Ground Truthing and Physiological Validation of Vis-NIR Spectral Indices for Early Diagnosis of Nitrogen Deﬁciency in cv. Barbera ( Vitis vinifera L.) Grapevines

: Several narrow or broadband spectral indices can be calculated at varying spatial and spectral resolution, which can then be correlated with the physiological and nutritional status of the leaves. In a three-year trial carried out on fruiting, potted cv. Barbera grapevines subjected to full (N + ) or no (N0) nitrogen supply, seasonal evolution of di ﬀ erent leaf spectral indices were correlated with non-destructive chlorophyll readings (Minolta SPAD meter), leaf gas exchange, and vine performance. Throughout the entire trial, N starvation resulted in greater-than-proportional limitation of vine yield as compared to vegetative growth (55% compared to 26% less than values measured on N + ). Indices calculated within the red-edge spectral domain had highest sensitivity to relative change between N + and N0, also indicating that the promptest response was recorded at the median shoot zone level. Twelve broadband indices were linearly correlated with leaf blade N concentration at veraison, indicating that N values ≤ 1.8% of dry matter identify a limiting N status. Any of these indices collected at the leaf level can be reliably used as a non-destructive predictor of N availability, albeit due to signiﬁcant between-year variation in their absolute values at a given N level, readings should always include a well-supplied N treatment.


Introduction
Nitrogen is a key element affecting canopy growth, yield formation, and fruit composition in the grapevine [1]. Overall, seasonal N demand function by the vine has been thoroughly characterized leading to the conclusions that the highest demand is around flowering and fruit set and, depending on crop level, a second smaller peak might take place slightly after veraison [2,3], coinciding with resumption of berry enlargement and a rapid increase of soluble sugars. However, likely due to high mobility of this nutrient in the soil and through the plant [4], growers still encounter difficulties at matching demand with supply at specific phenological stages. Cases of N-deficiency or excess are anything but rare. N-deficiency is especially harmful when occurring around flowering [3] as it can severely curtail fruit set of the current season, while having also negative effects on next-year bud induction [5]; N excess can likely be even more detrimental as it can lead to: (i) Too high shoot

Plant Material and Experimental Layout
The three-year trial (2016)(2017)(2018) was carried out at the Department of Sustainable Crop Production (DIPROVES) of the Università Cattolica del Sacro Cuore (45 • 02 N, 9 • 43 E, 54 m asl) using a batch of 25 three-year old cv. Barbera vines grafted onto 110 Richter (V. Berlandieri × V. Rupestris) grown outside in 14 L pots filled with a loamy soil with available water of 110 mm/m. The main soil characteristics were as follows: 12.1% and 24.4% and 1.46 g/cm 3 corresponding to the wilting point, the field capacity and the bulk density, respectively, according to Saxton and Rawls [33]. Chemical and physical properties of the soil used to fill the pots are reported in Table S1. Weather trends of the three growing seasons included in the trial are given in Figure S1. Barbera is one of the most cultivated varieties in North-Western Italy and it is often grafted to 110 R. Previous studies had demonstrated that V. Berlandieri × V. Rupestris rootstocks can be sensitive to N-deficiency [34].
Plants were head-trained and two 2-node spurs per vine were kept at winter pruning. After budburst (BBCH 15 according to Lorenz et al. [35]), vines were thinned to four main shoots while crop level was standardized to four clusters per vine by removing all distal inflorescences (BBCH 57). From the initial group, eight vines were assigned to a N-fertilized (N+) treatment whereas an equal number of vines was kept as an unfertilized N control (N0). Differential fertilization was performed in N+ plants by using ammonium nitrate (34%) at the dose of 0.5 g per application. Phosphorous and potassium were provided to both N+ and N0 vines by using a 0:52:34 (N:P:K) fertilizer at the rate of 0.5 g per application. Fertilization was carried out manually by dissolving fertilizers in 1 L of water per vine and by watering the top of the pot with the solution. Applications were performed three times per week between inflorescence swelling (BBCH 55) and beginning of ripening (BBCH 81). Such a schedule resulted in 30, 39, and 34 fertilization events in 2016, 2017, and 2018, respectively yielding a cumulated total of 51.5 g N/vine on the three-year span. Pots were painted white to minimize possible overheating of the root system. The number and intensity of daily irrigation events was calibrated to assure pot water capacity not to decrease below 90%; the total amount of daily irrigation was 3.50 L/pot that represents the 100% of the soil field capacity. Pots were provided by under-pots and plant protection was primarily focused against downy and powdery mildews and followed sustainable local practices. Each year the canopies were trimmed when shoots outgrew the top foliage wire to retain 13-14 main leaves.

Vine Measurements
On each vine (eight per treatment), two shoots (S1 and S2) were selected each spring season and three leaves were tagged on S1 to represent basal (4-5th node, cluster zone), median (8-9th node), and apical (11-12th node) shoot zones. On each leaf, the Greenness Index was determined by using a SPAD-502 portable meter (Konica Minolta, Osaka, Japan). For each leaf, five determinations were taken and averaged considering main and lateral lobes on a typically 5-lobe shaped Barbera leaf.
At the same dates and on the same leaves, the Greenness index (SPAD) was measured and assimilation (A, µmol m −2 s −1 ) and stomatal conductance (g s ) were assessed with the portable gas-exchange analyzer LCi-SD (ADC BioScientific Ltd., Hoddesdon, UK). All readings were taken in the morning hours (10:00-13:00) under clear sky and saturating light conditions. Previous studies have demonstrated that N deficiency exhibits different patterns depending on leaf age [14], hence the evaluation of physiological data against leaf position. Measurements were taken at ambient relative humidity, and the flow fed to the broad-leaf chamber (4.5 cm 2 window size) was 300 mL min −1 . For each trial year, monitoring of the above-mentioned parameters started prior to fertilization until pre-veraison and measuring dates were 7, 9, and 5 in 2016, 2017, and 2018, respectively. Agronomy 2019, 9, 864 4 of 20 Each year, at pre-veraison, leaves from nodes 4, 7, and 10 along the stem were sampled from all vine shoots except for S1. Thus, for each treatment × node position combination, 12 leaves were harvested. Thereafter, petioles were removed and leaf blades were washed twice in distilled water, dried in a forced air oven at 75 • C, finely ground, and sent to an external laboratory for mineral ion analysis.
In 2016, 2017, and 2018, physiological measurements were associated to Vis-NIR spectra collected on the same leaves with a spectroradiometer having a wavelength range of 325-1075 nm, an accuracy of ±1 nm and a resolution of <3 nm at 700 nm (ASD FieldSpec ® HandHeld 2 VNIR Spectroradiometer; Analytical Spectral Devices, Inc., Boulder, CO, USA). The instrument is equipped with a fibre-optic contact probe ending with a leaf clip allowing one-handed functionality for non-destructive contact readings on a portion of leaf area of 0.8 cm 2 . Single-wavelength reflectance rates were measured for each trial year; measurements were periodically conducted from the beginning of fertilization to veraison. Thereby, spectral data were elaborated using the Prospect package, version 0.1.3 [36]. On every spectral acquisition, the smoothing was applied, and bands were analysed by "resample2" function that uses gaussian models to resample the hyperspectral data. The FWHM values used to assess the new band positions and resolution were determined in order to reproduce the spectral configuration of Sentinel-2 (0.443, 0.490, 0.560, 0.665, 0.705, 0.740, 0.783, 0.842, 0.865, and 0.945 µm) [37]; broadband indices were then calculated. In addition to standard NDVI, twenty-nine relevant broadband vegetation indices were developed by modifying the base formula of the respective original index with different bands configurations. Acronyms, base formulas, references full name, and spectral bands used for each index are reported in Table S2 [38][39][40][41][42][43][44]. Twelve indices having higher significance early in the 2016 season and recorded from the basal tagged leaves, where then correlated with N content in dry leaf mass measured at veraison.

Grape Composition
At harvest occurring on DOY 256, 242, and 234 in 2016, 2017, and 2018, respectively, clusters from tagged shoots were individually picked, immediately weighed, and taken to the laboratory. The remaining clusters were harvested together, and their total number and mass recorded as well. Clusters from S1 and S2 were then destemmed, berries were counted and classified as a function of their size (i.e., normal and shot berries). Accordingly, cluster compactness was evaluated as cluster mass-to-rachis length. A sub-sample of 10 intact berries per cluster was taken and stored at −18 • C for subsequent determination of the berry mass and individual berry organs (skin, flesh, and seeds). Skin, flesh, and seed mass were then determined through the use of a razor blade and a small metal spatula. Seeds and flesh were carefully removed from each berry without rupturing any pigmented hypodermal cells, then the seeds carefully separated from the flesh with tweezers and the number of seeds per berry was counted. Both skins and seeds were rinsed in deionized water, blotted dry, and weighed. A second sub-sample of 50 healthy berries was stored at −18 • C and then used for the determination of anthocyanins and total phenolic concentration according to Iland et al. [45]. Remaining berries were crushed and the juice immediately processed to determine TSS using a temperature compensated refractometer whilst titratable acidity (TA) was measured by titration with 0.1 N NaOH to a pH 8.2 endpoint and expressed as g/L of tartrate equivalents. Must pH was measured simultaneously using a pH-meter CRISON GLP 22 (Crison, Barcelona, Spain). The concentration of tartaric and malic acids was determined by HPLC. The quantification of organic acids was performed by injecting 1:4 diluted must into HPLC after filtering through a 0.22 µm polypropylene filter. The identification was performed by external calibration with standards and concentration was calculated measuring the peak area and expressed in g/L. For this analysis an Allure Organic Acid column, 300 × 4.6 mm, 5 µm (Restek, Bellefonte, PA, USA) was used. Separation was performed in isocratic conditions using water, pH was adjusted at 2.5 by adding orthophosporic acid. The column temperature was maintained at 30 ± 0.1 • C, 15 µL of sample was injected. The elution was monitored at 200-700 nm and detected by UV-Vis absorption with DAD at 210 nm. At the end of the season, immediately after leaf fall, the Agronomy 2019, 9, 864 5 of 20 pruning weight of the one-year wood was recorded by separating main and lateral canes, and the yield-to-pruning weight ratio calculated.

Statistical Analysis
Plant growth, yield components, nutritional status, and fruit composition data were subjected to a two-way analysis of variance (ANOVA) assuming N supply as main factor (N+ versus N0) and year as a random factor. Treatments were compared by Student-Newman-Keuls test at p = 0.05. Treatment × year interaction was partitioned only in case of F test significance, and mean values compared by standard error. Spectral data, narrow and broad-band vegetation indices depending on N supply were compared by t-test at p = 0.05.

Leaf Nutrition, Vegetative Growth, Yield Components, and Grape Composition
Three-year data pooled over shoot positions and years showed that N+ vines had higher leaf blade N concentration at veraison (2.21% compared to 1.60% recorded in N0), whereas P, Mn, and Zn concentrations showed an opposite trend (Table 1).  Figure S2.
The main factor represented by leaf position along the shoot stem also played a role as N, P, Ca, Mg, and S concentration increased from the base to the shoot apex along a decreasing gradient of age, while Mn and Zn retained the highest concentration in their basal leaves. A few first order interactions occurred, indicating that ∆N between N+ and N0 increased proceeding from the base to the shoot apex ( Figure S2) whereas, within Y × T interactions, N0 had consistently high leaf P concentration that  Figure S2C). Multi-season N supply versus no N had a strong impact on vegetative growth (Table 2).  Figure S3.
Total pruning weight per vine, once averaged over the three trial years was reduced by 26% in N0 vines, with the greatest limitation observed for lateral growth (60% less than N+). The impact of no N supply was even greater on yield per vine, which decreased by 55% as compared to N+ along with all main yield components and the fraction of shot berries; lower fruit set and smaller berries allowed N0 vines to show a reduced cluster compactness; interestingly the Ravaz index (yield-to-pruning weight ratio) was significantly lowered by no N application.
The effect of N supply on vegetative growth and yield is also marked by a number of significant Y × T interactions that were partitioned in Figure S3. Vine vigor-assessed as total pruning weight and its components-showed a strong response in 2016 and 2018 and a milder one in 2017 ( Figure S3A-C); conversely, relative reduction of yield per vine and cluster weight of N0 vines was highest in 2017 ( Figure S3D-E). As a related result, yield-to-pruning weight ratio significantly increased in 2017 in N0 vines ( Figure S3F).
Growth of individual berry organs (skin, flesh, and seeds) was markedly affected by N supply (Table 3).
No N supply reduced flesh weight/berry, seed number and development, and increased both skin-to-berry and skin-to-flesh ratios. Interestingly, total skin weight per berry was not affected. A few parameters also showed significant Y × T interactions ( Figure S4) which, however, had a different nature. Conversely, in 2016 N0 had a higher skin/berry ratio, while the reverse was true for the two following seasons, with N+ taking over in 2017 ( Figure S4A). For flesh weight/berry, each season N0 had lower values that reached a maximum gap in 2017 ( Figure S4B).
Must composition at harvest was dramatically changed by N supply as N0 vines showed, on average, higher TSS (+2.1 • Brix as compared to N+), lower TA driven by a drastic decrease in malic acid and notably higher total anthocyanins and phenols concentrations (Table 4). Once again, significant Y × T interactions were found for several parameters ( Figure S5). Regardless of season, N0 strongly reduced malic acid concentration at harvest; by contrast TSS and total anthocyanins showed no response to N supply in 2016, while over the following years N0 reached significantly higher values for both parameters; tartaric acid at harvest was lower in 2016 in N0 vines while an opposite response was found in 2017 and 2018. Within column, in case of significant F test, mean separation was performed by Student-Newman-Keulstest. * = p < 0.05, ** p < 0.01, ns = not significant. When significant, partitioning of the T × Y (Treatment × Year) interaction is shown in Figure S4. Within column, in case of significant F test, mean separation was performed by Student-Newman-Keulstest. * = p < 0.05, ** p < 0.01, ns = not significant. When significant, partitioning of the T × Y (Treatment × Year) interaction is shown in Figure S5.

Leaf Physiology
Seasonal variation of the Greenness index taken with the SPAD 502 Minolta meter showed significant modifications between different shoot zones within the same year ( Figure 1). In 2016, regardless of leaf position along the stem, values measured in N0 were definitely similar to those measured in N+ for the first several sampling dates. Median and apical leaves of N0 vines were the quickest to show lower SPAD values versus N+ (i.e., 13-15 d after initial N supply), whereas basal leaves took longer to differentiate between treatments (28 d) ( Figure 1A-C). Such a pattern was significantly modified in 2017, when even the pre-fertilization measurement already showed lower SPADs in median and apical leaves of N0 ( Figure 1E-F). Then, over the remainder of the season, such differences broadened further with the largest gap reached during late season readings taken on Agronomy 2019, 9, 864 8 of 20 apical leaves. 2018 was overall confirmatory of previous years' assessment with an important change highlighting that, even in basal leaves, pre-fertilization SPAD rates were lower in N0 as compared to N+ vines ( Figure 1G). Agronomy 2019, 9, x FOR PEER REVIEW 8 of 21 apical leaves. 2018 was overall confirmatory of previous years' assessment with an important change highlighting that, even in basal leaves, pre-fertilization SPAD rates were lower in N0 as compared to N+ vines ( Figure 1G). Seasonal variation of leaf assimilation (A, µ mol m −2 s −1 ) taken each season at different dates on the same leaves previously used for SPAD readings provided a quite different scenario ( Figure 2). In 2016, on basal leaves only, mid-season A rates recorded on N0 vines were lower than rates taken on N+ vines; conversely, very similar A was measured during the season in the two treatments for either median and apical leaves (Figure 2A-C). In 2017, occasional and quite inconsistent differences were found for A measured in N0 and N+ at different positions along the stem and even fewer differences were found in 2018, albeit under a lower number of measuring dates ( Figure 2D-I). Seasonal variation of leaf assimilation (A, µmol m −2 s −1 ) taken each season at different dates on the same leaves previously used for SPAD readings provided a quite different scenario ( Figure 2). In 2016, on basal leaves only, mid-season A rates recorded on N0 vines were lower than rates taken on N+ vines; conversely, very similar A was measured during the season in the two treatments for either median and apical leaves (Figure 2A-C). In 2017, occasional and quite inconsistent differences were found for A measured in N0 and N+ at different positions along the stem and even fewer differences were found in 2018, albeit under a lower number of measuring dates ( Figure 2D-I).
A quite different behaviour was found for seasonal trends for leaf stomatal conductance (g s ) that, regardless of leaf position along the stem, showed on N0 values either similar or even significantly higher than those recorded on N+ (Figure 3).
Quite expectedly, no significant correlation was found between SPAD unit and A when data were pooled over years and position or, likewise, analysed separately within basal, median, and apical shoot portions (not shown). A similar poor correlation (R 2 ranging from 0.02 and 0.16) was found when, within each N supply level, leaf blade N concentration determined upon sampling performed at veraison was regressed over the closest measurement of leaf assimilation rates. A quite different behaviour was found for seasonal trends for leaf stomatal conductance (gs) that, regardless of leaf position along the stem, showed on N0 values either similar or even significantly higher than those recorded on N+ ( Figure 3).  A quite different behaviour was found for seasonal trends for leaf stomatal conductance (gs) that, regardless of leaf position along the stem, showed on N0 values either similar or even significantly higher than those recorded on N+ (Figure 3).

Spectral Indices and Relationships with Leaf Function
Leaf light reflectance within the Vis-NIR spectrum measured at various dates in 2016, 2017, and 2018 indicates large changes between seasons and, within each season, among different shoot zones (Figure 4). Differences in leaf reflectance rates between N0 and N+ evaluated as trespassing critical t-values at P 0.05 suggest that the green and red-edge domains were the most sensitive, whereas the red wavelength was overall less responsive. Within each shoot zone, leaf reflectance increased, in terms of earliness and intensity of response, in 2017 and 2018 as compared to 2016. Significant leaf reflectance differences between N0 and N+ started to become detectable on DOY 180, 159, and 172, for basal, median, and apical shoot zones in 2016 ( Figure 4A-C), respectively, whereas in 2017, in the same order, the same response was prompted on DOY 144, 129, and 144 (i.e., 36, 30, and 28 days earlier) ( Figure 4D-F). In 2018, in basal and apical leaves, significant leaf reflectance differences between the two N supplies were found on DOY 152, whereas in median leaves such a response was already reached on DOY 130 ( Figure 4G-I). Notably, regardless of season, median leaves were the promptest to show significant alteration of leaf reflectance properties due to N supply and, in 2017 and 2018, N0 and N+ were already different at the first spectral acquisition on DOYs 129 and 130 ( Figure 4E,H).
Quite expectedly, no significant correlation was found between SPAD unit and A when data were pooled over years and position or, likewise, analysed separately within basal, median, and apical shoot portions (not shown). A similar poor correlation (R 2 ranging from 0.02 and 0.16) was found when, within each N supply level, leaf blade N concentration determined upon sampling performed at veraison was regressed over the closest measurement of leaf assimilation rates.

Spectral Indices and Relationships with Leaf Function
Leaf light reflectance within the Vis-NIR spectrum measured at various dates in 2016, 2017, and 2018 indicates large changes between seasons and, within each season, among different shoot zones (Figure 4). Differences in leaf reflectance rates between N0 and N+ evaluated as trespassing critical tvalues at P0.05 suggest that the green and red-edge domains were the most sensitive, whereas the red wavelength was overall less responsive. Within each shoot zone, leaf reflectance increased, in terms of earliness and intensity of response, in 2017 and 2018 as compared to 2016. Significant leaf reflectance differences between N0 and N+ started to become detectable on DOY 180, 159, and 172, for basal, median, and apical shoot zones in 2016 ( Figure 4A-C), respectively, whereas in 2017, in the same order, the same response was prompted on DOY 144, 129, and 144 (i.e., 36, 30, and 28 days earlier) ( Figure 4D-F). In 2018, in basal and apical leaves, significant leaf reflectance differences between the two N supplies were found on DOY 152, whereas in median leaves such a response was already reached on DOY 130 (Figure 4 G-I). Notably, regardless of season, median leaves were the promptest to show significant alteration of leaf reflectance properties due to N supply and, in 2017 and 2018, N0 and N+ were already different at the first spectral acquisition on DOYs 129 and 130 ( Figure 4E,H).  Focusing on green and red-edge domains, seasonal variation of reflectance values averaged over the whole leaf sampling (n = 24, eight leaves per shoot zone) showed that, in 2016, at the second sampling date (DOY 159, i.e., 16 days after the beginning of the N supply), N0 vines already had higher reflectance than N+ and such gap broadened along the season for both waveband lengths ( Figure 4A-C). Robustness of such a response increased in 2017, when at the very first sampling date (DOY 129, i.e., 11 days after the beginning of the N supply) and, for all the following dates, N0 had higher leaf reflectance rates than N+ in both wavebands ( Figure 4D-F). In 2018, reflectance in the two wavebands started to differentiate between N0 and N+ beginning at the third date of sampling (DOY 152, i.e., 25 days after the beginning of the N supply) and then widened along the season ( Figure 4G-I). Figure 5 Shows t-values resulting from pair comparisons (N+ versus N0) in 2016 for all indices. Data are presented separately for basal, median, and apical shoot zones and, for each shoot section, top panels report data taken the same day the N supply started, whereas panels underneath shows data taken 14, 27, and 35 days later. It is quite apparent from the seasonal dynamic of spectral signatures that median shoot zone was prompter than basal and apical, reaching significant t-values already at DOY 159, i.e., two weeks after the beginning of N supply ( Figure 5E). higher leaf reflectance rates than N+ in both wavebands ( Figure 4D-F). In 2018, reflectance in the two wavebands started to differentiate between N0 and N+ beginning at the third date of sampling (DOY 152, i.e., 25 days after the beginning of the N supply) and then widened along the season ( Figure 4G-I). Figure 5. Shows t-values resulting from pair comparisons (N+ versus N0) in 2016 for all indices. Data are presented separately for basal, median, and apical shoot zones and, for each shoot section, top panels report data taken the same day the N supply started, whereas panels underneath shows data taken 14, 27, and 35 days later. It is quite apparent from the seasonal dynamic of spectral signatures that median shoot zone was prompter than basal and apical, reaching significant t-values already at DOY 159, i.e., two weeks after the beginning of N supply ( Figure 5E).  (Figures 6 and 7) show that, in 2017, the median leaves were especially reactive, as several indices already showed significant t-values for the N0 versus N+ comparison at the very first date of sampling (DOY 129), i.e., 11 days after the beginning of the N supply, ( Figure 6B) and such higher reactivity was maintained for the remainder of the season ( Figure  6D,F,I). In 2018 ( Figure 7) the pattern described in 2017 was essentially maintained.  (Figures 6 and 7) show that, in 2017, the median leaves were especially reactive, as several indices already showed significant t-values for the N0 versus N+ comparison at the very first date of sampling (DOY 129), i.e., 11 days after the beginning of the N supply, ( Figure 6B) and such higher reactivity was maintained for the remainder of the season ( Figure 6D,F,I). In 2018 ( Figure 7) the pattern described in 2017 was essentially maintained.
Criteria used to select, among the population of 29 broadband vegetation indices, those to be regressed against leaf N concentration determined at veraison is based on data shown in Figure 5, giving priority to those reaching, in 2016, significant t-values as related to the sampling date and position along the stem. Such criteria were met by readings taken in the median shoot zone on DOY 159 and indices having, at that date, a t-value ≥ than significant thresholds at P 0.05 are highlighted in bold character in Table 1. For the selected 12 broadband vegetation indices, within-year correlation between index reflectance and leaf N concentration was evaluated for data pooled over N+ and N0 (

Agronomic Vine Performance to N Supply
In this trial, the vine responses to N supply marked large differences between a so called "agronomical" behaviour (i.e., vegetative growth, yield and grape composition) and physiological leaf function (namely SPAD values and gas-exchange rates). In regard to the first group of variables, the vines indeed showed a fast response to the N0 and N+ treatments, beginning from the first trial year when leaf N concentration at veraison was already lower than N+ ( Figure S2B); moreover, as expected, leaf N increased linearly, according to a decreasing gradient of age from the base to apical part of the shoot as already shown in Poni et al., [14], who also suggested that such a behaviour reflects the seasonal increase, with age, of dry weight per unit leaf area. It is quite notable that, in N0, a leaf N concentration at veraison of 1.60% (DM basis) for data pooled over seasons and positions (Table 1) was low enough to drastically curtail yield per vine (55% less than N+) and such limitation was much stronger than that observed for total pruning weight per vine (−26% than N), leading to the ostensible paradox that a N deficiency actually leads to decreased Ravaz index ( Table 2). Such a severe yield reduction was contributed to by both fewer and smaller berries (−41% and −21%, respectively when compared to N+), while the fraction of shot berries increased by 270% in N0. Moreover, the alternate pattern of proportional berry organ growth suggests that vine vigour can affect the berry development. When pruning weight was severely impaired in N0 vines in 2016, it might have led to a much more open canopy with more exposed clusters, which, as a well-known defense mechanism, were prone to increased skin thickness [46]. The same mechanism did not occur in 2017 and 2018 because, with increasing N deficiency, this "microclimate" effect was fully offset by N starvation that is also quite critical for cell division hence number and thickness of skin cell layers. Although in this trial we did not assess leaf N earlier in the season, the above scenario suggests an inadequate N supply around flowering and fruit that might have exacerbated flower drop and impaired the fertilization process (hence the rise in shot berries and the drop in seed number and size), according to May et al. and Guilpart et al. [3,5]. This hypothesis is also inherently supported by the fact that a strong yield reduction was already recorded on year one of our trial when negative carryover effects on bud induction and differentiation from the previous season should be ruled out. Sensitivity of the grapevine reproductive behaviour to N supply was also reported in Keller et al. [47]. In their trial on cv. Müller-Thurgau comparing two rates of N supply (0 and 100 kg/ha), they found that no supply significantly decreased fruit-set, berries per cluster and berry weight, whereas inflorescence necrosis was increased. A three-year trial conducted in Pinot Noir grown in pots and subjected to different levels of N supply (i.e., 100% as control and reduced levels of 50%, 20%, and 10% of control) showed that vine vigour given as total dormant cane weight was reduced by 31% with 10% N replenishment as compared to full supply, whereas yield per vine was curtailed by 66%, thus confirming that reproductive behaviour might be more significantly affected than vegetative growth under N deficiency [48]. These authors concluded that N leaf blade values of 1.8% at veraison are required to maintain yield and this fits very nicely with the mean blade N concentration we found at veraison (1.6% for data pooled over seasons and leaf positions in N0 vines).

Physiological Vine Performance to N Supply
The study tracked the progressive inception of symptomless and then more visible N deficiency in N0 plots. Both SPAD and assimilation trends showed no difference between treatments in the initial sampling dates, hence initial leaf function on N0 vines was definitely not limited. Although it is well known that under N-deficiency, leaves decrease their chlorophyll content to avoid oxidative stress induced by a more pronounced drop in rubisco activity as compared to the decrease in the electron transport capacity [1], in our study no correlation was found between SPAD values and leaf A rates. Therefore, when using the greenness index measured by the Minolta SPAD 502, two different considerations are at hand: (i) SPAD is, in general, useful to detect relative differences between treatments and its sensitivity is generally quite good at discriminating either year-to-year variations or at identifying when, during the season, treatments subjected to different N supply start to show differences; (ii) such differences do not necessarily correspond to actual variations in leaf chlorophyll content and/or leaf assimilation rates. Although in our study we did not carry out destructive chlorophyll readings, it is apparent from data reported in Figure 1 that over 95% of the SPAD values were above the 25 SPAD unit threshold. Such a threshold is exactly matching the one above which Steele et al. [21] found quite poor correlation with chlorophyll values derived from wet chemical extractions.
A distinctive feature of our work was that plants subjected to N-deficiency showed a moderate decrease in assimilation rates that were slightly lower than well N supplied vines at few dates in both 2016 and 2017, whereas in 2018 treatments showed similar leaf assimilation (Figure 2). Such a response seems to contradict the strong limitation that no N supply exerted onto vine vigour and, especially, on yield performance. Very little information is available for the grapevine about long-term adaptation mechanisms to a low N supply. In their three-year study on Pinot noir, Schreiner et al. [48] found no difference in A rates among different N supply levels (100% down to 10%) over the first two years of the trial and only during the third year, 10% and 20% replenishment resulted in lower A than the 100% level. Their conclusion was that the lowest N concentration in leaves they observed (about 1.4%) was above the limiting level needed for high rates of carbon fixation in Pinot noir vines, suggesting caution in basing leaf tissue N guidelines on photosynthetic capacity of leaves. Thus, there is a good fit with our data since the lowest leaf N concentration measured in leaf blades at veraison was 1.48% (N0 in 2017, Figure S2B). A more complicated matter of discussion is related to: (i) Causes of lower A rates under N-deficiency, i.e., stomatal versus non stomatal and (ii) type of compensation mechanisms aiming at maximizing N utilization for photosynthesis. Both Keller et al. [1] and Schreiner et al. [48] agree that non-stomatal limitations are the primary cause for lower A under N-deficiency primarily in light of a rising sub-stomatal CO 2 concentration (c i ) and a parallel variation in A and mesophyll conductance. This explanation can also be extended to our work if we also consider that, in N0, g s was never reduced (Figure 3), supporting the hypothesis that the low N vines kept the stomata proportionally more open in an effort to maximise A. In regard to item ii), to the best of our knowledge, no specific information seems to be available for the grapevine. However, a case in point that is well adapted to our case is the one reported for maize plants subjected, for two years, to nil or 180 kg N ha −1 . Interestingly, leaf A rates were not at all affected, despite specific N content (g m −2 ) being reduced by 38% and, as a related consequence, photosynthetic N-use efficiency increased by 54% [49]. Their interpretation for such combined effects was that, under low-N stress, maize plants tended to invest relatively more N into bioenergetics to sustain electron transport and, conversely, N allocated to chlorophyll and light harvesting proteins was reduced to control excess electron production. Low N supply in grapevine could, indeed, cause a similar within-leaf N allocation mechanism. As a matter of fact, in the three-year trial, the different fertilization caused N concentration (g N m −2 ) change equal to 44%, interpolated using leaf dry matter m −2 . At the same time, the photosynthetic N-use efficiency, calculated using assimilation averaged data of three years, increased by 58%. However, data shown in Figure 2 for seasonal A rates' variation in 2016 according to leaf position (hence age) along the stem also suggest a between-leaf N allocation mechanism. Poni et al. [15] pointed out that, when leaf N content (mg/cm 2 ) is plotted versus leaf age-given as days after unfolding, the peak reached at around 50 days of age and then, with increasing age a diminishing pattern is quite apparent. Data reported in panels A, D, and G of Figure 2 highlight that, for older basal leaves, no N supply anticipated N export towards median and apical leaves to sustain or improve their assimilation capacity under a low N regime. As a matter of fact, at no dates in 2016 did leaf A differ between N0 and N+ for apical leaves (Figure 2C), i.e., those able to provide highest assimilation late in the season, i.e., after veraison. Interestingly, the described mechanism did not occur in 2017 and 2018, suggesting that an abrupt change in N supply is likely required to foster such a response before an equilibrium is reached.

Sensitivity of Canopy Reflectance Indices versus N Leaf Status and Vine Behaviour
Our large data set on seasonal variation of canopy reflectance indices calculated on Barbera potted vines subjected to regular or no N supply during the season confirm that combined indices processed within the green or the red-edge domains are the most reliable for early detection of inadequate N status in grapevines [26]. While Rustioni et al. [27] have recently successfully shown that reflectance spectra collected along veins, in interveinal areas and at leaf margins are able to discriminate among early iron, magnesium, nitrogen, and potassium deficiency without necessarily requiring the availability of a well-supplied control, our three-year work adds more about sensitivity of reflectance indices versus the dynamic of early N-deficiency in grapevine leaves. Contrariwise to what Rustioni et al., [27] suggested for initial N-deficiency symptoms, our 2016 campaign indicates that for 12 spectral indices, the median part of the shoot was the first to be affected in terms of significant spectra modifications against no N supply ( Figure 5E) and such a behaviour was confirmed with greater magnitude over the following seasons ( Figure 5H,M). Thus, interpreting N mobility within the shoot as a progressive and faster translocation from the older (basal) leaves towards the youngest apical leaves-especially when N supply is becoming increasingly deficient-seems to be a risky oversimplification. In fact, within a so called "basal", "median", and "apical" shoot zone we might have, depending upon sampling date and shoot development stage, a population of leaves greatly differing in age and, as a consequence, in terms of source-sink balance. In our study, all spectra readings, except for a quite late date (DOY 200) in 2018 corresponding to post-veraison, were taken between pre-flowering and lag-phase of berry growth. Throughout those dates, the sampled median leaves (8-9th along the shoot stem) were the most reactive in terms of reflectance in the red-edge to N supply ( Figure 4B,E,H). Thus, our hypothesis to explain why those leaves were the promptest to show early and significant changes in reflectance links to older data reported by Poni et al. [14] who showed, in field grown Sangiovese grapes, that, from flowering until veraison, N content (area basis) is highest in median leaves. It is possible that, within a scenario of progressive N starvation, those leaves having the highest N pool would actually favor translocation to either basal leaves, to prevent or slow down senescence and to the apical leaves to meet their higher sink demand. Indirect confirmation of such a hypothesis is also provided by t-values calculated for N0 versus N+ comparison of spectral signature performed on DOY 200 in 2018, i.e., post-veraison ( Figure 4I). On that specific occasion and for a quite late recording date, the apical shoot zone displayed highest spectral sensitivity to N supply; this is again fitting with the hypothesis built on the work of Poni et al. showing that, after veraison, the now mature apical leaves have the highest N content when given on a per leaf area basis.
A second interesting matter of discussion is if such detailed information provided by characterization of canopy reflectance properties can be practically used to guide N fertilization in the vineyard. Working for two years on two cultivars under either well-watered and water stressed conditions and employing either chlorophyll meters (SPAD 502 and CCM-200) and two active canopy sensors (Crop Circle ACS 210 and 430) Taskos et al. [28] concluded that such a task is still difficult to achieve due to both genotype-related variability and looser relationships with yield and grape composition later in the season. However, their leaf N concentrations for data pooled over the years and water regimes highlight a 1.78% and a 1.82% at veraison for cvs. Cabernet S. and Xinomavro, respectively, that, according to previous data and data presented in our study, do not seem to represent a severely limited status. Cases of correlations reported in Figure 8 suggest that in 2018 N deficiency was at the peak, leading to significant leaf discoloration or inception of necrosis and this phenomenon might have easily affected precision of the FieldSpec readings. However, these regressions provide two different and valuable guidelines in terms of management of N nutrition in cv. Barbera: (i) Regardless of season, all index values calculated for the N0 treatment correspond to a leaf N concentration at veraison ≤ 1.8%. Thus, such a threshold must be considered the upper boundary below which, in Barbera, an increasing limitation in yield and growth is expected; (ii) it is likewise true that, especially when referring to readings taken in 2017 and 2018 the same leaf N concentration might correspond to a quite different value of a specific index; yet such interference can be easily overcome by acquiring, in a given year, reflectance readings also on control leaves provided with adequate N supply.

Conclusions
In a three-year trail where fruiting cv. Barbera potted vines were subjected to standard (N+) or no (N0) nitrogen fertilization, it was apparent the N deficiency decreased yield more than proportionally when compared with its effects on vegetative growth by exerting severe limitations on berry set and size. Conversely, reduction of leaf assimilation in N0 vines was definitely milder or non-existent, suggesting that compensation mechanisms for photosynthesis under low N supply are triggered. Reflectance spectral characterization of the leaf nutritional status showed increased reflectance in green and red-edge bands under N-deficiency. Furthermore, some indices (CIre, RENDVI, OSAVI, GCI, MTCI, and REP), when opportunely adapted to use the bands within the red-edge domain, showed higher efficiency in detecting significant changes in leaf N concentration from the early stages of canopy growth, with leaves located in the median part of the shoots showing the highest level of promptness. Standard NDVI did not show sensitivity to leaf chlorophyll and N variations. Even when the formula was modified using red-edge bands, it showed a delayed response. Practical guidelines emerging from this work are that a leaf blade N concentration at veraison ≤ 1.8% identifies a threshold below which severe and increasing yield limitation is expected and that a number of broadband spectral indices, mostly falling in the red-edge domain, can be used to accurately and non-destructively estimate leaf N. Though, due to year-to-year variation in the values of each index, pair readings with control leaves supplied with adequate N is recommended. Further studies are needed to test the indices sensitivity in field conditions to investigate if the selected indices will show reliable responses in different environments and grapevine varieties.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/9/12/864/s1, Table S1. Chemical-physical properties of the soil used to fill the pots in trial. Table S2. Vegetation indices used in the study. Figure Figure S2. Partitioning of the interaction between leaf position (basal, median, apical) and fertilization treatment (N0, N+) for leaf N concentration (% DM) at veraison (panel A). Each treatment combination mean is calculated over the years and sub-replicates (n = 36). Vertical bars of each column represent standard error (SE). Partitioning of the interaction between years (2016, 2017, 2018) and fertilization treatment (N0, N+) for leaf N and P concentration (% DM) at veraison (panels B, C). Each treatment combination mean is calculated over leaf position and sub-replicates (n = 36). Vertical bars of each column represent standard error (SE). Figure S3. Partitioning of the interaction between years (2016, 2017, 2018) and fertilization treatment (N0, N+) for main, lateral, and total pruning weight per vine (panels A, B, C), yield per vine (panel D), cluster weight (panel E), and Ravaz index (panel F). Each treatment combination mean is calculated over sub-replicates (n = 8). Vertical bars of each column represent standard error (SE). Figure S4. Partitioning of the interaction between years (2016, 2017, 2018) and fertilization treatment (N0, N+) for skin weight, flesh weight and total seed weight per berry (panels A, B, C) and seed number per berry (panel D). Each treatment combination mean is calculated over sub-replicates (n = 8). Vertical bars of each column represent standard error (SE). Figure S5