Effect of Ozonated Avocado and High-Oleic Palm Oils on “Bolo Verde” Variety Squash

Abstract

Ozonated oils have promise as biostimulants, positively affecting physiological processes that promote plant growth and biomass accumulation. However, additional research is required to clarify their mechanisms of action, optimize dosages, and define effective application strategies. This study aimed to evaluate the biostimulant effect of three concentrations of two oils, avocado (Persea maricana Mill cv Hass) (50, 100, and 200 meqO₂ kg⁻¹) and high-oleic palm (Elaeis guineensis Jacq.) (5, 10, and 20 meqO₂ kg⁻¹), on the “Bolo Verde” squash Cucurbita moschata. The experiment followed a completely randomized design with a three-factor factorial arrangement: Factor I—type of ozonated oil; Factor II—application concentration (low, medium, and high); Factor III—application method (drench or foliar). The trial consisted of 15 experimental units, each with 32 plants, totaling 480 plants. Data were analyzed using SAS software. A one-way ANOVA was performed, and means were compared using Tukey’s test p ≤ 0.05. The drench application of high-concentration ozonated avocado oil (200 meqO₂ kg⁻¹) produced the most favorable biostimulant response, significantly increasing plant height, leaf number, root length, root volume, and total dry weight. This was followed by the drench application of low-concentration ozonated high-oleic palm oil (5 meqO₂ kg⁻¹), which yielded the highest dry matter accumulation. For the net assimilation rate (NAR) and leaf area index (LAI), the drench application of ozonated avocado oil at a high concentration resulted in 4.29 g cm⁻² day⁻¹ NAR and 7957.99 LAI, while low-concentration high-oleic palm oil recorded 4.36 g cm⁻² day⁻¹ NAR and 7208.40 LAI. Both treatments showed statistically significant differences (p < 0.05) compared to the control 2.35 g cm⁻² day⁻¹ NAR and 6780.24 LAI, indicating improved photosynthetic efficiency and leaf expansion. Similar trends were observed for crop growth rate (CGR) and relative growth rate (RGR). The drench application of high-concentration ozonated avocado oil yielded a CGR of 6.77 × 10⁻⁴ g cm⁻² day⁻¹ and RGR 0.0441953 g g⁻¹ day⁻¹. Low-concentration high-oleic palm oil drench application resulted in the highest CGR of 7.35 × 10⁻⁴ g cm⁻² day⁻¹ and RGR 0.0454216 g g⁻¹ day⁻¹. These values were significantly higher than those of the control (CGR 4.14 × 10⁻⁴ g cm⁻² day⁻¹; RGR 0.0357569 g g⁻¹ day⁻¹). These results suggest that the drench application of ozonated oils not only enhances photosynthesis and leaf growth but also favors the incorporation and accumulation of biomass in “Bolo Verde” squash.

1. Introduction

Squash has been a traditionally important crop worldwide, particularly in certain regions of the Americas, Asia, and Europe. Throughout history, it has served as a staple ingredient in numerous foods, as well as for medicinal and agroindustry purposes [1]. Squash is rich in carotenoids, pectins, and potassium, among other nutrients; due to its high nutritional content, it has maintained its popularity, and large areas have been dedicated to its cultivation [2]. As with any crop, squash has been subjected to agrochemical treatments and pesticide applications, which may pose environmental risks and impact human health.

Currently, organic agriculture faces multiple challenges, including pest and disease management, as well as the ongoing search for sustainable alternatives to traditional agrochemicals. It is essential to reduce environmental impacts and ensure safe food production, minimizing dependency on chemical pesticides and avoiding harmful residues on crops. In this context, ozonated oils emerge as an ecological alternative, contributing to improved soil conditions and promoting a healthy environment for plant growth [3].

Ozonated oils are substances obtained from lipid oxidation, which results from the reaction of ozone with fatty acids and other compounds present in vegetable oils [4]. When ozone reacts with the double bonds of triacylglycerols, various oxygen-derived products are formed, such as ozonides, hydroperoxides, aldehydes, peroxides, diperoxides, and 1,2,4-trioxolane rings [5].

Given the importance of vegetable oils in agricultural applications, Colombia is globally recognized as the fourth-largest producer of high-oleic palm oil [6] and the fourth-largest producer of avocado [7]. For the present study, avocado and high-oleic palm vegetable oils were selected due to their high content of monounsaturated and polyunsaturated fatty acids. These oils contain between 53% and 68% monounsaturated fatty acids, and between 10% and 12% polyunsaturated fatty acids. For the lipid ozonation process, it is essential that the oils are rich in monounsaturated and polyunsaturated fatty acids, as the presence of unsaturation makes them highly susceptible to reacting with ozone [8,9], and as a consequence, ozonized oils are produced.

These oils contain ozonides, hydrogen peroxide, and diperoxides, which possess fungicidal, bactericidal, and insecticidal properties [10]. Their high oxidative potential makes them ideal for controlling plant diseases caused by fungi in various crops [11] as well as for combating viruses and reducing damage caused by insects [12]. They have also been effective against both Gram-positive and Gram-negative bacteria [13]. When the oil comes into contact with a microorganism, adverse effects are observed in the cytoplasm, along with a reduction in nucleic acid content [14].

However, overdosing can cause abiotic stress, leading to tissue necrosis. On the other hand, applying appropriate doses of these oils favors vegetative growth by acting as a biostimulant, promoting increased oxygen availability to the plant, activating its defense mechanisms, and stimulating the development of roots, stems, leaves, and fruits, resulting in higher agricultural yield [15,16,17,18].

Studies indicate that ozone can be used in seedling management due to its oxidizing potential and its capacity to activate the antioxidant system, as well as in seed production and germination [3]. Additionally, the biostimulant effect contributes to improving production index, optimizing physiological responses, and increasing crop profitability [1,2,11,17,19,20].

Ozonated oil may demonstrate greater efficiency compared to conventional chemical pesticides in combating certain pests and diseases [21]. Moreover, it is generally considered less toxic and less harmful to the environment. When applied to the soil, ozonated oil promotes proper plant development by improving oxygen availability at the root level, a key element in various biochemical processes, including carbohydrate metabolism. It also supports protein renewal, facilitates nitrogen symbiotic fixation, and enhances nutrient absorption, all of which positively impact plant growth and development [22].

Although various ozonated oils have been used in agriculture, no reports were found on the specific use of ozonated avocado and high-oleic palm oils with microbial or biostimulant activity. This study aims to evaluate the effect of avocado and high-oleic palm ozonated oils on Cucurbita moschata “Bolo Verde” at three concentrations (low, medium, and high) to determine their influence on growth, development, and physiological performance.

2. Materials and Methods

2.1. Study Location

The study was conducted in the botanical garden of the University of Caldas, in the city of Manizales, located at an altitude of 2153 m above sea level, with an average temperature of 17 °C and relative humidity between 77% and 83%. Specifically, it was carried out in the hydroponic cultivation area, which has semi-controlled climatic conditions, i.e., with controlled exposure to sunlight and controlled irrigation.

2.2. Ozonation Process

An ozonation system from the brand Bioteco^® from Bioteco SAS, located in Medellin (Antioquia), Colombia was used, with an ozone gas flow rate of 120 L h⁻¹ (O₃ ≈ 35 g L⁻¹). Seven ozonation reactions were carried out using medical-grade oxygen as the gas source, with bubbling times of 18 h for avocado oil and 12 h for high-oleic palm oil. Each oil was processed in triplicate for each reaction time. A volume of 500 mL of vegetable oil was placed in a dark glass bubbling reactor positioned on a magnetic stirrer, with constant agitation at 1000 rpm to ensure sample homogeneity throughout the reaction. All reactions were conducted at room temperature (20 °C).

2.3. Peroxide Value (PV) Determination

This procedure was carried out using the iodometric method to determine the peroxide value in oils and fats. Standardizations were performed through visual endpoint detection following the NTC 236 standard [23] and the international reference standard ISO 3960:2017 [24]. The peroxide value is defined as “a measure of the amount of chemically bound oxygen in an oil or fat in the form of peroxides, particularly hydroperoxides,” and is expressed in milliequivalents of active oxygen per kilogram of oil (meqO₂ kg⁻¹).

2.4. Formulation of Ozonated Oil Concentrations

This study aims to determine the optimal concentration of ozonated oils to evaluate their biostimulant or phytotoxic effects on plants. To this end, plant tolerance to different doses was analyzed to establish the overdose threshold that could negatively affect plant development.

Ozonated palm oil was used at low concentrations (5–20 meqO₂ kg⁻¹), as preliminary studies indicated that its high antioxidant content makes it more resistant to the ozonation process. As a result, it generates fewer peroxides compared to avocado oil, which, due to its higher oxidative and degradative capacity, exhibits a significantly greater peroxide concentration expressed in meqO₂ kg⁻¹.

For this reason, in the present study, avocado oil was evaluated at concentrations ten-times higher than those of high-oleic palm oil, in order to determine how this difference in peroxide concentration influences its biostimulant or phytotoxic effect on “Bolo Verde” squash plants.

2.5. Experimental Design and Establishment

Two vegetable oils, avocado and high-oleic palm, were evaluated in a concentration ratio of 4:1, with avocado oil being at a higher concentration. From these concentrations, 1 L solutions were prepared at different levels: low, medium, and high. Each oil was applied in two ways, foliar and drench, in a factorial design (2 oils × 3 concentrations × 2 application methods). A completely randomized experimental design with three factors was employed. The first application was carried out when the plants were at the V-3 phenological stage (presence of two true leaves), and the second application was at the V-5 phenological stage (sprouting lateral branches or with five true leaves) (

Table 1. Application of two ozonated oils of avocado and high-oleic palm at three different concentrations with two types of application in two application stages in “Bolo Verde” variety squash plants.

			Concentration meqO2 kg−1
Treatment	Oil Type	Application Type		Application 1 (V3 Stage)	Application 2 (V5 Stage)
1	Avocado	Foliar	Low	50	50
2		Drench
3		Foliar	Medium	100	100
4		Drench
5		Foliar	High	200	200
6		Drench
7	High-oleic Palm	Foliar	Low	5	5
8		Drench
9		Foliar	Medium	10	10
10		Drench
11		Foliar	High	20	20
12		Drench
13	Commercial Oxyzhen	Foliar		Commercial dose 5 cm3 L−1	Commercial dose 5 cm3 L−1
14		Drench
15	Control (water)	---		---	---

). The application mixture of the ozonated oils included an adjuvant whose active ingredients were soybean oil 862 g L⁻¹ and sodium isooctyl polyethoxyethanol sulfonate 68 g L⁻¹ at a dose of 5 cc. For this trial, a commercial product (Oxyzhen^®) from Oxy Zhen SAS, located in Chía (Cundinamarca), Colombia, was used as a control. This ozonized oil was applied at the manufacturer’s recommended dose of 1 L per 200 L of water, using both foliar and drench methods. Irrigation water was used as an absolute control.

The experiment was established in five troughs, each measuring 4.5 m in length, 0.7 m in width, and 0.45 m in depth. The 15 treatments were randomly distributed across these five troughs. Each treatment consisted of 32 plants, totaling 480 plants for the entire experiment.

Sixteen-ounce containers were disinfected by washing them with soap and water, followed by immersion in a 5.25% NaClO solution. The substrate used was solarized to reduce the presence of fungal, bacterial, or nematode pathogens. Subsequently, the containers were filled with 454 g of moistened substrate, and two squash seeds were sown in each container. The seeds used were produced by the research unit of the National University of Colombia, Palmira campus, under ICA registration No. 2542 of 2004.

2.6. Crop Management

Following sowing, irrigation was consistently applied every three days. After germination, the frequency was increased to every two days due to the higher water requirements of the developing plants. Thinning was performed eight days after emergence when the seedlings had developed one true leaf. Once the cotyledons had emerged, an insecticide with the active ingredient Carbaryl at 80% was applied to protect the plants from cutworm pests. Starting eight days after emergence, fertigation was carried out twice a week for two weeks using a N-P-K fertilizer (13-6-40 and minor elements) at a dose of 0.15 g L⁻¹.

2.7. Evaluated Variables

For the evaluation of the variables, all 32 plants from each of the 15 treatments were considered, totaling 480 plants. The evaluated variables were as follows:

Plant Height: This variable was measured from the plant collar to the apex using a flexible measuring tape. The evaluation was conducted 60 days after sowing [25,26].

Number of Leaves: This was determined by manual in situ counting at 60 days after sowing [26].

Destructive Sampling: For destructive sampling, the 10 centrally located plants from each treatment were selected, totaling 150 plants across all treatments, to evaluate the following variables:

Leaf Area: This was determined using a CI-202 Portable Laser Leaf Area Meter, manufactured by CID Bio-Science, located in Camas (Washington), United States. The initial leaf area measurement was taken 20 days after sowing (DAS) on four additional plants that were removed during the thinning stage and transplanted into separate containers. These plants served as a baseline for initial data collection in the experiment and were not exposed to any of the treatments; they only received water. After sowing, when the plants showed one true leaf, a final measurement was taken before the destructive samplings were carried out 60 days after sowing.

Root Volume: This was determined by submerging the root system in water and measuring the volumetric displacement in a graduated cylinder [27].

Root Length: This was determined using a flexible measuring tape, measuring the plant collar to the root cap.

Dry Weight: This was obtained using a laboratory balance after drying the entire plant for 24 h at 72 °C in a botanical sample drying oven [28].

Growth Rates: Growth rates commonly used in crop physiology were calculated, including the crop growth rate (CGR), net assimilation rate (NAR), relative growth rate (RGR), and leaf area index (LAI) [28]. Figure 1 provides a detailed breakdown of the evaluations of the destructive and non-destructive variables.

2.8. Lipid Profile Using HS–SPME with GC–MS

A Shimadzu GCMS-QP2010 Plus gas chromatograph, of Shimadzu corporation, located in Kyoto, Japan, equipped with a split/splitless injector and coupled to a mass spectrometer, was employed following the methodology proposed by the authors, with modifications [29].

2.9. Statistical Analysis

The data collected in the field were integrated into databases and matrices using Microsoft Excel, Microsoft Office Professional Plus 2019 version. Once the various databases were compiled, they were processed using the SAS statistical software, 9.0 version. This involved testing the data for normality, conducting an analysis of variance (ANOVA), and performing Tukey’s honestly significant difference (HSD) post hoc test for mean comparisons (p ≤ 0.05).

3. Results

For plant height and the number of leaves at 60 days after sowing (DAS) and following a second application of the ozonated oils, it was observed that the ozonated avocado oil at the highest concentration, applied via drench, exhibited a height of 14.6 cm and an average of 5.2 leaves. This treatment showed the highest average compared to the others and presented significant differences in plant height relative to the remaining treatments, which ranged in height from 7.6 cm to 12.6 cm (

Table 2. Growth parameters of “Bolo Verde” variety squash plants with two applications of three different concentrations of two ozonated oils at 60 days after sowing, in Manizales, Caldas (Colombia).

Ozonated Oil Concentration		Application	Height (cm)	Number of Leaves	Root Length (cm)	Root Volume (cm3)	Total Dry Weight (g)
Avocado	Low	(1) Foliar	11.81 bc	4.66 bcde	36.45 abc	3.20 bcd	2.22 bcd
		(2) Drench	11.76 bcd	4.53 bcedf	41.50 abc	3.00 cd	1.92 cdef
	Average	(3) Foliar	10.83 bcde	4.50 cdef	36.80 abc	2.30 d	2.05 cdef
		(4) Drench	10.73 cde	4.56 bcdef	35.55 bc	3.30 bcd	2.05 cdef
	High	(5) Foliar	7.81 gh	2.84 h	34.15 bc	2.40 d	2.14 cde
		(6) Drench	14.64 a	5.19 a	44.55 ab	4.20 ab	2.60 ab
Palm	Low	(7) Foliar	11.05 bcde	4.16 efg	36.75 abc	2.90 cd	2.29 bc
		(8) Drench	12.68 b	5.00 abc	35.15 bc	4.20 ab	2.80 a
	Average	(9) Foliar	7.69 h	3.78 g	37.47 abc	3.20 bcd	2.27 bc
		(10) Drench	8.79 fgh	4.66 bcde	31.75 c	2.40 d	1.73 ef
	High	(11) Foliar	9.21 efgh	4.06 fg	37.43 abc	3.10 bcd	2.28 bc
		(12) Drench	9.86 def	4.34 def	33.35 c	3.60 bc	1.94 cdef
Commercial		(13) Foliar	9.68 efg	4.50 cdef	46.84 a	3.40 bcd	1.84 def
		(14) Drench	11.10 bcde	5.03 ab	30.92 c	5.10 a	2.21 bcd
Control			9.94 cdef	4.78 abcd	35.84 bc	3.90 bc	1.69 f

Means followed by the same letter in the column do not differ significantly from 1 according to Tukey’s HSD test (p ≤ 0.05). Control: absence of ozonated oil.

).

As shown in Table 2, the root length variable at 60 days after sowing indicated that the commercially available product via foliar application presented the highest values with 46.84 cm, showing no statistically significant difference from the ozonated avocado oil applied via drench at the highest concentration (44.55 cm) and the high-oleic palm oil via foliar application at the lowest concentration (36.75 cm). In contrast, the commercially available product applied via drench exhibited the lowest average compared to the other treatments, with a value of 30.1 cm, thus showing significant differences with the ozonated avocado oil at the highest concentration via drench and high-oleic palm oil at the lowest concentration via foliar application, with values of 44.5 cm and 46.84 cm, respectively. Thus, it is determined that ozonated avocado oil at a high concentration via drench and ozonated high-oleic palm oil at a low concentration via foliar application can have a beneficial effect on root growth variables. This demonstrates that the type of ozonated oil, the concentration, and the application method can have a differential effect on the growth of squash plants.

For the root volume variable at 60 DAS, the foliar application of ozonated avocado oil and the drench application of high-oleic palm oil presented the lowest averages with values of 2.3, 2.5, and 2.4 cm³, respectively, showing significant differences compared to the highest averages observed in the drench application of avocado oil at a high concentration and the foliar application of high-oleic palm oil, with the highest values ranging between 3.6 and 5.1 cm³ (Table 2).

The total dry weight with the use of ozonated avocado oil at the highest concentration and applied via drench showed the greatest accumulation with a value of 2.6 g, along with ozonated high-oleic palm oil at a lower concentration but via foliar application with a value of 2.8 g (Table 2). On the other hand, the control group presented the lowest values with 1.69 g in the total plant, representing the lowest average (Table 2).

For the crop growth rate (CGR), the ozonated avocado oil at a high concentration and applied via drench exhibited the best performance with a value of 0.000676692 g cm⁻² day⁻¹, similar to the ozonated high-oleic palm oil at a low concentration and applied via drench with a value of 0.000734529 g cm⁻² day⁻¹ (

Table 3. Growth rates of “Bolo Verde” variety squash plants with two applications of three different concentrations of two ozonated oils at 60 days after sowing, in Manizales, Caldas (Colombia).

Ozonated Oil Concentration		Application	Crop Growth Rate (g cm2 day−1)	Relative Growth Rate (g g day−1)	Net Assimilation Rate (g cm2 day−1)	Leaf Area Index
Avocado	Low	(1) Foliar	0.000566802 bcd	0.0405122 bc	2.62203 cd	4989.03 fg
		(2) Drench	0.000480046 cdef	0.0376485 cd	2.72693 cd	6509.65 bcde
	Average	(3) Foliar	0.00051764 cdef	0.0390575 cd	2.92534 bcd	6496.09 bcde
		(4) Drench	0.00051764 cdef	0.0391068 cd	2.94884 bcd	6573.79 bcde
	High	(5) Foliar	0.000543667 cde	0.040213 c	2.35326 cd	4645.19 g
		(6) Drench	0.000676692 ab	0.0441953 ab	4.29069 a	7957.99 a
Palm	Low	(7) Foliar	0.000587045 bc	0.0412687 bc	3.07454 bc	6048.57 cdef
		(8) Drench	0.000734529 a	0.0454216 a	4.36514 a	7208.40 ab
	Average	(9) Foliar	0.000581261 bc	0.0411 bc	3.18034 bc	6268.99 bcde
		(10) Drench	0.000425101 ef	0.0358173 d	2.10982 cd	5536.66 efg
	High	(11) Foliar	0.000584153 bc	0.0412926 bc	2.96081 bcd	5794.78 def
		(12) Drench	0.00048583 cdef	0.0383872 cd	2.86018 bcd	7104.83 abc
Commercial		(13) Foliar	0.000456911 def	0.0374252 cd	2.32656 cd	5814.03 def
		(14) Drench	0.00056391 bcd	0.0407485 bc	3.69219 ab	8075.03 a
Control			0.000413534 f	0.0357569 d	2.35711 cd	6780.24 bcd

Means followed by the same letter in the column do not differ significantly from 1 according to Tukey’s HSD test (p ≤ 0.05). Control: absence of ozonated oil.

). In contrast, the control group showed a lower growth rate with 0.000413534 g cm⁻² day⁻¹ compared to the other treatments, presenting significant differences. Thus, the use of ozonated avocado and high-oleic palm oils demonstrates greater efficiency in biomass production per unit of soil surface area, which translates to higher yields.

The relative growth rate (RGR) was higher with the use of ozonated avocado oil at the highest concentration applied via drench, with a mean value of 0.0454216 g g⁻¹ day⁻¹, and similar behavior was observed for the ozonated high-oleic palm oil at the lowest concentration and via foliar application, with a mean value of 0.0454216 g g⁻¹ day⁻¹ (Table 3), indicating greater increases in dry mass relative to the initial mass. The control group, along with the use of ozonated high-oleic palm oil applied via drench at a medium concentration, presented the lowest RGR values, each at 0.035 g g⁻¹ day⁻¹ (Table 3).

Regarding the net assimilation rate (NAR), the application of ozonated avocado oil at a higher concentration, ozonated high-oleic palm oil at a lower concentration, and commercial oil, all three applied via drench, were superior with values of 4.29069, 4.36514, and 3.69219 g cm⁻² day⁻¹, respectively, indicating a greater gain of dry matter per unit of assimilatory tissue per unit of time (Table 3). The control group presented the lowest net assimilation rate with a value of 2.35711 g cm⁻² day⁻¹, as did the use of ozonated avocado oil at a low concentration and a high concentration via foliar application (Table 3). A similarly low net assimilation rate was observed for ozonated high-oleic palm oil at medium and high concentrations, regardless of the application method (drench or foliar) (Table 3).

The leaf area index (LAI) in plants treated with ozonated avocado oil, high-oleic palm oil, and the commercial product, all applied via drench, exhibited the highest values with 7957.99, 7208.40, and 8075.03, respectively (Table 3). A lower effect is observed with the use of ozonated oils depending on the concentrations; thus, the leaf area index was lower with ozonated avocado oil at lower concentrations, while for ozonated high-oleic palm oil, it was lower at higher concentrations (Table 3). The lowest values were observed with the foliar application of ozonated avocado oil at a low concentration (4989.03), and for ozonated high-oleic palm oil, the lowest values were observed with the highest concentration and drench application (5536.66) (Table 3).

In the gas chromatography–mass spectrometry (GC-MS) analysis of virgin and ozonized vegetable oils—previously subjected to derivatization, typically via methylation of fatty acids to form their corresponding methyl esters—significant structural changes induced by ozonation were observed. The chromatographic profile of virgin oils revealed a high concentration of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2), in the form of their methyl esters, along with minor constituents such as tocopherols and sterols, which contribute to the oxidative stability and nutritional value of the oil.

In contrast, post-ozonation analysis showed a marked reduction in these unsaturated fatty acids, indicating cleavage of double bonds due to the oxidative action of ozone. This transformation led to the formation of various oxygenated compounds, including aldehydes (e.g., nonanal and octanal), ketones, hydroperoxides, and short-chain carboxylic acids such as acetic and oxalic acids—typical products of lipid oxidative degradation. These compounds not only alter the physicochemical properties of the oil but also exhibit biological activity, making ozonized oils of interest for pharmaceutical, cosmetic, and therapeutic applications.

Overall, the GC-MS results provide a clear qualitative and quantitative assessment of the compositional changes in the oils, confirming the efficacy of the ozonation process and its impact on the molecular structure of the constituent lipids.

4. Discussion

Ozone has several applications in agriculture as a biostimulant, supported by its potent oxidizing properties and its potential as a disinfectant. Ozone in plants induces the synthesis of plant hormones, stimulates the production of bioactive compounds, and enhances plant defense mechanisms. However, the environmental impacts of using ozone as a biostimulant include phytotoxicity and potential negative effects on crop yield. Although ozone shows promise in promoting plant growth and productivity, further research is necessary to fully understand its potential applications in sustainable agriculture.

Ozone functions as a biostimulant in plants by inducing the synthesis of plant hormones, stimulating the production of bioactive compounds, and enhancing plant defense mechanisms [30]. However, the environmental impacts of using ozone as a biostimulant include phytotoxicity and potential negative effects on crop yield. Although ozone shows promise in promoting plant growth and productivity, further research is necessary to fully understand its potential applications in sustainable agriculture. Consistent with the results of the plant growth parameters at 60 days after sowing, a positive effect was observed on the height, number of leaves, root length, and root volume of the plants treated with ozone (Table 2). Meanwhile, ozonated oils stimulate root growth, increasing root volume and facilitating a greater absorption of nutrients and minerals by the plant [19]. These findings align with the results obtained, where drench applications showed higher averages in root length and volume at 60 DAS in the destructive sampling, with the avocado oil at the highest concentration applied as a drench exhibiting the highest average. Biostimulant substances activate physiological mechanisms, stimulating the growth of plant organs [31]. This allows for better nutrient utilization, leading to increased plant growth and development, resulting in an increase in biomass, dry weight, and yield. Furthermore, these substances activate the plant’s response mechanisms, contributing to resistance against biotic and abiotic stress, which provides the plant with greater resilience to disease attack. The aforementioned aligns with the results obtained, as the drench applications of ozonated avocado oil resulted in greater plant height (Table 2).

These effects may be modulated by the molecular mechanisms of ozone’s biostimulant effects in plants, such as ozone inducing the synthesis of plant hormones like ethylene, salicylic acid, and jasmonic acid, which are necessary for plant growth, development, and defense responses [30,32]. It can also be associated with the production of reactive oxygen species (ROS) by ozone exposure, which plays a role in biochemical adjustments in plant leaves, leading to disruptive changes at the gene expression level and subsequent metabolic changes [33]. This is why the net assimilation rates and leaf area index were higher with the use of ozonated oil (Table 3). These responses of ozone in plants trigger a series of antioxidant and defensive indicators in plants, such as ascorbate, glutathione, salicylic acid, and jasmonic acid, which contribute to the plant’s response to ozone exposure [34]. Ozone exposure can stimulate the production of bioactive compounds in plants, such as hydroxycinnamic acids, rosmarinic acid, phenylpropanoids, and flavonoids, which are involved in adaptive responses [35]. A higher leaf area index is related to a larger photosynthetic area, greater transpiration, and increased respiration carried out by the plant, and therefore with the gas exchange and energy that the plant has with the environment [36,37].

The effects of ozone on plants are multiple and encompass physiological, molecular, and metabolic aspects [38]. From a physiological standpoint, this relates to positive effects on plant growth and development, and the connection to the underlying molecular mechanisms of its impact, as well as how ozone-induced oxidative stress affects plant metabolism and productivity [39]. On the other hand, ozone is a phytotoxic atmospheric pollutant that harms plants and agriculture, affecting various physiological and biochemical processes, crop growth, and yield [38]. Among the negative effects, ozone can decrease the yield of important crops, negatively affecting the fitness of native plant species and compromising current crop productivity [40]. Regarding the efficacy of ozone compared to other biostimulants in promoting plant growth, ozone has been shown to accelerate flowering in plants, potentially affecting interactions with pollinators and reproductive success [38]. Similarly, ozone treatment has been observed to increase the accumulation of bioactive compounds in plants, such as total phenols, antioxidant capacity, and total flavonoids, which may contribute to plant defense mechanisms against stress [15]. Pretreatment with ozone has also been found to contribute to the adaptation of sweet pepper hybrids to cold stress, resulting in an increased fruit set, early, total, and marketable fruit production, and improved fruit quality under adverse conditions [20].

It is worth noting that ozone has been studied for its potential as a biostimulant in agriculture, particularly to increase crop yield [41,42]. It exhibits physiological effects such as influencing plant morphology, growth, and productivity, with different plant species showing varying degrees of sensitivity to ozone exposure [40,43]. This is evident in our study, as the positive or negative response to the use of ozonated oils varies according to the concentration and application method, whether foliar or drench (Table 2 and Table 3). It is necessary to improve the understanding of the interaction between increased ozone concentration in oils and other aspects of climate change, such as drought, and to leverage the genetic variation observed in the response to ozone for crop improvement [41].

It is important to highlight that palm oil (Elaeis guineensis) is notable for its high concentration of bioactive compounds with antioxidant properties, including carotenoids, vitamin E (in the form of tocopherols and tocotrienols), sterols, phospholipids, glycolipids, and squalene [44]. These phytonutrients have demonstrated the ability to reduce lipid peroxidation and mitigate oxidative stress, suggesting a potential positive effect on plant growth and development—particularly in species of the genus Cucurbita—by minimizing oxidative damage at the cellular level.

Similarly, avocado oil possesses a lipid profile rich in unsaturated fatty acids, especially oleic acid, and contains antioxidants comparable to those found in palm oil [45]. These constituents may enhance nutrient uptake and modulate physiological responses to abiotic stress, thereby supporting optimal plant development [44,45].

Although direct empirical evidence regarding the biostimulant effects of palm and avocado oils on Cucurbita sp. is currently lacking, their phytochemical composition suggests the potential to elicit beneficial physiological responses. Theoretically, the synergistic action of antioxidants and fatty acids present in these oils could enhance plant performance by alleviating oxidative stress and improving nutrient assimilation. Although the potential of ozone as a biostimulant in agriculture is evident, more research is needed to fully understand its effects and optimize its application to enhance crop yield.

5. Conclusions

The drench application of high-concentration ozonated avocado oil (200 meqO₂ kg⁻¹) produced the most favorable biostimulant response, significantly increasing plant height, leaf number, root length, root volume, and total dry weight. This was followed by the drench application of low-concentration ozonated high-oleic palm oil (5 meqO₂ kg⁻¹), which yielded the highest dry matter accumulation. For the net assimilation rate (NAR) and leaf area index (LAI), the drench application of ozonated avocado oil at a high concentration resulted in 4.29 g cm⁻² day⁻¹ NAR and 7957.99 LAI, while low-concentration high-oleic palm oil recorded 4.36 g cm⁻² day⁻¹ NAR and 7208.40 LAI.

Regarding the crop growth rate (CGR) and relative growth rate (RGR), the drench application of high-concentration ozonated avocado oil yielded a CGR of 6.77 × 10⁻⁴ g cm⁻² day⁻¹ and RGR 0.0441953 g g⁻¹ day⁻¹. Low-concentration high-oleic palm oil drench application resulted in the highest CGR of 7.35 × 10⁻⁴ g cm⁻² day⁻¹ and RGR 0.0454216 g g⁻¹ day⁻¹. These values were significantly higher than those of the control (CGR 4.14 × 10⁻⁴ g cm⁻² day⁻¹; RGR 0.0357569 g g⁻¹ day⁻¹). These results suggest that the drench application of ozonated oils not only enhances photosynthesis and leaf growth but also favors the incorporation and accumulation of biomass in “Bolo Verde” squash.

Scaling up the production of ozonated oils for application in semi-commercial cropping systems is imperative. Concurrently, it is necessary to conduct comprehensive studies on additional agronomically and economically significant species to broaden the scope of potential applications. These investigations should encompass the entire phenological development and production cycle of the target species to rigorously assess the cost-effectiveness and agronomic viability of ozonated oil treatments. By enhancing crop productivity and resource-use efficiency, this approach presents a promising strategy for advancing sustainable and economically viable agricultural practices.