interesting 147 possible effects of mulching (Montanaro et al. 2017). Nonetheless, vine pruning waste’s 148 decomposition could compete with grapevines for nitrogen in the soil (Thomsen et al. 2008). 149 7 Furthermore, nutrient uptake is more influenced by the physical conditions of the soil, namely 150 moisture and temperature, than by nutrient availability in the soil (Pinamonti 1998). 151 Recently, López-Urrea et al. (2020) determined in a weighting lysimeter the short-152 term effects of covering the entire vineyard floor with vine pruning waste (organic mulching) 153 on the evapotraspiration of a fully irrigated vineyard and found that water use can be reduced 154 by 17%. This moderate decrease in water use could be particularly relevant under rainfed 155 conditions where vines normally experience more water stress that under irrigation and its 156 alleviation could be more important for improving vine physiology (Romero et al. 2010). 157 However, in rainfed vines, the effect of mulching on soil evaporation at mid-summer, when 158 there is a lack of rainfall and the soil is dry, may be minimal. Thus, the reduction effect of 159 mulching on ET is expected to be low in this period (Yunusa et al. 1997). In this sense, 160 previous studies under mulching have been carried out mainly under a standard watering 161 regime because they were more focused on exploring different soil management techniques 162 (Guerra and Steenwerth 2012; Bavougian and Read 2018; Gil et al. 2018). Nonetheless, 163 under a semi-arid climate, where vine water relations are a predominant factor affecting vine 164 performance (Mirás-Avalos et al. 2017), it is important to determine how soil management 165 with mulching is influenced by the watering regime, considering that drip irrigation only wets 166 a small portion of the entire soil allotted to each vine. 167 In this context, the aim of the current study was to assess the effects of the application 168 of an organic mulch (vine prunings) under no-tillage as compared to tillage, under two water 169 regimes (WR, rainfed and deficit irrigation) on soil physical properties, plant water and 170 nutritional status, yield and must composition of grapevine (Vitis vinifera L.) cv. Bobal 171 grown under the semi-arid hot-summer Mediterranean climate of Eastern Spain. The working 172 hypothesis was that mulching could improve vine water status and, particularly under rainfed 173 8 conditions, increase yield and WUE. In parallel, the potential effects of mulching on the 174 vineyard’s nutrient balance were assessed at the grapevine level by determining leaf nutrient 175 status and grape composition. Since the grapevine’s nutrient uptake varies according to 176 growth requirements, the response to the SM practice may differ between the different WR. 177 Moreover, SM could cause microclimatic changes which affect the vineyard water and 178 energy balances, and hence the grapevine’s response to water regime. Therefore, the possible 179 interaction between WR and SM was also explored. 180 181 2. Materials and Methods 182 2.1 Plant material and study site 183 The experiment was carried out during three consecutive seasons (2016 to 2018) in a 184 commercial vineyard located in Requena (39º 29’ N, 1º 13’ W, elevation 750 m, Valencia, 185 Spain) within the Designation of Origin (DO) Utiel-Requena. The vineyard was planted in 186 2002 with Vitis vinifera (L.) cv. Bobal on 110-R rootstock at a spacing of 2.6 by 1.4 m (2671 187 vines ha-1). Vines were trained to a bilateral cordon system leaving six two-bud spurs per 188 vine. Shoots were vertically trellised with a pair of steel catch wires. Rows were oriented 189 from north to south and followed the slope of the ground which was on average 3.2%. The 190 soil at this site was classified as a Typic Calciorthid according to the Soil Taxonomy (Soil 191 Survey Staff, 1999), with a clay loam to clay texture according to USDA classification, 192 highly calcareous (200 – 380 g kg-1), with a pH of around 8.5, an electrical conductivity 193 around 0.2 dS m-1, and low in organic matter (3 – 20 g kg-1) and nitrogen (0.4 g kg-1). The 194 available water capacity was ≈200 mm m-1 and the bulk density was 1.43 to 1.55 g cm-3. The 195 soil depth to the unaltered hard parent material (R horizon) exceeded 2 m. The climate of the 196 area was classified as semi-arid hot-summer Mediterranean (de Paz et al., 2004; Rodríguez-197 9 Ballesteros, 2016). The historical average annual rainfall was 390 mm and the reference 198 evapotranspiration (ETo) was 1120 mm (Supplementary Figure 1). Approximately 65% of 199 rainfall occurs during the dormant period. Budbreak for Bobal in this area usually occurs by 200 the end of April, flowering by June, veraison is reached by mid-August with harvest at the 201 beginning of October (Salón et al. 2005). 202 203 2.2 Experimental design 204 Two treatments were established in the vineyard following a split-plot design. A 205 given water regime (WR), either rainfed (R) or deficit-irrigated (I), was assigned to the main-206 plots, whereas a given soil management (SM), either tillage without mulching (T) or 207 mulching with no tillage (mulch, M), was assigned to the sub-plots with four replicates per 208 combination. Therefore, the combined treatments applied were RT, RM, IT and IM. Each 209 subplot or experimental unit (EU) consisted of five rows with nine vines per row. The vines 210 located in the center of the middle rows were used for measurements and samplings (21 211 vines), while the rest were left as buffers. 212 Deficit irrigation was applied in an attempt to maintain the midday stem water 213 potential (stem) of the IT treatment above the threshold values of -0.80 and -1.20 MPa at 214 preand post-veraison, respectively. These degrees of water stress were considered as targets 215 based onprevious research carried out in the area by Salón et al. (2005). The same irrigation 216 regime was applied to the M and T treatments. Organic mulching consisted in the application, 217 both in the rows and the inter-rows, of mechanically-chopped vine prunings corresponding 218 to the theoretical amount that would be produced over 10 years by each vine (Supplementary 219 Figure 2). That is, 4-5 kg of crushed pruning waste were spread over the 3.64 m2 of vine 220 16 In general, the contents of B, Cu, Fe, Mn and Zn in leaves did not show significant 360 differences between either the SM or WR treatments (Table 3), with some remarkable 361 exceptions depending on the season for specific elements. For instance, the contents of Mn 362 were higher under irrigation in two out of the three seasons studied. In contrast, the B and Zn 363 contents were lower in the vine leaves under irrigation in 2016. Note that in that season, the 364 leaf contents of both microlements were the highest in the whole trial. Differences in the 365 response to the SM were barely significant with the sole exception of B and Fe. The latter 366 was significantly higher in the mulching treatments in 2016 and 2017, whereas B was higher 367 only in 2016, when there was an interactive effect between factors for this element (Table 3). 368 369 3.2 Vine performance and berry composition 370 Pruning weight was greater in vines from the I treatments in most of the years studied. 371 However, no effect of the SM on pruning weight was observed (Table 4). The number of 372 clusters per vine was significantly greater in I than in R vines in 2017, while no differences 373 among treatments were observed in 2016 and 2018. In addition, cluster weight was 374 significantly higher in the I treatments, leading to higher yields. Cluster weight was also 375 significantly higher in the M treatments in 2017, although in 2018 the opposite was observed. 376 Berry weight was higher in the I treatments in 2016 but not in 2017 and 2018. No significant 377 differences were observed regarding SM practices. In 2017, WUE was significantly affected 378 by both SM and WR. In this season, significant interactions between SM and WR were 379 detected for cluster weight, yield and WUE (Table 4). 380 Berry composition differed primarily depending on the WR and secondarily on the 381 SM (Table 5). The TSS were lower in the irrigation treatments, while no differences were 382 found between SM treatments. In contrast, total acidity (TA) and pH behaved differently 383 17 depending on the season. The malic acid concentration was higher in the irrigation treatments 384 and also in the mulched ones in 2017. Tartaric acid concentration in berries was lower in the 385 I treatments in 2016 but higher in 2017, while no clear effects were observed in 2018. On the 386 other hand, SM caused a consistent reduction in tartaric acid concentration over the whole 387 study period. The maturity index (TSS-to-TA ratio) was significantly lower in 2017 in 388 response to I and M. Concentrations of phenolic compounds (total polyphenols and 389 anthocyanins) in most seasons were lower in both the I and M treatments. No significant 390 interactions between SM and WR were detected for any of the berry compositional traits 391 (Table 5). 392 393 3.3 Effects of mulching on soil properties 394 No significant differences were observed in soil surface basic properties such as 395 textural fractions and organic matter content between tillage and mulching under rainfed 396 conditions at the end of the experiment (Table 6). 397 Contrary to the basic soil properties, the bulk density, water content and saturated 398 hydraulic conductivity did differ between SM practices. The bulk density was significantly 399 higher under mulch at the soil surface by the end of the experiment with negligible 400 differences in deeper layers (Figure 3a and Supplementary Table 2). Also, the soil water 401 content was significantly higher under mulch in the surface layer with differences again 402 vanishing with depth (Figure 3b and Supplementary Table 2). The aggregate stability in the 403 soil surface layer was the same regardless of the soil management (Figure 3c). Interestingly, 404 the saturated hydraulic conductivity was the soil property for which differences between the 405 T and M treatments were larger. Specifically, its value under mulching was found to be one 406 order of magnitude lower than under tillage (Figure 3d). 407 18 408 4 Discussion 409 In this three-year study we focused on the effects of the soil and irrigation 410 management mainly on vine performance, plant water and nutrient status considering that at 411 the vine level it is possible to integrate both the effect of the soil resources availability and 412 the vine-environment interactions. While the effects of irrigation on the soil water balance 413 are easy to predict and assess, the implications that soil mulching and no tillage may have on 414 the vineyard water balance are more difficult to predict. This is because the soil management 415 strategies tested here can affect many components of the soil water balance including 416 evaporation, water infiltration, soil water holding capacity, vine microclimate, vineyard 417 energy balance and also vine root growth and activity and therefore the vine plant water and 418 nutrient uptake capacity. Indeed, the mulch application seemed to be effective for improving 419 grapevine water status, both under R and I conditions (Figure 2). This can be due to the fact 420 that mulching increased the soil water content at a depth of 0-5 cm as revealed by the 421 measurement made at the end of the experiment under rainfed conditions (Figure 3). This 422 can be attributed to the lower water losses through soil evaporation (Davies et al. 2011; 423 Myburgh 2013; Cao et al. 2012). Montoro et al. (2016), using a weighing lysimeter, estimated 424 that direct soil evaporation accounts for 26-31% of the vineyard evapotranspiration under 425 drip-irrigated conditions in a semi-arid region from South-Eastern Spain. Consequently, 426 employing mulches for covering vineyard soil surface may provide substantial water savings 427 (López-Urrea et al. 2020). Nonetheless, research on the assessment of the effects of mulching 428 on crop water use efficiency in grapevines provided contrasting results (Montoro et al. 2016). 429 In the case of vineyards, Pinamonti (1998) reported 2% increments of soil water availability 430 under mulching when compared to bare soil in a Merlot vineyard. Agnew et al. (2002) found 431 19 that mulches allowed for retaining soil moisture early in the season, reporting soil water 432 contents 5% higher under mulch in the first 30 cm of the soil profile. In our study, the increase 433 was as high as 35% on average in the first 20 cm. In Mediterranean vineyards, Medrano et 434 al. (2015) indicated that direct soil evaporation may account for 20% of water consumption, 435 so the reduction in evaporation observed in mulched soil could result in a greater water 436 availability for vines (Davies et al. 2011). In the current study, the water stress integral values 437 reflected an average improvement of 5% in vine water status over the growing season when 438 mulch was applied, in comparison with vines under tilled soil (Figure 1). Under irrigation, 439 the improvement was even higher: 13% on average for the three studied years. As expected, 440 greater improvements were observed in dry seasons (2016 and 2017). In addition, the 2017 441 season in which rainfall was highest during the off-season period, WUE was improved by 442 11% in mulch treatments (Table 4). These results are in accordance with previous research 443 on the effects of organic mulching on crop water use efficiency (Buckerfield and Webster 444 2001; Fourie 2011; Guerra and Steenwerth 2012; Nguyen et al. 2013). Moreover, an 445 improved vine water status coud be also due to differences in the root system provoked by 446 mulching, mainly due to the proliferation of fine roots (Gaiotti et al. 2017; Morlat 2008; 447 Linares-Torres et al. 2018). 448 On the other hand, both mulching and irrigation regimes can affect vine performance 449 by modifying the vineyard nutrient balance (Keller et al. 2005). In the present research, we 450 focused on determining the end effects at the vine level via a detailed analysis of the leaf 451 macroand micro-nutrient status. Despite other authors reported improvements in vine 452 nutrient status in response to mulch application (Agnew et al. 2005; Nguyen et al. 2013), leaf 453 nutrients did not show a consistent response to the treatments imposed in the current work 454 (Table 2). In fact, the effect of WR and SM on vine nutrition was minimal and not fully 455 20 consistent over the study period. For instance, deficit irrigation did not lead to nutritional 456 deficiencies due to increased vigor as compared to rainfed treatments. Only the slight 457 increases detected in P in response to the application of mulch may be linked to improved 458 soil water content in these treatments (Mpelasoka et al. 2003), rather than to any effect on 459 the incorporation of nutrients into the soil from pruning waste. This increase in P contents 460 allowed for correcting a nutritional deficiency in the soil that existed prior to the application 461 of mulching (Poni et al. 2003; Romero et al. 2005; Navarro et al. 2008; García-Escudero et 462 al. 2013). Leaf micronutrients behaved inconsistenly with SM and WR. It should be noted 463 that in 2017, the Cu values must have been affected by fungicide residues so they have no 464 physiological meaning. On the other hand, the high levels of B found in the first experimental 465 season, which were far from optimal (García-Escudero et al. 2013), may be due to the 466 application of manure in this season. Although the nutrient levels in the manure were standard 467 (Supplementary Table 3), it cannot be ruled out that the trial conditions favoured a high 468 absorption of B, which is an essentially passive nutrient in contrast with other compounds 469 such as Fe (Reid 2001). However, in some seasons, leaf contents of both nutrients were 470 increased by the effect of mulching, most likely due to the increased soil water content 471 (Keller et al. 2005). Other studies assessing the effect of vine pruning mulch on foliar nutrient 472 status showed similar results in Cabernet franc on 3309C rootstock in the medium-term 473 (Morlat 2008). Nevertheless, in the long-term (28 years) a trend towards a favorable influence 474 of mulching on grapevine nutrition was observed (Morlat 2008), likely due to the increase of 475 the soil organic matter content (Morlat and Chaussod 2008) which increased, in turn, the soil 476 water holding capacity and, consequently, improved nutrient uptake by plants. 477 In our study, the worsened soil hydrophysical properties under mulching were the 478 consequences of soil compaction, which was reflected in the increased bulk density at 0-5 479 21 cm (Figure 3), similar to the response to non-tillage reported by other authors (Álvaro-480 Fuentes et al. 2008a; Hansen et al. 2011). Contrary to the upper topsoil, the soil layers below 481 the depth reached by the cultivator’s tines, i.e., 10 cm, tended to be more compact under 482 tillage, which is an undesirable effect known to be caused by repeatedly ploughing at the 483 same depth (Tripplett and Dick 2008). The differences in bulk density in the 0-5 cm layer of 484 the soil were reflected in differences in the saturated hydraulic conductivity of the soil surface 485 (Figure 3), in accordance with previous studies (Curtis and Claassen 2009). However, in 486 treatments where infiltrability was increased, this had no consequences on the soil water 487 content below the surface layer (Figure 3). 488 Aggregate stability was not affected by soil management (Figure 3). The stability of 489 aggregates increase with the build-up of binding agents (Álvaro-Fuentes et al. 2008b; Virto 490 et al. 2012). In the Typic Calciorthid soil featured in the current study these binding agents 491 are mainly calcium carbonate and organic matter. Nevertheless, on the one hand, the calcium 492 carbonate content of soils under semi-arid Mediterranean climate only significantly changes 493 in the very long-term and, on the other hand, even though the organic matter had increased 494 due to mulching, as observed in other vineyards (Ferrara et al. 2012; Peregrina et al. 2012), 495 it may be also a very short time for structural stability to increase (Table 6). In order to be 496 able to change this parameter in a soil with poor aggregate stability as this, the mulching 497 should definitely increase the organic matter content more than 2 g kg-1. Perhaps it takes a 498 much longer time for the mulching to incorporate into the soil, since the functioning of 499 calcareous soils does not rapidly change in Mediterranean vineyards, thus limiting the effects 500 of soil improving practices (Salomé et al. 2016). 501 In addition to the effects on plant water and nutrient status, the present research 502 carried out a comprehsive agronomic assessment of vine performance and grape composition 503 22 in order to integrate the effects of the soil mulching and irrigation on soil characteristics and 504 vine physiology at the whole vine level. Although previous research showed that employing 505 mulches increased vine vegetative growth (Gaiotti et al. 2017; Pinamonti 1998; Agnew et al. 506 2002), no clear effects were observed in the current study (Table 4). Despite the reports 507 indicating that the use of organic mulches increases grape yields (Fourie 2011; Guerra and 508 Steenwerth 2012; Nguyen et al. 2013), in the current study, yield increased by irrigation but 509 not by the application of mulching. Nonetheless, cluster weight did increase in M treatments 510 in 2017, but the contrary was observed in 2018, with no effect in 2016. In 2017, there was an 511 interactive effect between SM and WR in yield and WUE, suggesting that the increase in soil 512 water content under mulching during the off-season period of 2017 was enough for enhancing 513 vine performance and WUE in the most stressed vines (rainfed) but not in irrigated vines. It 514 is noteworthy that this interactive effect did not occur in all seasons (Supplementary Table 515 1). These irreproducible effects indicate that the environmental conditions the grapevines 516 must cope within this semi-arid region are rather restrictive and the improvements generated 517 by mulching and no-tilling are not sufficient for having a consistent impact on grapevine 518 performance, at least in a three-year span. In addition, the beneficial increase in the soil water 519 content promoted by mulching could be offset by the detrimental soil surface compaction 520 effect due to no tillage methods (Figure 3). Although these findings contradict previous 521 research on which mulching clearly increased vine yield (Porter 1999; Agnew et al. 2002), 522 they may be explained by the different environmental conditions in which these studies were 523 conducted. Environmental conditions are of paramount importance on grapevine response to 524 management practices (Jackson and Lombard 1993) and, in fact, other studies reporting no 525 significant effects on vineyard yield are not rare. For instance, Ferrara et al. (2012) did not 526 23 observe significant effects of mulching application on grapevine yield after two years of 527 research, in accordance with the results found in the current study. 528 Grape composition parameters were more clearly affected by WR than by SM (Table 529 5). For instance, irrigation decreased TSS and increased malic acid concentration, which is 530 in accordance with previous research on irrigation effects on this variety (Salón et al. 2005). 531 In contrast, SM only consistently affected the concentration of tartaric acid in the grapes 532 which in turn showed an increased pH in 2016 and 2017. This contradicts previous works in 533 which the application of mulch significantly increased TSS and TA (Mundy and Agnew 534 2002; Varga and Májer 2004). These contrasting results among studies may depend on the 535 cultivar and the pedoclimatic conditions (Ferrara et al. 2012; Salomé et al. 2016). Notably, 536 both WR and SM affected grape phenolic composition, with deficit irrigation and mulching 537 reducing the concentration of polyphenols and anthocyanins in some seasons (Table 5). This 538 can be explained by the effects that I and M had on alleviating vine water stress (Fig.1) and 539 thus on regulating phenolic ripening (Castellarin et al. 2007; Romero et al. 2010). The 540 observed effect of mulching on phenolic compounds was in agreement with previous 541 evidences showing that organic amendments, such as crushed pruned vine-wood, decreased 542 grape phenolic compounds in the long-term (Morlat and Symoneaux 2008). 543 544 5 Conclusions 545 Yield components were mostly unaffected by the combined effects of mulching with 546 vine prunings and no-tillage under both water regimes. Vine nutritional status was not 547 consistently affected. However, vine water status was enhanced under mulching, leading to 548 water stress integral values over the season that were 5 and 13% lower than those from the 549 tilled soil under rainfed and irrigation regimes, respectively. This enhancing effect, which is 550 24 a result of the higher soil water content under mulching and no-tillage, resulted in reductions 551 in grape phenolic composition. In one of the studied seasons, the soil management and water 552 regime had an interactive effect on water use efficiency, highlighting the importance of 553 environmental conditions on vine response to management practices. At the end of the 554 experiment, however, soils from the mulched and no-tilled treatments also showed a higher 555 bulk density in the shallower soil layer, along with a lower saturated hydraulic conductivity. 556 According to these results, combining an organic mulch and no-tillage seems to have been 557 useful in reducing direct soil water loss and limiting early transpiration losses, which were 558 eventually revealed by the better vine water status. Nevertheless, the final higher compaction 559 and lower infiltration ability of soils under mulching and no-tillage suggests that these 560 positive effects may be unrepeatable along different seasons and therefore, complementary 561 soil improvement practices should be adopted. Furthermore, the amount of material needed 562 for mulching and its cost of establishment are additional factors that might constrain the use 563 of pruning waste as organic mulching. 564 565 Acknowledgements 566 This work was supported by the Spanish Ministry of Economy and Competitiveness 567 (MINECO) with FEDER co-financing [grant number AGL2017-83738-C3-3] and the EU by 568 H2020 project SHui [grant number 773903]. Cajamar and Lucio Gil de Fagoaga for 569 facilitating the experimental field. 570 571 Conflict of interest 572 The authors declare that they have no conflict of interest. 573 574 25 References 575 Agnew RH, Mundy DC, Spiers TM (2002) Mulch for sustainable production. Malborough 576 District Council Sustainable Management Fund Project 4123. 52 pp. Christchurch, 577 New Zealand. 578 Agnew RH, Mundy DC, Spiers TM, Greven MM (2005) Waste stream utilization for 579 sustainable viticulture. 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RT, Rainfed Tilled; RM, Rainfed Mulched and no-tilled; IT, Deficit Irrigated 874 Tilled; IM, Deficit Irrigated Mulched and no-tilled. 875 Treatment 2016 2017 2018 Rainfall Irrigation Rainfall Irrigation Rainfall Irrigation RT 166.0 0 118.6 0 231.9 0 RM IT 166.0 259.8 118.6 120.4 231.9 68.9 IM Off-season 109.0 - 383.8 - 175.3 - 876 877 39 Table 2. Contents of N, Ca, K, Mg and P at veraison in leaf blades from Vitis vinifera (L.). 878 cv. ‘Bobal’ onto 110-R under two different soil management and water regime strategies 879 during 2016, 2017 and 2018. RT, Rainfed and Tilled; RM, Rainfed and Mulched and no-880 tilled; IT, Deficit Irrigated and Tilled; IM, Deficit Irrigated and Mulched and no-tilled. 881 Parameter Year Water Regime (WR) Significance of effects R I R I Soil Management (SM) T M WR SM WR × SM N (g kg-1 DW) 2016 19.4 21.8 19.3 21.7 0.007 0.854 0.941 20.6 20.5 2017 17.9 18.1 18.7 19.7 0.311 0.009 0.241 18.0 19.2 2018 18.0 19.0 18.9 19.6 0.178 0.282 0.858 18.5 19.2 Ca (g kg-1 DW) 2016 26.3 30.5 27.8 32.5 0.003 0.004 0.491 28.4 30.1 2017 29.0 35.6 33.3 32.2 0.116 0.678 0.008 32.3 33.3 2018 27.9 24.9 24.7 31.0 0.610 0.279 0.008 26.4 27.8 K (g kg-1 DW) 2016 6.7 6.1 6.6 6.8 0.684 0.279 0.150 6.4 6.7 2017 4.5 5.4 6.4 6.3 0.247 0.006 0.203 5.0 6.4 2018 6.1 6.4 5.8 7.8 0.097 0.412 0.237 6.2 6.8 Mg (g kg-1 DW) 2016 3.3 3.9 3.2 4.1 0.009 0.663 0.577 3.6 3.6 2017 3.7 4.2 3.6 3.4 0.358 0.078 0.145 4.0 3.6 2018 2.9 3.0 2.8 3.2 0.540 1.000 0.098 3.0 3.0 P (g kg-1 DW) 2016 0.9 1.2 0.9 1.3 < 0.001 < 0.001 < 0.001 1.1 1.1 2017 0.6 0.9 0.8 1.1 0.005 0.006 0.834 0.8 1.0 2018 0.7 0.8 0.7 1.0 0.087 0.033 0.331 0.7 0.9 Statistical significance effect of SM, WR and their interaction is also indicated by means of p-values. SM = Soil Management; WR = Water 882 regime. 883 884 885 40 Table 3. Contents of B, Cu, Fe, Mn and Zn at veraison in leaf blades from Vitis vinifera (L.). 886 cv. ‘Bobal’ onto 110-R under two different soil management and two water regime strategies 887 during 2016, 2017 and 2018. RT, Rainfed and Tilled; RM, Rainfed and Mulched and no-888 tilled; IT, Deficit Irrigated and Tilled; IM, Deficit Irrigated and Mulched and no-tilled. 889 Parameter Year Water Regime (WR) Significance of effects R I R I Soil Management (SM) T M WR SM WR × SM B (mg kg-1 DW) 2016 129.5 102.6 161.1 110.0 0.004 0.002 0.020 116.0 135.5 2017 21.3 22.5 22.4 24.3 0.081 0.118 0.671 21.9 23.3 2018 20.9 19.8 18.4 24.4 0.269 0.028 0.418 20.3 21.4 Cu (mg kg-1 DW) 2016 4.6 5.0 5.0 6.0 0.142 0.105 0.389 4.8 5.5 2017 41.5 63.9 57.1 48.5 0.475 0.992 0.055 52.7 52.8 2018 4.5 3.9 3.1 5.6 0.433 0.874 0.111 4.2 4.3 Fe (mg kg-1 DW) 2016 139.1 134.0 443.7 193.8 0.099 0.023 0.088 136.6 318.7 2017 101.7 124.6 130.8 135.5 0.246 0.029 0.244 113.2 133.1 2018 115.1 78.5 86.3 105.4 0.648 0.964 0.235 96.8 95.8 Mn (mg kg-1 DW) 2016 97.7 146.3 99.3 167.0 0.004 0.157 0.215 122.0 133.1 2017 93.2 146.9 124.5 130.3 0.021 0.511 0.062 120.1 127.4 2018 91.4 94.1 76.5 112.9 0.144 0.795 0.053 92.8 94.7 Zn (mg kg-1 DW) 2016 20.1 13.7 18.5 14.4 0.015 0.553 0.139 16.9 16.4 2017 14.5 19.3 18.2 18.7 0.018 0.285 0.151 16.9 18.4 2018 13.5 12.4 13.5 13.3 0.660 0.832 0.825 13.0 13.4 Statistical significance effect of SM, WR and their interaction is also indicated by means of p-values.SM = Soil Management; WR = Water 890 regime. 891 892 41 Table 4. Pruning weight and yield components in a Bobal onto 110-R vineyard under two 893 soil management and two water regime strategies over the 2016-2018 growing seasons. RT, 894 Rainfed and Tilled; RM, Rainfed and Mulched and no-tilled; IT, Deficit Irrigated and Tilled; 895 IM, Deficit Irrigated and Mulched and no-tilled. 896 Parameter Year Water Regime (WR) Significance of effects R I R I Soil Management (SM) T M WR SM WR × SM Pruning weight (kg/vine) 2016 0.30 0.61 0.30 0.73 0.023 0.204 0.181 0.46 0.52 2017 0.52 0.76 0.55 0.89 0.043 0.071 0.341 0.64 0.73 2018 0.58 0.77 0.51 0.80 0.114 0.671 0.323 0.68 0.65 Clusters per vine 2016 10.3 12.6 9.7 12.4 0.108 0.265 0.845 11.3 10.9 2017 7.3 13.1 8.0 12.4 0.004 0.963 0.145 10.0 10.0 2018 8.7 10.4 9.1 9.6 0.356 0.622 0.172 9.5 9.3 Yield (kg/vine) 2016 2.1 6.7 2.3 7.1 0.044 0.623 0.896 4.2 4.4 2017 2.5 6.3 3.3 6.0 0.010 0.134 0.013 4.3 4.5 2018 3.1 4.5 2.9 4.0 0.088 0.133 0.594 3.8 3.4 Cluster weight (g) 2016 211.7 538.3 234.2 591.0 0.029 0.283 0.629 361.7 393.4 2017 337.7 479.6 404.6 494.4 < 0.001 0.005 0.038 403.8 444.9 2018 354.7 431.6 312.5 396.6 0.046 0.025 0.761 390.2 349.1 Berry weight (g) 2016 1.41 3.20 1.47 3.56 0.001 0.117 0.252 2.30 2.51 2017 3.44 3.26 3.55 3.49 0.323 0.114 0.578 3.35 3.52 2018 2.63 3.04 2.66 3.21 0.069 0.450 0.581 2.84 2.94 WUE (kg/m3) 2016 3.5 4.4 3.8 4.6 0.069 0.706 0.945 3.9 4.1 2017 5.8 7.3 7.5 6.9 0.035 0.025 0.008 6.5 7.3 2018 3.7 4.1 3.5 3.7 0.160 0.160 0.773 3.9 3.5 Statistical significance effect of SM, WR and their interaction is also indicated by means of p-values. SM = Soil Management; WR = Water 897 regime.898 48 Supplementary material 931 Supplementary Figure 1. Monthly averages of reference evapotranspiration and total 932 rainfall in Requena, Valencia, Spain for the 2001-2015 period. 933 934 935 49 Supplementary Figure 2. Detail of the soil mulching applied in the cv. Bobal vineyard 936 located in Requena, Valencia, Spain. 937 938 939 50 Supplementary Table 1. Results of the ANOVA (p value) conducted to assess the effects of the soil management (SM), water regime 940 (WR), year of study and their interaction on the parameters assessed on Vitis vinifera (L.). cv. ‘Bobal’ grafted onto 110-R. 941 Variable SM WR Year SM x WR SM x Year WR x Year SM x WR x Year Elements in leaves N 0.073 < 0.001 < 0.001 0.721 0.249 0.052 0.797 Ca 0.951 0.002 0.026 0.832 0.339 0.428 0.001 K 0.066 0.111 < 0.001 0.919 0.062 0.204 0.110 Mg 0.185 0.007 < 0.001 0.390 0.342 0.185 0.350 P < 0.001 < 0.001 < 0.001 0.770 < 0.001 0.074 0.278 B 0.001 < 0.001 < 0.001 0.002 < 0.001 < 0.001 0.016 Cu 0.977 0.607 < 0.001 0.387 0.998 0.873 0.384 Fe 0.010 0.049 < 0.001 0.024 < 0.001 0.014 0.031 Mn 0.898 < 0.001 < 0.001 0.266 0.300 0.008 0.006 Zn 0.301 0.155 < 0.001 0.224 0.806 < 0.001 0.383 Yield components and vegetative growth Pruning weight 0.216 < 0.001 < 0.001 0.100 0.383 0.250 0.981 Clusters per vine 0.483 < 0.001 < 0.001 0.229 0.840 < 0.001 0.438 Yield 0.937 < 0.001 < 0.001 0.201 0.198 < 0.001 0.226 Cluster weight 0.224 < 0.001 < 0.001 0.866 < 0.001 < 0.001 0.161 Berry weight 0.082 < 0.001 < 0.001 0.319 0.875 < 0.001 0.907 WUE 0.197 0.005 < 0.001 0.038 0.041 0.460 0.047 Berry composition TSS 0.073 < 0.001 0.047 0.452 0.857 0.009 0.434 TA 0.578 0.070 < 0.001 0.574 0.375 < 0.001 0.645 pH 0.002 0.156 < 0.001 0.187 0.895 < 0.001 0.539 Malic acid 0.155 < 0.001 < 0.001 0.912 0.018 < 0.001 0.045 Tartaric acid < 0.001 < 0.001 < 0.001 0.891 0.883 < 0.001 0.560 TSS/TA 0.115 < 0.001 0.008 0.902 0.236 < 0.001 0.252 Total Polyphenols < 0.001 0.006 < 0.001 0.688 0.619 < 0.001 0.732 Anthocyanins 0.001 < 0.001 < 0.001 0.861 0.344 < 0.001 0.803 Bold values indicate statistically significant effects for each factor on a given parameter. WUE = Water use efficiency; TSS = Total soluble solids; TA = Total acidity 942 943 944 51 Supplementary Table 2. ANOVAs conducted to assess the effect of the soil management, soil depth and their interaction on the bulk 945 density and soil water content under the rainfed treatment in the plantation of Vitis vinifera (L.). cv. ‘Bobal’ grafted onto 110-R 946 Source of variance Sum of squares Degrees of freedom Mean squares F p-value Bulk density ----------------------------------------------------------------------------------------- --------------------- Soil management 0.0062 1 0.0062 0.2124 0.647 Soil depth 0.1217 2 0.0609 2.0899 0.136 Interaction 0.2060 2 0.1030 3.5373 0.038 Residual 1.2233 42 0.0291 Total 1.5572 47 Soil water content ---------------------------------------------------------------------------------- --------------------- Soil management 0.0045 1 0.0045 5.1641 0.028 Soil depth 0.0072 2 0.0036 4.1693 0.022 Interaction 0.0039 2 0.0020 2.2729 0.116 Residual 0.0362 42 0.0009 Total 0.0518 47 947 948 949 52 Supplementary Table 3. Nutritional composition of the buried manure applied at the beginning of the experiment in a Vitis vinifera 950 (L.). cv. ‘Bobal’ vineyard grafted onto 110-R. 951 Parameter Manure N (% DW) 2.5 P (% DW) 1.35 K (% DW) 2.79 Ca (% DW) 6.3 Mg (% DW) 1.33 Na (% DW) 0.58 B (mg kg-1 DW) 53.4 Fe (mg kg-1 DW) 4036 Cu ( m g k g -1 DW ) 50,0 Mn (mg kg-1 DW) 198 Zn (mg kg-1 DW) 273 952