The Effects of Different Mineral Clay Particles on Olive Yield and Olive Oil Quality of Two Cultivars Under Rainfed or Irrigated Conditions

Abstract

The olive tree is the emblematic tree of the Mediterranean basin, enduring intense irradiance and heat stress during prolonged dry summers. Particle film technology is a relatively new tool for mitigating both biotic and abiotic stress factors. In the present trial, two Greek olive cultivars, i.e., rainfed ‘Megaron’ and irrigated ‘Koroneiki’, were used to test the efficacy of kaolin, talc, and, for the first time, attapulgite clay particles as single and double foliar applications on the quantitative and qualitative traits of production. Clay particle treatments generally increased yield, resulting in higher olive oil production per tree. Oil quality parameters remained within the standards for extra virgin olive oil across all treatments. Talc differed from the other clay minerals, particularly in its effect on the free acid composition of the oil. Attapulgite application enhanced yield and oil production in ‘Koroneiki’, especially when compared to double kaolin application. Conversely, kaolin double application resulted in the highest yield and oil production in the ‘Megaron’ cultivar. These findings indicate that the efficacy of particle film treatments varies depending on multiple factors, yet they remain a valuable tool for mitigating the adverse effects of climate change on olive production. As this is the first study to test talc and attapulgite on olive trees, further research is required to fully elucidate the potential of particle film technology.

1. Introduction

Over the past decade, extreme weather phenomena have become increasingly frequent, largely attributed to climate change, which has escalated into a climate crisis in some areas [1]. Certain regions of the planet are particularly vulnerable to such weather extremes, including the Mediterranean basin [2,3]. According to some studies, the mean annual temperature in this region has risen by nearly 1.5 °C compared to the previous century [1,4]. This temperature increase, driven by elevated atmospheric CO₂ levels, has intensified extreme events, such as floods, prolonged droughts, heat waves, reduced rainfall, and increased vapor pressure deficit, which now occur with growing frequency [1].

The agricultural sector is highly susceptible to weather extremes, with significant losses in agricultural productivity recorded worldwide. Water scarcity, often coupled with high irradiance and temperatures in summer, induces various physiological malfunctions in plants. These include stomatal closure, reduced photosynthesis, increased production of reactive oxygen species (ROS) leading to oxidative damage, leaf wilting and loss, yield reduction, and quality degradation. In more severe cases, plant mortality has also been documented [1].

Perennial species are particularly vulnerable, as they endure multiple abiotic stress factors throughout their lifespan and cannot escape unfavorable conditions [1,5]. Among the most significant trees in the Mediterranean region is the olive tree, one of the oldest and most emblematic species of the region, holding immense economic and cultural value for the people of the corresponding countries [6,7]. The olive is a xerophytic, sclerophyllous species capable of tolerating harsh conditions, such as drought, salt stress, and high temperatures. However, under conditions of water scarcity combined with extreme heat and high irradiance, olive trees experience significant stress, resulting in reduced stomatal aperture, impaired photosynthesis, and, ultimately, yield losses [7,8]. Therefore, developing eco-friendly strategies to enhance plant adaptation to such challenging conditions is essential.

At the same time, consumer awareness of pesticide residues is increasing, fueling a global trend toward reduced pesticide use and greater reliance on products that enhance plant defense mechanisms, such as biostimulants and biopesticides [9,10]. One of the most exciting and emerging multi-functional, environmentally friendly technologies for alleviating abiotic stresses while functioning as pest protection is the adaptation in the agricultural sector of particle film technology (PFT) [9,11,12,13,14,15]. This technology involves the use of aqueous formulations of chemically inert mineral particles treated in such a way as to produce a continuous coating over plant surfaces, functioning as protective films [14,16].

Effective PFT products possess beneficial properties, including high transmission of photosynthetically active radiation (PAR), while reflecting ultraviolet and infrared radiation. This reduces heat accumulation and solar damage to leaves and fruit [12,14,17]. Additionally, these particles can camouflage plant tissues, making them less recognizable to pests [14]. By forming a white mineral barrier on plant surfaces, they create an unfavorable environment for pests, leading to reduced infestation rates [18].

Among the most used minerals in PFT are clays, such as kaolinite, attapulgite, montmorillonite, talc, and pyrophyllite [14]. Kaolinite has been extensively used after proper treatment, giving rise to kaolin, a commercially available product. There are numerous studies on the use of kaolin clay particles in various plant species (pomegranate, olive, apple, grapefruit, grapes, coffee, walnut, tomato, etc.), aiming at alleviating the adverse effects of abiotic and/or biotic stress factors. In olive trees, kaolin has demonstrated promising results in enhancing both physiological and yield-related parameters under stress conditions. These improvements are achieved through an increased photosynthetic rate, improved water use efficiency, and the alleviation of oxidative stress, among other beneficial effects [6,7,8,14,19]. To our knowledge, talc has not been tested on olive trees, although it has been used on pomegranates to prevent fruit sunburn [20]. Similarly, attapulgite has not been reported to have been applied to olive trees or any other species as a foliar application, and thus no data exist on its efficacy.

This study aimed to evaluate the impact of particle film technology on olive tree productivity, both quantitatively and qualitatively, in terms of olive oil yield and quality. Three commercially available agricultural products containing kaolin, talc, and attapulgite particles—two of which were tested on olive trees for the first time—were applied to two olive oil cultivars in central and southern Greece.

2. Materials and Methods

2.1. Test Site Location—Plant Material

The trial was conducted in two different regions in Greece in monovarietal olive groves. The first one took place in central Greece, Viotia County, in the region of Vathi Avlidas (38°24′33.06″ North, 23°36′49.11″ East, altitude 65 m) in 2019–2020 (Figure 1). The mean annual temperature in the region is approximately 16.7 °C, while the average temperature is around 27.2 °C in July and around 27.1 °C in August with minimal rainfall. The mean monthly total solar irradiation on a horizontal level in the area is 220.0 kWh m⁻² in July and 204.0 kWh m⁻² in August (data from the Greek Ministry of Environment and Energy, https://www.helapco.gr/ims/file/installers/totee-klimatika.pdf, visited on 6 March 2025). The second one took place in southern Greece, on the island of Crete in Heraklio County, in the region of Kato Kastelliana (35°1′56.813309″ North, 25°16′10.86″ East, altitude 450 m) in 2022–2023. The mean annual temperature in the region is approximately 17.8 °C, while the average temperature is around 26.1 °C in July and around 25.9 °C in August. The mean monthly total solar irradiation on a horizontal level in the area is 227.1 kWh m⁻² in July and 207.0 kWh m⁻² in August (data from the Greek Ministry of Environment and Energy, https://www.helapco.gr/ims/file/installers/totee-klimatika.pdf, visited on 6 March 2025). The cultivars used were ‘Megaron’ (synonym ‘Megareitiki’ or ‘Megaritiki’) in Viotia (60-year-old rainfed trees) and ‘Koroneiki’ in Crete (30-year-old drip-irrigated trees), both olive oil cultivars of significant importance for Greece as well as for the entire world (regarding ‘Koroneiki’). All trees in each olive grove were selected based on uniform growth and similar expected yield, without any visible symptoms of either nutrient deficiencies or disease infection. All the necessary cultivation practices were carried out by the farmers (weed, pest, disease control, etc.) to ensure unhindered tree growth and fruit production.

During the first year in Viotia County, two commercial products were used, i.e., Surround^® WP (kaolin—aluminum silicate 95% w/w) (Al₂Si₂O₅(OH)₄ 95%) (Tessenderlo Kerley, Inc., Phoenix, AZ, USA), distributed in Greece by Hellafarm SA, as recommended by the supplier at a registered dose rate of 3 kg 100 L⁻¹, and Invelop^® White Protect (Talc E553b) (Mg₃Si₄O₂₁H₂₀ 100% w/w) (COMPO EXPERT GmbH, Münster/Westphalia, Germany), distributed in Greece by Compo Hellas, as recommended by the supplier at a registered dose rate of 3 kg 100 L⁻¹. Two schemes of spray application took place, i.e., a single application in August (18 August 2019—BBCH crop growth stage 77–78) and a double application both in July (BBCH crop growth stage 76–77) and August (13 July 2019 and 18 August 2019); thus, a total of five treatments were applied (including control) (Figure 2). Four trees were used per treatment, i.e., a total of twenty (20) trees.

During the second year in Crete (Heraklio County), three commercial products were used. Along with the two previously described ones, i.e., Surround WP and Invelop, a third one, Aglev^® SI 300 (attapulgite 100%) ((Mg,Al)₂ Si₄O₁₀(OH)₄(H₂O)) (Geohellas SA, Athens, Greece), was applied as recommended by the supplier at a registered dose rate of 2 kg 100 L⁻¹. The timing of spray applications was similar to those described above, i.e., a single application in August (21 August 2022—BBCH crop growth stage 76–77) and a double application both in July (BBCH crop growth stage 74–75) and August (21 July 2022 and 21 August 2022). Thus, a total of seven treatments were applied (including control). Four trees were used per treatment, resulting in a total of 28 trees.

The schedule of two applications in July and August was chosen for two reasons. First, it ensured that the trees remained covered by particle films during the harsh summer conditions in Greece, a period when olive fruits are growing and oil accumulation in the fruit is occurring (Figure 3). Typically, no rainfall is expected in these areas during this time, as was the case throughout the experimental period. Second, a single application was tested to evaluate its efficacy, potentially reducing the cost of particle film application for farmers. This was timed for August, when oil accumulation in the fruit begins to increase. In all spray applications, a surfactant (Haiten Plus 15SL, Phytorgan SA, Kato Kifissia, Greece, at the dose rate of 25 mL 100 L⁻¹) was added to the tank mix. It is not expected to have an influence on the efficacy of the treatments, as the dose rate is too small to expect any effect.

At harvest (24 November 2019 and 19 November 2022 in Viotia and Heraklio County, respectively), each tree was harvested separately, and the yield was recorded. Approximately 1.5 kg of free-from-leaves, healthy olive fruits were randomly sampled from each plot and transferred to the laboratory for further processing. A total of four samples (1.5 kg each) per treatment were assayed.

2.2. Olive Oil Extraction Procedure

The maturity index of the fruits was determined in the laboratory in a sample of 100 randomly selected olive fruits based on the color of their skin and pulp. The free-from-leaves and other debris olives were crushed using an Abencor-type olive mill (Callis S.A., Athens, Greece). The crusher operated at 2800 rpm, and the paste produced was malaxed for 30 min at 50 rpm and 25–27 °C in the malaxer. Following malaxation, 50 g of the paste was transferred to a 50 mL Falcon tube and centrifuged at 2500× g for 15 min to accurately determine the oil percentage in the paste. The remaining paste was centrifuged at 1400× g for 3–4 min using the semi-industrial centrifuge of the Abencor-type olive mill, and the oil produced was stored in filled bottles at 4 ± 2 °C until analysis.

2.3. Olive Oil Analyses

The determination of olive-oil-free acidity, peroxide index, and ultraviolet absorption at 232 and 270 (K232 and K270, respectively), as well as ΔK (delta kappa), was conducted according to the European Official Methods of Analysis 2016/1784.

2.4. Determination of Total Phenols

Phenolic compounds were extracted by adding 5 mL of hexane into 2.5 g of olive oil. After vortexing, 5 mL of methanol–water solution (60:40) was added, and the mixture was vortexed for 2 min. The sample was then centrifuged at 2500× g for 10 min, and the upper phase was discarded. The lower phase was used for the determination of the different phenol fractions. The total phenols, the total o-diphenols, and the total flavonoids were determined as described by Roussos et al. [21] and expressed as mg gallic acid equivalents (GAEs) kg⁻¹ olive oil, mg caffeic acid equivalents (CAEs) kg⁻¹ olive oil, and mg catechin equivalents (CtEs) kg⁻¹ olive oil, respectively.

Individual phenolic compounds were detected in the same phenolic extract by high-performance liquid chromatography (HPLC) (Shimadzu Nexera X2, Shimadzu Europa GmbH, Albert-Hahn-Str. 6-10, Duisburg, Germany) using a diode array detector operating at 260 nm, 280 nm, 325 nm, and 360 nm. A sample of 40 μL was injected in a Luna LC column 250 × 4.6 mm 5 μm C18 (2) 100 A (Phenomenex, 411 Madrid Avenue, Torrance, CA 90501-1430, USA) thermostated at 20 °C. The different phenols were eluted by a gradient program using two mobile phases, i.e., (A) methanol:acetonitrile (1:1) and (B) 1% v/v acetic acid in water. The flow rate was adjusted at 1 mL min⁻¹, and the gradient profile was as follows for solvent A: 10% from 0 to 5 min, 20% from 10 to 35 min, 25% from 45 to 55 min, 30% at 70 min, 40% at 100 min, and 70% from 106 to 123 min. A total of eleven (11) phenolic compounds were detected based on their retention time and spectra similarity with the corresponding standards. The phenolic compounds detected were hydroxytyrosol, tyrosol, vannilic acid, caffeic acid, vannilin, p-coumaric acid, ferulic acid, oleacein, oleocanthal, luteolin, and apigenin. The standards were purchased from Sigma-Aldrich (Sigma-Aldrich, Inc., PO Box 14508, St. Louis, MO, USA), and their concentration was expressed in mg kg⁻¹ olive oil.

2.5. Antioxidant Capacity

The antioxidant capacity of the oils was determined in the same extract of phenolic compounds based on two different assays, i.e., the diphenyl picryl hydrazyl (DPPH) and the ferric reducing antioxidant power (FRAP) assays, according to Roussos et al. [21]. The antioxidant capacity was expressed as μmol Trolox equivalent kg⁻¹ olive oil.

2.6. Fatty Acids and Squalene Determination

Fatty acid composition, expressed as a percentage of corresponding methyl esters (FAMES), was determined by diluting 0.5 g of olive oil in 2 mL of GC-grade hexane and vigorously shaking, followed by the addition of 1 mL of a 0.2 N solution of KOH. The methyl esters were analyzed by gas chromatography (Shimadzu GC-2030) (Shimadzu Europa GmbH, Albert-Hahn-Str. 6–10, 47269 Duisburg, Germany) equipped with an autosampler (AOC-20i+s), and a flame ionization detector (FID). The analysis was performed using a fused silica capillary column (Supelco SP^®-2340 60 × 0.32 mm × 0.2 μm film thickness) (Sigma-Aldrich, Inc., PO Box 14508, St. Louis, MO 63178, USA). A split ratio of 80.0 was used, and the injector temperature was set at 249 °C, while the detector temperature was set at 249 °C. Then, 10 µL of the sample was injected at an initial column temperature of 150 °C, which was held for 18 min. The temperature was then increased at a rate of 2 °C min⁻¹ to 175 °C, held for 5 min, and then raised at a rate of 5 °C min⁻¹ to a final temperature of 200 °C, which was held for 22 min. In oils from both cultivars, the following free fatty acids were determined: palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2), linolenic (C18:3), arachidic (C20:0), and gadoleic (C20:1) acids. In the ‘Koroneiki’ oil samples, heptadecanoic (C17), heptadecenoic (C17:1), behenic (C22), and lignoceric (C24) acids were also detected in small amounts. Squalene (Sigma-Aldrich, Inc., PO Box 14508, St. Louis, MO 63178, USA) concentration was determined by a five-point standard curve of the corresponding analytical standard and expressed as mg 100 g⁻¹ of olive oil.

2.7. Statistical Analysis

The trial was designed with four replicates of one tree in each orchard, i.e., four trees per treatment. The four plots (trees) within each orchard were randomly selected in each orchard’s area (the trial followed the completely randomized design). Significant differences among treatments for the same trial area (Viotia and Heraklio County) were determined based on the Tukey HSD test at a = 0.05 after checking the normal distribution of raw data using standard skewness, standard kurtosis, and the homogeneity of variances, and a suitable transformation was performed in those datasets that did not follow normal distribution. Hierarchical cluster analysis (Ward method) of raw data (olive oil quality indexes, FAMES content, phenolic compounds concentration, squalene, and antioxidant capacity as well as yield, oil percentage per fruit, and oil quantity per tree) was performed to obtain descriptive information on the effects of the various treatments. Constellation plots were also constructed to depict possible similarities of the various treatments based on the measured variables. The statistical software JMP 13.0 (SAS Institute) and Statgraphics Centurion XV (Statgraphics Technologies, Inc., The Plains, VA, USA) were used for the aforementioned analyses.

3. Results

3.1. Effects of the Treatments on Yield Components

The treatments applied in cv. ‘Megaron’ in Viotia County had a significant effect on the yield components (

Table 1. Effects of the various treatments on olive fruit yield, olive oil percentage, oil produced per tree, and fruit maturity index at harvest separately per cultivar.

Treatment	Yield (kg Tree−1)	Oil Percentage per Fruit	Oil per Tree (kg)	Maturity Index
	cv. ‘Megaron’ in Viotia County
Control	23.0 b	28.35 b	6.50 c	3.01 b
K-JA	30.0 b	27.47 b	8.25 bc	2.76 b
K-A	48.0 a	25.20 b	11.95 a	2.52 b
T-JA	26.9 b	33.22 a	8.91 b	2.52 b
T-A	24.3 b	29.20 ab	7.01 cd	4.13 a
	cv. ‘Koroneiki’ in Heraklio County
Control	36.1 ab	25.5 a	9.12 ab	2.66 a
At-JA	57.9 a	24.1 a	13.90 a	2.51 a
At-A	43.4 ab	24.3 a	10.67 ab	2.91 a
K-JA	30.1 b	22.3 a	6.52 b	3.06 a
K-A	46.2 ab	21.5 a	9.92 ab	2.83 a
T-JA	41.4 ab	20.9 a	8.72 ab	2.91 a
T-A	43.8 ab	25.5 a	10.77 ab	2.63 a

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: K, kaolin; T, talc; JA, application in both July and August; A, application in August; At, attapulgite.

). Kaolin application in August resulted in the highest yield and oil production per tree, reaching 48 kg of olive fruits and 11.95 kg of oil per tree. The lowest oil production per tree was determined under control conditions (only 6.50 kg per tree). The highest oil percentage was achieved in trees treated with talc both in July and August (33.22%), followed by talc treatment only in August (29.20%). The maturity index was highest under talc applied in August.

The treatments in cv. ‘Koroneiki’ in Heraklio County had a slightly different effect. The highest yield of fruits and the highest olive oil per tree was produced after applying attapulgite both in July and August, followed by the single application of kaolin in August (fruit yield) and a single talc application in August (olive oil production). The maturity index as well as the oil percentage per fruit was similar under all treatments.

3.2. Effects of the Treatments on Olive Oil Quality Indices

The single kaolin application in July resulted in the lowest free acidity of ‘Megaron’ oil, followed by the double kaolin application (

Table 2. Effects of the various treatments on oil-free acidity, peroxide index, K232, K270, and ΔK separately per cultivar.

Treatment	Free Acidity (g Oleic Acid 100 g−1)	Peroxides (meq O2 kg−1)	K232	K270	ΔK
cv. ‘Megaron’ in Viotia County
Control	0.59 a	15.62 a	1.82 a	0.15 a	0.006 a
K-JA	0.38 bc	13.12 ab	1.76 a	0.12 a	0.009 a
K-A	0.35 c	10.0 b	1.64 a	0.12 a	0.005 a
T-JA	0.55 ab	10.62 ab	1.81 a	0.14 a	0.011 a
T-A	0.59 a	12.50 ab	1.88 a	0.14 a	0.005 a
cv. ‘Koroneiki’ in Heraklio County
Control	0.40 a	8.75 a	1.61 ab	0.16 a	−0.06 bc
At-JA	0.40 a	12.5 a	1.90 a	0.20 a	0.005 ab
At-A	0.39 a	12.5 a	1.84 a	0.19 a	−0.025 abc
K-JA	0.42 a	8.75 a	1.97 a	0.20 a	0.025 ab
K-A	0.40 a	10.0 a	1.11 b	0.11 a	−0.127 c
T-JA	0.40 a	11.25 a	2.04 a	0.22 a	0.052 a
T-A	0.44 a	11.25 a	1.68 a	0.20 a	−0.015 ab

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: K, kaolin; T, talc; JA, application in both July and August; A, application in August; At, attapulgite.

). Peroxide number was highest under control conditions and lowest under the single kaolin application, while there were no other differences regarding K232, K270, and ΔK.

The treatments did not differ regarding free acidity, peroxide number, and K270 of the oil produced by ‘Koroneiki’, while the lowest K232 was determined under the single kaolin application in August.

3.3. Effects of the Treatments on Olive Oil Phenol Content and Antioxidant Capacity

The double kaolin application (July and August) in ‘Megaron’ resulted in oil rich in total phenols and with high antioxidant capacity based on the FRAP assay (

Table 3. Effects of the various treatments on olive total phenols, o-diphenols, and flavonoids concentration and antioxidant capacity separately per cultivar.

Treatment	Total Phenols (mg GAE kg−1)	o-Diphenols (mg CAE kg−1)	Total Flavonoids (mg CtE kg−1)	FRAP (μmol Trolox Equiv. kg−1)	DPPH (μmol Trolox Equiv. kg) −1
	cv. ‘Megaron’ in Viotia County
Control	245.5 b	139.5 a	127.0 b	1300.5 b	195.6 b
K-JA	500.9 a	159.8 a	254.3 ab	2153.9 a	636.6 ab
K-A	446.4 ab	150.9 a	271.7 a	1871.4 ab	739.2 a
T-JA	469.1 ab	109.1 a	250.1 ab	1883.2 ab	607.1 ab
T-A	415.5 ab	102.0 a	222.7 ab	1492.4 ab	665.9 ab
	cv. ‘Koroneiki’ in Heraklio County
Control	595.2 a	61.4 ab	590.1 a	2345.1 a	2618.2 a
At-JA	525.6 ab	124.7 a	473.4 ab	1768.7 ab	2134.8 a
At-A	497.2 ab	64.7 ab	404.5 ab	1747.9 ab	2069.7 a
K-JA	521.8 ab	55.0 b	369.2 ab	1642.2 b	1788.8 a
K-A	480.0 b	36.5 b	390.9 ab	1505.6 b	1898.1 a
T-JA	468.0 b	69.1 ab	355.7 ab	1598.0 b	1997.9 a
T-A	468.1 b	38.5 b	349.8 b	1525.4 b	1764.7 a

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: K, kaolin; T, talc; JA, application in both July and August; A, application in August; At, attapulgite.

). On the contrary, control oil exhibited the lowest total phenol concentration and antioxidant capacity (based both on FRAP and DPPH assays). Kaolin single application resulted in oil rich in flavonoids and of high antioxidant capacity (based on DPPH assay).

On the other hand, ‘Koroneiki’ control trees produced oil rich in total phenols and flavonoids and of high antioxidant capacity (based on FRAP assay). There was no significant difference among treatments regarding antioxidant capacity measured by the DPPH assay, while the double attapulgite application resulted in high o-diphenol concentration in the oil.

The phenolic compound with the highest concentration in olive oils from both cultivars was oleocanthal, followed by oleacein, tyrosol, and hydroxytyrosol (

Table 4. Effects of the various treatments on the concentration of individual phenolic compounds detected in olive oils in mg kg⁻¹ separately per cultivar.

Treatment	Hydroxytyrosol	Tyrosol	Vanillic Acid	Caffeic Acid	Vanillin	p-Coumaric Acid	Ferulic Acid	Oleacein	Oleocanthal	Luteolin	Apigenin
	cv. ‘Megaron’ in Viotia County
Control	23.38 a	28.00 a	0.27 b	0.02 a	0.09 a	0.98 a	0.15 bc	42.7 a	152.7 ab	9.5 ab	1.23 ab
K-JA	13.61 b	23.78 ab	0.76 a	0.03 a	0.07 a	0.87 a	0.25 a	23.5 bc	185.6 a	11.2 ab	1.54 a
K-A	13.72 b	16.84 c	0.89 a	0.02 a	0.06 a	0.59 ab	0.20 ab	19.1 bc	178.1 a	11.7 a	1.52 a
T-JA	16.14 ab	18.77 bc	0.29 b	0.03 a	0.06 a	0.71 ab	0.13 c	36.1 ab	92.3 bc	6.5 bc	0.84 bc
T-A	7.54 b	8.11 d	0.15 b	0.02 a	0.08 a	0.30 b	0.06 d	13.2 c	49.8 c	1.9 c	0.19 c
	cv. ‘Koroneiki’ in Heraklio County
Control	13.49 a	24.75 a	0.43 a	0.02 ab	0.03 ab	0.39 a	0.05 a	18.0 a	169.0 a	15.7 a	2.95 a
At-JA	11.91 a	23.71 a	0.70 a	0.01 b	0.04 a	0.26 bc	0.05 a	28.4 a	158.3 a	9.1 ab	2.54 ab
At-A	13.17 a	20.63 ab	0.34 a	0.01 b	0.03 ab	0.20 c	0.04 a	26.1 a	142.7 a	9.4 ab	1.99 ab
K-JA	8.16 a	21.27 ab	0.52 a	0.04 a	0.02 b	0.33 ab	0.03 a	14.0 a	185.6 a	10.3 ab	1.76 ab
K-A	10.13 a	18.47 b	0.72 a	0.01 b	0.03 ab	0.37 a	0.06 a	22.0 a	137.9 a	8.5 b	2.08 ab
T-JA	9.47 a	17.29 b	0.76 a	0.01 b	0.03 ab	0.32 ab	0.04 a	18.6 a	160.7 a	10.0 ab	1.66 b
T-A	7.34 a	21.21 ab	0.69 a	0.01 b	0.03 ab	0.34 ab	0.04 a	20.1 a	141.6 a	9.0 ab	1.82 ab

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: K, kaolin; T, talc; JA, application in both July and August; A, application in August; At, attapulgite.

). Olive oil from the control treatment of cv. ‘Megaron’ exhibited the highest concentration of hydroxytyrosol, tyrosol, p-coumaric acid, and oleacein. The double application of kaolin clay particles resulted in high concentrations of vanillic acid, p-coumaric acid, ferulic acid, oleocanthal, and apigenin, while the single application produced oils rich in vanillic acid, oleocanthal, and luteolin.

There were not many differences regarding the concentration of phenolic compounds found in oils derived from ‘Koroneiki’ under the influence of the treatments imposed. Oils from the control treatment presented high concentrations of tyrosol, luteolin, and apigenin, while the double attapulgite application resulted in oils with high tyrosol and vanillin concentrations. Kaolin double application resulted in a high caffeic acid concentration in the oil, while the single one resulted in a high p-coumaric acid concentration. As with ‘Megaron’, oils produced after talc application in most cases presented low phenolic compound concentrations.

3.4. Effects of the Treatments on Olive-Oil-Free Fatty Acid Content

Significant differences were detected among the olive oils of cv. ‘Megaron’ produced under the influence of the different treatments regarding the free fatty acid content (

Table 5. Effects of the various treatments on the content of free fatty acids and on various groups of free fatty acids (determined as FAMEs—%) and squalene concentration in the oil of cv. ‘Megaron’ in Viotia County.

Treatment	C16	C16:1	C18	C18:1	C18:2	C20	C18:3	C20:1
Control	16.99 b	1.62 a	2.05 b	63.65 a	14.71 b	0.23 b	0.59 a	0.14 a
K-JA	17.33 ab	1.70 a	2.16 b	62.22 a	15.59 ab	0.27 ab	0.59 a	0.15 a
K-A	16.74 b	1.72 a	2.46 a	64.33 a	13.85 b	0.29 a	0.53 a	0.13 a
T-JA	17.14 b	1.59 a	2.20 b	63.13 a	14.85 b	0.25 ab	0.58 a	0.16 a
T-A	17.87 a	1.68 a	2.52 a	59.59 b	17.27 a	0.28 a	0.59 a	0.15 a
	Various groups of free fatty acids and squalene
	SFA	MUFA	PUFA	UFAs	MUFA/PUFA	SFA/UFA	C18:1/C18:2	Squalene (mg 100 g−1)
Control	19.3 b	65.4 a	15.3 b	80.7 a	4.27 a	0.24 b	4.32 ab	414.4 a
K-JA	19.8 b	64.0 a	16.2 ab	80.2 a	3.99 ab	0.25 b	4.03 b	398.6 a
K-A	19.5 b	66.0 a	14.4 b	80.5 a	4.59 a	0.24 b	4.66 a	386.3 a
T-JA	19.6 b	65.0 a	15.4 b	80.4 a	4.23 a	0.24 b	4.26 ab	402.8 a
T-A	20.7 a	61.4 b	17.9 a	79.3 b	3.44 b	0.26 a	3.145 c	344.9 a

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: K, kaolin; T, talc; JA, application in both July and August; A, application in August.

). Single talc application resulted in oils rich in C16, C18, C18:2, and C20 and low in C18:1. Similarly, the single kaolin application resulted in oils also rich in C18 and C20, but they were rich in C18:1 (without any difference from the control or the double application of either kaolin or talc). As a result of these differences, oils produced under the single talc application were rich in SFAs and PUFAs, with a high ratio of SFA/UFA but a low C18:1/C18:2 one. All other treatments resulted in oils rich in MUFAs and UFAs with a high ratio of MUFA/PUFA (except for the double kaolin application). There were, however, no differences concerning squalene concentration.

There were not so many differences in fatty acid concentration in the oils of the ‘Koroneiki’ cultivar (

Table 6. Effects of the various treatments on the content of free fatty acids and on various groups of free fatty acids (determined as FAMEs—%) and squalene concentration in the oil of cv. ‘Koroneiki’ in Heraklio County.

Treatment	C16	C16:1		C17		C17:1	C18		C18:1		C18:2	C20		C18:3		C20:1		C22	C24
Control	12.74 a	0.86 a		0.026 a		0.036 a	2.69 b		76.32 a		5.96 ab	0.41 c		0.54 a		0.25 a		0.12 a	0.03 a
At-JA	12.44 a	0.77 bc		0.028 a		0.036 a	2.74 ab		77.05 a		5.51 b	0.43 abc		0.56 a		0.27 a		0.12 a	0.03 a
At-A	12.71 a	0.81 ab		0.022 a		0.036 a	2.76 ab		76.17 a		6.11 ab	0.42 bc		0.54 a		0.26 a		0.12 a	0.03 a
K-JA	12.34 a	0.74 bc		0.029 a		0.036 a	2.93 ab		75.90 a		6.56 ab	0.44 ab		0.58 a		0.26 a		0.13 a	0.02 a
K-A	12.38 a	0.71 c		0.023 a		0.035 a	2.93 ab		76.34 a		6.08 ab	0.45 ab		0.59 a		0.27 a		0.13 a	0.03 a
T-JA	12.52 a	0.75 bc		0.025 a		0.040 a	3.05 a		75.52 a		6.64 a	0.46 a		0.58 a		0.26 a		0.12 a	0.02 a
T-A	12.61 a	0.78 abc		0.028 a		0.042 a	2.83 ab		75.83 a		6.41 ab	0.44 abc		0.58 a		0.27 a		0.12 a	0.03 a
	Various groups of free fatty acids and squalene
	SFA		MUFA		PUFA			UFAs		MUFA/PUFA			SFA/UFA		C18:1/C18:2		Squalene (mg 100 g−1)
Control	16.03 a		77.46 a		6.50 ab			83.96 a		11.92 ab			0.16 a		12.82 ab		170.6 a
At-JA	15.79 a		78.12 a		6.07 b			84.20 a		12.89 a			0.19 a		14.02 a		209.2 a
At-A	16.07 a		77.27 a		6.65 ab			83.92 a		11.66 ab			0.19 a		12.52 ab		185.5 a
K-JA	15.90 a		76.94 a		7.15 ab			84.09 a		10.79 b			0.19 a		11.59 ab		174.0 a
K-A	15.95 a		77.36 a		6.68 ab			84.04 a		11.58 ab			0.19 a		12.55 ab		188.0 a
T-JA	16.20 a		76.57 a		7.22 a			83.79 a		10.64 b			0.19 a		11.42 b		151.1 a
T-A	16.07 a		76.93 a		6.98 ab			83.92 a		11.16 ab			0.19 a		12.03 ab		185.1 a

Means within the same column and for the same county/cultivar followed by the same letter do not differ significantly based on the Tukey HSD multiple range test at a = 0.05. Abbreviations: At, attapulgite; K, kaolin; T, talc; JA, application in both July and August; A, application in August.

). Oils produced under control conditions presented a high content of C16:1, while the double talc application resulted in high C18, C18:2, and C20 content. As a result, the PUFA content was high in oils produced under the influence of the double talc application compared to the double attapulgite application, while the ratios MUFA/PUFA and C18:1/C18:2 were higher under the double attapulgite application, with a significant difference from the double talc application.

3.5. Hierarchical Cluster Analysis Results

Taking into account all the harvest parameters and oil quality indices of the ‘Megaron’ cultivar, it is obvious that the single talc application separates from the other treatments, characterized by high levels mainly of free fatty acids (C16, C18:2, PUFAs, SFAs, C18, and C20), low levels of phenolic compounds (such as o-diphenols, oleocanthal, oleacein, apigenin, etc.) and squalene, and low yield and low oil production (Figure 4). On the other hand, kaolin, either single or double, seems to give similar results. Interestingly, when the analysis took place, taking into consideration only the products used and not the number of spray applications, talc was separated from kaolin and control treatments, which were located close to each other in the constellation plot (Figure 5). Talc application, regardless of the times it was applied, resulted in a high olive percentage and fruit maturity index as well as a high content of some free fatty acids, as previously described for the single talc application. At the same time, it resulted in oils with low phenolic compounds and squalene as well as oleic acid (C18:1).

In the ‘Koroneiki’ cultivar, the hierarchical clustering analysis produced less distinctive results (Figure 6). Talc treatments seem to lead to similar results concerning harvest data and oil quality indices, as both treatments resulted in low levels of many of the attributes tested in this trial. The double application of attapulgite was grouped quite close to the control treatment. Irrespective of the application number a product was applied, the analysis produced quite clear results (Figure 7). All treatments were separated from each other, as depicted by the constellation plot. It is quite interesting to see that attapulgite treatment resulted in high yield, peroxide number, oil production, oleacein, and o-diphenols concentration, as well as high content of some free fatty acids (C18:1, MUFAs, and UFAs) and squalene. The control treatment was characterized by a high oil percentage in the fruits and some phenolic compounds (vanillin, total phenols, total flavonoids, apigenin, and luteolin) and antioxidant capacity (DPPH and FRAP). Kaolin resulted in high maturity index olive fruits and oils high in some free fatty acids (C22, C18, C20, C20:1, and C18:3). Application of talc, on the other hand, resulted in oils with high free acidity, such as K232 and K270, and free fatty acids, like C18:2, C17:1, and SFAs.

4. Discussion

The hierarchical clustering of the two trials revealed both differences and similarities among treatments in terms of production traits and olive oil quality. As this is the first report on the use of talc and attapulgite on olive trees, interpretations of their mode of action are based on current knowledge of how PFT works.

Under rainfed conditions, where the ‘Megaron’ cultivar was grown, kaolin—followed by talc to a lesser extent—significantly improved fruit yield and oil production per tree. Additionally, double talc application enhanced oil percentage per fruit mass. In the irrigated ‘Koroneiki’ cultivar, clay particle treatments slightly increased yield and oil production, except for double kaolin application. The maturity index remained unchanged in ‘Koroneiki’ but was advanced in ‘Megaron’ with a single talc application, probably a result of the low efficacy of the single talc application to alleviate stress, resulting in an acceleration of maturation compared to other particle films [22] or a result of the chemical and/or optical properties of talc, which should be further examined.

Particle film technology reduces leaf temperature, maintaining stomatal function under conditions that would otherwise impair photosynthesis and transpiration [9]. Kaolin applications have been shown to decrease olive leaf temperature by approximately 2.5 °C, with irrigated trees generally exhibiting lower leaf temperatures than rainfed ones [7,8], alleviating the heat stress and allowing the unhindered function of the photosynthetic mechanism. Such a function of kaolin particle film has been described in several species, such as apple, pomegranate, tomato, coffee, etc. [9,14,23,24,25]. The increase in photosynthesis rate under stress conditions, due to the protective film formed on leaves, results from a multi-level alleviation effect. It is expected that, as stomata remain functional to a certain degree, this will influence transpiration rates and, consequently, water and nutrient uptake. Improved [9,14,23,24,25] hydration and nutrition could explain the higher yields recorded under rainfed conditions in the present experiment. Furthermore, it has been found that a kaolin film mitigates oxidative stress under high heat loads, enabling plant functions to operate more effectively than under control conditions, thus supporting growing fruit and enhancing yield [26]. Similar benefits are anticipated from other clay particles, such as talc and attapulgite, which form reflective films on leaf surfaces. However, as this is the first report on the effects of talc on olive trees, it would be premature to assume that the reflective capacity and overall properties of films formed by these clay materials are identical. However, under irrigated conditions, the effectiveness of particle films tends to be lower [11,12], as irrigation itself helps alleviate heat stress to some extent. This was observed in the present trial, where particle clay treatments slightly increased yield and oil production, suggesting that irrigation alone may not sufficiently mitigate heat stress under Crete’s summer conditions [27].

The lower yield and oil production in ‘Koroneiki’ under double kaolin application is consistent with prior reports of reduced growth or biomass accumulation when particle films are applied under optimal irrigation conditions [7,11,12,26]. However, genotype-specific responses to particle film treatments should also be considered, as previously reported in both olive [7,8,28,29] and other species [11,30]. The ‘Megaron’ cultivar is characterized by medium-sized, longitudinally shaped leaves, while ‘Koroneiki’ has smaller leaves and is considered more tolerant to drought stress [31]. These morphological differences may have influenced the varying effectiveness of the clay particles in this study.

Regarding oil quality, a single kaolin application in ‘Megaron’ resulted in superior olive oil attributes, including lower free acidity and peroxide values, compared to both the control and talc treatments. Low free acidity is highly desirable in the olive oil industry, and kaolin has been shown to contribute to reduced acidity in multiple cultivars such as ‘Konservolia’ [26], ‘Zard’ [32], and ‘Zeity’ [19]. These improvements in oil quality could be attributed to kaolin’s ability to reflect UV radiation, reduce fruit temperature, and enhance tissue hydration. According to several authors [33,34], under warm conditions with low precipitation, the olive oil produced is characterized by higher acidity than that produced under cooler and more humid environments. Thus, kaolin-treated olive canopies, as mentioned above, could have been cooler than the canopies of control trees [7,8], thereby preserving low oil acidity. At the same time, kaolin protects the fruits from olive fruit fly infestation [18], which is known to be a major factor contributing to increased oil acidity due to hydrolytic and oxidative reactions following fly attacks [35]. Furthermore, as previously suggested, kaolin application on olive trees alters the plant’s oxidative status [36,37], resulting in olive oils with reduced free acidity [26].

This trend was evident in the irrigated ‘Koroneiki’ cultivar, where no significant effects of clay treatments were observed on free acidity or peroxide values, except for slight reductions in K232 and ΔK under single kaolin application. This could be partly attributed to the already alleviated heat stress by irrigation, combined with the additional alleviation provided by kaolin clay particles, as discussed above and by other researchers [33,34]. Similar non-significant changes in acidity [13,19] and peroxide number [6,13] have been reported under clay particle applications, likely due to factors such as environmental conditions, genotype, spray application timing, frequency, and severity of abiotic stress. Regardless, all produced oils met the criteria for extra virgin olive oil (EVOO).

Kaolin treatments also enhanced the functional properties of ‘Megaron’ oil, leading to increased total phenol and flavonoid concentrations and higher antioxidant capacity, as reported in previous studies [13,32]. This effect is likely due to kaolin’s UV-reflecting properties, which lower fruit temperatures. Particle films applied to stressed olive trees, such as rainfed ‘Megaron’, significantly influence phenol biosynthesis, though the specific impact varies based on the mineral clay used [13].

Nonetheless, there are reports where olive oil phenolic content is related more to environmental conditions than to kaolin treatment [11]. Interestingly, the irrigated ‘Koroneiki’ oils from control and attapulgite-treated trees had the highest phenol concentrations and antioxidant capacities, while talc-treated trees produced oil with the lowest phenolic content. This suggests that both genotype and cultivation conditions (irrigation) significantly affect phenol content and antioxidant capacity. Phenolic compounds act as non-enzymatic antioxidants under stress, mitigating oxidative damage [30,38]. This could justify the increase in phenol concentration in oils from control ‘Koroneiki’ trees grown under irrigated conditions, as the applied water was probably not enough to minimize leaf and fruit heat load under summer conditions in Crete. Applying clay particles on the trees resulted in an alleviation of heat load stress, leading to the generation of fewer phenolic compounds [28,30]. This was most evident in irrigated ‘Koroneiki’ under talc and kaolin treatments, suggesting a potentially higher efficacy of these two clay materials in alleviating heat stress. Nonetheless, further research is needed to elucidate the distinct effects of these clay particles and their impact on leaf and whole-plant physiology. Conversely, when stress exceeds the plant’s capacity to regenerate antioxidant molecules, the rate of phenolic compound oxidation surpasses their rate of biosynthesis, leading to a reduction in their tissue concentration. This may be the case with the ‘Megaron’ cultivar under control conditions. Trees likely experienced severe drought and heat stress, unable to maintain phenol production rates sufficient to match phenol oxidation rates, resulting in oils with low phenolic content. Supporting this hypothesis are the results following kaolin application, where the oils produced exhibited higher phenolic content than those from control trees, indicating that kaolin effectively reduced heat and drought stress in ‘Megaron’ trees. Under these conditions, it could be expected that the need for phenols acting as antioxidant substrates to protect physiological functions would be diminished, thereby resulting in a higher concentration in the oil produced.

The applied clay particles had a significant impact on the concentration of the individual phenolic compounds detected in the produced olive oils. A high concentration of hydroxytyrosol, tyrosol, oleacein, and p-coumaric acid was determined in oils derived from the control treatment in the ‘Megaron’ cultivar. Kaolin treatment, on the other hand, resulted in high concentrations of vanillic and ferulic acids and oleocanthal, while talc application resulted in the lowest values. These differences align with previous findings [13], which attribute variations in phenolic profiles to the mineral properties of the clay particles, their ability to filter UV radiation, and their effects on leaf and fruit temperature and hydration [13].

This study is the first to evaluate attapulgite as a foliar spray, and its efficacy appears distinct from other clay minerals. Similarly, talc was less effective in irrigated ‘Koroneiki’ trees under Crete’s summer conditions. However, it must be noted here that the pulp moisture of the fruit greatly affects phenol transferring from the olive paste to the oil [6] and phenols’ loss in the aqueous phase after centrifuge. Future studies should measure fruit water content under different treatments to better explain the observed differences in phenolic content and antioxidant capacity. Additionally, it would be interesting to test, apart from the reflecting and physical properties of the clay minerals on plant tissue surfaces, their ability to supply mineral nutrients (such as aluminum, sulfur, and magnesium, which is not a component of kaolin in contrast to talc and attapulgite) to the plants, minerals found in different concentrations and forms in these products. Possible differences in mineral nutrient concentration and availability may have an effect on plant metabolism and phenol biosynthesis and explain the detected differences.

A noticeable effect of the single talc application on ‘Megaron’ was the lowest concentration of C18:1 in the oil produced, with a simultaneous increase in C16, C18, C18:2, C18:3, and C20, compared to the control. It seems that talc treatment increased the saturated fatty acids of the oil and decreased the mono-unsaturated fatty acids, also resulting in the lowest ratio of C18:1/C18:2. Many pre-harvest factors influence this ratio, favoring either C18:1 or C18:2 [39,40]. As all ‘Megaron’ trees were cultivated in the same orchard, cultivation practices and pedoclimatic factors should be ruled out. However, based on data regarding the effect of air temperature on these two fatty acids, it has been reported that under warm conditions, the C18:1 and monounsaturated fatty acid (MUFA) content decreases while the C18:2 content increases, whereas under cooler conditions, the opposite occurs [22,40,41,42,43,44]. Given this evidence, it is reasonable to conclude that a single talc application in August failed to sufficiently lower fruit and canopy temperatures to prevent the observed decrease in C18:1 content and the subsequent increase in C18:2 content. This further supports the advanced maturity index observed with the single talc application, likely resulting from severe stress that accelerates peel color development and fruit maturation [22]. On the other hand, the differences induced by the application of clay particles were quite few in ‘Koroneiki’. The double talc application resulted in higher C18 and C20 concentrations compared to the control and kaolin single application in lower C17:1. According to Khaleghi et al. [32], kaolin and, to our belief, all the other mineral clay particles influence fatty acid composition by modulating the expression of genes involved in the biosynthesis of fatty acids through the alleviation of heat stress. Consequently, kaolin clay particles altered the composition of fatty acids in olive oil, decreasing C16:0, C18:2, and C18:3 and increasing [22,40,41,42,43,44] C18:1, MUFAs, and the ratio of C81:1/C18:2 [32]. On the other hand, Rotondi et al. [13] reported an increase in C18:2 and decreased C18:3 content. Such effects of kaolin were not observed in the present trial, similar to that reported by Taskin and Ertan [17] and by Karaat and Denizhan [45] in almonds treated with kaolin and other reflecting materials. These discrepancies may be attributed to multiple factors, including genotype, environmental conditions during growth [13], application timing, fruit maturity at harvest, and the severity of stress [46]. The olive tree is an emblematic species for the Mediterranean basin, and although quite tolerant, climate change greatly influences its growth and production by disrupting key physiological mechanisms (photosynthesis, transpiration, nutrition, and antioxidant mechanisms), with substantial impacts on fruit production and product quality [47,48,49,50,51]. Therefore, further research is needed to refine the application of particle film technology in olive cultivation and optimize its effects on olive fruit and oil quality, particularly in the context of the unfolding climate crisis scenarios.

5. Conclusions

This study marks the first investigation into the use of talc and attapulgite as a foliar spray and their effects on olive tree production. The application of all three mineral clay particles led to varying degrees of improvement in olive yield and oil production while influencing oil quality and functional properties to some extent. However, further research is necessary to evaluate the effectiveness of these mineral clays across a wider range of cultivars, pedoclimatic conditions, and cultivation practices, as it is evident that no single clay material performs optimally under all conditions.