**3. Results**

#### *3.1. Sampling Issues*

Along the four years of the current study, two harvest seasons (2016/2017 and 2018/2019) have been characterized by intense pest attacks and/or other agro-climatic issues, such as late frost, which destroyed almost all the production [36]. In these seasons, only two samplings (S1 and S2) out of the four planned were carried out. As a consequence, the discussion of the results will be mostly based on those obtained during the harvest seasons 2017/2018 and 2019/2020 although all the available results are reported.

#### *3.2. Drupes Characteristics*

The ripening process of the drupes was measured as Pi and it is reported in Figure 1. As a first glance, it appears the maturity pattern was different among the harvest seasons. Depending on the year, the drupes started with different values of Pi. In 2018/2019 and 2019/2020, the drupes started totally green (Pi = 0) while they were slightly spotted in the case of 2016/2017 and 2017/2018 (Pi around 1, i.e., <50% black skin with white flash).

It is worthy to highlight that the first sampling (S1) fell in all the studied seasons around the middle of October. Following the evolution, the drupes reached a Pi of about 2 (≥50% black skin with white flash) in all the years, with exception of the season 2018/2019. This stage (Pi = 2) correspond to the stage commonly considered the physiological maturity (half-veraison of the fruits) and expected to be the stage in which the oil accumulation reached its maximum (i.e., technological maturity).

Thereafter, an important gap among the years 2017/2018 and 2019/2020 was recorded at S3. In the latter, the full maturity seemed almost reached and, from this point on, only a slight increase in Pi was recorded. Differently in 2017/2018, the Pi remained practically unchanged between S2 and S3 while the most significant jump was observed from S3 to S4. On the whole, the ripening process proceeded in a more regular way during the harvest season 2019/2020.

**Figure 1.** Trends in the Pi (mean ± SD, *n* = 3) of the Oliva Rossa drupes during ripening in four consecutive harvest seasons. Different letters for each harvest year indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S1–S4: four subsequent samplings.

As evidenced by Camposeo et al. [37], the observed differences are likely related to the climatic conditions and crop load, which, among other factors, strongly influence drupe ripening. In this regard, the lower temperatures recorded during the ripening period in 2017 with respect to 2019 (±2–3 ◦C less, Figure S1) could be responsible for the observed delay in the pigmentation pattern.

From a technological point of view, it should be stressed how these differences were reflected in the drupes and in the oil characteristics. The evolution of the oil content during maturation is reported in Figure 2.

**Figure 2.** Oil content on dry weight (mean ± SD, *n* = 3) of the Oliva Rossa drupes during ripening in four consecutive harvest seasons. Different letters for each harvest year indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S1–S4: four subsequent samplings.

The oil content at S1 ranged between 25 and 26 g 100 g−<sup>1</sup> on dry weight (2018/2019 and 2019/2020) and goes up to 45 g 100 g−<sup>1</sup> in 2016/2017. The season 2017/2018 lied in between the others, showing an initial oil content of 33 g 100 g<sup>−</sup>1. The very high oil content of the harvest season 2016/2017 should be considered as an exceptional case, confirming that such a season was an outlier with respect to the general pattern. In 2019/2020, the evolution of oil content was very similar to that of the Pi in Figure 1. Indeed, a regular accumulation of the oil was observed with a positive slope of about 5 g 100 g−<sup>1</sup> each 14 days from S1 to S3. Thereafter, a small significant decrease in oil was reported. Such a slight decrease was likely linked to the natural variability of the sampled fruits among different trees and canopy positions. During 2017/2018, the most significant phase of oil accumulation occurred between S2 and S3, moving from 32 g 100 g−<sup>1</sup> up to 40 g 100 g−1. Such an increase in oil was not reflected in the Pi, which was unaffected when moving from S2 to S3 (Figure 1). Afterward, the oil content remained constant. As a most important finding, in both the harvest seasons for which the full maturity pattern was followed, S3 corresponded to the maximum oil content. Unfortunately, the Pi did not reflect directly such a conclusion. In fact, at the half-veraison of the drupes (i.e., Pi = 2), generally considered as the moment of physiological maturity, the fruits could still be in the stage of intense oil accumulation, as observed in the 2019/2020 crop year. Figure 3 shows the evolution of the olives TPC.

**Figure 3.** Total phenolic content (TPC) on dry weight (mean ± SD, *n* = 3) of the Oliva Rossa drupes during ripening in four consecutive harvest seasons. Different letters per each harvest year indicate significant differences according to one-way ANOVA followed by Fisher's LSD post-hoc test (α = 0.05). S1–S4: four subsequent samplings.

During fruit ripening the TPC decreased, as is already known [38]. Further, in the harvest seasons 2016/2017 and 2018/2019, the negative effect of the pest attacks and late frost on the phenolic content of the drupes was easily observed. A high TPC was observed in 2017/2018, which was around 30 g kg−<sup>1</sup> and about 25% less than in 2019/2020. The TPC of the Oliva Rossa drupes was similar to that reported for the Frantoio cultivar [38], while less than what was reported for the Sardinian cultivars [16]. Despite the initial gap at S1 between 2017/2018 and 2019/2020, the TPC tended to become similar during ripening and in particular at S3 the differences were negligible. The sharp TPC decrease from S1 to S2 in 2017/2018 could be linked to the abundant rainfalls recorded in October (Figure S1). Indeed, the tree water status is reported to be inversely correlated with the phenolic content [39]. It is noteworthy pointing out that, at S3, the phase of the more intense

oil accumulation was already over (Figure 2) and the fruits also had comparable TPC. Thus, based on the available results, it would be reasonable considering S3 as the best moment for harvesting. Nonetheless the main issue of a feasible and reliable way of determining such a moment is still of concern considering that S3 corresponded to the stage in which the difference in the Pi was the biggest among the considered harvest seasons (Figure 1). The phenolic content of the drupes represents an important feature for the future VOOs and it should be considered that only 1–2% of the olives phenols moves into the oily phase during extraction [40], which may, in turn, depend on the fruit ripening [41].
