Leaf blade and petiole nutritional evolution and variability throughout the crop season for Vitis vinifera L. cv. Graciano

An adequate nutritional state of a crop can be kept by means of a well-designed fertilization plan based on the assessment of the nutrient availability throughout the growing season. The objective of this study was to determine the reliability of leaf blade and petiole diagnosis and the period of validity of their references at both flowering and veraison by means of systematic monitoring throughout the complete growing season. The study was carried out in two plots planted with Vitis vinifera L. cv. Graciano within the AOC Rioja (Spain). Blades and petioles were collected throughout a growing season (2006) and total N, P, K, Ca, Mg, Fe, Mn, Zn, Cu and B concentrations were analyzed in both tissues. Results suggest, in general, that petioles have higher variability and lower analysis reproducibility than blades. Therefore, blade could be a more appropriate tissue to evaluate N, P, K, Ca, and Mg at both flowering and veraison in this variety. Micronutrients in blade and petiole showed different variability behaviour in each of the vineyards studied, therefore, based on our results, it was difficult to determine which one could be the best tissue for the nutritional diagnosis of the ‘Graciano’ variety. Seasonal changes of nutrient concentration in both tissues also confirmed the need for reference values for each tissue and each phenological stage. Additional key words: nutrition; grapevine; leaf analysis; nutritional references. Abbreviations used: AOC (appellation d’origine contrôlée or appellation of controlled origin); CV (coefficient of variation percentage, %); DOP (deviation from optimum percentage method); DRIS (diagnosis and recommendation integrated system); DW (dry weight); PC (principal component); PCA (principal components analysis); RA (Rioja Alta vineyard); RM (Rioja Media vineyard). Citation: Domínguez, N.; García-Escudero, E.; Romero, I.; Benito, A.; Martín, I. (2015). Leaf blade and petiole nutritional evolution and variability throughout the crop season for Vitis vinifera L. cv. Graciano. Spanish Journal of Agricultural Research, Volume 13, Issue 3, e0801, 17 pages. http://dx.doi.org/10.5424/sjar/2015133-5142. Received: 15 Oct 2013. Accepted: 24 Jun 2015 Copyright © 2015 INIA. This is an open access article distributed under the Creative Commons Attribution License (CC by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain) (Project SCOO-016); Regional Government of La Rioja (Spain) (Regional Project PR-04-06). Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Ignacio Martín: ignacio.martin@icvv.es or Natalia Domínguez: ndominguezm@larioja.org (shared corresponding authors)


Introduction
Fertilization is one of the farming techniques which the wine-grower uses to modify the nutritional status of the plant in search of the correct balance between yield and crop quality. Nevertheless, an inadequate application of fertilizers can unbalance the availability of nutrients, negatively affecting the nutritional status of plants, the final harvest quality and even the environment. The current nutritional status of plants is key in designing an adequate fertilization plan. The knowledge of the desirable level for each nutrient in representative tissues allows to define its excess or deficiency in the plant and the design of a correct fertilization program. Therefore, nutrition can be monitored by the analysis of the chosen tissues, applying the different available methodologies of plant diagnosis such as diagnosis and recommendation integrated system (DRIS), deviation from optimum percentage (DOP), sap analysis from conductive tissues, flower analysis, analyses of active metabolites (e.g. N-NO 3in petiole), or even studies of specific enzymatic activity (Cook & Kishaba, 1956;Bonilla et al., 1980;Lucena, 1997;Robinson, 2005).
Many of the diagnosis methodologies of leaf tissue use the individual total nutrient concentrations in dry tissue, comparing those values to a reference. However, the physical and chemical characteristics of the 2 Argentina. 'Graciano' is also grown in the French-Midi and South Africa (cv. Morrastel among other synonyms), and Portugal (cv. Tinta Miuda).
The objective of this study was to determine the reliability of leaf blade and petiole diagnosis of 'Graciano' variety by means of systematic monitoring studies throughout a complete growing season. This procedure also allows us to establish the period of validity of a reference suggested for both flowering and veraison phenological stages, the periods for which fertilization references are usually set. A year-long (2006) monitoring study was carried out in two AOC Rioja vineyards. Both were representative of an average vineyard within the Rioja region and their yield and wine quality were within the usual values for the AOC Rioja. Vineyards were located in Haro, sited at the Rioja Alta subzone (RA) within the AOC, and Logroño, sited at the Rioja Media subzone (RM) (La Rioja, Spain). They were more than five years old and had similar agronomic characteristics.

Material and methods
The vineyards were planted with cv. Graciano (Vitis vinifera L.) grafted on 110 Richter rootstock. Plants were trained to a single cordon on a vertical-shootpositioned (VSP) trellis system and pruned leaving 10 to 12 buds per vine. Planting density was 3,100 vines/ ha for RA and 3,350 vines/ha for RM. The experimental design divided each vineyard in three homogeneous subplots, with 108 vines per subplot distributed in three consecutive rows, to obtain the three necessary replicates to perform statistical analyses.
The recorded annual rainfall was 468 mm in RA and 405 mm in RM. The vineyards were not irrigated and both were managed according to the ordinary practices used by growers in the region.
The grape juice quality parameters were optimal at harvest with values within those common for the AOC Rioja. The RA vineyard showed an average bunch weight of 400 g, while grape juice showed a sugar concentration of 17.8 ºBrix (refractometer), titratable acidity was 7.28 g/L tartaric acid, and pH was 3.09 (pH meter). On the other hand, for the RM vineyard the average bunch weight was 344 g, and the grape juice showed a sugar concentration of 25.0 ºBrix, titratable acidity was 6.78 g/L tartaric acid and pH was 3.32.
The RA soil was classified as typic Calcixerepts while the RM soil was classified as typic Xerofluvents (Soil Survey Staff, 2010). Both soil textures were loamy in the topsoil (A P ) and in the subsurface horizon (B). Physical and chemical properties are shown in Table 1. soil, rootstock, variety, climatic conditions, cultural practices, possible diseases, training system, sampling time and the analyzed organ could modify the mineral composition of the plant (Delas, 1990;Benito et al., 2013;Romero et al., 2013) reducing the accuracy of these methods. Thus, it is essential to standardize the sampling method for nutritional diagnosis (Delas, 2000) and a wide range of criteria are used with respect to the sampling time, the analyzed tissue, or its position on the plant (Romero et al., 2010Benito et al., 2013).
Current bibliography shows a disparity of opinions with regard to the tissue and the ideal moment for sampling with diagnostic purposes. Concerning the organ to be analyzed, initially the whole leaf (blade and petiole together) was used, before analyzing the leaf blades and petioles separately. With respect to sampling time, different papers propose two phenological stages: end of flowering or fruit-set, and/or veraison. For instance, Failla et al. (1995) used the leaf blade at flowering and veraison; Navarro et al. (2008) analyzed the leaf blade at veraison; García-Escudero et al. (2002) and Wolpert & Anderson (2007) chose flowering and veraison phenological stages analyzing both leaf blades and petioles separately; Christensen (1984) and Robinson (2005) analyzed petioles at flowering, while Delas (2000) used petioles at veraison.
Previous studies with cv. 'Tempranillo' and cv. 'Red Grenache' showed the usefulness of carrying out monitoring studies throughout the complete crop season in order to determine the reliability of leaf blade and petiole diagnosis and the period of validity of their references (Benito et al., 2013;Romero et al., 2013). According to these and other studies (Fraguas et al., 2003;Wolpert & Anderson, 2007;Benito et al., 2013;Romero et al., 2013), the higher variation for nutrient concentrations in petiole implied, in general, a lower reproducibility for this tissue. Comparing the analysis of blade and petiole, Christensen (1984) and Champagnol (1990) indicated that the variation intervals of phosphorus (P), potassium (K) and magnesium (Mg) in petiole were higher than in leaf blade. Nevertheless, Champagnol (1990) suggested that the significance of a result obtained from both tissues would be identical; also other studies showed that the individual variability for each nutrient was sometimes different between different vine varieties (Benito et al., 2013;. On the other hand, the nutrition level for each element can change widely among different vineyards, especially with respect to K and Mg nutritional status in the vines (Champagnol, 1990;Loué, 1990).
'Graciano' is a grapevine variety with a Spanish origin which is currently grown in other winemaking regions, e.g. for example Australia, California and 3 Leaf blade and petiole analyses for 'Graciano' grapevines Barcelona, Spain) at 70ºC for 48 hours. Finally, dried samples were ground and homogenized with an ultracentrifugal mill (ZM1; Retsch, Haan, Germany), in order to pass through a 0.5 mm mesh.
For total N determination (N-organic+N-NH 4 + ) in leaf blades and petioles, 0.20 g of the ground sample was used to carry out a wet digestion using the Kjeldahl method (Jones et al., 1991). For the remaining nutrients (P, K, Ca, Mg, Fe, Mn, Zn, Cu and B), 0.20 g was used to carry out a wet digestion with 95% H 2 SO 4 and 30% H 2 O 2 (Hoenig et al., 1998) and afterwards were analyzed by inductively coupled plasma-optical emission spectrometry (Optima 3000DV, Perkin Elmer, Norwalk, CT, USA). Double deionised water (Milli-Q; Milli-pore, Bedford, MA, USA) was used for all dilutions. Concentrations were expressed on a dry weight (DW) basis.

Statistical analysis
A principal components analysis (PCA) was performed to detect the vectors that differ from the ten dependent variables (N, P, K, Ca, Mg, Fe, Mn, Zn, Cu, B) data. A non-rotated component matrix was used as factor analysis and the extraction method was performed by a scree plot with eigenvalue>1. The variables were adequately represented within the model. Furthermore, comparisons for equality of means, between blade and petiole tissues at each sampling time, were performed using the parametric t-Student test.
Nutrient trends were fitted with Tablecurve 2D (vers. 5.1; SYSTAT Software Inc., San Jose, CA, USA). The coefficient of variation (CV) percentage between replicates was analyzed for each nutrient, tissue, and sampling date as a measurement of the reproducibility of the analysis within each vineyard.
Finally, the differences between consecutive sampling dates were determined by means of a monofactorial ANOVA followed by a post-hoc test (Tukey, p≤0.05) in order to define the periods of validity for references at different phenological stages. The ANOVA procedure was performed by comparing groups of five consecutive samplings, as a moving window throughout the growing season, displacing the ANOVA procedure throughout the period to the next sampling and discarding the first sampling of the previous ANOVA to assemble the new group of five samplings to perform the next ANOVA procedure. Furthermore, when a long period with no significance differences was detected, the first sampling of the first window was additionally compared with the last four samplings of the last window to confirm the absence of differences over the whole period. This procedure prevents error accumulation as it increases the number of comparisons within the ANOVA.

Sampling
An exhaustive leaf sampling, according to the sampling intervals shown in Table 2, was carried out from pre-flowering to post-harvest. Thirty complete, diseases free and non-senescent leaves were sampled in each replicate. Sampling was carried out between 10 a.m. and 12 a.m., taking one leaf per plant, proceeding from fruit-bearing shoots of average vigour, out of 30 vines chosen at random within each replicate. The leaves collected were opposite to the first bunch from pre-flowering until pre-veraison, and opposite to the second bunch from veraison to ripening (Romero et al., 2010). Sunlight exposure over the canopy was also considered at sampling time and, therefore, leaves were sampled alternatively on both sides of the trellis. Samples were codified using the percentage of time that had elapsed in each phenological stage (Table 2).

Principal components analysis
PCAs are employed to reduce a large set of intercorrelated variables to a smaller number of uncorrelated variables. When applying this method, if the variables are highly correlated, some of the first components are able to describe most of the data variability. In our study three principal components (PC) contained approxi-mately 83% of the total variance of the ten original variables (Table 3). The first PC (PC1) explained 41% of the total variance for both vineyards. The second component (PC2) explained 30% of the variance for the RA and 32% for the RM dataset. The third component (PC3) explained 12.6% of the variance for RA and 10% for RM.
The highest component loadings for the RA vineyard were: P, K, Ca, Mn and B for PC1, N, Mg, and Fe for PC2, and Zn for PC3. On the other hand, the highest component loadings for the RM vineyard were: K, Mg, Fe, Zn, and Cu for PC1, N, P, Ca, and Mn for PC2, and B for PC3. The PCA for the RA dataset showed the existence of a strong relationship between PC1 and the phenological stage as well as between PC2 and the analyzed tissue (Fig. 1A). Inversely, for the RM dataset, PC1 showed a strong relationship with the tissue and    PC2 with the phenological stage (Fig. 1B). These two components together explained more than 71% of the total variance in both cases. The positive correlations between the original nutrient concentrations and PC2 for RA, and between those concentrations and PC1 for RM, indicated that nutrient concentration in blade was higher than in petiole (Table 3). Therefore, blades showed higher concentrations than petioles for N, Fe and Cu, and slightly higher for Ca and B (Figs. 2 & 3; Table 4). Consequently, petioles had higher concentrations of K, Mg, and, to a lesser extent, Mn, and Zn. Phosphorus did not show clear behaviour as it had higher concentration in blades from RA but lower in blades from RM (Table 4; Figs. 2C,D).
Concerning the phenological stage, component loadings of the original nutrient concentrations for PC1 for RA, and for PC2 for RM (Table 3) indicated that the nutrients which positively correlated with the PC showed an accumulating trend in the analyzed tissue throughout the growing cycle (Table 3; Figs. 1C,D). That was the case for Ca, Mg, Fe, Mn, and Cu; while N, P, K, and B showed a negative correlation with the PC and, therefore, a reduction in the concentration in both tissues throughout the growing season. Finally, Zn showed a low correlation with respect to the PC, which indicates that the Zn requirements were relatively unsteady throughout the crop season in both tissues, mainly for the RA vineyard (Table 3; Figs. 3E,F).
The third principal component (PC3) explained 12% and 10% of the global variance, showing a low influence in the total variability compared to the other PCs, phenological stage and tissue (Table 3).

Trends in nutrient concentrations
Nitrogen showed maximum concentration at prebloom sampling. Its concentration decreased over the course of the growth cycle until a stabilization trend for blade was noted around the veraison phenological stage (Figs. 2A,B). Phosphorus also showed a declining pattern of concentration from flowering to harvest (Figs. 2C,D).
Potassium showed a declining pattern for blades (Figs. 2E,F). However, petioles only showed this pattern for RA vineyard (Fig. 2E), while petioles for RM showed an irregular trend throughout the growth cycle (Figs. 2F). On the other hand, Ca and Mg showed an increasing concentration in both tissues (Figs. 2G-J). In general, macronutrients showed a nutrient evolution adjusted to a logarithmic curve in blade and petiole, with a decreasing slope until values near to zero when the veraison phenological stage was reached (Figs. 2A-J).
With respect to micronutrients, trends were not as easily recognizable as for macronutrients (Fig. 3). Some micronutrients showed a logarithmic trend (B, Fe and Zn in petiole for RM); others showed a polynomial fitting (Fe and Cu in petiole and Zn in blade for RA, as well as Cu for RM); and the others fitted to a sigmoidal curve (Fe, Mn, and B in blade and Zn in petiole for RA). Those nutrients with a sigmoidal curve showed two periods where the slope tends to zero, the first around flowering and the second at veraison (Fig. 3).
Iron concentration increased throughout the cycle except at the beginning of bloom and at veraison for both tissues (Figs. 3A,B). Manganese concentration increased in both tissues until veraison, and then its concentration remained stable until the end of the growth cycle (Figs. 3C,D).
Zinc concentration in RA decreased until half of the berry growth phenological stage, increasing thereafter until the end of veraison and then maintaining concentration levels up to harvest in leaf blade and petiole (Fig. 3E). However, Zn concentration in blades from the RM vineyard maintained its levels after fruit set until the end of veraison, while the petiole showed a constant Zn concentration increasing from half of the berry growth until the end of veraison which denoted a different behaviour (Fig. 3F).
Copper maintained relatively constant levels of concentration in both leaf blades and petioles until flowering and even to early pea-sized berries. However it was noted that the application of phytosanitary sprays with a copper-based formulation prompted an increase in blade Cu concentration in the early maturation stage (Figs. 3G,H).
Finally, B concentration increased at the beginning of the cycle, afterwards showing a sharp decrease in blades (Figs. 3I,J).

Stability periods
A stability period is defined as the period where the nutrient concentration remains stable, allowing its comparison with a reference which, therefore, would be reliable for that complete period. However, the length of each stability period is difficult to be delimited only since the slope. Due to it, an stability period could also be defined by the absence of statistical differences between consecutive samplings. Different stability periods of various lengths in time, were obtained for each nutrient and tissue because of the different behaviour of the nutrient concentration throughout the season. From these different stability periods, the period of validity for references proposed for flowering or veraison stages was determined. Furthermore, a period of sampling for blade or petiole could be also delimited for a common diagnosis of all the analyzed nutrients.   Blade results showed that there was no period which included the complete flowering phenological stage for any nutrient in the RA vineyard (Fig. 4) because of the absence of sufficient samplings within this phenological stage for this vineyard (Table 2). Nevertheless, for N, P, K, Mn, Zn and B a period which included the

11
Leaf blade and petiole analyses for 'Graciano' grapevines the end (M100) of this phenological stage due to the change in the concentration of these elements in petioles (Fig. 5).

Coefficient of variation and analysis of reliability
The CV is used to determine the most appropriate tissue for nutritional diagnosis. The maximum CV values for macronutrients were, in general, greater in petioles than blades. The greatest differences between both tissues were found mainly at veraison especially for N, K and Ca in the RM (Figs. 6B,F,H). At flowering, differences between both tissues were often lower than at veraison (Fig. 6).
Micronutrients did not show clear differences between blades and petioles. The maximum CV values were usually lower than 50% and, in general, higher in petioles than blades (Fig. 7). Furthermore, differences were found between vineyards. Iron, Mn and Zn showed greater CV in petioles from RA vineyard throughout the crop season (Figs. 7A,C,E) whereas results from RM did not show such marked differences.

Principal components analysis
The differences between both tissues for N has been described for varieties such as 'Tempranillo' , 'red Grenache' (Benito et al., 2013) and others (Christensen, 1984;Champagnol, 1990;Kliewer, 1991). Besides this, the higher concentrations in blades for Ca, Fe, B and Cu have also been observed in previous reports (Christensen, 1984;Benito et al., 2013;Romero et al., 2013). This is also the case for the higher concentration in petioles described in the bibliography for K (Christensen, 1984;Schreiner & Scagel, 2006), Mg (Schreiner & Scagel, 2006), Mn (Romero et al., 2010) and Zn (Christensen, 1984;Romero et al., 2013). However, the differences between both tissues were not so marked for K, Mn and Zn in the RA vineyard or for B in the RM plot. Finally, Atalay (1978) found a higher P concentration in petiole from vineyards with high P concentration in the soil, while Benito et al. (2013) observed that P concentration was higher in blades than in petioles. These results show the importance of proposing specific reference levels for blade and petiole tissues, separately.
The variation of the nutrient content in blade tissues throughout the crop season has been widely discussed complete flowering stage was found for the RM vineyard. This period was limited to flowering for N and P, and was slightly broader for K and B. For Mn, this period was extended to K75, and even until complete veraison for Zn due to the high variability of the Zn concentration in blade at some moments during the growing cycle (Fig. 4). This latter fact was the origin of the absence of significant differences between samplings for Zn at RM.
The RM vineyard showed a period of stability which included the whole of veraison (the last sampling time of this vineyard) for N, P, K, Mn, Zn and B. However, the stability period for Ca ranged from 25% to 100% of veraison, and for Mg and Fe it comprised 50% to 100% of veraison (Fig. 4).
On the other hand, in the RA vineyard only Fe and Mn had a stability period that lasted all veraison while the other nutrients showed stability periods which ranged, in general, between M50 (50% of veraison) and N50, or 50% of ripening (Fig. 4).

Leaf petioles
The stability periods for petioles at both vineyards are shown in Fig. 5. For petioles at flowering, the nutrient concentration and thus the stability periods behaved similarly to blades. Petiole results showed that there was not a period which included the complete flowering phenological stage for N, P, Ca, Fe and B in the RA vineyard (Fig. 5). However, the absence of sampling for H100 and I40 from this vineyard (Table 2) rules out an adequate evaluation of the flowering stability period length for the RA vineyard. On the other hand, Ca, Mg and Fe had stability periods that did not include the complete flowering stage in the RM vineyard (Fig. 5). Therefore, Ca, Mg and Fe would need one reference at the beginning of flowering and another at the end due to the rapid change in the concentration of these elements in petioles (Fig. 5).
With respect to veraison, the RA vineyard showed a stability period which included this phenological stage completely for all nutrients except Ca, which ranged from M15 to 50% of ripening (N50). Nevertheless, the RM vineyard only showed a stability period for the complete veraison phenological stage for N, P and Fe. Furthermore in the RM vineyard, Zn and B showed a stability period from M15 until the end of veraison (the last sampling time of this vineyard), K from M50 to the end of veraison, and finally Ca, Mg and Mn only showed a stability period within M15 to M75 phenological stages (Fig. 5). In these cases, a reference obtained at M50 for Ca, Mg and Mn would not be valid for a sampling at the beginning (M5) or at 12 Figure 5. Stability periods for leaf petiole nutrients concentration in RA (Haro) and RM (Logroño). Different letters among phenological stages express significant differences at p ≤ 0.05 (Tukey test). Shaded periods show common periods for a general diagnosis of all nutrients for 'Graciano'.  13 Leaf blade and petiole analyses for 'Graciano' grapevines  With respect to Cu, Schreiner (2005) observed a decreasing trend for Cu concentration in blade throughout the season for 'Pinot noir' cultivar while, in this study, blade increased Cu concentration in the early maturation stage. Therefore, the application of fungicides impeded the study of Cu behaviour (Benito et al., 2013;Romero et al., 2013).
Finally, the differences between nutrient concentration for leaf blades and petioles, as well as the seasonal changes throughout the growing season, confirm that it is necessary to design specific reference levels for each tissue and phenological stage.

Evaluation of two sampling periods for nutrient diagnosis purposes
The most adequate moment for nutritional diagnosis would correspond to the period in which nutrient concentrations remain highly stable for some time in the tissue analyzed allowing a reliable comparison with their references. Therefore, the best sampling times are those where the concentration trend shows a slope as near as possible to zero. Most of the trends, mainly for macronutrients, showed curves with slopes near to zero at veraison (Fig.  2), making that phenological stage an adequate moment to propose useful references. Furthermore, those nutrients with sigmoidal behaviour trends presented a shorter stability period at flowering (Figs. 2 & 3).
Flowering and veraison are the preferred phenological stages to evaluate the grapevine nutritional status (Robinson, 2005). Therefore, the available bibliography offers different reference values for nutritional diagnosis in grapevine tissues at these two phenological stages. However, the length of each stability period is not fully defined and it is difficult to delimit it considering only the slope, especially at flowering. Therefore, a statistical determination of the differences between sampling times becomes necessary to enclose the validity period for each nutrient reference at flowering and veraison.
Considering all constraints, the best time to perform a multi-element analysis of leaf blades and petioles during flowering for the 'Graciano' variety is at 100% flowering as it is an easy recognizable phenological stage, and references provided for this moment remain valid until fruit set. This recommendation agrees with results obtained for other varieties (Benito et al., 2013;Romero et al., 2013).
Regarding veraison, the recommendable period to carry out a general nutritional diagnosis of the ten nutrients studied for blades extends from 50% veraison to beginning of ripening, except for K. This situation is not similar to recommendations obtained for other by different authors (Colugnati et al., 1992;Parejo et al., 1992;Benito et al., 2013;Romero et al., 2013). In a PCA when one of the PC is highly related to the phenological stage, the original variables (nutrients) with sharp changes in concentration over the growing season are related to that PC.
Therefore, in our study, those elements showing slight concentration changes over the growing season present a weak relation with PC1 for RA and with PC2 for RM (Table 3). This result shows the importance of proposing specific reference levels for different phenological stages, mainly for those nutrients with strong evolutionary behaviour over the crop season.
In addition, our results showed that the vineyard origin and/or the year effect could modify the nutrient concentration. Other studies showed that the year effect was low on the total variance of datasets obtained for single vineyards throughout different years (Benito et al., 2013;Romero et al., 2013).

Trends in nutrient concentrations
Most of the trends, mainly for macronutrients, showed curves with slopes near to zero at veraison (Fig. 2). Furthermore, some nutrients with sigmoidal behaviour curves presented two periods with slopes near to zero, one at the flowering phenological stage, which was shorter than that found at veraison (Fig. 3).
Regarding micronutrients, the increases in Fe and Mn, agree with previous reports for other cultivars (Schreiner, 2005;Benito et al., 2013;Romero et al., 2013). The different trends observed by the Zn were probably due to either different climatic conditions and/ or the soil effect (Figs. 3F,G). The trend observed by the Zn in RA was observed by Christensen (1984). Finally, the B pattern was also reported by Failla et al. (1995). 16 varieties in longer monitoring studies, where the stability period covers all the veraison period or even allows an early diagnosis, up to a month before the start of this phenological stage (Benito et al., 2013;. Therefore, the results suggest the need for more years of study to avoid the year and/or the soil effect on the nutrient concentrations in blades. With respect to petioles, the optimum period for a common diagnosis at veraison ranged from two weeks after closed bunch until one week before the end of the veraison (75% of the berries coloured), except for K. Benito et al. (2013) and Romero et al. (2013), for 'red Grenache' and 'Tempranillo' varieties respectively, showed that the diagnosis in veraison with respect to a reference for 50% of coloured berries (M50), ranged from two weeks before the veraison to the beginning of ripening. This difference might be due to variety behaviour. However it is also necessary to extend the years of study to adjust these results for petiole. Finally, phytosanitary treatments did not permit a correct copper diagnosis throughout the growing season.

Coefficient of variation and analysis of reliability
To evaluate the general nutritional status of a vineyard, the vine grower usually carries out a single analysis that compares the results to the standards. Therefore, an adequate tissue for nutritional diagnosis should provide acceptable analysis reproducibility (Wolpert & Anderson, 2007). A high variability between replicates from a uniform vineyard implies that obtaining representative nutrient concentrations is difficult. Due to that, the CV is used for the evaluation of the analysis reproducibility in both tissues at each phenological stage throughout the growing season.
Considering the diagnosis of individual nutrients, results suggest that blade was a more appropriate tissue than petiole to compare N, P and K concentrations with respect to their references (Figs. 6A-F). Calcium and Mg did not show clear differences between blades and petioles throughout the growing season, and CV in both cases was lower than 20%, but results suggest that blade would be preferable at flowering and veraison phenological stages (Figs. 6G-J). Some of these results agreed with those found for 'Tempranillo'  as for N and K at both phenological stages and for P and Mg at veraison. Furthermore, results are also in agreement with those found for N, P and K for 'red Grenache' cultivar (Benito et al., 2013).
The greater CV values in petioles implied a lower reproducibility of the analysis that may lead to diagnosis errors when the vinegrower compares a routine single analysis with the macronutrient reference values (Benito et al., 2013). Furthermore, CV result also suggest that the analysis of blade and petiole separately would be more appropriate than whole leaf since one tissue will always show a lower CV.
With regard to the micronutrients, results found for both vineyards are contradictory and therefore it is not possible to establish a blade vs. petiole preference sampling policy (Fig. 7).
In summary, the higher variability within petiole nutrient concentrations for the cv. Graciano suggests that, for routine single analysis, leaf blades would be preferable to petioles for N, P, K, Ca and Mg diagnosis at both flowering and veraison. With regard to micronutrients, the differences found between both vineyards do not allow to define the best tissue to analyze micronutrients. In general, references for blade and petiole at full flowering could be valid for diagnosis of all nutrients even in late samplings at fruit set. On the other hand, references obtained at 50% veraison, would allow the simultaneous diagnosis for leaf blades of all the studied nutrients between the middle of veraison and ripening. For petioles, the simultaneous diagnosis period ranges from the beginning of veraison until one week before the end of this phenological stage.
The methodology employed is appropriate in establishing the most adequate tissue and moment for the diagnosis of ten different nutrients; however, it is necessary to extend this study for a longer period to include data from different pedoclimatic subzones, in order to obtain conclusive results for 'Graciano' and to evaluate the local effects on nutrient behaviour.