Modiﬁcations Induced by Rootstocks on Yield, Vigor and Nutritional Status on Vitis vinifera Cv Syrah under Hyper-Arid Conditions in Northern Chile

: Hyper-arid regions are characterized by extreme conditions for growing and lack of water (<100 mm annual rainfall average), where desertiﬁcation renders human activities almost impossible. In addition to the use of irrigation, different viticultural strategies should be taken into account to face the adverse effects of these conditions in which rootstocks may play a crucial role. The research aim was to evaluate the effects of the rootstock on yield, vigor, and petiole nutrient content in Syrah grapevines growing under hyper-arid conditions during ﬁve seasons and compare them to ungrafted ones. St. George induced lower yield than 1103 Paulsen. Salt Creek induced higher plant growth vigor and Cu petiole content than ungrafted vines in Syrah, which was correlated to P petiole content. However, Salt Creek and St. George rootstocks induced an excess of vigor in Syrah according to Ravaz Index. Rootstocks with V. berlandieri genetic background should be used in Syrah growing under hyper-arid conditions to maintain adequate levels of plant vigor and to avoid deﬁciencies or toxicity of macro and micronutrients.


Introduction
Chilean viticulture was developed for many years with ungrafted (own-rooted) vineyards, unlike what occurs in other viticultural countries with long winemaking tradition, such as Portugal, Spain, Italy, or France that were forced to use rootstocks mainly due to the presence of phylloxera (Daktulosphaira vitifoliae) [1][2][3]. Since this root parasite is not yet reported in Chile, there is limited local experience in the use of rootstocks in the national viticulture for wine grapes [4][5][6]. However, plant-parasitic nematodes are a main constraint in the national viticulture, affecting vine root development [7]. These nematodes may cause several damages that are typically reflected in lower production and, in some cases, total crop loss [7]. Rootstock adoption in Chile is justified not only by the need for resistance against nematodes or the need to replace old or unproductive vineyards but also by the need to overcome soil limitations, especially in the arid, northern region of the country [4][5][6].
Grafting is a millennial horticultural technique performed in perennial and annual crops, which is used to join scion and root from two plants, allowing one to enable the desirable traits of these different genotypes to be combined in a single plant [8]. Grafting allows some grape grower control over important agronomic traits and provides flexibility in growing a particular scion or cultivar across diverse soil and environmental conditions [9].

Study Site and Plant Material
The field trial was conducted in an experimental vineyard located at the Vicuña Experimental Center belonging to the Instituto de Investigaciones Agropecuarias (INIA) (30 • 02 S, 70 • 41 W, 630 m above sea level; Coquimbo region, Chile) in five consecutive seasons (2003-2004, 2004-2005, 2005-2006, 2006-2007, and 2007-2008). Syrah grapevines own-rooted and grafted onto 1103 Paulsen, 140 Ruggeri, 99 Richter, Freedom, Harmony, Salt Creek, SO4 (Selection Oppenheim 4), and St. George (Rupestris du Lot) were established in 2001 in a replanting soil previously planted with avocado (Persea americana). The vines were grafted using the Omega technique following the procedure stated by Ibacache and Sierra (2009). The vines were planted at a distance of 1.5 m × 2.5 m, formed in bilateral cordon, trained on a vertical shoot position trellis system, and spur pruned to two buds. Syrah grapevines were managed according to the standard viticultural practices used in commercial vineyard in terms of fertilization, pruning, pest, and disease in each growing season. Briefly, fertilization program consisted of applications of N, P 2 O 5, and K 2 O (90, 50, 70 kg ha −1 ) by means of fertigation in spring and early summer.
Soil conditions were previously reported by Ibacache and Sierra [20]. The vineyard soil is a loamy alluvial Entisol and has a flat topography (<1% slope). The soil has moderate depth (>50 cm), alkaline pH (7.9), 2.4% of organic matter, and an electrical conductivity of 1.0 dS m −1 in saturated paste. The mineral N, P Olsen, and exchangeable K levels were 9, 12, and 237 mg kg −1 , respectively. Before the vineyard establishment, a nematological analysis was performed into the vineyard soil, reporting a presence of Pratylenchus, Meloidogyne, Criconemoides, Pratylenchus, and Tylenchulus semipenetrans species lower than 18 individuals per 250 g of soil.
To characterize the vineyard climatic conditions in terms of temperature and precipitation during the seasons, an automatic weather station (AWS) located at 100 m from the experimental vineyard was utilized. Based on the data provided by the AWS, different bioclimatic indices, such as growing season temperature (GST), cool night index (CI), heliothermal index (HI), growing degree days (GDD), biologically effective degree days (BEDD), mean spring temperature summation (SON Mean), maximum spring temperature summation (SON Max), and the accumulated precipitation from May (year n) to April (year n + 1), were calculated and are shown in Table 1. GST: growing season temperature [21]; CI: cool night index [22]; HI: heliothermal Index [23]; GDD: growing degree days [24]; BEDD: biologically effective degree days [25]; SON Mean: mean spring temperature summation [26]; SON Max: maximum spring temperature summation [26]. PP May-April: accumulated precipitation from May (year n) to April (year n + 1). a Mean of 1985-2015 years.
The zone under study is classified as hyper-arid (dry index = 0.05) based on the United Nations Environmental Program [27]. Due to the low rainfall that is recorded during the season and the high vapor pressure deficit, it is necessary to apply water through irrigation. In this regard, the vines were drip irrigated using one irrigation line per row with emitters supplying water at a rate of 4 L h −1 spaced at 1 m. The reference evapotranspiration (ET 0 ) during the five seasons varied between 1127 and 1162 mm (September-April). Annual ET 0 ranged between 1368 and 1420 mm (January-December).

Experimental Design
Syrah grapevines own-rooted and grafted onto 1103 Paulsen, 140 Ruggeri, 99 Richter, Freedom, Harmony, Salt Creek, SO4, and St. George rootstocks were assigned in a completely randomized design at planting. The experimental design consisted in nine treatments (one: own-rooted and eight: grafted vines) and four replicates of four adjacent vines each to cope for soil variability along the vineyard. The descriptions of the rootstocks under study are shown in Table 2.  Harvest was carried out when berries reached a total soluble solids content between 22 and 23 • Brix. All bunches of the replicates were manually harvested and weighed in a digital weight scale, recording yield by vine (kg vine −1 ) and the number of bunches per vine. The bunch weight (g) was obtained, dividing the yield by the number of bunches per plant. During spring of each season, budburst percentage (ratio of number of buds that burst vs. total number of buds left at pruning) and fruitfulness percentage (ratio of number of bunches vs. total number of buds left at pruning) were measured on each plant.

Vine Vigor and Balance
Vines of each replicate were manually pruned in winter and the pruning weight (kg vine −1 ) was determined. Based on yield and pruning weight obtained, the Ravaz index was calculated as the ratio between yield and pruning weight, representing the balance between vine reproductivity and vegetative activity. Scion trunk circumference (cm) was measured at the end of each season (April) at 30 cm above the ground using a metric tine.

Petiole Nutrient Content
Petioles of leaves opposite to the bunches were collected at flowering stage of each study season. Forty petioles per treatment and per replicates were sampled. The samples were dried in an oven at 65 • C until a constant mass was achieved, then samples were milled and sieved through a 1 mm mesh. Then, concentrations of macro (N, P, K, Ca, and Mg) and micro (Zn, Mn, and Cu) nutrients were analyzed at the Foliar Analysis Laboratory of Vicuña Experimental Center. The Kjeldahl method was used to analyze N content of leaf petioles according to methodology described by Nikolaou et al. [28]. The Olsen colorimetric method was utilized to analyze P content in leaf petioles using a Spectronic 21 spectrophotometer (Spectronic Instruments, Garforth, UK) at 440 nm. K was determined by atomic absorption spectrophotometry (Unicam 929, Unicam Ltd., Cambridge, UK) according to the method described by Garcia et al. [29]. Ca and Mg were determined by atomic absorption. Zn, Mn, and Cu concentrations were analyzed by sample calcination and atomic absorption spectrophotometry. Macronutrient concentration was expressed in terms of percentage (w w −1 ), while micronutrients were expressed in ppm.

Statistical Analysis
The variables were analyzed considering a completely randomized design with factorial arrangement, accounting for nine treatments (eight rootstocks plus own-rooted) by five study seasons. Variables were subjected to an analysis of variance (ANOVA). The significance of the differences was determined by Tukey's test (p ≤ 0.05). Additionally, a principal component analysis (PCA) was performed to determine relationships among variables according to treatment (rootstocks) and season, using the average data in each case. Both analyses were performed using the Xlstat Software version 2020.3.1 (Addinsoft SARL, Paris, France).

Yield and Its Components
Season factor significantly affected yield, number of bunches per vine, bunch weight, and percentage of budburst and fruitfulness, while rootstock factor only affected yield in Syrah grapevines growing under hyper-arid conditions (Table 3). Syrah grapevines grafted onto 1103 Paulsen reached a higher yield (7.9 kg) than vines grafted onto St. George

Vine Vigor and Balance
Both rootstock and season factors affected pruning weight, Ravaz index, and scion trunk circumference in Syrah grapevines cultivated under hyper-arid conditions (Table 4)
Rootstock affected significantly N, P, K, Ca, Mg, Zn, Mn, and Cu petiole content. Syrah ungrafted grapevines presented lower N petiole concentration than Harmony and St. George. Syrah ungrafted grapevines presented lower P petiole content than most of the rootstocks with the exception of Harmony. Ca petiole content was lower in Syrah ungrafted grapevines than in most of the rootstocks, with the exception of 99 Richter, Freedom, and Salt Creek (Table 5). Syrah grapevines grafted onto Harmony presented the highest K petiole content and lower Zn petiole content than most of studied rootstocks with the exception of 1103 Paulsen and 140 Ruggeri. The rootstocks induced lower Mg petiole content compared to ungrafted vines in Syrah. Vines grafted onto Harmony presented lower Mn petiole content than vines grafted onto 140 Ruggeri and St. George, whereas vines grafted onto Salt Creek showed higher Cu petiole content than vines grafted onto 99 Richter, Freedom, Harmony, SO4, and St. George rootstocks.   Table S1).

Principal Component Analysis (PCA)
In order to classify the different rootstocks assessing their influence on productivity, viticultural parameters, and petiole nutrient content, a principal component analysis (PCA) was performed (Figure 1). Principal component 1 (PC 1) explained 43.13% of the variance, and principal component 2 (PC2) explained 25.44%, representing 68.57% of all the variance. PC 1 was most closely correlated (−) with N, Ca, and (+) to number of bunches per vine and yield, while PC 2 was most closely (+) correlated with Zn. Ungrafted vines were inversely correlated with P petiole content and pruning weight, and these variables were inversely correlated with Ravaz index. According to PC 1, St. George and Harmony rootstocks were positively correlated with N, Ca, and K petiole content, and these variables were negatively correlated with number of bunches per vine, yield, Ravaz index, and percentage of fruitfulness. According to PC 2, Salt Creek rootstock was inversely correlated with Mg petiole content and scion trunk circumference. Pearson correlations confirmed some relationships (Supplementary Table S2  In order to classify the different seasons and to assess their influence in viticultural productivity parameters and petiole nutrient content, a PCA was also performed (Figure 2).  Table S3). N petiole content was positively related to Cu (r = 0.88). P petiole content was directly related to Zn (r = 0.93). K petiole content was inversely related to yield (r = −0.96) and Ravaz index (r = −0.96). Ca petiole content was directly related to Cu (r = 0.97). Mn petiole content was inversely related to scion trunk circumference (r = −0.90).

Discussion
This five year study evaluated scion performance and petiole nutrient content effects of ungrafted Syrah young grapevines in comparison to eight rootstocks in a hyper-arid irrigated vineyard. St. George induced lower yield than 1103 Paulsen rootstock in Syrah, and there were no statistical differences among the rest of the rootstocks and the ungrafted vines. Previous studies showed that rootstocks significantly affected scion yield [30][31][32][33][34]. Bascuñán-Godoy et al. [32] reported that yield was related to number and weight of bunches and pruning weight in vines grafted onto different rootstocks growing under hyper-arid conditions. These results partially coincided to those presented here, since yield was positively related to number (r = 0.96) and weight (r = 0.73) of bunch (Supplementary  Tables S2 and S3), but these components were not affected by rootstock factor and were not related to pruning weight (Table 3). Recently, Clingeleffer et al. [35] showed that scion-rootstock interactions affected yield, yield components, pruning weight, and Ravaz index in vines grafted onto different rootstocks. These findings indicate that specific rootstocks are required to optimize scion variety performance under our studied conditions. Agreeing with our results, some reports evidenced that rootstocks did not affect scion yield components in different grapevine varieties growing under cold and tropical climate conditions [36,37]. In this way, season was the most important factor influencing yield components in Syrah grapevines growing under hyper-arid climatic conditions ( Table 3). As expected, annual precipitation was correlated with vine yield (r = 0.71), whereas mean (SON Mean) and maximum (SON Max) spring temperature summation were correlated with budburst percentage (r = 0.75 and r = 0.82, respectively) (data not shown). These last bioclimatic indices were elaborated upon based on heat summation method during bud break and flowering [26]. Mean and maximum springtime temperatures modulate timing of bud break as well as growth rate throughout the season [26,38,39]. Number of bunches per vines was correlated with different bioclimatic indices such as growing season temperature (GST) (r = 0.74), heliothermal index (HI) (r = 0.69), and growing degree days (GDD) (r = 0.75) (data not shown). Recently, it was reported that bud fruitfulness was mostly influenced by bud light interception, while inflorescence primordia size was positively correlated with shoot growth capacity and carbohydrate level of buds [40].
Rootstocks have the ability to modulate yield by modifying several plant traits [41,42]. In this fashion, water and nutrient uptake were identified as two key processes that differ between own-rooted and grafted plants [8,20,41]. Indeed, Salt Creek induced higher plant growth vigor than ungrafted vines in Syrah measured as pruning weight (Table 4), which was also correlated with P petiole content (r = 0.79) (Supplementary Table S2). A previous study showed that Salt Creek induced an increment in N and P concentrations in aerial portion of Red Globe vines, which could be positively related to plant growth vigor [20]. Phosphorus is an essential element for plant growth and is involved in many fundamental processes, including photosynthesis, biosynthesis, and respiration because of its role in energy generation via adenosine triphosphate [8,43]. Low P availability in vines may limit cell division, restricting leaf initiation of shoot apical meristem, expansion of newly developed leaves, and lateral shoot growth [44,45]. P deficiency may decrease root hydraulic conductance so that reduction in water supply to growing organs restricts cell expansion, which in turn strongly limits leaf expansion [45,46]. Despite the above, ungrafted vines presented the highest Ravaz index, defined as the ratio yield of pruning wood weight, and that value indicated that these vines were vegetatively balanced. In this way, Syrah grapevines grafted onto Salt Creek and St. George rootstocks held an excess of vigor according to their Ravaz index (Table 4), confirming previous rationale. K petiole content was inversely correlated with yield and Ravaz index according to Figure 2. Based on our results, the vines that reached high K levels in their leaves tissues presented lower yield (Tables 4 and 5). In addition, in most of the samples, K content in petioles was above the optimal nutritional content (>1.8%) [45] (Table 5). K is the most abundant cation in grape berry [47]. Plant growth and development require large K fluxes to provide this ion to growing tissues, and deficiencies in K may reduce xylem sap flow, limiting shoot and fruit growth and, by consequence, affecting yield [45,47]. Nevertheless, according to our results, K management should be considered a priority in the nutritional management program by vine growers of Northern Chile to avoid problems of excesses of this nutrient. Scion trunk circumference (STC) was higher in ungrafted vines than in vines grafted onto Freedom rootstock, and STC increased as seasons passed ( Table 4). The vines under study were young, thus STC was strongly influenced by the season since vines were actively growing season to season. Most of published studies showed that STC allowed one to characterize vine vigor, which was strongly related to pruning weight [48,49]. Nevertheless, in our study, vines were young, and STC was not related to pruning weight (r = 0.14) or yield (r = 0.46), and possibly other variables may affect their changes, such as Mg petiole content (r = 0.67). In this regard, graft union may play a key role in water transport [41] and thus in vigor, especially in the first years of vine development. A successful graft union must differentiate functional phloem and xylem connections across graft surface with the aim to uptake and transport water, nutrients, and photo-assimilates [41,45]. Grafting can have a negative impact on hydraulic conductivity and therefore on development and lifespan of the scion [41,50]. Therefore, it is possible that, during first years of development, ungrafted vines do not possess these limitations and can grow quickly compared to grafted vines.
Normally, the region under study presents a significant amount of alkaline soils that have a high pH and presence of CaCO 3 , which affects essential nutrient availability for crop development [51]. These soils can display an accumulation of soluble salts and a high content of exchangeable Na, resulting in micronutrient deficiencies such as Fe, Zn, Mn, and Cu in crops [52][53][54]. However, according to nutritional standards for viticultural management [55], Syrah ungrafted and grafted grapevines under study exhibited adequate levels in all nutrients analyzed in petioles with the exception of ungrafted vines for P (deficient), St. George for N (excess) and K (excess), and Harmony for K (excess). St. George and Harmony rootstocks induced higher petiole N concentration than ungrafted vines in Syrah (Table 5). Similar results were found in Thompson Seedless and Red Globe varieties cultivated under hyper arid conditions [20]. St. George is a very vigorous rootstock and presents a long vegetative cycle and delays scion maturity [56][57][58]. Due to this, St. George is not suitable for varieties with irregular set, such as Carmenère, Pinot Noir, Chardonnay, or Merlot [56]. Concerning N, this essential macronutrient was inversely correlated with bunch numbers per vines (r = −0.85), yield (r = −0.85), Ravaz index (r = −0.81), and fruitfulness percentage (r = −0.70). Guilpart et al. [59] reported that, during a critical period of vine growing (400-and 700-degree days), water and N stress drove bud fertility and berry number and determined 65-70% of vine yield in the next season.
Syrah ungrafted grapevines exhibited lower P and Ca petiole content than most of the grafted vines and the highest Mg petiole content, whereas St. George induced high P petiole content, while Harmony induced high Ca petiole content in Syrah grapevines (Table 5). Vines grafted onto St. George in high P soil availability induced less shoot dry weight than those grafted onto Freedom, which were greatly inhibited by low P availability in soils [60]. St. George seemed to use P inefficiently for vine growth under these soil conditions. Recently, it was reported that genetic background of vine rootstock may affect petiolar nutrient concentration in scions [8,61]. Rootstocks that present V. riparia genetic background confer low P and Mg petiolar concentrations [8]. These findings are similar to our results, since SO4 rootstocks leant to induce low P and Mg petiolar content compared to the rest of the rootstocks tested in Syrah grapevines (Table 5). Vines growing under low P soil availability showed symptoms of Mg deficiency that were relieved by application of P fertilizer [62]. This lack of P uptake capacity could also affect uptake of Mg by rootstocks that present V. riparia genetic background and its subsequent translocation to shoots [8]. St. George and Harmony rootstocks induced higher K petiole content than the rest of the rootstocks in Syrah grapevines (Table 5). Rootstocks differed significantly in petiole K content at flowering, and rootstocks that presented V. berlandieri genetic background contained lower levels of petiole K [63], which was in agreement in most cases with our results (Table 5). High levels of K uptake may increase pH, leading to wine instability and altering taste, acidity, and color in wines [47]. Based upon these findings, it is important to select rootstocks that confer low potassium absorption, even more in vines growing under hyper-arid climatic conditions, probably by using Vitis berlandieri rootstocks. Season factor also affected most of the nutrient petiole content in Syrah grapevines ( Table 5). The accumulation of K, Mg, and especially Ca is higher in berries that have higher transpiration rates, even at veraison [45,64]. In addition, Ca content increased from anthesis to ripeness in the whole berry [65]. Confirming these findings, our results showed that, in 2006-2007 season, Syrah grapevines presented higher content of several nutrients in petiole, including Ca, and the highest SON Max, thus nutrient petiole content could be determined by temperature accumulation between budburst and flowering.
Salt Creek rootstocks induced higher Zn and Cu and lower Mn petiole content than most of grafted vines in Syrah, although 140 Ruggeri induced higher Mn petiole content than most of the rootstocks in Syrah (Table 5). High Cu concentration in vineyard soils may provoke increases in Cu toxicity symptoms in young vines [66]. Cu concentrations may affect Mn, Zn, and Fe concentrations in vine plant tissues, but effects provoked by Cu toxicity on micronutrients concentrations (Mn and Zn) depend on rootstock genotype [67]. Season also affected the content of most micronutrients in petioles of Syrah grapevines (Table 5). Commonly, concentrations of ions increased across berry development and ripening, but Zn and Mn accumulated before veraison [45,68]. Salt Creek could be a rootstock that should not be used in Syrah due to its high Cu uptake, which could limit uptake of other micronutrients of importance to vines.

Conclusions
Rootstocks and seasons affected vigor and petiole nutrient content in Syrah grapevines grown under hyper-arid conditions. As for the yield components, the season influenced all the parameters tested, while the rootstock only affected the yield per vine. Therefore, in our experimental conditions, the genotype of the root system did not show to significantly affect number of bunches, bunch weight, budburst, and fruitfulness. St. George rootstock resulted in lower yield than 1103 Paulsen in Syrah grapevines. Salt Creek induced higher plant growth vigor than ungrafted vines, which was correlated with P petiole content. However, Salt Creek and St. George rootstocks induced unbalanced vines with an excess of vigor according to their Ravaz index values. St. George and Harmony rootstocks induced a higher petiole N concentration than ungrafted vines in Syrah, whereas Salt Creek rootstocks induced high Zn and Cu petiole content than most of the grafted vines in Syrah. In summary, Vitis berlandieri rootstocks induced intermediate and low levels of K concentration in petioles of Syrah grapevines. The use of St. George, Salt Creek, and Harmony rootstocks in Syrah grapevines growing under hyper-arid conditions should be limited with the aim to avoid irregular set and imbalances of some nutrients. According to our results, rootstocks that present Vitis berlandieri genetic background should be used in Syrah grapevines grown under hyper-arid conditions. These results are relevant for wine industry and may contribute to viticultural management of vines cultivated under hyperarid climatic conditions. In this way, future research should be carried out considering the effect of rootstocks on oenological variables in order to have more information about its effects on the Syrah variety cultivated under hyper-arid conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11050979/s1, Table S1: Interaction effect of rootstock and season for Mg (%) petiole concentration. Table S2: Pearson's correlation obtained from principal component analysis of rootstock. Table S3

Data Availability Statement:
The data presented in this study are available on request from the corresponding and the first author.