In the last decade, the need for suitable, innovative, and non-chemical strategies for agrifood production and plant protection has given rise to new tools for microbial decontamination, plant resistance induction, and enhancement of growth and nutraceutical properties of products. Low-temperature plasma is proposed as a revolutionary tool in modern agriculture, offering versatile applications that span from seed treatment to soil improvement [1]. This novel approach involves subjecting agricultural systems to non-thermal plasma, generating reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). These plasma-induced reactive species have shown great potential in influencing various aspects of plant growth, stress response, and overall crop performance [2–4]. However, plasma treatments are mainly confined to the surface of materials, and therefore not able to interact with all plant tissues. Thus, different strategies are required for applications, such as the production and usage of plasma-activated media.

In conjunction with the advancements in low-temperature plasma applications, Plasma Activated Water (PAW) has emerged as a cutting-edge and transformative tool in modern agriculture, offering promising potential to enhance crop productivity and address various challenges faced by the industry. This novel approach involves subjecting water to non-thermal plasma exposing it to ROS and RNS [5] and inducing changes in the physicochemical properties of the liquid. These changes can be tailored for different applications [6,7]. Reactive species in PAW impart beneficial effects on plants inducing physiological and biochemical changes with positive effects on growth and stress tolerance [6]. Thus, PAW can be used for watering crops, soaking seeds, and spraying on foliage, being a sustainable and environmentally friendly alternative to traditional chemical fertilizers and pesticides. Recent research on PAW has shown its potential in agriculture. Studies have found that PAW can enhance seed germination, plant growth, and yield across various crops like wheat, cucumber, and tomato [8,9]. It also improves photosynthetic efficiency, and antioxidant enzyme activities [10], and induces self-defense mechanisms in plants [11,12]. However, further research is needed to fully harness its potential.

In this investigation, we studied the potential of PAW to promote the growth of tomato plants under both normal and virus-induced stress conditions, caused by Tomato mottle mosaic virus (ToMMV) infection. This dual-pronged approach allowed us to unravel the biological and physiological mechanisms underlying the multifaceted effects of PAW on the growth of both healthy and virus-inoculated plants.

In the examination of healthy plants, our study employed a comprehensive set of parameters to assess the impact of PAW. We closely monitored the growth of the tomato plants, by measuring parameters such as stem length, diameter and leaf area, and the influence of PAW on key components of the plant’s antioxidant defense system, including ascorbate, glutathione, chlorophylls, and carotenoids content. Additionally, the gene expression of four different defense-related genes [phenylalanine ammonia-lyase (PAL), pathogenesis-related protein 1 (PR-1), lipoxygenase (LOX), and catalase (CAT)] was analyzed to explore tomato plant responses after PAW irrigation and ToMMV inoculation. Expanding our focus to plants subjected to ToMMV-induced stress, we implemented a comparative analysis employing micro X-ray fluorescence (μ-XRF) spectroscopy to visualize the changes in the distribution of micro and macro-nutrients in the plant leaves, unveiling insights into the nuanced responses mechanisms of virus-inoculated plants to PAW treatment.

In this paper, we evaluated Plasma Activated Water (PAW) as a novel irrigation medium to promote growth and defense responses in tomato seedlings, either healthy or inoculated with ToMMV. Plasma-based technologies could pose an interesting and viable alternative to traditional fertilizing methods, by their nature as on/off sources that do not require the employment of chemicals and eliminate the need for stockage [32].

PAW was produced by exposing double-distilled water to a VDBD plasma in a custom-made reactor. The “activation” process involves chemical species that are produced at the onset of the plasma discharge. The main reactants in the gas phase that are exposed to plasma are molecular oxygen and nitrogen, from the supplied air, and water vapor. When the discharge is ignited, these molecules are immediately vibrationally excited, and the energy provided by the plasma breaks them apart and fosters a series of reaction pathways that are not yet fully understood. For example, from the spectroscopic observations we reported, we can infer the formation of NO, atomic oxygen, and hydroxyl radicals near the discharge filaments. These species are then dissolved in the water bulk through the water-gas interface, where they continue to undergo a series of reactions and chemical transformations. As reported by Lukes et al. [33], the transient species produced in the discharge (namely, OH, NO, and NO₂ radicals) are converted into more stable chemical products once they are dissolved in water, such as H₂O₂, NO 2 − and NO 3 − , acidifying the produced PAW. At the acidic pH values that are typical of the produced PAW, moreover, a conversion mechanism of H₂O₂ and NO 2 − into NO 3 − through the peroxynitrite intermediate is established [33], leading to the depletion of hydrogen peroxide and nitrite ion and an increase in the concentration of nitrate ion at longer treatment times. A week after the PAW production, this reaction results in the complete disappearance of NO 2 − , a decrease of the H₂O₂ content in the PAW, and conversely, an increase in NO 3 − . The acidity (pH 3.0 ± 0.1) after complete treatment of the solution can be attributed mainly to nitric acid. However, the acidic nature of PAW is not a problem when used to water plants grown on soil or a solid substrate since soils and substrates usually have a buffer capacity that prevents substrate acidification or strongly limits pH variations, such as in the case of our study. A proper selection of soil and substrate, as it is usually done in horticulture, is therefore important to get a final pH that is suitable for the growth of the selected crop. In addition, NO 3 − uptake by plant roots occurs through a symport transport with H⁺ ions which causes an increase of pH of the growth medium [34]. Being nitrogen mainly in the form of NO 3 − in PAW, such physiological process contributes to balance PAW acidity. Differently, more attention should be paid when PAW is used in hydroponic systems. In this case, the solution should be properly buffered (e.g., by adding other mineral nutrients as base salts or metals with catalytic properties for nitrogen fixation [35,36]) before use.

The measured ORP of 506 ± 11 mV, which suggests an oxidizing character of the PAW, is caused by the presence of all the above-mentioned species, known to possess highly oxidative properties. The typical ORP value of the PAW could prove useful in the decontamination of soils against plant pathogens to prevent and manage soilborne diseases and could also put the plant cell into mild oxidative stress, unlocking new metabolic pathways for growth and defense responses conferring resistance to biotic and abiotic stresses.

Biometric measurements were performed on plants after 21 days of irrigation for the four different treatment conditions. Results showed a statistically different behavior of PAW irrigated samples with respect to other conventional treatments and untreated ones. In particular, the growth of PAW-irrigated seedlings was enhanced by at least 10% for all the measured parameters. Moreover, to evaluate the effect of PAW on the fitness of the seedlings after 21 days of treatment, antioxidant molecules such as ascorbic acid and glutathione and pigments such as chlorophylls and carotenoids were also monitored.

Chlorophylls and carotenoids are pigments that with proteins form the light-harvesting complexes that are part of photosystems, functional units of photosynthesis [37]. Although WNPK and WAN induced an increase in both chl a and b implying a higher photosynthetic activity, the higher increase of chl a than chl b in the PAW treatment suggests in the latter an easier translocation of energy from chl b to chl a. This trend facilitates the irreversible direction in the energy capture process making the energy transfer of light excitation to the reaction center more efficient.

As a consequence, this could lead to higher production of NADPH and ATP in the light-dependent reactions (first stage of the photosynthesis) and their increased availability for the Calvin cycle (second stage of the photosynthesis) with higher production of photosynthates (such as sucrose), necessary for plant growth and productivity. The sugar sucrose is a source of glucose which is the substrate, in the Smirnoff-Wheeler pathway, for vitamin C biosynthesis in plants [38] that, in our study, increased in PAW-treated seedlings. The presence of ascorbate is associated with preserving the photosynthetic process [39] and limiting ROS-mediated damage and leaf senescence [40]. The decrease in chlorophyll a was linked to reductions in AsA content, leading to decreased RuBisCO activity and CO₂ assimilation [40].

It is known that an augmented carotenoid level could be correlated to counteract the damage to the photosynthetic apparatus induced by UV light [41]. The fact that an increase of carotenoid content in PAW was not observed as compared to WAN, could therefore suggest the absence of relevant perturbations in the light-harvesting complex and the energy transfer towards the reaction center [37].

Ascorbate and glutathione are part of the ascorbate-glutathione cycle [42] and are the most abundant low molecular-weight antioxidants in plant cells. They regulate the concentration of the reactive oxygen species in plant cells, particularly hydrogen peroxide [38]; however, they are multifaceted molecules involved in various processes such as plant growth and defense [43]. The higher content of AsA in tomato seedlings irrigated with PAW strongly suggests the role of PAW in promoting the production of this antioxidant compound. On the other hand, the content of reduced glutathione (GSH - another important antioxidant metabolite of the plant) and glutathione pool (GSH + GSSG) remained unchanged in the presence of PAW with respect to the NTDDW control, suggesting a more relevant involvement of AsA rather than GSH in the PAW-modified plant metabolism, confirming the prominent role of ascorbate in the plant defense responses [44,45]. AsA, counteracting the oxidative stress in cells, underlines its key role played in the tolerance to several biotic and abiotic stresses [46]. The decreased GSH content in both WNPK and WAN samples with the contemporaneous higher GSSG production contributed to a shift in the redox state of this metabolite towards the oxidative form with consequent higher oxidized cell status [47] in those seedlings.

The possible induction of genes implicated in plant defense by irrigation of tomato seedlings with PAW was investigated at different time points. Transcript changes of the analyzed genes, including PR-1 and PAL with a key role in defense responses to pathogens, LOX, and the antioxidant enzyme CAT, were compared between treated and untreated control plants. In particular, PR-1 is a molecular marker for the salicylic acid-activated pathway that is rapidly and strongly induced as a response to pathogen infections [48]. All four genes were not activated, in our experiments, during the early stages after plant exposure to PAW. However, the plants irrigated with PAW showed a significant upregulation of the majority of the genes, slightly predominant for PR-1, following inoculation with the virus ToMMV, suggesting an effect of priming of induced defense responses by the treatment [49]. This could suggest that the activation of plant defense responses, possibly primed by reactive species in PAW, should allow the treated plants to be more promptly reactive against pathogens. In the uninoculated condition, downregulation of the same genes in treated vs. untreated samples could be likely due to the activation in treated plants of primary metabolism responsible for plant growth and development instead of that related to defenses [31].

In the cultivar Regina we used for the test, which is a traditional, long-lasted local variety in our region, the symptoms expression appeared very late in the time frame chosen for our experimental plan and, thus, it could be considered tolerant to ToMMV infection. Significant symptoms we observed, such as green mottling, shoe-string leaves, and stunting, started to be visible at least 3 weeks after inoculation. The comparison between I-PAW and I-NTDDW showed indeed a higher virus accumulation rate, with an increase in the former rather than in the latter. It can be hypothesized that the infection of a tobamovirus, having such an active replication and accumulation in the infected tissues, is somehow favored by an overall better nutritional status and nutrient mobilization as promoted by the PAW treatment. It could mean that the plants watered with PAW uptake higher amounts of nutrients (e.g., mineral elements) to support and even enhance the virus replication in well-nourished tissues.

In plants, disorders in element homeostasis at the leaf level are often correlated to nutritional deficiencies, the presence of pathogens (including viruses), environmental stresses, etc. Observing the changes in the spatial distribution of certain macro and/or micronutrients can provide valuable information for understanding the type and the impact of the stressor. For such investigations, μ-XRF is an essential and effective technique [31]. Significant changes in elemental distribution were observed in μ-XRF maps for P, S, Ca, and K in the virus-inoculated sample watered only with distilled water (I-NTDDW) as compared to a nutrient-sufficient uninoculated plant (WNPK), showing several spotted depletion zones. The lack (or lower concentration) of these important macronutrients in many leaf cells provides evidence of the virus infection, resulting in the already mentioned visual symptoms such as stunting, shoe-stringed and mottled leaves, and generalized yellowing of the foliage. Yet, some inhomogeneities in element distribution could be also observed in the plants watered only with double-distilled water (NTDDW), suggesting nutrient deficiencies, as compared to the nutrients-sufficient control (WNPK). However, the virus inoculation appeared to worsen the pre-existing nutritional stress status, with an evident increase in the size and number of nutrients-depleted areas (Figure 13). Such inhomogeneous distributions are not evident in the PAW watered samples, thus suggesting that PAW can provide a sufficient level of nutrients to tomato plants, similar to what happens to the plants irrigated with a solution containing the chemical fertilizer (WNPK). In addition, the irrigation of virus-inoculated plants with PAW (I-PAW) appeared to almost completely restore the mineral nutrients distribution in tomato leaves, becoming more similar to that of the control healthy plant (WNPK).

Finally, the S distribution in PAW-treated plants, either inoculated or uninoculated, showed that S concentration increases in leaf veins, suggesting a mobilization of S-containing compounds (e.g., glutathione) from plant leaves through the phloem. Indeed, S assimilated in leaves is exported via the phloem to sites of protein synthesis (shoot and root apices, and fruits) mainly as glutathione [34] which can also act as a non-enzymatic antioxidant that can be activated to overcome oxidative perturbations [50] like those induced by PAW.

The results of this study regarding the PAW’s effects on plant growth and defense responses are in accordance with previous studies [9–12]. Generally, the results indicate the presence of a better physiological status of the PAW-treated plants to cope with oxidative stress and maintain cellular homeostasis. The augmented chlorophyll a content with the increase of antioxidant molecules such as the ascorbate suggests that PAW treatment can potentiate the plant defense and contribute to higher sugar production, necessary for plant growth and productivity. In agreement with our results, an enhancement of plant defense responses induced by PAW treatment has been also reported by several authors [12,51]. Overall, these findings highlight the potential of PAW as a sustainable and innovative irrigation medium for enhancing plant performance and resilience in the face of environmental challenges. However, additional studies regarding the involvement and effect of PAW on other antioxidant systems (such as enzymatic activity, and phenols) and metabolic pathways (such as sugar synthesis) are necessary to better understand the potential of PAW in the plant systems.

This thorough exploration, encompassing both healthy and virus-stressed conditions, provides a more detailed understanding of the dynamic interplay between PAW and tomato plant growth. Our findings aim to contribute not only to the growing body of knowledge surrounding the applications of PAW but also to the specific insights into its potential as a stress-alleviating agent in the context of viral infections. Through the examination of plant development, biochemical responses, nutrient dynamics, and gene expression patterns, our research provides further evidence of the role of PAW as a sustainable alternative to conventional chemicals used in agriculture. Furthermore, the over-expression of the defense-related genes of plants irrigated with PAW suggests that the crop resilience to pathogenic challenges could be enhanced potentially reducing crop losses on the field.