*2.2. Environmental Electronic Microscope (ESEM) Observations*

Leaf and fruit samples treated with different particle films were collected during the study according to the methodology reported by Lanza and Di Serio [43]. Samples were observed by ESEM (Zeiss, EVO LS 10, Oberkochen, Germany).

To assure a homogeneous distribution of the particle films on the olive surface, preliminary observations were carried out by ESEM. Generally, obtaining good coverage is mandatory when using non-systemic products, such as zeolitite or kaolin. This is because only the "covered" areas of the canopy surface are protected [44]. To this aim, the droplet size distribution during atomization is very important because it affects the biological activity and the spray drift [45]. Study by Skuterud et al. [46] showed that, when applying contact products such as zeolitite, it is important to use fine (60 μm) or medium-sized (60–200 μm) droplets. The final coverage is also affected by the spray type: high application volumes can result in product run-off, which leads to considerable losses. On the other hand, low volume spraying leads to very poor coverage of the leaf surface and hence loss of efficacy [45]. Considering also the lower concentration of zeolite compared to the concentration of kaolin it was necessary to identify the right diameter of nozzles to guarantee a homogenous coating. This was achieved through several ESEM observations and measurements of the distance between the crystals (Figure 1A). These observations and measurements have confirmed that good coverage was achieved when spraying CHAzeolitite utilizing a handgun sprayer with 0.2 mm diameter nozzles. These nozzles cause a dispersion of the product characterized by a distance among crystals under tenths of millimetres which is far smaller than the area interested by oviposition puncture of *Bactrocera oleae* (triangular slot of 1–1.5 millimetres long).

**Figure 1.** ESEM observations of CHA-zeolitite leaf coating to check the distribution protocol. (**A**) Measurements of the distance between CHA-zeolitite particles; (**B**) CHA-zeolitite particles' composition by EDS-microanalysis.

To establish the exact nature of the observed particles, semi-quantitative EDS (energy dispersive spectroscopy) microanalysis systems were carried out randomly to determine particles' composition (Figure 1B).

#### *2.3. Chemical Analysis on Leaves and Soil Samples*

A total of 50 g of leaves and 500 g of soil samples were analysed for total C and N and the relative isotopic signature (δ13C and δ15N) with an Elementar Vario Micro Cube Elemental Analyzer (EA) in line with an ISOPRIME 100 Isotopic RatioMass Spectrometer (IRMS) operating in continuous-flow mode (Elementar Analysensysteme GmbH, Langenselbold, Germany). Soil samples were additionally processed for X-ray fluorescence (XRF) analysis on powder pellets, using a wavelength-dispersive automated ARL Advant'X spectrometer (Thermo Electron SA, Ecublens, Switzerland). The organic matter of soil samples was measured by quantifying the weight loss after combustion at 550 ◦C.

#### *2.4. Ecophyisiological, Optical Properties and Color Leaf Measurements*

Leaf gas exchange measurements: photosynthesis (A), stomatal conductance (g), intercellular CO2 concentration (Ci), transpiration rate (E) and intrinsic water use efficiency calculated as the ratio of photosynthesis rate to transpiration rate (WUE), were measured during a clear sky using a Li-Cor portable photosynthesis system (LiCor 6400, Lincoln, NE, USA) operating at 400 μmol m−<sup>2</sup> s−<sup>1</sup> flow rate. Measurements were taken in the morning (10:00 a.m to 12:00 p.m.), according to the protocols of Denaxa et al. [47] and Jifon and Syvertsen [36], on undamaged mature sun leaves located at the central part of the one-year-old shoot of the marked branches, according to Larbi et al. [48].

Total directional-hemispherical reflectance of the upper and lower leaf surface was measured with a calibrated spectroradiometer LiCor 1800 (Li-Cor, Nebr, Lincoln, NE, USA) able to scan from 300 to 1100 nm connected to a Li-Cor 1800-12 integrating sphere. To prevent spectral changes due to water losses and metabolic modification, spectral measurements were made immediately after the leaves were picked, according to Baldini et al. [42].

Leaves' colour was measured on the upper surface of one-year leaf using a Konica Minolta CR-400 Chroma Meter (Konica Minolta, Inc., Osaka, Japan) calibrated with a standard white plate at room temperature. The data collected were L\* (lightness) and a\* (red-green scale) recorded at three random locations on each leaf on twenty leaves collected from the olive trees submitted to different treatment (T, Z and K).

All leaf f measurements (ecophyisiological, optical properties and color surface) were carried out on 8 July, 24 August and 20 September 2019.

#### *2.5. Olive Analyses and Olive Oils Sensory Evaluation*

Considering that the optimal ripening index (RI) for the Correggiolo cultivar is included in the range 2–2.5 of the Jaén index [49], the RI was monitored for each treatment according to the method developed by the Agronomic Station of Jaén defining the RI as a function of fruit colour in both skin and pulp [50]. On the same samples, each olive fruit was examined for the presence of *Bactrocera oleae* infestation, dissecting the fruits to determine the percentage of total infestation (egg, larva or pupa, sting scar, exit holes). Olive water content was gravimetrically determined placing olive samples in oven at 60 ◦C for 8 days. Olive firmness was determined using a *penetrometer* (PCE-FM 200, PCE Group, Lucca Italy); it was measured at two points on each fruit, and the average readings were reported in g/mm2 as exerted pressure.

The total production of the selected trees for each treatment was handpicked; an amount of 50 kg was transformed into oil. Olives were defoliated, washed and milled using a low scale continuous mill (Oliomio®; Toscana Enologica Mori, Firenze, Italy) equipped with blade crusher, horizontal malaxator and a two-phase decanter. Olive samples were processed within 24 h of harvest. For each sample the technological settings (temperature (below 27 ◦C) and the time of malaxation (20 min), the speed of the decanter (4200 rpm) and the flux of water in the separator (0.8 L h<sup>−</sup>1)) were standardized in order to minimize

the variability due to the extraction procedures. Oil samples were filtered through cotton filters, poured into dark glass bottles, keeping the headspace to a minimum, and stored in a temperature-controlled cupboard set at 15 ± 1 ◦C until analysis.

Sensory analyses were carried out by a fully-trained analytical taste panel recognized by the International Olive Oil Council (IOOC) of Madrid and by the Italian Ministry of Agricultural, Food and Forestry Policies. The panel evaluated all oil samples following an incomplete randomized block design. Olive oil samples were placed in blue tasting glasses and the temperature of samples was kept at 15–18 ◦C. A panel test was established for the present study using a standard profile sheet (IOOC/T20) modified by IBIMET-CNR [51] that allows the obtaining of a more complete description of the organoleptic properties of the oils. The tasters evaluated direct or retronasal aromatic olfactory sensations (olive fruity, green/leaf and secondary positive flavours), gustatory sensations (olive fruity, bitterness and secondary positive flavours) and tactile/kinesthetic sensation (pungency). The tasters had to rate the intensity of the different descriptors on a continuous 0–10 cm scale. Values of the median of sensory data and robust standard deviation were calculated.

### *2.6. Statistical Analysis*

The data collected were elaborated using Microsoft® Excel 2007/XLSTAT© (Version 2009.3.02, Addinsoft, Inc., Brooklyn, NY, USA). The significant differences among means at a 5% level were determined by ANOVA followed by a Tukey's Honestly Significant Difference (HSD) test. Principal component analysis (PCA) has been performed to explore data distribution patterns using physiological data.

#### **3. Results and Discussion**

#### *3.1. ESEM Observations*

Particles of both treatments (K and Z) were more homogenously distributed on the leaves' surface rather than on the surface of the olive. This higher attachment onto the leaves' surface is due to their peculiar morphology, characterized by overlapped stellar trichomes, particularly frequent on the lower surface (Figure 2A).

Since the first foliar application, a good distribution of both K and Z products was observed on the upper surface of the leaves, compared to the test which lacked particles on its surface (Figure 2B). In K treatment, kaolin appeared as a continuous layer and it was not possible to recognize the underlying star hairs (Figure 2C), while in Z treatment, the CHAzeolitite film was more discontinuous and star hairs were still recognizable (Figure 2D). The same differences were also noted on the lower surface of the leaves.

This difference in the surface coverage is attributable to both higher amounts of kaolin sprayed at each application compared to the CHA-zeolitite and to the different morphology of kaolin and CHA-zeolitite particles (lamellar vs. pseudo-cubic).

Due to the particular morphology of the olive, characterized by a smooth and curved surface, the adhesion of the particles was less uniform than that observed on the leaves. This difference was observed from the first application and increased in subsequent applications, thanks to the accumulation of the deposited kaolin and CHA-zeolitite particles.

On the surface of the untreated olives (T), the epicuticular waxes arranged in crystalloid structures (membranous platelets) were well recognizable (Figure 3A), as observed in Carboncella olives by Lanza and Di Serio [43]. Micro-changes of epicuticular waxes, which occur with the progressing of ripening [43], were well visible in the T olives while in K and Z olives these micro-changes were hidden by particle accumulation, especially at the end of the experimentation.

**Figure 2.** ESEM observations of treated and untreated olive leaves. (**A**) Accumulation of CHA-zeolitite on stellar trichomes that cover the lower surface of the olive leaves; (**B**) Upper surface of test olive leaf; (**C**) Olive leaf treated with kaolin; (**D**) Olive leaf treated with CHA-zeolitite.

**Figure 3.** ESEM observations of treated and untreated olives. (**A**) Untreated (T) olive surface; (**B**) Olive surface treated with CHA-zeolitite (Z treatment, first application); (**C**) Olive surface treated with kaolin (K treatment, first application); (**D**) Olive surface treated with CHA-zeolitite (Z treatment, last application); (**E**) Olive surface treated with kaolin (K treatment, last application); (**F**) Morphology of CHA-zeolitite particles; (**G**) Morphology of kaolin particles; (**H**) Olive surface where kaolin appears to be incorporated by waxes.

As reported for the leaves, olives coverage was greater and more homogeneous in K treatment compared to Z treatment (Figure 3B,C); these differences were accentuated due to the accumulation of particles as the experiment progressed (Figure 3D).

ESEM observations after light rain events highlighted the tendency of K microaggregates to disperse and to form a continuous layer (macroscopically visible) on the surface of leaves and olives. With the growth of the olive tissues, the continuous K layer tends to fissure, leaving some areas uncovered (Figure 3E). The different aspect of K and Z films is linked to the specific morphology of the kaolin and CHA-zeolitite particles. CHA-zeolitite particles are mainly pseudo-cubic (Figure 3F) [52] while Kaolin is shaped as sheets/lamellae/irregular flakes (Figure 3G) [53].

Sample observations after heavy rain events showed that both K and Z coatings were well preserved, with the difference that the CHA-zeolitite particles kept their original shape and "anchored" themselves to the waxes, whereas those of kaolin appeared incorporated in waxes (Figure 3H); the same results were observed in apples treated with kaolin [54].

#### *3.2. Chemical Analysis on Leaves and Soil Samples*

The chemical composition of the soil between the various treatments was very similar in terms of soil organic matter, total N and C, major and trace elements (Supplementary Tables S2 and S3). The study area can be thus considered homogeneous in terms of soil chemistry and N availability to plants. Also, no significant differences were accounted in terms of total C, N and relative isotopic signature of the leaves at the end of the experimentation (Table 1).

**Table 1.** Results of leaves analysis (EA-IRMS) from each experimental plant treated with kaolin (K), CHA-zeolitite (Z) and the control (T). TN and TC are the total nitrogen and carbon content measured by EA analysis, δ15N and δ13C are the isotopic signatures expressed as delta notation by IRMS. Values are expressed as the mean of three replicates ± standard deviation. The same letters in the same column express no significant differences (*p* > 0.05) as results of ANOVA and Tukey's (HSD) tests.

