Ten batches of 110 Richter rootstock ( V. berlandieri x V. rupestris clone 237) woody canes were obtained from 10 mother plant production fields in 2019. Six of the ten mother fields were located in Spain and four in France (Figure 1). The cultivation system of the rootstock mother plant production fields was spread over the soil surface, and the plants were pruned as a low head-trained system (“tête de saule”) at a space of 3 × 3 m. Irrigation was carried out to achieve maximum vegetative growth following the common practices used in the area. Weed management of the vine plantation was conducted by applying herbicide (glyphosate 36 %) in winter. Located in limestone-calcareous soils, the mother fields differ highly in their climate conditions, as summarised in Table 1. The Huglin Index (HI) classifies Vendée and Camiran as temperate (1800<HI<2100); Chaborra, Sansuañe-1, Sansuañe-2, Aubignan and Carcassonne as warm-temperate (2100<HI<2400); and Villena, Montesa and Albaida as warm regions (2400<HI<3000). Annual rainfall varies from 336 mm in Villena to 950 mm in Camiran. These ten cane production areas were grouped according to their agroclimatic characteristics: North Atl (N Atl), South Atl (S Atl), North Med (N Med) and South Med (S Med). These four groups are hereafter referred to as “production origins”.

Table 1. Location of the mother fields and weather conditions at each production origin in 2019.

⁽¹⁾ Data retrieved from French (www.meteo60.fr) and Spanish (https://eportal.mapa.gob.es/websiar) national climatic websites. ⁽²⁾ Heliothermal index calculated according to Huglin (1978) ⁽³⁾ Growing season is considered as being from 1 April to 31 October.

Figure 1. Location of rootstock cane production origins in France and Spain (Google Inc., 2015).

Rootstock cane batches were collected in all the mother fields following the same sampling protocol: ten vines arranged diagonally within the production field were selected and, from each vine, six shoots were chosen at intervals of ± 60 degrees, with the aim of covering all possible orientations and thus avoiding discrimination between shoots more or less exposed to light. In December 2019, once pruned, the material was cut to form 1 to 2 m-long canes and sent to the facilities of Vitis Navarra (Larraga, Spain). Upon reception, this material was cut to form 38-40 cm-long rootstock canes (from which buds had been manually removed), sprayed with fungicide and kept in cold storage at 4 °C (TARRE, Noain, Spain) until grafting. At the time of grafting, 15 rootstock canes from each batch were randomly sampled and used for cane size and anatomical characteristics, nutritional status and carbon and nitrogen stable isotopes ratio.

As a preliminary step, an RT-PCR analysis was carried out to evaluate the presence of the five major grapevine viruses in Europe (ArMV, GFLV, GFkV, GLRaV-1 and GLRaV -3), following the procedure detailed in Crespo-Martínez et al. (2023). Similarly, an analysis for the prevalence of pathogens associated with fungal trunk diseases (Petri and Black foot diseases, and Botryosphaeria dieback) was carried out applying the method described in Ramsing et al. (2021). All batches of rootstock canes proved to be free of the viruses mentioned above and only a sporadic presence of the fungal species Cadophora luteo-olivacea (6 %) was detected in the canes from the Montesa (S Med) and Vendée (N Atl) mother fields. Therefore, the influence of viruses or trunk disease-causing pathogens can be considered negligible. The methods for characterising the canes are described in the following subsections.

Cane size was assessed by measuring the weight (CW) of the canes with a digital scale (Sartorius BSA 822-CW, Goettingen, Germany). The cane cross-sectional area (CCSA) was estimated by measuring the diameters of both phyllotaxis planes in the cane middle-zone internode with a digital calliper (Mitutoyo CD67-S15PP, Kanagawa, Japan) (at approximately 25 cm from the base of the rootstock cane). In a first step, 5 cm segments of each rootstock cane sample were fixed in ethanol (95 %) /acetic acid 3:1 (v/v) over 24 h, and then transferred to ethanol (70 %) at 4 ˚C for conservation ( Irisarri et al., 2019). Subsequently, semi-thin free-hand sections of 1 mm thickness were cut with a blade and transferred into a Petri dish where they were stained for 30 seconds with 0.2 % toluidine blue O solution. Anatomical observation was carried out with a stereoscopic microscope equipped with a digital camera (Stemi 305/Axiocam ICc 3, Carl Zeiss). For each segment, xylem total area (XA) and conductive area (CA) and the distribution of xylem vessels according to their diameter, in 40 μm increments, were determined. Counts and measurements were performed using ImageJ image analysis software (v. 1.53k, National Institutes of Health, USA) and Microsoft Excel® analysis software. Image contrast and saturation were adapted to maximise the differentiation between occluded and non-occluded xylem vessels. The regions of interest were then measured (number, surface areas and diameters) using the ‘Analyse Particles’ command in ImageJ. Anatomical information was used to estimate the theoretical hydraulic conductivity (K _h) based on the Hagen-Poiseuille law ( Scholz et al., 2013).

A subsample of each rootstock cane was dried and ground to assess cane nutritional status by determining starch, glucose, sucrose, and fructose. Ethanolic extraction was carried out as described by Hendriks et al. (2003), and sucrose, glucose and fructose were measured in the ethanolic supernatant and by HPLC ( Hendriks et al., 2003; Prodhomme et al., 2019). Starch content was determined as follows: the ethanol extract was dissolved by heating it to 95 °C in 0.1 M NaOH for 30 min; after acidification to pH 4.9 using HCl/sodium-acetate, part of the suspension was digested overnight with amyloglucosidase and α-amylase, and the glucose content of the supernatant was then used to assess the starch content of the sample ( Hendriks et al., 2003). Finally, total protein content was measured using the method described in Bradford (1976).

Another subsample of each rootstock cane was oven-dried at 60 °C for 72 h and ground to a homogeneous fine powder. Then 2 mg of the powder was analysed for δ ¹⁵N and δ ¹³C using an elemental analyser (NC2500, Carlo Erba Reagents, Rodano, Italy) coupled to an isotopic mass spectrometer (Thermoquest Delta Plus, ThermoFinnigan, Bremen, Germany). The isotope ratios were expressed as parts per thousand deviations from that standard using the equations included in Santesteban et al. (2015).

The woody canes obtained from the batches of different geographic origin were transferred to Vitis Navarra SA.T. Nursery in December 2019 and grafted on 15 April 2020. For each batch, 100 plants were grafted, using V. Vinifera cv. Tempranillo (clone VN69) as the scion. The grafting process was carried out following the commercial protocol used in Vitis Navarra SA.T. Nursery. In brief, all the plant material was kept in cold until grafting; then it was soaked for 24 h in water at room temperature, and one-bud dormant hardwood scions were omega-grafted to the de-budded rootstock canes. The grafted area was immediately waxed and placed in the stratification chamber at 20-25 °C and 90-95 % relative humidity for ± 20 days. Upon successful callusing, the bases of the grafted canes were soaked with rooting hormone and re-waxed before planting in the nursery on 28 April 2020. The plants grew for one season in the nursery and were dug up after leaf fall (on 23 December 2020). The climatic conditions during nursery production are detailed in Figure S1.

At the end of the season, the grafted plants were mechanically uprooted and transported to the nursery for evaluation. To evaluate success rate (SR) per batch of wood, all plants were individually inspected, and those not fulfilling the technical features of a successful grafted plant were discarded. The technical features considered were (i) lack of vegetative development, (ii) poor root development, and (iii) lack of resistance to the “thumb test”, a manual test used to evaluate the mechanical strength of the union ( Real Decreto, 2003, 2003). Success rate was calculated as the ratio between the number of grafts fulfilling the technical criteria and the total number of grafts produced. Successful grafted plants were characterised measuring the cross-sectional area of the rootstock (RCSA) and the shoot (SCSA) using a Mitutoyo CD67-S15PP digital calliper, and the plant weight (PW) was calculated with a digital scale (Sartorius BSA 822-CW, Goettingen, Germany).

Three parameters related to the anatomic characteristics of the cane were measured: cutting cross-sectional area, total xylem area and conducting area. Despite the significant variability observed in the cutting cross-sectional area (55.2 to 87.5 mm ²), the weights of the canes from the ten wood production fields were similar, ranging from 23.2 to 35.2 g (Table 2). These values correspond to the standards established by European regulations for the production of grapevine plants in nurseries ( Real Decreto, 2003, 2003) and, therefore, are comparable to standard production practices in nurseries. The area of the xylem varied from 33.5 mm ² to 55.8 mm ², while the conducting area ranged from 6.8 mm ² to 11.3 mm ² and was calculated by measuring the diameter of the xylem vessels. In relative terms, the area of the xylem represented a minimum of 60 % and a maximum of 63 % of the total cross-sectional area of the cane. In light of these results, we consider the cuttings to be mature since, according to Dardeniz et al. (2007), well-grown and mature canes are well lignified when their piths are narrow and the xylem tissues are wide. Conducting vessels accounted for between 8 % and 20 % of the total surface area and between 13 % and 33 % of the xylem area, in agreement with the value range observed in Carlquist (1985).

The batches differed markedly in terms of the canes’ anatomic characteristics: the biggest cuttings were those in the batches from Vendée and Sansuañe-1, and the smallest were those from Villena, Montesa and Albaida. Within the conductive area, Montesa and Vendée batches exhibited the highest values (11.3 and 9.7 mm ², respectively). Nonetheless, the canes in the batch from Sansuañe-1 displayed a reduced surface area of conducting vessels (7 mm ²–13 % of the xylem area), while those from the Albaida field exhibited lower values (6.8 mm ²–20 % of the xylem area). This result highlights disparities in hydraulic conductivity, as it varied by several orders of magnitude from the minimum value obtained in cuttings from Albaida (39.3 mm ⁴/MPa/s) to the value reached in the cuttings from Vendée (142.9‧10 ³ mm ⁴/MPa/s). Cuttings from Vendée notably exceeded all other production fields in this regard, compared to Sansuañe-1 which, despite having the highest xylem area-to-CCSA ratio (63.7), had smaller vessels and thus a lower hydraulic conductivity value. This significant effect is associated with differences in the density and diameter of the xylem vessels. The fact that vessels developed under different environmental conditions and practices would explain this difference in xylem anatomy traits ( Pouzoulet et al., 2020).

Table 2. Cutting weight and cane anatomical characteristics of 110R rootstock batches of ten different geographical origins. Values represent means (n=15).

Within each column, different letters indicate significant differences (Tukey test; p > 0.05).

The counting and analysis of all the vessels revealed a significantly different vessel distribution between the batches from the production fields considered (Figure 2). The diameters measured in our work ranged from 0-40 µm to 280 µm.

Although for all the batches > 35 % of the xylem vessels were found in the range of 0-40 µm, the canes from Albaida stood out with 48.5 % within this range, while in those from Chaborra it was 38 % (Figure 2A). This size distribution agrees with the general anatomical structure of many vines and climbers, which exhibit a significant presence of narrow vessel elements alongside some vessels with large diameters ( Carlquist, 1985; Peccoux, 2011; Schultz & Matthews, 1993). When the hydraulic conductivity values were estimated from vessel number and diameters (Figure 2B), the batches with the highest proportion of large vessels were found to also have the highest conductivity ( Sperry et al., 2006): although most of the vessels (85.4 %) were small, they only contributed to 28 % of total hydraulic conductivity; meanwhile, the largest diameter categories (> 160 µm) were responsible for 70 % of conductivity, despite numerically representing only 12 % of the total vessels. A similar phenomenon of dominance by wide vessels in terms of total hydraulic conductivity has also been recorded in other species and in stems of grapevine and V. riparia ( Hargrave et al., 1994; Lobos-Catalán & Jiménez-Castillo, 2023; Munitz et al., 2018; Tibbetts & Ewers, 2000).

Regarding the diameter and frequency of the xylem vessels (Figure 2A), the canes from Sansuañe-1 and Albaida showed a higher proportion of small vessels (91.2 and 90.6 % of 0-160 μm, respectively) than the batch from Vendée (78.2 %). It is important to note that, except for Vendée, the rootstock canes analysed here did not exhibit the bimodal distribution of vessel size that is typical of vines and climbers ( Carlquist, 1985), and is characterised by numerous small vessels and numerous large vessels, with a decrease in the frequency of medium-sized vessels (60-140 µm).

Regarding the distribution of conductivity by xylem vessel size (Figure 2B), the Sansuae-1 and Albaida batches showed a higher proportion of conductivity associated to the small vessels (37.1 and 45.7 % respectively for vessel diameter in the 0-160 μm range) than the batch from Vendée (12.7 %). By contrast, the conductivity associated to the large vessels was lower in the former than in the latter batches (59 and 54 % vs. 87.2 % for 160 to 400 μm-diameter vessels). On average, conductivity peaked at a single point between the diameters of 160 and 200 µm. By contrast, the canes from Vendée reached their maximum percentage of conductivity at around 200 to 240 µm, which was due to their higher percentage of vessels within that diameter range compared to the other batches. It should be noted that cuttings from the same V. berlandieri x V. rupestris cross acquire very different structural and physiological traits when grown under different weather conditions and soils. The proportions of the different xylem vessel sizes can affect the sap flow pattern ( McElrone et al., 2021; Tyree & Zimmermann, 2002) and can be explained by factors such as salinity ( Quintana-Pulido et al., 2018), different growing conditions ( Olson & Rosell, 2013), radiation intensity, shoot orientation, and water stress or water deficit ( Lovisolo et al., 2010), the latter being related to a considerable reduction in the average vessel size ( Pire et al., 2007). However, while stressful habitats have been reported to induce changes in xylem development in roots ( Rajaei et al., 2013) and stems ( Lovisolo & Schubert, 1998) of V. vinifera, to date, no such data is available for Vitis sp. rootstocks. As already mentioned, the batches of cuttings from the "drier" climatic zones ( i.e., Albaida) had a higher percentage of narrow vessels than those from the “wetter” areas ( i.e., Vendée). Additionally, the high-water availability at the beginning of the growing season in Vendée may have led to vigorous vegetative growth with wider vessels and higher hydraulic conductivity, as observed by Munitz et al. (2018) and Netzer et al. (2019) for Merlot and Cabernet-Sauvignon.

Figure 2. Hydraulic characteristics of 110Richter rootstock batches from ten different geographical origins. Distribution of cane (A) xylem vessels and (B) hydraulic conductivity according to diameter classes (μm).

Starch accounted for the majority of carbohydrate reserve compounds measured in the canes in all the analysed batches, followed by sucrose, glucose, and fructose in that order (Table 3). Starch concentration values varied from 159 mg/g to over 200 mg/g depending on the sample. These values are within the range reported by Phillips et al. (2015), and correspond to a slightly broader range than the ones observed by Hunter et al. (2003) and Somkuwar et al. (2017). Total soluble sugar concentrations ranged from 32.6 to 52.1 mg/g and accounted for an average of around 20 % of the total CHO, which is very similar to the percentage reported in Phillips et al. (2015). The different soluble sugar concentrations were as follows: 27.8 mg/g sucrose, 8.4 mg/g glucose, and 8.2 mg/g fructose. Sucrose represented, on average, 69 % of the soluble sugars. The total CHOs content in the samples varied from 194 to 264 mg/g. Consistent with the findings of Phillips et al. (2015); Wample and Bary (1992); Winkler and Williams (1945), while high levels of total CHO were associated with high starch concentrations, there was a weak relationship between soluble sugars and total CHO. Significant differences in carbohydrate concentrations were observed among production fields. The highest starch concentration was found in the batch from Villena, followed by those from Sansuañe-1 and Montesa, all of which exhibited above-average starch values (172.4 mg/g). By contrast, the batches from Aubignan and Albaida had notably low starch content, with values of 159.1 and 160.4 mg/g, respectively. This trend was consistent for the other analyzed sugars, except in Sansuañe-1, which showed values below the average for glucose, fructose, and sucrose. In contrast, Albaida had above-average values for these parameters.

The range of the soluble sugars to starch ratio was between 0.19 and 0.37, which was lower than that reported by Todic et al. (2005) (0.30 to 0.55), and therefore corresponds to high cane maturity and conservation status. The sucrose concentration positively affected this parameter, with higher for the batch from Albaida (0.37) and lower ratios for Sansuañe-1 and Sansuañe-2 (0.19 and 0.21, respectively). This indicates that the Sansuañe-1 and Sansuañe-2 rootstock canes exhibited a higher degree of wood maturation than Albaida, as their soluble sugar content was 1.5 times higher, especially the sucrose content.

The protein content differed among the batches, ranging from 4.8 to 8.6 mg/g (Villena and Sansuañe-2, respectively). The protein content of each of the batches from Villena, Albaida and Aubignan was below the average value, while those of the other batches exceeded a mean of 7.13 mg/g. These values are much lower than those reported by Somkuwar et al. (2017) for 110 Richter rootstock canes in India, but the reason for this difference is unknown to us.

The variability of the nitrogen isotope ratio was also high, with values ranging between -0.6 and 5.2 ‰ and with a mean of 2.1 ‰, which are similar to those achieved in Tempranillo canes ( Santesteban et al., 2024). Interpreting δ ¹⁵N values is complex, because plant δ ¹⁵N values reflect various interactions between nitrogen sources and isotope fractionations that vary over time and across different compartments ( Stamatiadis et al., 2007). A likely explanation for the high δ ¹⁵N values in the canes of the Chaborra, Camiran and Aubignan batches (5.22, 4.84 and 4.62 ‰, respectively) was that the plants had relied more on organic sources of nitrogen for their nutrition ( Bateman & Kelly, 2007). In contrast to the latter canes, the values of those from the Villena, Carcassonne, Albaida, and Sansuañe-2 batches were below 0 ‰. This may indicate that an increase in available inorganic nitrogen made plants more dependent on inorganic sources, resulting in lower δ ¹⁵N values. Additionally, the soils in these regions may have had low organic matter content, limiting the availability of organic nitrogen ( Santesteban et al., 2024).

Canes are not usually considered a sensitive organ to detect differences in water status though the measurement of the carbon isotope ratio, since they are majorly formed before water deficit arises ( Santesteban et al., 2014). Thus, samples from vegetative tissues (roots and wood) typically exhibit a ¹³C isotope content lower than that in berries ( Taskos et al., 2020; Zyakun et al., 2013). In our study, there were relevant differences in the isotope ratio of ¹³C among the production fields, for which the values ranged between -25.3 and -27.6 ‰ (Table 3), which represent a significant range of variation that should correspond, in our case, to differences in the water status of the production fields. ¹³C isotope values of close to and above -26 ‰ were reported for the batches from Camiran, Aubignan and Carcassonne, which indicates lower water availability during the growing season, while the values of the batches from Vendée, Albaida, Sansuañe-2 and Montesa (< -27 ‰) correspond to conditions of less water stress ( Taskos et al., 2020). These differences in water status probably resulted from a combination of weather conditions and the use of irrigation, since the least stressful conditions are not only associated with the rainier area of Vendée, but also with areas with the driest climates, where irrigation is systematically used to ensure wood production.

On the whole, the range of the cane nutritional status is relatively broad. The reasons behind this diversity cannot be determined with our dataset, but they may be partly due to the variability in meteorological conditions of the mother field sites or to differences in soil composition, fertilisation and irrigation management ( Bianchi et al., 2020). The differences observed for both carbon and nitrogen isotope ratios support the hypothesis of relevant differences in the growing conditions of the mother vines.

Table 3. Starch, soluble sugars (mg/g of dry material), total carbohydrates (CHOs), soluble sugar-to-starch ratio, proteins (mg/g of dry material) and nitrogen and carbon isotope ratios (‰) of 110 Richter rootstock batches from ten different geographical origins. Values represent means (n=15) ± SEM.

Within each column, the different letters indicate significant differences (Tukey test; p > 0.05).

Correlations between rootstock cane characteristics were assessed using a correlation heatmap (Figure 3). Regarding the relationships between meteorological parameters (Huglin Index (HI) and rainfall) and cane characteristics, HI showed a significant positive correlation with cane glucose content. Albeit not statistically significant, the same trend was found for the other measured sugars, except for fructose. By contrast, a negative significant relationship was observed between HI and the cutting size, estimated as cutting weight (CW). Our dataset did not allow us to find a relationship between rainfall and cane characteristics. Although wetter conditions can lead to a higher sugar concentration and a higher sugar-to-starch ratio ( Liu et al., 2018) and promote the formation of wider vessels ( Tyree & Zimmermann, 2002), the interaction of irrigation with rainfall probably prevented us from finding any such relationship.

Regarding the cane anatomical characteristics, the cutting weight (CW), cane cross-sectional area (CCSA), and xylem area (XA) were positively correlated. As expected, given that vessel diameter is a key determinant in the Hagen-Poiseuille equation for assessing theoretical hydraulic conductance ( Tombesi et al., 2010), high conductivity values were associated with a high proportion of wider vessels (> 120 µm).

When cane nutritional variables were compared, significant positive correlations were observed between starch and total carbohydrates. A similar trend was observed between sucrose and soluble sugar content, as well as between sucrose and the ratio of soluble sugars to starch. Cane protein content exhibited a negative correlation with all measured carbohydrates, notably with sucrose content, soluble sugars, and total carbohydrates. Regarding nitrogen and carbon isotopic ratios, no significant correlation was observed.

An overall comparison of all rootstock cane traits revealed that a superior conductive area (CA) is conducive to the development of larger conducting vessels (VD > 120 µm) and improves hydraulic conductivity. Lastly, improvements in anatomical characteristics seem to reduce the glucose content in the propagation wood, with a significant interaction with the cutting weight (CW).

Figure 3. Correlation matrix of all the variables measured in 110R rootstock batches from ten different geographical origins. Circle size and colour represent R values for Pearson's correlation analysis *, ** and *** indicate significance at probability levels of 0.05, 0.01 and 0.001, respectively.