*3.3. Ecophysiological Parameters and Optical Properties*

After the first two foliar applications, no significant changes in the photosynthetic rate (A) were observed between the treatments (Table 2). After the 7th application (20th September), a significant decrease of A and stomatal conductance (g) in K plants was observed (Table 2): K plants showed photosynthesis and stomatal conductance values 27% and 55% lower than those of the test plants, respectively. Similar results were found in bean plants by Tworkoski [55], whereas Jifon and Syvertsen [36] observed that the increasing leaf whiteness after kaolin sprays on grapefruit reduced the leaf temperature and increased stomatal conductance and net CO2 assimilation rates. At first measurements (8th July), plants belonging to K, Z and T treatments did not show any difference in leaf transpiration (E) but, as the treatments continued (increasing particle accumulation), E decreased significantly in K plants and consequently, the WUE was significantly higher (Table 2). The effect of the kaolin accumulation on physiological parameters is possible to see in the PCA analysis where a clustering of the last date of the kaolin treatment occurs (Supplementary Figure S1). Similar results were observed by Jifon and Syvertsen [36] where WUE in kaolin sprayed leaves of grapefruits was 25% higher than that of control leaves.


**Table 2.** Ecophysiological parameters measured after each foliar application of K (kaolin), Z (CHA-zeolitite), and T (control). Data are presented as mean ± standard deviation. Different letters (a,b,c) indicate significant differences according to ANOVA and Tukey's HSD test (*p* < 0.05) at each application date.

<sup>1</sup> A is the net photosynthetic rate; <sup>2</sup> g is stomatal conductance; <sup>3</sup> Ci is the intercellular CO2 concentration; <sup>4</sup> E is the transpiration; <sup>5</sup> WUE is the water use efficiency calculated as the ratio of photosynthesis rate to transpiration rate.

> No differences were observed in E and WUE between Z and T plants, while g was higher after the last two applications in the Z treatment (22 and 19%, respectively) without, however, influencing the photosynthetic rate (Table 2). Similar results were observed in soybean plants coated with kaolin, where the net radiation was reduced by 8% and short-wave irradiation was reduced by 20%, suggesting a potential reduction in transpiration and water use [34]. Also, Le Grange [56] reported a reduction in photosynthetic rates in kaolin sprayed leaves attributable to increased reflection and absorption of light reduced by 20–40%. Some authors [57,58] reported that kaolin treatment did not reduce the photosynthesis of single leaves but increased the photosynthesis of the whole canopy and therefore the productivity. In rainfed olive trees, Brito et al. [59] demonstrated that kaolin treatment counteracted the effect of water shortage and high light intensity on leaf sclerophyll and on stomatal density. Still in rainfed olive orchards, kaolin application contributed to keep a better water status by creating a specific microclimate around the leaves; moreover, it alleviated the adverse effect of summer stress through distinct physiological and biochemical responses [59].

> In our study, the positive effect of kaolin was not observed because the olive trees are grown in environmental conditions (high rainfall and low temperatures) that do not lead to stress conditions; on the contrary, the abundant covering of the kaolin film had a negative effect on photosynthesis, that decreased during the delicate ripening phase of the olive fruits. Stomatal conductance and transpiration were also significantly reduced in K trees at the end of the experiment. This was probably the result of the abundant accumulation of kaolin on the leaf surfaces, leading to obstruction of stomata, with an alteration of leaf gas exchanges.

> The authors are aware of the fact that, in these environmental conditions, a lower amount of kaolin or less frequent applications would have been sufficient (the concentration of kaolin was five-fold higher than that of CHA-zeolitite), but we aimed at reproducing the operative protocols commonly adopted for olive fruit fly defense. In the several Italian regions where olive cultivation is practiced, indeed, standardized protocols for protection from the olive fruit fly are used, regardless of the different climate conditions. A differentiation for kaolin-based treatments would be necessary and specific protocols should be developed for each different cultivation environment. These protocols must guarantee an adequate level of defense against fly attacks without significantly altering the physiological parameters of the plant.

> Since the 4th application, a significant increase in Ci (CO2 inside the lamina) was observed in Z plants compared to the other treatments. It has been reported that zeolites can adsorb carbon dioxide molecules and release them slowly into the environment; also, it has been suggested that when zeolites are spread on plant leaves, they may increase the

amounts of CO2 near the stomata, concomitantly increasing the photosynthesis rate [60]. In our experiment, however, we have observed no significant effect on the photosynthesis rate in Z plants. On the contrary, K leaves showed lower Ci that is in agreement with the observed decrease in A. Farquhar and Sharkey [61] indeed asserted that where CO2 diffusion limits A, a decrease in Ci would also occur.

In our study, the upper and lower sides of K leaves showed a significant increase in reflectivity compared to the other treatments at all dates (Figure 4). The reflectance is the ability to reflect part of the incident light on a given surface and its effectiveness in reflecting radiant energy. Similar results were observed in grapefruit leaves coated with kaolin, which showed a higher reflectance compared to control leaves [36].

In Z leaves, the reflectance was similar to the T during the summer; only after the last application (29th September) did it increase (Figure 4). The different reflective capacity of the two films is attributable both to the different colour of the powders, white for kaolin (higher light reflectance) and light brown for CHA-zeolitite (lower light reflectance) and to the morphology of the particles, lamellar for kaolin (higher reflectance) vs. pseudo-cubic for CHA-zeolitite (lower reflectance). Furthermore, leaf reflectance data (Figure 4) showed that there is no difference when the number of treatments increases.

Colour measurement carried out on leaves treated with Kaolin showed a greater lightness (L\*) compared to the test and CHA-zeolitite leaves at all dates (Figure 5). This difference in L\* value between kaolin and CHA-zeolitite is due to the different conformation and colour of the kaolin (phyllosilicate) and natural zeolite (tectosilicate). After the 4th foliar application (24th August), the L\* value of K leaves was greater by 22% and 19% with respect to those of T and Z leaves, respectively. Colour measurements performed on K leaves after the 3rd application showed lower L\* values than the previous measurements, while no differences were accounted in T and Z treatments. The lower L\* values observed in K treatment were probably due to kaolin leaching due to rainfall occurring during the previous days (Supplementary Figure S1). At the last measurement (performed on the 20th September), L\* values were higher in both K and Z treatments compared to the T, suggesting that an accumulation of both kaolin and CHA-zeolitite on leaves occurred. Our data agrees with Jifon and Syvertsen's [36] measurements on grapefruit leaves treated with kaolin. Regarding the a\* value, in the first two measurements T leaves showed higher values than those recorded in K and Z treatments, that were similar (Figure 6). It is interesting to note that contrary to L\*, the a\* value was not affected by the rainfall. After

the 3rd measurement, a\* decreased in all the treatments; this was probably caused by the leaf seasonality, since a reduction in the chlorophyll content in the leaf is expected at the end of the hot season [62].

**Figure 5.** Changes in L\*color values measured on the upper part of the leaves of T (control), K (kaolin) and Z (CHA-zeolitite) treatments. Error bars represent standard deviation. Different letters above the bars for each group of histograms indicate significant differences according to ANOVA and Tukey's HSD test (*p* < 0.05).

**Figure 6.** Changes in a\*colour values measured on the upper part of the leaves of T (control), K (kaolin) and Z (CHA-zeolitite) treatments. Error bars represent standard deviation. Different letters above the bars for each group of histograms indicate significant differences according to ANOVA and Tukey's HSD test (*p* < 0.05).

#### *3.4. Olive Analyses and Olive Oil Sensory Evaluation*

In a year characterized by lower temperature (2019) (Supplementary Figure S2) with a high risk posed by the olive fruit fly, zeolite and kaolin sprays have significantly reduced the incidence of *Bactrocera oleae*; in fact, olives produced by Z and K olive trees present a decrease (over 40%) of infestation compared to control (Table 3). Water content of olive treated with kaolin was higher than the water content of olive treated with zeolitite while the olive from untreated trees showed similar value to both treatments (Table 3). The olive firmness did not statistically differ within the treatments, and the same results were observed in pear fruits treated with kaolin [63].

**Table 3.** Ripening index (RI), percentage of olive fruit fly infestation, water content and fruit firmness in olive from trees treated withK (kaolin), Z (CHA-zeolitite), and T (control). Data are presented as mean ± standard deviation. Different letters in the same column (a,b,c) indicate significant differences according to ANOVA and Tukey's HSD test (*p* < 0.05).


<sup>1</sup> express as g/mm2.

The sensory profiles of olive oils extracted from plants treated with Kaolin (K), CHAzeolitite (Z) and control (T) are shown in Figure 7. On a sensory level, the differences found in the oils were slight: Z and T olive oils showed a higher intensity of olfactory olive fruity than K olive oil. For the hint of bitterness K and T olive oil showed higher intensity than Z while for the hint of pungency test olive oil had higher intensity compared to K and Z olive oil. Test oil showed a lower intensity in olfactory secondary flavours while Z oil had a higher intensity in both olfactory and gustatory secondary flavours. Detailed examination of the pleasant flavours (Figure 8) revealed that, at the olfactory level, oils produced from both treatments had an artichoke scent and were perceived as fresher with respect to the oils produced by the test, that smelled of ripe tomato.

**Figure 7.** Sensory profiles of olive oils produced by plants treated with CHA-zeolitite (Z), Kaolin (K) and untreated plants (T).

**Figure 8.** Pleasant flavors of olive oils produced by plants treated with CHA-zeolitite (Z), Kaolin (K) and untreated plants (T).

At the gustatory level, no differences were observed between the T and K oils, for which the tasters only perceived the hint of green almond. On the other hand, it is interesting to underline that the oil produced from olive trees treated with zeolite presented, in addition to the prevailing hint of almond, a note of artichoke which gave the oil a note of freshness compared to others.

De la Roca [30] found that kaolin application against the olive fruit fly significantly reduced the percentage of infested olives. Saour and Makee [31] showed that a kaolinbased particle film formulation significantly reduced fruit infestation levels; the authors hypothesized that adult flies may fail to recognize kaolin sprayed olive trees, and the gravid females are repelled due to the tactile unsuitable surface texture of particle filmtreated olives.

#### **4. Conclusions**

In the scenario of sustainable and environmentally friendly olive oil production, both treatments represent valid alternatives to chemical insecticide. From an economic point of view, CHA-zeolitite represents an advantage because the recommended application rate is five times lower than that commonly used for kaolin. Moreover, CHA-zeolitite volcanic rocks abound in Central Italy and are already exploited for several purposes, including the production of micronized powder from the granular material resulting from building block cutting in quarries. CHA-zeolite supplying is thus relatively less impactful from an environmental point of view, with respect to other types of powders that are quarried and manufactured in foreign countries.

ESEM observation performed on leaf and olive surfaces highlighted microstructural differences between the two tested particle films which influenced some ecophysiological parameters. The intercellular CO2 concentration was positively influenced by CHA-zeolitite application while kaolin application decreased photosynthesis, stomatal conductance and transpiration rates compared to the other foliar treatments. Therefore, in hot environments, the use of kaolin has the dual function of protecting the olive tree both from high temperatures and from the olive fly but the resulting impactful coating caused a reduction of photosynthesis that can, however, be compensated by an increase in WUE due to the reduced transpiration. The continuous layer of kaolin on leaf surface has also significantly influenced the leaf reflectance thanks to its crystal morphology, colour and application rate.

In a cold and humid environment (such as our experimental conditions), CHA-zeolitite was found to be the ideal compound because it exerted a protective effect against olive fruit fly attack, similar to kaolin, but left the leaf gas exchanges unaltered. Moreover, oils obtained from CHA-zeolitites showed higher intensities of gustatory and olfactory pleasant flavours than olive oils produced from kaolin and untreated trees, thus enhancing the quality and sustainability characteristics of this product.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/foods10061291/s1, Supplementary Table S1: Mineralogical composition (XRPD Rietveld-RIR method) of the kaolin and CHA-zeolitite supplied by Balco s.p.a and used in the experimentation. Data from the product's technical sheet supplied by the company; Supplementary Table S2: Results of soil analysis (oven-combustion, EA-IRMS) from each experimental plant treated with kaolin (K), CHA-zeolitite (Z) and the control (T); Supplementary Table S3: Results of soil analysis (2 replicates) by X-Ray Fluorescence (XRF) from each experimental plant treated with kaolin (K 1 and 2), CHAzeolitite (Z 1 and 2) and the control (T 1 and 2); Supplementary Figure S1: PCA of the ecophysiological parameters measured after the foliar applications of K (kaolin), Z (CHA-zeolitite), and T (control); Supplementary Figure S2: Minimum, mean and maximum temperature (◦C) and rainfall (mm) recorded in the period 1st Julay-31st October.

**Author Contributions:** Conceptualization, A.R.; methodology, L.M. and O.F.; formal analysis, L.M.; investigation, L.M., A.R. and O.F.; writing—original draft preparation, A.R.; writing—review and editing, L.M. and G.F.; supervision, B.F. and M.C.; project administration, A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors gratefully thank Matteo Mari for technical support, Barbara Alfei and Panel of ASSAM Marche for sensory analysis, the CNR-ISAC and CNR-ISP staff for the meteorological data and a special acknowledgment to Franco Corticelli for ESEM-EDX analysis support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

