## 3 Results and Discussion
### Effect of Pre-Harvest YE Application on Carotenoid Content  
According to the choice of the main figures of merit reported elsewhere [28], the adopted HPLC-DAD method was validated for the target analysis of carotenoids in terms of linearity, LOD, LOQ, and precision (Supplementary Materials, Table S1). The calibration curves were obtained by the internal standard method to give determination coefficients (R\({}^{2}\)) which were higher than 0.99; moreover, the detection and quantification limits were found to be under 2 and 5 ug/kg, respectively. As regards precision tests, intra-assay and inter-assay coefficients of variation were calculated, with retention times ranging between 0.11% and 0.13%, and 0.37% and 0.50%, respectively, while corresponding measures for W\({}_{1/2}\) were between 2.9% and 15%, and 2.8% and 12%, respectively. These results confirmed the accuracy and reproducibility of data obtained by the analytical method for carotenoid analysis. Tables 2 and 3 listed the changes in caroten and xanthophyll concentrations (expressed in ug/kg of berries) during the grape ripening of the two varieties, Negro Amaro and Primitivo, in control and YE-treated samples. As expected, total carotenoids significantly decreased from veraison to maturity, even more consistently in Negro Amaro (F = 34.06, \(p<0.001\)), due to the higher accumulation of the compounds in the first sampling date, respective to Primitivo (F = 19.54, \(p<0.001\)) (Figure 4). Regarding the effect of the elicitor (YE), the treatment factor (T) as well as its interaction with ripening (TxR) resulted in a more pronounced effect in the variation of total carotenoid content in Primitivo (T: F = 29.11, \(p<0.001\); TxR: F = 7.06, \(p<0.001\)) than Negro Amaro (T: F = 5.55, \(p=0.0047\); TxR: F = 6.05, \(p=0.0021\)). This difference was mostly influenced by the anomalous behavior of the P0813 control sample; the lowest carotenoid value could be ascribed to an experimental drawback that occurred during the sampling and/or extraction phase, as suggested by the extraction yields in the four replicates, which were low (Supplementary Materials, Figure S1). Figure 3: UV-Vis spectra of (**a**) violaxanthin and (**b**) (9\({}^{\prime}\)Z)-neoxanthin (3 ug/mL) treated with tartaric acid (15 g/L). \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & Sampling Date & **09/13/19** & **09/26/19\({}^{\text{\textregistered}}\)** & **09/04/19** & **09/19/19** & **AC\({}^{\text{\textregistered}}\)** \\ \hline  & **C** & **YE** & **C** & **YE** & **C** & **YE** & **C** & **YE** & **C** & **YE** \\ \hline
**TSSP** & 175\(\pm\) 1.0\({}^{\text{\textregistered}}\) & 170 \(\pm\) 2.0 & 187 \(\pm\) 1.5 & 200 \(\pm\) 2.2 & 226 \(\pm\) 4.2 & 227 \(\pm\) 1.4 & 236 \(\pm\) 5 & 229 \(\pm\) 15 & \\ \hline
**pH** & 2.98 \(\pm\) 0.02 & 2.97 \(\pm\) 0.03 & 319 \(\pm\) 0.02 & 322 \(\pm\) 0.02 & 340 \(\pm\) 0.01 & 335 \(\pm\) 0.08 & 345 \(\pm\) 0.01 & 340 \(\pm\) 0.07 & \\ \hline
**TX** & 112 \(\pm\) 0.4 & 131 \(\pm\) 0.6 & 68 \(\pm\) 0.3 & 7.03 \(\pm\) 0.15 & 62 \(\pm\) 0.4 & 67 \(\pm\) 0.3 & 57 \(\pm\) 0.4 & 61 \(\pm\) 0.4 & \\ \hline
**TSSP/TA** & 157 \(\pm\) 0.5 & 125 \(\pm\) 0.4 & 27 \(\pm\) 1.3 & 28 \(\pm\) 0.7 & 363 \(\pm\) 1.9 & 34 \(\pm\) 3 & 41 \(\pm\) 2 & 39 \(\pm\) 3 & \\ \hline
**Compounds** & & & & & & & & & \\ \hline violasanthin like structured\({}^{\text{\textregistered}}\) & 5 \(\pm\) 20\({}^{\text{\textregistered}}\) & 21 \(\pm\) 6\(\pm\) 30 \(\pm\) 9\(\pm\) 9\(\pm\) 25 \(\pm\) 7\(\pm\) 30 \(\pm\) 7\(\pm\) 22 \(\pm\) 6\(\pm\) & 30 \(\pm\) 6\(\pm\) 6\(\pm\) 17 \(\pm\) 50 & \\ \hline violasanthin & 31 \(\pm\) 19d & 144 \(\pm\) 6\(\pm\) 60 & 160 \(\pm\) 30a & 129 \(\pm\) 9\(\pm\) 9b & 155 \(\pm\) 18 ab & 115 \(\pm\) 17 bc & 132 \(\pm\) 18 ab & 87 \(\pm\) 12c & \\ \hline
**(R’R’-a-neckome\({}^{\text{\textregistered}}\)** & tr & tr & tr & 32 \(\pm\) 0.5 & tr & tr & tr & tr \\ \hline
**(R’-20-a\(\beta\)-carotene and (all\(E\))-lutein were the main compounds in terms of concentration in all analyzed samples.