**1. Introduction**

The olive fruit fly (*Bactrocera oleae*) is worldwide considered a major harmful pest of the olive agroecosystem. Under certain environmental conditions (high humidity and precipitations and temperature below 28–30 ◦C), the fruit fly is responsible for large infestations that seriously compromise olive yield and oil quality [1].

The many olives strongly attacked by flies produce oxidized oils with a reduced quantity of phenolic substances, which therefore are unlikely to live up to the EFSA health claim [2]. In Italy, the fruit fly infestation is traditionally countered by spraying chemical insecticides such as dimethoate (in integrated regime) or by applying organic formulations (organic farming) [3]. However, taking into account the recent ban of dimethoate [4] and the increasing awareness of consumers of food sustainability, the interest in developing natural and chemical-free alternatives to pesticides, such as organic agrochemicals or the use of geologic material as particle film, is rising [5].

Agronomic practices are also one of the keys to allow the development of extra virgin olive oil (EVOO) market niches, guaranteeing high and constant quality standards [6].

The spraying of "rock dust" (e.g., kaolin) as foliar treatment in organic agriculture to reduce the negative impact of environmental stresses and to protect fruits from insect pests is a well-established approach [7]. Kaolinite (Al2Si2O5(OH)4) is an aluminium–silicate clay mineral composed of a layered silicon-oxygen tetrahedron and a layered aluminium– oxygen octahedron [8,9]; the commercial term "kaolin" refers to a rock whose percentage of kaolinite is higher than 50% [10]. In kaolin, kaolinite is often associated with other minerals

**Citation:** Rotondi, A.; Morrone, L.; Facini, O.; Faccini, B.; Ferretti, G.; Coltorti, M. Distinct Particle Films Impacts on Olive Leaf Optical Properties and Plant Physiology. *Foods* **2021**, *10*, 1291. https://doi.org/ 10.3390/foods10061291

Academic Editors: Cristina Alamprese, Emma Chiavaro and Francesco Caponio

Received: 27 April 2021 Accepted: 31 May 2021 Published: 4 June 2021

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such as quartz, feldspar and various phyllosilicates (such as muscovite and illite) [11]. Contrary to other clay minerals, such as smectites, kaolinite is characterized by a relatively low cation exchange capacity (CEC) (0.38 meq/g) [12]. The size of kaolinite particles can reach a colloidal level after milling and grinding during mineral processing [13].

Similarly, natural zeolites represent another geologic material that can be used as particle films for crop protection [14].

Zeolites are crystalline aluminosilicates composed of a 3D framework of linked [SiO4] <sup>4</sup><sup>−</sup> and [AlO4] <sup>5</sup>−. The framework delimits open cavities in the form of channels and cages in which H2O molecules and extra-framework cations can be reversibly exchanged. The most important properties of zeolite minerals are (i) high cation-exchange capacity, (ii) reversible dehydration and (iii) molecular sieve. Nowadays, more than sixty types of natural zeolites have been described by researchers (http://www.iza-online.org/natural/ default.htm (accessed on 15 March 2021)), each differing in terms of framework structure, mineral chemistry and ion exchange capacity, but only a few occur in sufficient amounts and purity to be considered as exploitable natural resources [15]. Among them, clinoptilolite is the most frequent and abundant sedimentary zeolite in nature, followed, in the order, by mordenite > chabazite > phillipsite > erionite [16]. Natural zeolites are often constituents of volcanic tuffs [17]; thus, the term "natural zeolites" is inappropriate from a geological perspective and it should be substituted by rocks or tuffs rich in zeolite. Analogously to kaolin, if the zeolite content is greater than 50%, the rock can be classified as "zeolitite", specifying the main zeolite constituent (e.g., chabazitic-zeolitite) [16].

Chabazite zeolite (CHA), although less abundant than clinoptilolite, is particularly attractive for agricultural and industrial applications because of its very high CEC (3.84 meq/g) and easiness in sorption and subsequent release of NH4 <sup>+</sup> ions [18,19]. The "honeycomb" framework of zeolite minerals, together with their carbon dioxide sorption and heat stress reduction capacity, makes them suitable as leaf coating products. Furthermore, their reversible dehydration makes them effective against fungal disease and insect pests [14]. Zeolite tuffs are most commonly used in agricultural practices as a soil amendment and for improving the nitrogen use efficiency (NUE) by crops because of their high affinity with NH4 <sup>+</sup> ions [20,21]. Recently, Italian CHA-zeolitite was used as a soil amendment in a long-term field experiment [22–24]. Laboratory incubations highlighted the positive effects of CHA-zeolitite on soil N and C gaseous emissions and microbial biomass [25,26]. The same rock was used for removing N and Na from animal liquid manure and low-quality irrigation waters, with promising results [27–29].

Studies on the use of powders for contrasting olive fly are fairly recent, and showed that kaolin application on the olive fruit fly significantly reduced the percentage of infested olives [30,31].

Rumbos et al. [32] studied the insecticide potential of zeolite formulations against stored grain insects but, to the best of our knowledge, zeolite tuffs have not yet been studied as a defense tool against the olive fly.

Regions characterized by arid climate and low rainfall regimes are the most suitable for this technology due to the reduced temperature of the leaves and the wash-off risk for the particle films. High rainfall regimes may lead to the necessity of multiple applications, increasing the costs (for materials and manpower) and hence significantly decreasing the attractiveness of the methodology [33].

Besides the effectiveness against the fruit fly, it is crucial to understand if the particle films interfere with the physiological activity of the plants, as the literature shows contrasting evidence on this subject. Some authors reported that kaolin film causes a reduction in leaf temperature, transpiration and water use efficiency (WUE) in soybean plants [34], as well as in apple leaves [35]. Contrarily, Jifon and Syvertsen [36] reported that the WUE of the kaolin-treated citrus leaf was higher than untreated leaves because photosynthesis was increased without an increase in leaf transpiration. In apple trees, the lower leaf temperature of kaolin-treated plants increased photosynthesis and stomatal conductance [37].

As mentioned above, the effect of zeolitite particle film on plant physiology is mostly unknown due to its recent application in agriculture. Besides the reduction of heat stress, zeolitites may also be used to reduce water stress. The adsorption selectivity of zeolites for water is greater than any other minerals [38], leading to an adsorption capacity that may reach up to 30% of the zeolite weight without any volume modification, depending on the zeolite type [39]. Thanks to these properties, together with the relatively low-cost and high abundance, zeolite attractiveness for agricultural utilizations has recently risen, overcoming that of kaolin.

According to Reddy et al. [40], the application of particle films over the stomata is known to increase resistance to water vapor losses. Moreover, particulate sprays modify the leaf optical properties, increasing foliage reflectivity and modifying plant physiological processes such as photosynthesis, morphogenesis and water balance [41].

The olive leaves are covered by trichomes, which may directly influence the diffusion boundary layer of the leaf surface, increase leaf reflectance for all wavelengths of solar radiation between 400 and 300 nm and restrict radiation absorbance, resulting in a reduction of the leaf load [42].

The experiments presented here were carried out in order to test the effectiveness of different particle films in a cold and humid environment, typical of northern Italy, where the olive fly attack is increasingly worrying. Here, small-scale, high quality olive oil production is carried out on the Emilia-Romagna Appennine hillsides.

In addition, this study aims to evaluate and compare the effects of two different particle films (kaolin and zeolitite) on leaf optical properties, leaf gas exchange and on the incidence of the olive fruit fly attack. ESEM observations allowed us to investigate the microstructural differences of the particle film on leaf and olive surfaces. Olive fruit analyses and sensory characterization of olive oils produced by the different treatments were also performed, in order to establish if the influence of foliar application on the ecophysiological parameters could affect harvest quality.

#### **2. Materials and Methods**

#### *2.1. Treatments and Sampling*

The study was carried out in 15-year-old commercial olive (*Olea europeae*) cv Correggiolo plants located in Bologna hills (Italy). One third of the olive orchard was submitted to kaolin treatment (K), 1/3 to zeolitite treatment (Z) and in the last 1/3 of orchard no applications were made (T). Two olive trees for each thesis were chosen, four branches for each tree were marked in different cardinal points, and for each branch three leaves were sampled. Twenty-four leaves for each thesis were considered for physiological, optical, ESEM and color leaf measurements. The tested treatments were:


The kaolin and the CHA-zeolitite were supplied by Balco s.p.a company. The mineralogical composition of both products is reported in Supplementary Table S1.

The tested application dosages were chosen according to the guidelines provided by the producer. Kaolin and CHA-zeolitite were applied by covering the total foliage using a mounted sprayer (flow max 50 L/min, capacity 200 l-Idromeccanica Bertolini-Reggio Emilia Italy) equipped with a handgun sprayer and testing different nozzle diameters. The average particle size of both kaolin and CHA-zeolitite was 6–10 μm.

The foliar applications started at the beginning of the summer, when olive fruits were developed enough to be attacked by *Bactrocera oleae*, and applications were repeated approximately every 20 days (13 June, 3 July, 21 July, 17 August, 5–12–19–29 September 2019), the applications were repeated after heavy precipitations (September) to guarantee sufficient coverage until the end of the growing season. Conventional orchard agronomic practises, pruning and winter treatment based on Bordeaux mixture, were applied for all thesis. Environmental temperatures and rainfalls were monitored using a weather station IRDAM WST 7000 C (IRDAM SA, Yverdon-les-Bains, Suisse).

50 g of leaves were randomly sampled from each olive plant to carry out elemental and isotopic analysis of C and N to check for possible differences in C-N composition between the studied plants. Once in the laboratory, the leaves were washed with deionized water, dried for three days at 60 ◦C and then ground to a fine powder. Additionally, to gain information on the soil environment, soil samples from the first 0.3 m depth were collected using a manual auger (Eijkelkamp). To address spatial variability, three logs per plant were mixed to form a global sample; each one was then sieved at 5 mm and air-dried before further analyses.
