Next Article in Journal
Peanut Growth Promotion and Biocontrol of Blight by Sclerotium rolfsii with Rhizosphere Bacterium, Bacillus siamensis YB-1632
Previous Article in Journal
Construction of a Multi-Source, Heterogeneous Rice Disease and Pest Knowledge Graph Based on the MARBC Model
Previous Article in Special Issue
Fly High: Volatile Organic Compounds for the Early Detection of the Seed-Borne Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agronomy
Volume 15
Issue 3
10.3390/agronomy15030567
agronomy-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

On the Effectiveness of Ozone Treatments: A Silver Bullet for Plant Health?

by
Chiara Pastacaldi
1,2,*,
Dario Gaudioso
1,
Cosimo Beltrami
1,
Benedetta Gunnella
1 and
Stefania Tegli
1,2,*
1
Laboratorio di Patologia Vegetale Molecolare, Dipartimento di Scienze e Tecnologie Agrarie, Alimentari Ambientali e Forestali (DAGRI), Università degli Studi di Firenze, 50019 Florence, Italy
2
Joint Lab Ozone Plant Health, Dipartimento di Scienze e Tecnologie Agrarie, Alimentari Ambientali e Forestali (DAGRI), Università degli Studi di Firenze, 50019 Florence, Italy
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(3), 567; https://doi.org/10.3390/agronomy15030567
Submission received: 30 December 2024 / Revised: 18 February 2025 / Accepted: 22 February 2025 / Published: 25 February 2025
(This article belongs to the Special Issue Phytoalexins, Resistance Inducers, and Sustainable Control Measures in Crop Protection Strategies—2nd Edition)
Browse Figures
Versions Notes

Abstract

:
The development of innovative and eco-friendly strategies to protect plant health is one of the main challenges for the agricultural sector to respond to the increasing global food demand. In this contest, ozone (O3) could be a promising sustainable alternative to current pesticides, since it is a powerful oxidizing agent and does not leave residues in the environment. However, the molecular mechanisms involved in its potential bioactivity as a plant defense inducer are still poorly known. Thus, this study aimed to understand the role of O3 in plant defenses, as well as in plant growth, development and physiology, by a multidisciplinary approach. Here, O3 was applied as ozonated water to the soil in field experiments or to the nutrient solution of hydroponically grown plants. Experiments were carried out on different plant species, including the model plant Nicotiana tabacum and agronomically important crops such as lettuce, bean, and tomato. The analysis of several physiological parameters, such as plant weight, chlorophyll content, and stomatal conductance, indicated that O3 effects are species-specific. Moreover, the expression analysis of specific defense-related genes showed that O3 induced substantial changes in key hormonal and defense signaling pathways. Overall, O3 was demonstrated to trigger plant defenses, mainly mediated by pathogenesis-related proteins, mimicking a pathogen attack.

1. Introduction

Climate change is one of the main threats to contemporary agriculture, which can, in turn, affect global food security. The world population is expected to rise to approximately 10 billion by 2050 [1], driving a significant increase in food demand. Consequently, the agricultural sector must face several challenges to meet this demand by increasing food production while minimizing environmental impacts [2,3]. Therefore, it is mandatory to avoid the pressure on the agricultural sector to sustain the growing food demand leading to an increased use of agrochemicals, the overexploitation of natural resources, and an unchecked deforestation for livestock and crop production [3].
Pesticides are chemical substances, either single or in mixtures, used in agriculture to protect plants from harmful pests, thereby ensuring crop production as well as food quality and quantity [4,5]. In 2022, the global pesticide market reached nearly 4 million tons, doubling since 1990 [6]. However, these products have several well-known negative effects on human health and the environment, including soil, water, air, and ecosystems, where they can also impact non-target species, leading to a reduction in biodiversity. Additionally, pesticides can bioaccumulate in animal fatty tissues, remaining persistent in the food chain for long periods [5,7]. Lastly, the extensive use of these chemicals has led to the development of pesticide-resistant strains in many pathogens, making their application ineffective [5]. Thus, the development of innovative, eco-friendly, and sustainable strategies to protect plant health is urgently needed. In this context, ozone (O3) could be a feasible alternative.
O3 is a gas that is naturally part of the atmosphere, and it is made up of three atoms of oxygen. O3 is a very reactive and unstable molecule that rapidly decomposes in oxygen and OH radicals when dissolved in water (i.e, half-life of 20 min at 20 °C) [8,9] without leaving any harmful by-product in the environment. Moreover, it is a powerful oxidizer that targets several biological processes in microorganisms [10]. Consequently, it is widely used as a broad-spectrum and eco-friendly disinfectant agent in wastewater treatment and to sanitize the surface of fruits and vegetables [10,11,12]. In recent years, O3 has also gained increasing interest in the agricultural sector as a suitable alternative to traditional agrochemicals. Preliminary studies suggested that at low concentrations, O3 can trigger plant defense mechanisms and stimulate the production of secondary metabolites [13]. Moreover, when applied to soil or nutrient solutions, it increases the amount of oxygen, which in turn enhances root respiration [10,14]. At these concentrations, soil enzymatic activities and microbial communities were not affected by O3 application, suggesting that this molecule does not compromise soil health or fertility [15,16]. Conversely, at higher concentrations, O3 has been shown to negatively affect plant growth and development, primarily by reducing photosynthesis and stomatal conductance [13,17,18]. Therefore, understanding the optimal O3 doses that benefit plants is crucial for developing innovative and eco-friendly pesticides.
The positive effects of O3 can be achieved by treating plants with ozonated water, which can be applied directly to the soil (e.g., through soil drench), or sprayed on plant organs [13], or by fumigating gaseous O3 directly onto plants, seeds, or soil [10,13].
To date, ozonated water has gained the most interest as a potential sustainable strategy to improve crop yield and reduce the use of conventional pesticides. Indeed, treatment with ozonated water has been shown to increase biomass, yield, and fruit quality in tomatoes [19], as well as when plants were cultivated in nutrient-deficient soils [20]. Similarly, fruit yield in cucumber was improved under drought stress after ozonated water treatment [21]. Irrigation with ozonated water could induce the expression of genes involved in antioxidant activity, nitrogen uptake, and chlorophyll synthesis in tomato seedlings, thereby improving their physiology [22]. Concerning plant protection, ozonated water was shown to efficiently control Ralstonia solanacearum and Alternaria solani infections in tomato [23]. Moreover, O3 has been shown to modulate plant defense mechanisms, protecting tomato plants against the root-knot nematode Meloidogyne incognita [24,25]. Lastly, O3 has also been shown to induce callose deposition in tomato plants through the activation of the ABA signaling pathway, leading to a reduction in the fitness of Bemisia tabaci [26].
However, the molecular pathways underlying the beneficial effects of O3 are still poorly understood. The present study aimed to uncover the biological mechanisms triggered by O3 on plant growth, development, and defense. Here, O3 was applied as ozonated water to the soil of plants grown in pots in field experiments and directly to the nutrient solution of hydroponically grown lettuce. The results indicate that O3 affects plant morphology, physiology and defense in a species-specific manner with the effects also depending on the mode of O3 application. Furthermore, gene expression analysis suggests that O3 may be involved in defense mechanisms mediated by salicylic acid (SA), as evidenced by the upregulation of genes encoding pathogenesis-related (PR) proteins.

2. Materials and Methods

2.1. Experimental Design and O3 Treatments

To evaluate the effects of ozonated water on plant growth, development, and defense mechanisms triggering, open-field experiments were carried out at the Laboratorio di Patologia Vegetale Molecolare (DAGRI, University of Florence, Italy), in Sesto Fiorentino (43°49′12.7″ N 11°11′38.1″ E), from June to August 2024. Plants were grown in pots containing approximately 300 kg of sandy soil per pot with the following characteristics: pH 8.3, electrical conductivity (EC) 66 µS/cm, and water retention capacity of 34%. Plants were irrigated (9 L/pot) two to three times per week, depending on the season.
The plant species analyzed in this study include Nicotiana tabacum var. White Burley, Lactuca sativa var. Canasta, Solanum lycopersicum var. cerasiforme, and Phaseolus vulgaris var. Supernano Giallo. Tomato and tobacco plantlets were transplanted into the pots at 6 weeks of age, lettuce at 4 weeks, and beans at 2 weeks. The tomato, tobacco, and bean seedlings were propagated from seeds, which were sown in a high-quality professional potting mix composed of 23% coconut granules (0–5 mm), 27% Irish peat (0–5 mm), 14% volcanic pumice (3–8 mm), 36% superfine peat (0–10 mm), and slow-release fertilizers (pH [H2O] 6.0–7.0, and EC 0.30–0.40 dS/m). The seeds were grown under controlled conditions (24 °C ± 2 °C, 16 h light/8 h dark cycle) until they reached the required developmental stage. Conversely, lettuce plantlets were sourced from the market at 4 weeks of age. All seeds and plantlets were purchased from the Agricultural Consortium of Florence.
Ozonated water was prepared by insufflating O3 directly into a tank containing 9 L of tap water. O3 was diffused in water through an air stone to create microbubbles and increase mass transfer. In addition, tap water was kept in agitation by a magnetic stirrer during O3 treatment to ensure solution homogeneity. For all experiments, O3 was generated using a prototype of the Defender 1000™ system, developed by TEA Group srl (Signa, Florence, Italy), which transforms ambient oxygen into O3 through corona discharge capable of producing 20 g of O3 per hour, which is equivalent to approximately 9400 ppm/h. Two different ozonated water treatments were applied by insufflating O3 for 30 min (T1) and 1 h (T2). Immediately after, the concentration of dissolved oxygen (DO) was measured with a dissolved oxygen meter (DO-9100, Hedao, Fujian, China). The T1 treatment resulted in 0.3 mg/L DO, while the T2 treatment resulted in 0.5 mg/L DO. The ozonated water was applied within 5 min of preparation. As a control, plants were irrigated with tap water. Twelve plants per treatment were used for each species. Plants were harvested 28 days after potting for lettuce and 45 days for the other species. Specifically, the third youngest leaves of three plants per treatment were collected for each species and immediately frozen in liquid nitrogen (N2) for subsequent gene expression analysis. Furthermore, both the aerial parts and the roots were collected and analyzed separately to assess several physiological parameters.
Regarding the hydroponic experiment, two separate trials with L. sativa var. Canasta under nutritional stress were conducted in controlled conditions. Specifically, 4-week-old lettuce plantlets were cultivated in tanks containing 4.5 L of Hoagland nutrient solution [27] (Table 1), continuously oxygenated for 14 h a day with specific air pumps, and renewed every 2 weeks. In each tank, three plantlets were placed after their roots were washed with tap water to remove any soil. They were then allowed to acclimate to the hydroponic conditions for one week before starting the O3 treatments. The experiment was conducted into a growth chamber, set to 24 °C ± 2 °C and with a photoperiod of 14 h light/10 h dark.
Nutritional stress was induced by cultivating plants in a 20% Hoagland nutrient solution (pH 7.5, EC ca. 260 µS/cm) (hereafter, C20%). Then, O3 was applied by insufflating the gas directly into the nutrient solution for either 15 or 30 min (T1 and T2 treatment, respectively), with each treatment repeated twice a week. As a positive control, lettuce plants were cultivated in full-strength Hoagland solution (pH 7, EC ca. 1200 µS/cm) (hereafter, C100%). After 28 days, lettuce plants were harvested and analyzed as described above. Additionally, half of the roots of each plant were immediately frozen in liquid N2 for subsequent gene expression analysis.
In both the experiments conducted on plants grown in soil and those in hydroponics, each sample consisted of six biological replicates, and two independent experiments were carried out. Specifically, for each independent experiment, four plants per pot were sampled (i.e., pseudoreplicates), with three pots per plant species per treatment. To avoid data loss, more than four plants were planted per pot (i.e., an average of 5–6, depending on the species), but only four were randomly sampled and considered as one replicate. Similarly, for hydroponically grown lettuce plants, three tanks per plant species per treatment were used in each experiment, with three plants per tank randomly sampled, giving a total of six replicates.

2.2. Analysis of Plant Physiological Parameters

Before sampling the potted plant, stomatal conductance and photosystem II (PSII) activity were measured with a porometer/fluorometer LICOR-600 (Li-Cor, Lincoln, NE, USA). Moreover, the fourth and fifth youngest leaves of three plants per treatment were collected and used to determine chlorophyll (chl) a and b content, following the method described by Wang et al. [28]. Briefly, 0.2 g of leaves (fresh weight, FW) were incubated in 10 mL (V) of 80% acetone for 24 h in the dark at room temperature. The chl content was determined by measuring the optical density at 663 nm (OD663) and 645 nm (OD645) for chl a and b, respectively, with a UV spectrophotometer and multimode plate reader Infinite® 200 PRO (Tecan, Zürich, Switzerland). Afterward, the chl concentration was calculated using the following formulas:
c h l   a   m g / g = 12.72 × O D 663 − 2.59 × O D 645 × V 1000 × F W
c h l   b   m g / g = 22.88 × O D 645 − 4.67 × O D 663 × V 1000 × F W
Subsequently, the plants were removed from the pots, and the aerial part (hereafter referred to as ‘leaf’) was separated from the roots, which were thoroughly washed to remove all soil. After measuring fresh weight, the roots and aerial parts were incubated at 60 °C for 2–3 days to determine the dry weight.
The same analyses have been performed on hydroponically grown lettuce plants.

2.3. RNA Extraction and Plant Gene Expression Analysis

Plant samples were ground with liquid N2, and RNA extraction was performed using the NucleoZOL reagent (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer’s instructions. The total RNA was eluted in 30–60 µL of nuclease-free water. RNA concentration and quality were assessed using both a NanoDrop Spectrophotometer ND-100 (Nanodrop Technologies Inc., Wilmington, DE, USA) and gel electrophoresis (1% agarose gel in 1X TAE). To eliminate any genomic DNA (gDNA) contamination, the total RNA samples were further purified using the RapidOut DNA Removal Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Then, cDNA was synthesized using the RevertAid RT Kit (ThermoFisher Scientific) following the manufacturer’s protocol.
PCR primers to be used in gene expression analysis have been designed and optimized for each plant species using the Primer-BLAST tool (National Center for Biotechnology Information, NCBI) [29] and the “PCR Primer Stats” tool of The Sequence Manipulation Suite. The primers were synthesized by Eurofins Genomics (Ebersberg, Germany) and are listed in the Supplementary Materials (Tables S1–S4). The expression profile of the following genes was analyzed: auxin-responsive genes 1 (ARF1), transport inhibitor response 1 (TIR1), auxin efflux carrier PIN1, phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), jasmonoyl—l-amino acid synthetase (JAR1), and pathogenesis-related proteins (PR1, PR3 and PR5).
Real-time quantitative PCR (RT-qPCR) was performed with the SsoFast™ EvaGreen® Supermix (Bio-Rad Laboratories, Hercules, CA, USA), according to Tegli et al. [30]. The expression of the target genes was normalized using two housekeeping genes for each plant species analyzed. In particular, EF1α and L25 were used for tobacco, Ubi3 and Act for tomato, IDE and Act11 for common bean, and APT1 and TIP41 for lettuce.
The RT-qPCR data were analyzed using CFX Maestro 1.0 software (Bio-Rad). Relative gene expression was determined using the 2−ΔΔCt method [31]. The data from the treated plants were compared with those from the controls. CFX Maestro 1.0 was also used to perform statistical analysis with a significance threshold set at a p-value < 0.05. For each sample, three technical replicates were processed in each of the three independent RT-qPCR runs carried out.

2.4. Statistical Analysis

The statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Boston, MA, USA). After assessing normality, data were analyzed using either one-way ANOVA or Kruskal–Wallis tests with a significant threshold of p < 0.05. Afterwards, a post hoc analysis using either Tukey’s or Dunn’s test was performed by comparing the mean of each treatment with the mean of any other treatment for each plant species.

3. Results

3.1. Effects of Ozonated Water on the Development and Physiology of Potted Plants

The effects of ozonated water treatments on plant growth and development varied depending on the plant species tested with lettuce being demonstrated as the most sensitive to the O3 treatments (Figure 1). Concerning the aerial part, a statistically significant increase in leaf dry weight compared to the control was observed only in lettuce, and not in the other plant species, following the T1 treatment, while no differences were observed with the T2 treatment (Figure 1B). Similarly, in tobacco, the leaf’s dry weight was slightly increased after ozonated water treatments, being more pronounced after the T2 treatment, as shown in Figure 1E. Conversely, root development was negatively affected by ozonated water in lettuce, with both T1 and T2 treatments causing a statistically significant reduction in root dry weight compared to the control (Figure 1C). In contrast, root development was positively influenced by ozonated water treatments in tobacco plants, showing a statistically significant increase following the T1 treatment (Figure 1F). In tomato, plant development was barely influenced by the treatment of ozonated water. Indeed, the leaf dry weight was slightly increased after both T1 and T2 treatments, while a trending reduction in root dry weight was observed compared to the control (Figure 1H,I). No statistically significant differences in leaf and root dry weight were observed between treated and control plants in bean (Figure 1K,L).
Photosynthetic parameters were also affected differently by ozonated water treatments, depending on the plant species. Regarding chl a content, the values found in the untreated controls were comparable among the plant species tested (Figure 2). An increase in chl a content was observed in both lettuce and tomato following O3 treatments compared to the control. In lettuce, both T1 and T2 treatments were effective, while in tomato, the increase in chl a content was primarily observed with the T2 treatment (Figure 2A and Figure 2C, respectively).
A statistically significant decrease in chl a content was observed in both tobacco and bean plants after the T1 treatment (Figure 2B and Figure 2D, respectively). Conversely, the chl a content in T2-treated plants was significantly increased compared to T1-treated plants and was comparable to the control. Data on chl b content were comparable to those for chl a across all the plant species here tested.
Regarding stomatal conductance and PSII activity, these photosynthetic parameters were not affected by O3 treatments on potted lettuce and bean plants. Conversely, stomatal conductance was significantly affected in both tobacco and tomato plants, albeit differently. In particular, a statistically significant reduction in stomatal conductance was found in tobacco after irrigation with ozonated water, being more pronounced following the T1 treatment (Figure 3A). In contrast, a significant increase was observed in tomato following the T2 treatment, while it was slightly reduced in T1-treated plants (Figure 3B).
As far as PSII activity was concerned, a significant increase was observed in tobacco after the T2 treatment compared to the control (Figure 4A). Similarly, in tomatoes, a slight increase was observed after the T2 treatment, whose results were statistically significant compared to the T1-treated plants (Figure 4B).

3.2. Impact of Ozonated Water on the Expression of Defense-Related Genes in Potted Plants

Ozonated water treatments also affected the expression of defense-related genes, depending on the plant species tested. Concerning the auxin pathway, ARF1 and TIR1 genes were upregulated in common bean after the T1 treatment but downregulated in tomato and common bean after the T1 and T2 treatments, respectively (Figure 5C,D). Moreover, the relative expression of the PIN1 gene was slightly increased in tomato and common bean after the T2 treatment, while an opposite result was observed in tobacco (Figure S1).
The relative expression levels of PAL were significantly increased in tobacco and common bean after the T1 and T2 treatments, respectively (Figure 5B,D).
Interestingly, the PR1 gene was upregulated in all the plants tested after ozonated water treatments (Figure 5). Notably, the increased expression of PR1 was significant after T1 treatment in tobacco and lettuce (Figure 5A,B) and after the T2 treatment in common bean (Figure 5D). Moreover, the PR3 was significantly upregulated in tomato and in lettuce after T1 treatment and in common bean after T2 treatment, as shown in Figure S1. On the contrary, the T1 treatment induced the downregulation of the gene in common bean (Figure S1).
Lastly, JAR1 expression was significantly induced in tobacco after ozonated water treatments (Figure 5B). A similar trend was also observed in lettuce, as shown in Figure 5A. In contrast, JAR1 was significantly downregulated in tomato plants after the treatments (Figure 5C). No variations in the expression of this gene were observed in the treated plants compared to control in common bean (Figure 5D).

3.3. Effects of Ozonated Water on the Development and Physiology of Hydroponically Grown Lettuce Under Nutritional Stress

Concerning hydroponically grown lettuce, the reduction in the Hoagland solution (up to 20%) did not statistically affect its growth compared to the control plants grown in a full-strength nutritional solution (i.e., C100%). Only a slight reduction in leaf dry weight was observed in C20% samples compared to C100%. Similarly, neither the T1 nor the T2 O3 treatments applied to plants under nutritional stress caused any alterations in root and leaf dry weight data in comparison to the C20% control (Figure 6). Stomatal conductance was slightly increased in the T1-treated plants compared to C20%, while the PSII activity was not affected by nutritional deprivation or O3 (Figure S2).

3.4. Impact of Ozonated Water on the Expression of Defense Related Genes in Hydroponically Grown Lettuce

In hydroponic lettuce, defense-related genes were regulated differently in leaves and roots by O3 treatments. Indeed, most of the genes analyzed were significantly overexpressed in the nutritionally stressed plants (C20%) both with and without O3 treatment of the 20% nutrient solution for 30 min compared to the C100% control plants except for the CHS, PAL, PR3, and JAR1 genes (Figure 7A). Interestingly, both the O3 treatment for 15 or 30 min caused an overexpression of the PR5 gene in comparison to C20%, comparable to C100%, suggesting a recovery from a potential trade-off in plant fitness (Figure S3). Conversely, the O3 treatment of the 20% nutrient solution for 15 min appeared to alleviate plants from this abiotic stress, with most of the genes analyzed expressed as in the C100% control plants, with the exception of PIN1 and JAR1 genes (Figure 7A).
Surprisingly, the highest overexpression of defense-related genes was observed in the lettuce roots (Figure 7B). Following the treatment of the 20% nutrient solution with O3 for 15 min, a statistically significant increase was induced in the relative expression levels of PIN1, PR3, and PR5 compared to both the C100% control (Figure 7B) and the C20% control (Figure S3). A recovery in the defense response of C20% control plants was observed for ARF1 and JAR1 genes in root samples, following treatment with the 20% nutrient solution and O3 for 30 min (Figure S3).

4. Discussion

The development of innovative and eco-friendly strategies to protect plant health is currently one of the main challenges for the agricultural sector, aiming to integrate several issues, such as boosting crop production and addressing the increased risk of epidemic outbreaks due to ongoing global changes while reducing the use of traditional agrochemicals for their well-known negative impacts on human health and the environment [5,7]. In this general frame, O3 has emerged as a potential eco-friendly alternative to pollutant conventional plant protection products [10,13]. In recent times, it has been demonstrated that O3 is an environmentally friendly option, because it rapidly decomposes into active oxygen species, such as hydroxyl radicals (OH-) and superoxide anions (O2−), in aqueous solutions, leaving no harmful residues behind [32]. However, the molecular mechanisms involved in its bioactivity are not completely understood.
In this study, we evaluated the effects of O3 applied as ozonated water to soil cultivated with different plant species as well as insufflated into the nutrient solution of hydroponically grown lettuce. The main variables assessed in this study are related to plant growth and development, photosynthetic parameters, and changes in the expression of genes associated with the activation of plant defense mechanisms. Firstly, our results demonstrate that the effects of O3 treatments on plants are species-specific and depend on both O3 concentration and application methods.
Plant growth and development were affected differently by ozonated water treatment across the plant species tested. Soil-grown lettuce has been demonstrated to be particularly sensitive to treatments with ozonated water where leaf biomass was significantly increased while roots dry weight was reduced after O3 treatments. In tobacco, irrigation with ozonated water enhanced leaf and root biomass. Overall, these data align with previous studies on tomato, non-heading Chinese cabbage, and pepper [19,23,33]. Moreover, the growth of tomato and bean plants was not negatively affected by ozonated water irrigation, which was consistent with findings by Veronico et al. [24]. Interestingly, the growth and development of hydroponically grown lettuce were not significantly affected when O3 was directly insufflated into the nutrient solution, as also reported by Zheng et al. [34]. Concerning plant physiology, ozonated water induced an increase in chl content in tomato and lettuce, as also shown by Guo et al. [23], where chl content was slightly enhanced after treatment with 6 mg/L ozonated water. The measurement of PSII activity reflects the status of the photosynthetic machinery [23]. In tobacco, an increase in PSII activity could be correlated with the enhanced plant biomass. Stomatal conductance, which measures the rate of gas exchange and transpiration through leaf stomata, provides a relative indication of stomatal aperture [35]. In tobacco, both ozonated water treatments resulted in a significant reduction in stomatal conductance by the end of the experiment, suggesting increased stomatal closure. In contrast, in tomato plants, the T2 treatment led to an increase in stomatal conductance. In hydroponic lettuce, however, O3 did not negatively affect plant physiology, as no statistically significant differences in stomatal conductance or PSII activity were observed between the control and treated plants.
Although atmospheric O3 is one of the major air pollutants that negatively impacts plant growth and development, the results presented here further support the beneficial effects of O3 when applied as ozonated water to plants. It is well known that long-term exposure to low O3 concentrations reduces photosynthetic rates and plant growth in addition to causing premature senescence [16,36]. However, in this study, the application of ozonated water did not negatively affect plant growth; in fact, in some plant species, growth was enhanced, as demonstrated for lettuce and tobacco.
Once O3 enters plant tissues, it begins triggering various defense signaling pathways involving the production of reactive oxygen species (ROS), second messengers (e.g., calcium), and phytohormones [36]. To date, most research on the molecular mechanisms involved in O3 activity has focused on analyzing antioxidant enzymes, including catalase, superoxide dismutase, ascorbate peroxidase, and peroxidase [22,24,34,37]. Here, we analyzed the expression profiles of defense-related genes involved in various pathways, such as secondary metabolites synthesis (i.e., PAL, CHS), and the signaling pathways for auxin (i.e., ARF1, TIR1, PIN1), jasmonic acid (JA) (i.e., JAR1, PR3) and salicylic acid (SA) (i.e., PR1, PR5). The results indicated that the expression of these genes was differently regulated among the plant species tested. Notably, in hydroponically cultivated L. sativa, distinct expression profiles were also observed between leaves and roots. These contrasting expression profiles suggest that O3 may stimulate defense mechanisms differently in various plant organs [25]. Additionally, in leaves, the expression levels of the defense-related genes showed a comparable pattern between C100% plants and those treated with 15-min O3 exposure. This suggests that plants under nutritional deficiency (C20%) may experience higher stress levels and that O3 treatment could have partially alleviated this stress. In particular, in the experiments carried out on hydroponically grown L. sativa under nutritional stress, the trigger of the crosstalk existing between the auxin and SA pathways was very well demonstrated with PIN1 affecting auxin distribution and influencing the plant’s ability to activate SA-mediated defense responses, including the expression of defense-related genes like PR3 and PR5. Ozone is able to stimulate plant defenses responses through the SA signaling pathway [25,38,39]. Interestingly, PR1 and PR5 genes were significantly upregulated in plants treated with ozone both in soil and hydroponic systems. This can suggest the activation of systemic acquired resistance (SAR) [40]. Moreover, in tobacco, the increase in PR1 expression after the T1 treatment, along with elevated SA levels, could be linked to a reduction in stomatal conductance, leading to stomatal closure [40,41]. The rise in SA could be also associated with the increased expression of PAL observed in tobacco and lettuce after the T1 treatment and in beans after the T2 treatment. Indeed, PAL is a key enzyme in the phenylpropanoid biosynthesis pathway [42], and it is also involved in SA biosynthesis [43]. The PAL gene was significantly upregulated in P. vulgaris after the T2 treatment, as also reported by Paolacci et al. [44]. A similar trend was also observed in L. sativa grown in pots, particularly after the T1 treatment. In hydroponically grown lettuce, the PAL gene was also slightly upregulated in the roots. This could suggest an increase in the biosynthesis of secondary metabolites, such as flavonoids and lignin, which are known to promote pathogen resistance [45]. Moreover, the enhanced biosynthesis of secondary metabolites could also improve the content of bioactive compounds, as demonstrated in red-leaf lettuce [46] and Salvia officinalis [47]. Regarding the auxin pathway, we evaluated the expression profiles of ARF1 and TIR1 genes. ARFs are transcriptional factors that form a complex with the Aux/IAA proteins and bind to auxin-responsive elements (Aux-REs) in the promoter regions of auxin-responsive genes when auxin levels are low [48]. When auxins increase, they bind to the TIR1/AFB receptor and form a complex with the Aux/IAA, leading to its degradation through the proteasome [48]. ARF1 is an auxin-induced gene [49] and is reported as a repressor of auxin-responsive genes [48,50]. In the present work, ARF1 was upregulated in tobacco and beans after the T1 treatment as well as in hydroponic lettuce roots after 30 min of O3 exposure. These results could suggest an increase in auxin levels in these plants. Moreover, the TIR1 gene was upregulated in bean and hydroponic lettuce roots after the T1 treatment and 30-min O3 exposure, respectively. These data further support the potential increase in auxin content in the treated plants. Conversely, the TIR1 gene was significantly downregulated in tobacco and tomato plants after the T1 treatment as well as in T2-treated bean plants. This downregulation of TIR1 is consistent with the increased expression of PR1, suggesting an increased SA content and a consequent inhibition of auxin responses [51,52]. Indeed, other studies have highlighted the antagonistic crosstalk between SA and auxin in plant defense with SA known to repress auxin-related genes, including TIR1 [51,52].
Concerning the JA signaling pathway, the JAR1 gene was significantly downregulated in tomato with ozonated water, while it was upregulated in tobacco. A similar trend was also observed in lettuce cultivated in pots. JAR1 (jasmonate resistant 1) is a key enzyme in the JA signaling pathway, and it is responsible for producing the JA–Ile conjugate [53]. The increase in JAR1 expression could enhance plant defenses against herbivores, nematodes, and necrotrophic pathogens [54]. Furthermore, the PR3 gene was upregulated after both ozonated water treatment in tomato and lettuce cultivated in pots. Its expression levels were also significantly increased in T2-treated bean plants and in the roots of hydroponic lettuce when O3 was applied for 15 min. The PR3 gene is generally induced by necrotrophic pathogen through the activation of the JA signaling pathway [40]. The PR3 encodes for chitinases, which hydrolyze chitosan, a major component of fungal cell walls. The upregulation of PR3 suggests that ozonated water may enhance resistance to fungal pathogens [40,55].
Taken together, these results indicate that ozonated water affects plant growth, development, and physiology as well as defense mechanisms in a species-specific manner. Interestingly, ozonated water triggered plant defenses particularly through the SA signaling pathway and the induction of PR proteins. Therefore, ozone could serve as a potential elicitor of plant defenses. However, further studies are needed, especially in more complex plant–pathogen systems, to fully understand its potential and to successfully tailor O3 treatment on the different plant species.

5. Conclusions

In conclusion, the results of this study suggest that ozonated water has a significant impact on plant growth, development, physiology, and defense mechanisms, although its effects vary depending on plant species, O3 concentration, and application method. O3 was shown to stimulate plant defenses mainly through the induction of PR proteins and the activation of salicylic acid signaling pathway. While ozone shows promise as an eco-friendly alternative to traditional pesticides, further research is necessary to fully understand its molecular mechanisms. This knowledge could enable the application of O3, mainly as ozonated water, as a sustainable tool in crop protection strategies.

Supplementary Materials

The following supporting information can be downloaded at www.mdpi.com/article/10.3390/agronomy15030567/s1, Tables S1–S4: Primers designed and used for L. sativa, N. tabacum, S. lycopersicum and P. vulgaris; Figure S1: Gene expression analysis of defense-related genes in potted plants irrigated with ozonated water (30 min, T1 treatment; 1 h, T2 treatment): (A) L. sativa; (B) N. tabacum; (C) S. lycopersicum; (D) P. vulgaris. Error bars represent the standard error of the mean (SEM). * p-value < 0.05. Figure S2: Stomatal conductance (A) and photosystem II activity (ϕPSII) (B) of L. sativa plants cultivated in the hydroponic system after treatment of the nutrient solution with O3 for 15 min (T1) and 30 min (T2). Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s post hoc tests (p-value < 0.05). Figure S3: Gene expression analysis of defense-related genes in leaves (A) and roots (B) of L. sativa hydroponically grown plants under nutritional stress treated with O3 for 15 min (T1) and 30 min (T2). As a control for each gene, the C20% samples were used. Error bars represent the SEM. * p-value < 0.05.

Author Contributions

Conceptualization, C.P., D.G., C.B. and S.T.; methodology, C.P., D.G., C.B., B.G. and S.T.; software, C.P.; validation C.P.; formal analysis, C.P. and B.G.; investigation, C.P. and B.G.; resources, S.T.; data curation, C.P. and B.G.; writing—original draft preparation, C.P.; writing—review and editing, C.P., D.G. and S.T.; visualization, C.P. and S.T.; supervision, S.T.; project administration, S.T.; funding acquisition, S.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by MUR and EU-FSE, which financially supported the PhD fellowship of Chiara Pastacaldi (D.M. 1061/2021, PON Research and Innovation 2014–2020), XXXVII Cycle.

Data Availability Statement

The original contributions presented in the study are all included in the paper; further inquiries can be directed to the corresponding authors.

Acknowledgments

Chiara Pastacaldi’s PhD grant was supported by MUR, PON “Ricerca e Innovazione” 2014–2020, Action IV.5 “Dottorati su tematiche green”, DM 1061/2021 (DOT13NTSF8-3, B11B21004830007), in collaboration with the TEA group srl. This study contributed to the research carried out within the Agritech National Research Center, European Union Next-GenerationEU, PNRR CN00000022, 10.13039/501100000780. Special thanks are due to Elisa Masi and her staff for their support in the analysis of stomatal conductance and PSII activity.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. World Population Prospects—Population Division—United Nations. Available online: https://population.un.org/wpp/graphs?loc=900&type=Probabilistic%20Projections&category=Population&subcategory=1_Total%20Population (accessed on 21 November 2024).
  2. Anderson, R.; Bayer, P.E.; Edwards, D. Climate Change and the Need for Agricultural Adaptation. Curr. Opin. Plant Biol. 2020, 56, 197–202. [Google Scholar] [CrossRef]
  3. Arora, N.K. Impact of Climate Change on Agriculture Production and Its Sustainable Solutions. Environ. Sustain. 2019, 2, 95–96. [Google Scholar] [CrossRef]
  4. Nicolopoulou-Stamati, P.; Maipas, S.; Kotampasi, C.; Stamatis, P.; Hens, L. Chemical Pesticides and Human Health: The Urgent Need for a New Concept in Agriculture. Front. Public Health 2016, 4, 148. [Google Scholar] [CrossRef] [PubMed]
  5. Vlaiculescu, A.; Varrone, C. Chapter 14—Sustainable and Eco-Friendly Alternatives to Reduce the Use of Pesticides. In Pesticides in the Natural Environment; Singh, P., Singh, S., Sillanpää, M., Eds.; Elsevier: Amsterdam, The Netherlands, 2022; pp. 329–364. ISBN 978-0-323-90489-6. [Google Scholar]
  6. FAOSTAT. Available online: https://www.fao.org/faostat/en/#data/RP/visualize (accessed on 21 November 2024).
  7. Mahmood, I.; Imadi, S.R.; Shazadi, K.; Gul, A.; Hakeem, K.R. Effects of Pesticides on Environment. In Plant, Soil and Microbes; Hakeem, K.R., Akhtar, M.S., Abdullah, S.N.A., Eds.; Springer International Publishing: Cham, Switzerland, 2016; pp. 253–269. ISBN 978-3-319-27453-9. [Google Scholar]
  8. Ozone Properties. Available online: https://ozonesolutions.com/blog/ozone-properties/ (accessed on 21 November 2024).
  9. Von Sonntag, C.; Von Gunten, U. Chemistry of Ozone in Water and Wastewater Treatment: From Basic Principles to Applications; IWA Publishing: London, UK, 2012; ISBN 978-1-78040-083-9. [Google Scholar]
  10. Remondino, M.; Valdenassi, L. Different Uses of Ozone: Environmental and Corporate Sustainability. Literature review and case study. Sustainability 2018, 10, 4783. [Google Scholar] [CrossRef]
  11. Martínez, S.B.; Pérez-Parra, J.; Suay, R. Use of Ozone in Wastewater Treatment to Produce Water Suitable for Irrigation. Water Resour. Manag. 2011, 25, 2109–2124. [Google Scholar] [CrossRef]
  12. Botondi, R.; Barone, M.; Grasso, C. A Review into the Effectiveness of Ozone Technology for Improving the Safety and Preserving the Quality of Fresh-Cut Fruits and Vegetables. Foods 2021, 10, 748. [Google Scholar] [CrossRef] [PubMed]
  13. Risoli, S.; Lauria, G. Ozonated Water Application as an Innovative Tool for Elicitation of Plant Defense Response: A Minireview. Curr. Opin. Environ. Sci. Health 2022, 28, 100375. [Google Scholar] [CrossRef]
  14. Ohashi-Kaneko, K.; Yoshii, M.; Isobe, T.; Park, J.-S.; Kurata, K.; Fujiwara, K. Nutrient Solution Prepared with Ozonated Water Does Not Damage Early Growth of Hydroponically Grown Tomatoes. Ozone Sci. Eng. 2009, 31, 21–27. [Google Scholar] [CrossRef]
  15. Díaz-López, M.; Siles, J.A.; Ros, C.; Bastida, F.; Nicolás, E. The Effects of Ozone Treatments on the Agro-Physiological Parameters of Tomato Plants and the Soil Microbial Community. Sci. Total Environ. 2022, 812, 151429. [Google Scholar] [CrossRef] [PubMed]
  16. Díaz-López, M.; Galera, L.; Bastida, F.; Nicolás, E. Tomato Growth and Physiology as Well as Soil Physicochemical and Biological Properties Affected by Ozonated Water in a Saline Agroecosystem. Sci. Total Environ. 2024, 906, 167472. [Google Scholar] [CrossRef] [PubMed]
  17. Ainsworth, E.A. Understanding and Improving Global Crop Response to Ozone Pollution. Plant J. 2017, 90, 886–897. [Google Scholar] [CrossRef] [PubMed]
  18. Jaspers, P.; Kollist, H.; Langebartels, C.; Kangasjärvi, J. Plant Responses to Ozone. In Antioxidants and Reactive Oxygen Species in Plants; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 2005; pp. 268–292. ISBN 978-0-470-98856-5. [Google Scholar]
  19. Tahamolkonan, M.; Ghahsareh, A.M.; Ashtari, M.K.; Honarjoo, N. Tomato (Solanum lycopersicum) Growth and Fruit Quality Affected by Organic Fertilization and Ozonated Water. Protoplasma 2022, 259, 291–299. [Google Scholar] [CrossRef] [PubMed]
  20. Labair, H.; Nemmich, S.; Chiali Charif, K.; Ramdani, N.; Nassour, K.; Reguig, M.; Tilmatine, A. Investigating the Synergistic Impact of Ozonated Water Irrigation and Organic Fertilization on Tomato Growth. Emir. J. Food Agric. 2024, 36, 1–9. [Google Scholar] [CrossRef]
  21. Najarian, M.; Mohammadi-Ghehsareh, A.; Fallahzade, J.; Peykanpour, E. Responses of Cucumber (Cucumis sativus L.) to Ozonated Water under Varying Drought Stress Intensities. J. Plant Nutr. 2018, 41, 1–9. [Google Scholar] [CrossRef]
  22. Xu, J.-P.; Yu, Y.-C.; Zhang, T.; Ma, Q.; Yang, H.-B. Effects of Ozone Water Irrigation and Spraying on Physiological Characteristics and Gene Expression of Tomato Seedlings. Hortic Res 2021, 8, 180. [Google Scholar] [CrossRef]
  23. Guo, Z.; Wang, Z.; Li, Y.; Wang, Q. Effect of Different Concentrations of Ozone on in Vitro Plant Pathogens Development, Tomato Yield and Quality, Photosynthetic Activity and Enzymatic Activities. Ozone Sci. Eng. 2019, 41, 531–540. [Google Scholar] [CrossRef]
  24. Veronico, P.; Paciolla, C.; Sasanelli, N.; De Leonardis, S.; Melillo, M.T. Ozonated Water Reduces Susceptibility in Tomato Plants to Meloidogyne incognita by the Modulation of the Antioxidant System. Mol. Plant Pathol. 2017, 18, 529–539. [Google Scholar] [CrossRef] [PubMed]
  25. Prigigallo, M.I.; Melillo, M.T.; Bubici, G.; Dobrev, P.I.; Vankova, R.; Cillo, F.; Veronico, P. Ozone Treatments Activate Defence Responses against Meloidogyne incognita and Tomato Spotted Wilt Virus in Tomato. Pest Manag. Sci. 2019, 75, 2251–2263. [Google Scholar] [CrossRef] [PubMed]
  26. Guo, H.; Sun, Y.; Yan, H.; Li, C.; Ge, F. O3-Induced Priming Defense Associated with the Abscisic Acid Signaling Pathway Enhances Plant Resistance to Bemisia tabaci. Front. Plant Sci. 2020, 11, 93. [Google Scholar] [CrossRef]
  27. Hoagland, D.R.; Arnon, D.I. The Water-Culture Method for Growing Plants Without Soil. Circular. Calif. Agric. Exp. Stn. 1950, 347, 32. [Google Scholar]
  28. Wang, J.; Lu, W.; Tong, Y.; Yang, Q. Leaf Morphology, Photosynthetic Performance, Chlorophyll Fluorescence, Stomatal Development of Lettuce (Lactuca sativa L.) Exposed to Different Ratios of Red Light to Blue Light. Front. Plant Sci. 2016, 7, 250. [Google Scholar] [CrossRef]
  29. Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T.L. Primer-BLAST: A Tool to Design Target-Specific Primers for Polymerase Chain Reaction. BMC Bioinform. 2012, 13, 134. [Google Scholar] [CrossRef] [PubMed]
  30. Tegli, S.; Bini, L.; Calamai, S.; Cerboneschi, M.; Biancalani, C. A MATE Transporter Is Involved in Pathogenicity and IAA Homeostasis in the Hyperplastic Plant Pathogen Pseudomonas savastanoi pv. nerii. Microorganisms 2020, 8, 156. [Google Scholar] [CrossRef] [PubMed]
  31. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  32. Gardoni, D.; Vailati, A.; Canziani, R. Decay of Ozone in Water: A Review. Ozone Sci. Eng. 2012, 34, 233–242. [Google Scholar] [CrossRef]
  33. Colunje, J.; Garcia-Caparros, P.; Moreira, J.F.; Lao, M.T. Effect of Ozonated Fertigation in Pepper Cultivation under Greenhouse Conditions. Agronomy 2021, 11, 544. [Google Scholar] [CrossRef]
  34. Zheng, L.; Liu, C.; Song, W. Effect of Ozonated Nutrient Solution on the Growth and Root Antioxidant Capacity of Substrate and Hydroponically Cultivated Lettuce (Lactuca sativa). Ozone Sci. Eng. 2020, 42, 286–292. [Google Scholar] [CrossRef]
  35. Berry, J.A.; Beerling, D.J.; Franks, P.J. Stomata: Key Players in the Earth System, Past and Present. Curr. Opin. Plant Biol. 2010, 13, 232–239. [Google Scholar] [CrossRef]
  36. Vainonen, J.P.; Kangasjärvi, J. Plant Signalling in Acute Ozone Exposure. Plant Cell Environ. 2015, 38, 240–252. [Google Scholar] [CrossRef] [PubMed]
  37. Martínez-Sánchez, A.; Aguayo, E. Effects of Ozonated Water Irrigation on the Quality of Grafted Watermelon Seedlings. Sci. Hortic. 2020, 261, 109047. [Google Scholar] [CrossRef]
  38. Sudhakar, N.; Nagendra-Prasad, D.; Mohan, N.; Murugesan, K. Induction of Systemic Resistance in Lycopersicon esculentum Cv. PKM1 (Tomato) against Cucumber Mosaic Virus by Using Ozone. J. Virol. Methods 2007, 139, 71–77. [Google Scholar] [CrossRef]
  39. Sharma, Y.K.; Léon, J.; Raskin, I.; Davis, K.R. Ozone-Induced Responses in Arabidopsis thaliana: The Role of Salicylic Acid in the Accumulation of Defense-Related Transcripts and Induced Resistance. Proc. Natl. Acad. Sci. USA 1996, 93, 5099–5104. [Google Scholar] [CrossRef]
  40. Ali, S.; Ganai, B.A.; Kamili, A.N.; Bhat, A.A.; Mir, Z.A.; Bhat, J.A.; Tyagi, A.; Islam, S.T.; Mushtaq, M.; Yadav, P.; et al. Pathogenesis-Related Proteins and Peptides as Promising Tools for Engineering Plants with Multiple Stress Tolerance. Microbiol. Res. 2018, 212–213, 29–37. [Google Scholar] [CrossRef] [PubMed]
  41. Meddya, S.; Meshram, S.; Sarkar, D.; S, R.; Datta, R.; Singh, S.; Avinash, G.; Kumar Kondeti, A.; Savani, A.K.; Thulasinathan, T. Plant Stomata: An Unrealized Possibility in Plant Defense against Invading Pathogens and Stress Tolerance. Plants 2023, 12, 3380. [Google Scholar] [CrossRef] [PubMed]
  42. Vogt, T. Phenylpropanoid Biosynthesis. Mol. Plant 2010, 3, 2–20. [Google Scholar] [CrossRef]
  43. Tamaoki, M. The Role of Phytohormone Signaling in Ozone-Induced Cell Death in Plants. Plant Signal. Behav. 2008, 3, 166. [Google Scholar] [CrossRef]
  44. Paolacci, A.R.; D’Ovidio, R.; Marabottini, R.; Nali, C.; Lorenzini, G.; Abenavoli, M.R.; Badiani, M. Research Note: Ozone Induces a Differential Accumulation of Phenyalanine Ammonia-Lyase, Chalcone Synthase and Chalcone Isomerase RNA Transcripts in Sensitive and Resistant Bean Cultivars. Funct. Plant Biol. 2001, 28, 425. [Google Scholar] [CrossRef]
  45. Pazarlar, S.; Cetinkaya, N.; Bor, M.; Ozdemir, F. Ozone Triggers Different Defence Mechanisms against Powdery Mildew (Blumeria graminis DC. Speer f. sp. tritici) in Susceptible and Resistant Wheat Genotypes. Funct. Plant Biol. 2017, 44, 1016. [Google Scholar] [CrossRef] [PubMed]
  46. Lee, J.-H.; Goto, E. Ozone Control as a Novel Method to Improve Health-Promoting Bioactive Compounds in Red Leaf Lettuce (Lactuca sativa L.). Front. Plant Sci. 2022, 13, 1045239. [Google Scholar] [CrossRef] [PubMed]
  47. Marchica, A.; Ascrizzi, R.; Flamini, G.; Cotrozzi, L.; Tonelli, M.; Lorenzini, G.; Nali, C.; Pellegrini, E. Ozone as Eustress for Enhancing Secondary Metabolites and Bioactive Properties in Salvia officinalis. Ind. Crops Prod. 2021, 170, 113730. [Google Scholar] [CrossRef]
  48. Del Bianco, M.; Kepinski, S. Context, Specificity, and Self-Organization in Auxin Response. Cold Spring Harb. Perspect. Biol. 2011, 3, a001578. [Google Scholar] [CrossRef]
  49. Waller, F.; Furuya, M.; Nick, P. OsARF1, an Auxin Response Factor from Rice, Is Auxin-Regulated and Classifies as a Primary Auxin Responsive Gene. Plant Mol. Biol. 2002, 50, 415–425. [Google Scholar] [CrossRef] [PubMed]
  50. Ellis, C.M.; Nagpal, P.; Young, J.C.; Hagen, G.; Guilfoyle, T.J.; Reed, J.W. AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2regulate Senescence and Floral Organ Abscission in Arabidopsis thaliana. Development 2005, 132, 4563–4574. [Google Scholar] [CrossRef] [PubMed]
  51. Wang, D.; Pajerowska-Mukhtar, K.; Culler, A.H.; Dong, X. Salicylic Acid Inhibits Pathogen Growth in Plants through Repression of the Auxin Signaling Pathway. Curr. Biol. 2007, 17, 1784–1790. [Google Scholar] [CrossRef] [PubMed]
  52. Kazan, K.; Manners, J.M. Linking Development to Defense: Auxin in Plant–Pathogen Interactions. Trends Plant Sci. 2009, 14, 373–382. [Google Scholar] [CrossRef] [PubMed]
  53. Staswick, P.E.; Tiryaki, I. The Oxylipin Signal Jasmonic Acid Is Activated by an Enzyme That Conjugates It to Isoleucine in Arabidopsis. Plant Cell 2004, 16, 2117–2127. [Google Scholar] [CrossRef]
  54. Campos, M.L.; Kang, J.-H.; Howe, G.A. Jasmonate-Triggered Plant Immunity. J. Chem. Ecol. 2014, 40, 657–675. [Google Scholar] [CrossRef] [PubMed]
  55. Dos Santos, C.; Franco, O.L. Pathogenesis-Related Proteins (PRs) with Enzyme Activity Activating Plant Defense Responses. Plants 2023, 12, 2226. [Google Scholar] [CrossRef] [PubMed]
Agronomy 15 00567 g001
Figure 1. Plants of L. sativa (A), N. tabacum (D), S. lycopersicum (G), and P. vulgaris (J), cultivated in pots in an open-field experiment, and irrigated with water ozonated for 30 min (T1 treatment) or for 1 h (T2 treatment). Dry weights of treated and control plants: L. sativa leaves (B) and roots (C), N. tabacum leaves (E) and roots (F), S. lycopersicum leaves (H) and roots (I), P. vulgaris leaves (K) and roots (L). Error bars represent the standard error of the mean (SEM). Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Figure 1. Plants of L. sativa (A), N. tabacum (D), S. lycopersicum (G), and P. vulgaris (J), cultivated in pots in an open-field experiment, and irrigated with water ozonated for 30 min (T1 treatment) or for 1 h (T2 treatment). Dry weights of treated and control plants: L. sativa leaves (B) and roots (C), N. tabacum leaves (E) and roots (F), S. lycopersicum leaves (H) and roots (I), P. vulgaris leaves (K) and roots (L). Error bars represent the standard error of the mean (SEM). Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Agronomy 15 00567 g001
Agronomy 15 00567 g002
Figure 2. Chlorophyll a content in plant leaves after irrigation with ozonated water (30 min, T1 treatment; 1 h, T2 treatment) in (A) L. sativa, (B) N. tabacum, (C) S. lycopersicum, and (D) P. vulgaris Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Figure 2. Chlorophyll a content in plant leaves after irrigation with ozonated water (30 min, T1 treatment; 1 h, T2 treatment) in (A) L. sativa, (B) N. tabacum, (C) S. lycopersicum, and (D) P. vulgaris Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Agronomy 15 00567 g002
Agronomy 15 00567 g003
Figure 3. Stomatal conductance of plants irrigated with ozonated water T1 (30 min treatment) and T2 (1 h treatment) at the end of the open-field experiment measured with a porometer/fluorometer LICOR-600: (A) N. tabacum; (B) S. lycopersicum. Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s post hoc tests (p-value < 0.05).
Figure 3. Stomatal conductance of plants irrigated with ozonated water T1 (30 min treatment) and T2 (1 h treatment) at the end of the open-field experiment measured with a porometer/fluorometer LICOR-600: (A) N. tabacum; (B) S. lycopersicum. Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s post hoc tests (p-value < 0.05).
Agronomy 15 00567 g003
Agronomy 15 00567 g004
Figure 4. Photosystem II activity (ϕPSII) of plants irrigated with ozonated water T1 (30 min treatment) and T2 (1 h treatment) at the end of the open-field experiment measured with a porometer/fluorometer LICOR-600: (A) N. tabacum; (B) S. lycopersicum. Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Figure 4. Photosystem II activity (ϕPSII) of plants irrigated with ozonated water T1 (30 min treatment) and T2 (1 h treatment) at the end of the open-field experiment measured with a porometer/fluorometer LICOR-600: (A) N. tabacum; (B) S. lycopersicum. Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s or Dunn’s post hoc tests (p-value < 0.05).
Agronomy 15 00567 g004
Agronomy 15 00567 g005
Figure 5. Gene expression analysis of defense-related genes in potted plants irrigated with ozonated water (30 min, T1 treatment; 1 h, T2 treatment): (A) L. sativa; (B) N. tabacum; (C) S. lycopersicum; (D) P. vulgaris. Error bars represent the SEM. * p-value < 0.05.
Figure 5. Gene expression analysis of defense-related genes in potted plants irrigated with ozonated water (30 min, T1 treatment; 1 h, T2 treatment): (A) L. sativa; (B) N. tabacum; (C) S. lycopersicum; (D) P. vulgaris. Error bars represent the SEM. * p-value < 0.05.
Agronomy 15 00567 g005
Agronomy 15 00567 g006
Figure 6. Dry weights of leaves (A) and roots (B) of hydroponically grown L. sativa plants after treatment of the C20% nutrient solution with O3 for 15 min (T1) and 30 min (T2). Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s post hoc tests (p-value < 0.05).
Figure 6. Dry weights of leaves (A) and roots (B) of hydroponically grown L. sativa plants after treatment of the C20% nutrient solution with O3 for 15 min (T1) and 30 min (T2). Error bars represent the SEM. Letters indicate statistically significant differences between treatments assessed with Tukey’s post hoc tests (p-value < 0.05).
Agronomy 15 00567 g006
Agronomy 15 00567 g007
Figure 7. Gene expression analysis of defense-related genes of leaves (A) and roots (B) of L. sativa hydroponically grown plants under nutritional stress treated with O3 for 15 min (T1) and 30 min (T2). As a control for each gene, the C100% samples were used. Error bars represent the SEM. * p-value < 0.05.
Figure 7. Gene expression analysis of defense-related genes of leaves (A) and roots (B) of L. sativa hydroponically grown plants under nutritional stress treated with O3 for 15 min (T1) and 30 min (T2). As a control for each gene, the C100% samples were used. Error bars represent the SEM. * p-value < 0.05.
Agronomy 15 00567 g007
Table 1. Composition of Hoagland solution.
Table 1. Composition of Hoagland solution.
ComponentsVolume (mL)
1 M KH2PO41.0
1 M KNO36.0
1 M Ca(NO3)2·4H2O4.0
1 M MgSO4·7H2O2.0
Micronutrient solution *1.0
Fe-EDDHA 60 g/L1.0
Distilled H2OUp to 1000
* 2.86 g H3BO3; 1.81 g MnCl2·4H2O; 0.22 g ZnSO4·7H2O; 0.08 g CuSO4·5H2O; 0.02 g H2MoO4·H2O.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Pastacaldi, C.; Gaudioso, D.; Beltrami, C.; Gunnella, B.; Tegli, S. On the Effectiveness of Ozone Treatments: A Silver Bullet for Plant Health? Agronomy 2025, 15, 567. https://doi.org/10.3390/agronomy15030567

AMA Style

Pastacaldi C, Gaudioso D, Beltrami C, Gunnella B, Tegli S. On the Effectiveness of Ozone Treatments: A Silver Bullet for Plant Health? Agronomy. 2025; 15(3):567. https://doi.org/10.3390/agronomy15030567

Chicago/Turabian Style

Pastacaldi, Chiara, Dario Gaudioso, Cosimo Beltrami, Benedetta Gunnella, and Stefania Tegli. 2025. "On the Effectiveness of Ozone Treatments: A Silver Bullet for Plant Health?" Agronomy 15, no. 3: 567. https://doi.org/10.3390/agronomy15030567

APA Style

Pastacaldi, C., Gaudioso, D., Beltrami, C., Gunnella, B., & Tegli, S. (2025). On the Effectiveness of Ozone Treatments: A Silver Bullet for Plant Health? Agronomy, 15(3), 567. https://doi.org/10.3390/agronomy15030567

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop