The Influence of Some Reactive Oxygen Species Treatments on the Yield and Changes in the Chemical Composition of Potato Tubers (Solanum tuberosum L.)

Abstract

In agriculture, there are various types of progress, and trends in agricultural production include the minimized use of chemicals and the production of food with favorable taste and increased nutritional value. In this study, during a three-year field experiment on potato cultivar Agria, ozone was applied in a fumigation process with an invented device, and hydrogen peroxide was applied as a foliar spray. The yield of ozone-treated plants was lower than of the control plants. Hydrogen peroxide treatment did not affect the yield of potatoes. Ozone fumigation had a positive effect on the chemical composition of tubers, while in the case of hydrogen peroxide, this effect occurred only in one year of research and only for selected parameters. The antioxidant response of plants, which is a defense response to artificially induced oxidative stress, contributes to the production of antioxidant-like metabolites by increasing the antiradical activity of crops by 3–23%, increasing the total content of polyphenolic compounds by 12–108%, and increasing the total antioxidant capacity by 23–42%. The obtained results confirmed that appropriate doses of ROS can be used in the field cultivation of potatoes to increase the nutritional value of tubers.

1. Introduction

One of the main markers of abiotic stress leading to yield reduction is the overproduction of reactive oxygen species (ROS) [1,2,3]. ROS occur in both radical and non-radical forms, which are more toxic due to their high reactivity. Radicals include superoxide anions (O₂•), hydrogen peroxide (H₂O₂), singlet oxygen (¹O₂), and hydroxyl radicals (OH•), which can be harmful and cause various physiological, molecular and biochemical responses [4,5,6]. ROS are also important signaling molecules that alert plants to adjust their metabolism [7] and adapt to adverse conditions. Data are available on the production of secondary metabolites such as phenol compounds, flavonoids, tannins, saponins, and anthocyanins in various plant species subjected to abiotic stresses [8]. It is possible to distinguish positive or negative changes, depending on a number of factors, such as the plant species, developmental stage, and duration of stress [9]. Protective strategies, including ionic homeostasis, activation of antioxidant enzymes, and production of various compatible organic solutes, are among the strategies adopted by plants to defend themselves against unfavorable changes [10,11].

Various biotic and abiotic stress factors affect the growth of plants under field conditions throughout their life cycle. Evolution has evolved a strategy for the adaptation of these organisms to environmental stressors. From the other site, plants have developed mechanisms regulating stress factors [12,13]. Abiotic stresses, including water deficiency and heat stress, lead to the formation of ROS through electron misdirection in photosystems. Plants respond to water deficiency conditions by increasing the production of osmolytes, which in turn increases the osmotic potential in the cell. Heat stress in plants causes the accumulation of ROS and damage to the cell membrane [14,15,16,17]. The main effect of a metabolic imbalance is oxidative stress caused by the excessive production of reactive oxygen species (ROS). Therefore, to maintain normal growth and development, plants organize a myriad of stress-responsive metabolites, such as proline and polyamines, along with several antioxidant enzymes involved in ROS detoxification. Recent studies have also revealed the ability of ROS to act as signalling molecules in activating defence responses [18,19,20]. Thus, ROS are now considered not only as toxic byproducts of oxygen metabolism with tightly controlled cellular levels but also as signalling factors regulating many biological processes and exerting pleiotropic effects [21,22,23].

Ozone is a molecule composed of three oxygen atoms. It is a powerful oxidizer that can be obtained from portable generators. Its advantages are the low cost of the materials used in the ozonation process and the absence of harmful residues—the half-life to atmospheric oxygen ranges from a few seconds to a few hours. The high reactivity of the ozone molecule plays a dual role in the atmosphere. The layer that protects life on Earth from harmful cosmic radiation is formed from stratospheric ozone molecules, while tropospheric O₃ is considered a major factor reducing the productivity of crops, including potatoes [24,25,26,27]. Exceeding the 40 ppb ozone threshold in the atmosphere is a critical value for crop plants, and above this level, this molecule can negatively affect growth and yield. Differences in the antioxidant activity of enzymes in the cell also affect the tolerance of plant genotypes to ozone existing in their growth environment. [28]. The ozone concentration, exposure time and the pool of alternative oxidase (AOX) affect its cytotoxicity. Plans tissue necrosis on the surface of plants can be a symptom of damage caused by ozone. However, without any visible markers, this gas can cause physiological damage and disruption of the stomata, resulting in reduced photosynthetic activity [29,30]. Significant damage (a high dose of O₃ in a short period of time) resembles a hypersensitivity response (HR), in which the effect is similar to fungal elicitors, leading to programmed cell death (PCD) and leaf damage in susceptible species [30,31,32]. In contrast, chronic damage (a low dose of O₃ over a long period of time) causes a decrease in the rate of photosynthesis, stunted growth, and leaf senescence, leading to a decrease in plant productivity, without visible damage to plant tissue [33,34,35,36,37]. Reactive oxygen species (ROS) produced in apoplasts, which are important mediators of signal transduction pathways, are closely related to the plant response to ozone. O₃ diffuses into plants through the stomata and further penetrates the apoplast, where it is rapidly converted into ROS that can react with cell wall and membrane components [38]. There is no doubt that, in some cases, ozone is a toxic factor for plants due to its strong oxidative properties, leading to plant growth inhibition [39], but it can be used as an alternative and environmentally friendly biocide; however, in plant protection, a proper dosage and exposure time for O₃ should be considered [40,41].

Hydrogen peroxide is a topical oxidizing agent that involves the formation of atomic oxygen during decomposition. It decomposes into non-toxic byproducts (water and oxygen). It is an inorganic chemical compound of the peroxide group, classified along with ozone as reactive oxygen species. H₂O₂, obtained, among other things, from the conversion of ozone, has an essential role in the coordination of physiological reactions occurring in the plant cell. In plant cells, H₂O₂ is also produced during their life processes. Its level increases in stressful situations and has an essential role in processes related to physiology (aging, movement of the stomatal apparatus, photorespiration, and photosynthesis) [42,43,44]. Hydrogen peroxide is transported across the cell membrane, alters gene expression, and modulates transcription factors [39]. It also participates in the regulation of plant growth and development, acts as a signaling molecule, and regulates the stress adaptation and programmed cell death (PCD) [45]. Yield loss is affected by stresses caused by environmental factors and responsible for ROS accumulation [4,42]. In case of high levels of ROS, a process of plant cell damage is possible, which may cause the destruction of the plant [46,47]. The biological effect depends on the location of the crop, the stage of development, and the previous occurrence of different types of stresses. The appearance of H₂O₂ at low concentrations can help plants tolerate abiotic and biotic stresses, but when concentrations increase and exceed high levels, it leads to PCD, which is important in developmental processes and environmental responses [42,46,48]. In addition, H₂O₂ works as a mild oxidant of various protein thiols, generating various types of oxidative posttranslational modifications, such as sulfenylation (-SOH), sulfinylation (-SO₂H), and sulfonylation (-SO₃H) [49,50]. Hydrogen peroxide is also an essential part of the signal transduction cascade, playing a role in plant adaption to high-stress situations and protection [51]. Changes in osmotic pressure make it possible to control the process of H₂O₂ diffusion. In turn, the process of hydrogen peroxide transport across plasma membranes is made possible by specific channels—aquaporins. [52,53]. It also plays an important role in transmitting the stomatal apparatus closure signal, which is induced by abscisic acid [51,54]. The process of strengthening cell walls also takes place through the involvement of H₂O₂.

Ozone and hydrogen peroxide, compounds that leave no chemical residue, can potentially be used on organic farms, producing high-quality food.

Potato (Solanum tuberosum L.) is a staple food with great economic and nutritional value. Among agricultural crops, it ranks fifth after rice, wheat, corn, and sugarcane [55,56,57]. The biodiversity of species and subspecies plays an important role in food security, because different varieties of the same species may contain different nutrients and biocompounds [58]. Potatoes are edible tubers and are available worldwide throughout the year. Potato varieties can be divided into several categories based on the texture and functionality of components. They are characterized by a large variety of forms with different botanical and functional characteristics. Potato tubers are cultivated for direct consumption [59], for starch isolation [60], and as a raw material for the production of processed foods [61,62]. Due to the widespread use of potato tubers, it is essential to obtain a high yield with adequate quality and nutritional value; so, this study was undertaken to investigate the effects of ozone and hydrogen peroxide applied in field cultivation on the yield and nutrient contents of potatoes.

2. Materials and Methods

2.1. Description of the Experimental Model

The experiment was performed in 2016–2018 in Lancut, Poland (50°03′08.7″ N, 22°12′20.9″ E, 254 m MSL). A single-factor microplot trial was set up using the randomized block method in four replicates. The area of individual plots was 6 m². The distance between individual factors was 2.5 m and between repetitions was 1.5 m. The experimental factor was the type of reactive oxygen form used in potato cultivation: I—control trial, II—hydrogen peroxide, and III—ozone.

Gaseous ozone was applied at a concentration of 5 ppm (fumigation time of 5 min). Hydrogen peroxide was applied as a 1% solution prepared immediately before spraying, based on demineralized water. Doses of reactive oxygen species were selected on the basis of a previous plot experiment as the maximal doses not causing phytotoxic effects (plant infestation and leaf damage).

Spraying was carried out with a handheld sprayer until the leaves were thoroughly covered with the solution, and the applied spray rate was 1.2 L per plot (converted to 2000 L∙ha⁻¹). Fumigation treatments were carried out using the device described below. Treatments were carried out every 10 days in the afternoon in order to reduce the infestation of leaves with ozone irritation under high air temperature conditions.

Mineral fertilization was determined based on soil analyses conducted annually before the experiment was established. Soil samples were taken in accordance with the Polish standard [63]. Soil reactions and the abundance of basic nutrients were analyzed using the following analytical methods: determination of the granulometric composition by the Casagrande areometric method modified by Proszynski, measurement of the reaction (pH) in water and a 1 mol·dm⁻³ KCl solution by the potentiometric method, determination of hydrolytic acidity by the Kappen method in 1 mol·dm⁻³ sodium acetate, determination of bioavailable forms of phosphorus and potassium by the Egner-Rhiem method (both components were extracted from soil buffered to pH = 3.6 with a calcium lactate solution at a concentration of 0.04 mol·dm⁻³; after extraction, phosphorus was determined calorimetrically on a Shimadzu UV-VIS UV-2600 spectrophotometer (SHIMADZU Excellence in SCIENCE, New York, NY, USA), while potassium was determined on a Hitachi Z-2000 atomic absorption spectrometer (TELEDYNE Teledyne LABS, Mason, OH, USA) by the emission method (EAAS)).

The soil abundance of the bioavailable forms of phosphorus and potassium was evaluated based on the criteria (limit numbers) specified in the Polish standards [64], and the bioavailable magnesium content was determined by the Schachtschabel method. After the extraction of soil with a 0.0125 mol·dm⁻³ CaCl₂ solution, with a ratio of soil to solution of 1:10, magnesium in the resulting extract was determined by atomic absorption spectrometry (FAAS) using a Hitachi Z-2000 apparatus. The soil abundance of bioavailable forms of magnesium was assessed based on the guidelines (limit numbers) contained in Polish standards [64], the nitrogen content was determined by the Kjeldahl method in an automated Kjeltec Foss 2300 kit (FOSS analytical, Hillerød, Denmark), and Na⁺, NH₄⁺, K⁺, Mg²⁺, Ca²⁺,NO₃⁻, and SO₄²⁻ in soil aqueous extracts were determined by ion chromatography using an ICS DIONEX 5000+ apparatus (Thermo Fisher Seientific, Waltham, MA, USA).

The forecrop for potatoes was spring oats. The planting of tubers was carried out by machine using a two-row planter, maintaining the same plant density in plots at a row spacing of 75 cm, with a row every 27 cm. Planting was carried out on 21 May 2016, 17 May 2017, and 12 May 2018. The experiment used certified material—potato tubers of the Agria variety. The soil for cultivation was prepared in accordance with the principles of good agricultural practice for the species. Experimental plots were set after plant emergence in order to maintain an identical planting density. Weed control in the plots was carried out mechanically (by hand), as was the harvesting of potato beetles. Potato harvesting was carried out manually on 28.09 in 2016, 30.09 in 2017 and 18.09 in 2018. All potatoes harvested from the experimental plots were evaluated annually for marketable yield. The fraction of tubers less than 30 mm and tubers with defects were separated from the samples, and the remaining tubers were weighed and the percentage of the total yield from the plot was determined [65].

The first year of running the experiment was treated as a pilot. The manufactured device used for the fumigation of plants was checked under field conditions, and yield analyses were limited only to the determination of yield and starch and vitamin C contents.

2.2. Plant Material

Potatoes of the Agria variety were used in the experiment. The characteristics of this variety are as follows: earliness—medium–early, shape—oblong–oval, skin color—yellow, flesh color—yellow, culinary type B—universal variety, and starch content—13%. The selection of the variety was made guided by its use in organic cultivation, as well as taking into account its strongly developed aboveground part, allowing us to observe the reaction of plants to possible infection caused by the use of ozone and hydrogen peroxide. The potatoes were planted mechanically with the use of a two-row planter to a depth appropriate to the degree of soil cultivation each year, about 8 cm. Certified seed material (class A, caliber 35–55 mm) with a plant passport was purchased from a local seed potato seed supplier.

2.3. Conditions for Conducting the Experiment

2.3.1. Soil Conditions

The field trial was conducted on brown soil of granulometric formula of clay loam. The characteristics of the soil before the establishment of the experiment are shown in

Table 1. Selected physicochemical and chemical properties of the soil before the establishment of the experiment (average over the years of research).

Granulometric composition	clay loam
pH	pH: H2O 5.25 ± 0.11, KCl 4.72 ± 0.17
Hydrolytic acidity	3.2 ± 0.12 mmol(+)∙100 g−1
Total nitrogen	≤1 g∙kg−1 soil
Phosphorus	14.2 ± 0.36 mg P2O5 100 g−1 soil
Potassium	16.7 ± 0.19 mg K2O∙100 g−1 soil
Magnesium	9.24 ± 0.32 mg∙100 g−1 soil
Contents of P, K, and Mg	average by abundance classes

and

Table 2. Contents of inorganic ions in water extracts of soil before the establishment of the experiment (mg∙100 g⁻¹) (average of the years of research).

	Na+	NH4+	K+	Mg2+	Ca2+	NO3−	SO42−
30 cm	0.07 ± 0.02	0.04 ± 0.01	0.58 ± 0.03	0.05 ± 0.01	0.25 ± 0.03	2.57 ± 0.30	0.42 ± 0.02
60 cm	0.16 ± 0.01	0.05 ± 0.03	0.20 ± 0.04	0.09 ± 0.03	0.63 ± 0.19	1.70 ± 0.15	1.01 ± 0.10
90 cm	0.21 ± 0.05	0.03 ± 0.01	0.04 ± 0.01	0.06 ± 0.01	0.37 ± 0.03	2.00 ± 0.17	1.31 ± 0.16

.

Before the experiment and on the basis of the analyses performed, the soil was characterized by the average amounts of assimilable phosphorus, potassium, and magnesium. The determined values of pH and hydrolytic acidity indicated its acid reaction. The determined average content of total nitrogen in the soil before the establishment of the experiment did not exceed 1 g·kg⁻¹. Each year before planting potatoes in individual microplots, the following fertilizer was applied: nitrate (27% N, 2% CaO, and 4% MgO) at a rate of 270 kg∙ha⁻¹ and ammonium nitrate (37% N) at a rate of 200 kg∙ha⁻¹, adjusted to the nutritional needs of this plant.

2.3.2. Weather Conditions

Data relating to weather conditions (Figure 1) were obtained from the Meteorological Station located in Zadabrowie (49°53′37.2″ N 22°49′17.6″ E). On their basis, monthly totals of temperature and precipitation were calculated for the years of the field experiments.

The growing seasons were characterized in terms of water–thermal conditions on the basis of the Sielianinov hydrothermal coefficient (k) (

Table 3. Sielianinov hydrothermal coefficient during the growing season of plants.

	May	June	July	August	September	Average
2016	0.83 d	0.58 vd	1.86 rh	2.18 h	1.80 rh	1.45 o
2017	1.02 rd	2.05 h	1.71 rh	1.48 o	0.91 d	1.43 o
2018	1.02 rd	2.05 h	1.71 rh	1.48 o	0.91 d	1.43 o
1981–2010	1.93 rh	1.71 rh	1.61 rh	1.26 rd	1.92 rh	1.69 rh

0.4 < k ≤ 0.7—very dry (vd), 0.7 < k ≤ 1.0—dry (d), 1.0 < k ≤ 1.3—relatively dry (rd), 1.3 < k ≤ 1.6—optimal (o), 1.6 < k ≤ 2.0—relatively humid (rh), 2.0 < k ≤ 2.5—humid (h) [67].

). The ratio is expressed as the quotient of total precipitation and total monthly temperatures minus a factor of ten [66]:

$$k={\displaystyle \frac{P}{({Ʃ}_t·0.1)}}$$

where

k—Sielianinov coefficient;

P—sum of monthly precipitation (mm);

Σ_t—sum of monthly daily temperatures (°C).

2.4. Device for Conducting the Plant Ozonation Process

In order to carry out the field fumigation of plants, the following necessary devices for the experiment were designed and manufactured. The set created consisted of a portable power generator, an electrical voltage stabilizer, an ozone generator, an ozone concentration sensor, and a fumigation device. The innovative device for treatments, with a cuboid shape, was manufactured on a steel frame with wheels, allowing it to be moved along the plot. The walls of the device were made of a steel sheet (1st version) and polycarbonate (2nd version), allowing us to significantly reduce the weight of the device, as well as to observe the moving and fumigation processes. On the outlets of the device and on the sides, just near the ground surface, plastic aprons were placed to prevent the escape of gas. At the top of the device, a system has been installed that is responsible for the even distribution of the ozone stream fed from the generator to the inside of the device. The width of the device corresponds to the width of two ridges. The device is equipped with casters that allow it to be moved manually along the rows; it is moved and operated by two people. Before starting the fumigation process of successive plots, the device operated in free space in order to achieve the optimal concentration of gas inside.

During the course of conducting ozone fumigation on experimental plots, in the first year of conducting the study, an agrotechnical problem was encountered when carrying out treatments with the manufactured device. Damage to the aboveground parts of plants while carrying out treatments became problematic. Originally, the device was used for the simultaneous fumigation of plants within a single furrow. Therefore, a new device was designed and manufactured with a width that allowed the fumigation process to be carried out simultaneously on two ridges while retaining the volume of the previous tunnel. The problem was significantly reduced after the change in the device, which made it possible to reduce losses caused by damage to aboveground plant parts. In the second and third seasons of the experiment conducted, there was no significant reduction in yield for the ozonated trial, which occurred in 2016 and was probably caused by significant damage to the green parts of the crops.

Treatments were carried out on a specific area of ridges placed in the center. The device was then moved to cover another identical area with its working range. An ozone generating device (CSI, Ekotech, Warsaw, Poland) placed on the fumigation device was powered by a portable power generator (Nutool, Nu Air, Warsaw, Poland). To maintain constant conditions during the treatments, an ozone concentration sensor (2B-Technologies, Boulder, CO, USA) and a current voltage stabilizer (SVR, Westlands, Melle, Germany) were used to power the generator.

2.5. Laboratory Evaluations

2.5.1. Starch Content

The starch content of potatoes was determined using the Ewers polarimetric method [68]. It is based on the extraction of starch with dilute hydrochloric acid and a measurement of the degree of twist of the plane of polarized light in the solution clarified by rebinding. The determination was performed in triplicate.

The starch content of the tested potato samples was calculated based on the following formula:

$$C={\displaystyle \frac{(100·A)}{183.7·l}}$$

where

C—starch content (g∙100 cm³)⁻¹);

A—the read angle of torsion of the plane of polarized light;

l—length of the polarimetric tube (mm);

183.7—specific torsion of starch.

2.5.2. Vitamin C Content

The vitamin C content was determined as the sum of L-ascorbic acid and dehydroascorbic acid contents determined by the Tillmans method [69], based on extraction of the vitamin with oxalic acid, followed by the oxidation of L-ascorbic acid to dehydroascorbic acid using 2,6-dichlorophenolindophenol. The determination was carried out in triplicate.

The vitamin C content was calculated according to the following formula:

$$c={\displaystyle \frac{v·M·v_{km}}{v_p·m}}$$

where

c—amount of vitamin C (mg∙100 g⁻¹);

v—volume of 2,6-dichlorophenolindophenol solution used for titration (cm³);

M—titration of 2,6-dichlorophenolindophenol solution;

Vkm—volume of the flask (cm³);

Vp—volume of the filtrate (cm³);

m—weight of the balance (g).

2.5.3. Analysis of the Antioxidant Activity by the ABTS and DPPH Methods

The determination of the antioxidant activity against ABTS and DPPH involves measuring the degree of scavenging of synthetic radicals by the antioxidant. During the analysis, radicals are neutralized by antioxidant compounds present in the test substances, which is observed as a change in the coloring of the reaction mixture. The degree of this change can be determined using spectrophotometric methods and with reference to standard curves. ABTS and DPPH tests were determined by the methods described by Yen and Chen [70] and Re et al. [71] in triplicate. The absorbance of solutions determined by the ABTS method was measured at λ = 734 nm and by the DPPH method at λ = 515 nm. Results are expressed as Trolox equivalents (mg) in 100 g of potatoes.

2.5.4. Total Content of Polyphenolic Compounds

The determination of the total content of polyphenolic compounds was performed using the Folin–Ciocalteu method [72]. This method measures the absorbance of the complex that is formed in the reduction reaction of molybdenum, contained in the Folin–Ciocalteu reagent, in an alkaline medium. Absorbance measurements were made at λ = 690 nm. Results are expressed as gallic acid equivalents (mg) contained in 100 g of potatoes. The measurements were made in three repetitions.

2.5.5. Total Antioxidant Capacity

Evaluations of the total antioxidant capacity were carried out using the CUPRAC method, utilizing the reduction of copper ions bound in a complex with neocuproine occurring in an inert environment [73]. Changes in color intensity were measured using a spectrophotometric method. The absorbance of the solutions was measured at λ = 450 nm. The analysis was carried out in triplicate. The total oxidative capacity was reported as Trolox equivalents (mg) in 100 g of potatoes.

2.6. Statistical Analysis

Statistical analysis was performed using Statistica 13.1 software. Analysis of variance (ANOVA) was performed according to the adopted methodology of the field experiment, and data were synthesized according to the experimental layout. A Tukey post hoc test was performed at a significance level of 5% to determine and verify relationships. To indicate statistically significant differences in the evaluation of potato blight, analysis of variance with repeated measures was performed with Tukey’s post hoc test for different Ns.

3. Results

3.1. Yield Analysis

Figure 2A shows the yields of potato tubers obtained in each growing season, depending on the experimental factor used. There were no significant differences between the yields of potato tubers obtained from plots where hydrogen peroxide was applied and control plots. Specifically, its value ranged from 4.99 (kg·m²)⁻¹ to 5.91 (kg·m²)⁻¹ and 6.12 (kg·m²)⁻¹ for the O₃, H₂O₂ and control treatments, respectively. The decrease in the yield obtained from plots where ozone was applied in the first year of the study (2016) was undoubtedly attributed to the significant damage to potato leaves due to the design of the fumigation device. This agrotechnical problem was eliminated in the next two seasons of conducting the study. The forced reconstruction of the plant fumigation device resulted in smaller differences between the yield obtained from the plots subjected to these treatments compared to the control trial. By increasing the width of the device, it was possible to conduct ozone fumigation treatments on two ridges simultaneously. At the same time, the cubic capacity of the device was preserved, which made it possible to conduct treatments under conditions identical to those of the 2016 season.

In the second year of conducting the study, as in the earlier growing season, there were no significant differences between the yield obtained from the plots where hydrogen peroxide spraying was applied and the control plots. Specifically, its value ranged from 5.47 (kg·m²)⁻¹ to 6.17 (kg·m²)⁻¹ and 6.28 (kg·m²)⁻¹ for the O₃, H₂O₂ and control treatments, respectively.

In 2018, due to a very large expansion of late potato blight on the green parts of the crops, the yield obtained from the experimental plots was about 50% lower compared to earlier growing seasons, regardless of the experimental factor. It was observed that this decrease occurred regardless of the factor used, and in the control trial, no significant differences in yield were observed between the experimental conditions and the control. Specifically, its value ranged from 2.67 (kg·m²)⁻¹ to 2.71 (kg·m²)⁻¹ and 2.73 (kg·m²)⁻¹ for the O₃, H₂O₂ and control treatments, respectively. Briefly, the potato tuber total yield was significantly affected only by the action of ozone, which, however, did not affect the marketable yield.

Figure 2B shows the share of marketable yield in the total yield of potato tubers, depending on the growing season and the experiment factor. The applied ROS did not significantly affect the marketable yield in 2016, 2017 and 2018. Differences were observed in 2018, when the marketable yield from all plots was significantly higher compared to previous years, but also with no differences between factors and the control. However, it should be noted that these values are higher/better compared to the percentages obtained in earlier growing seasons. It could be concluded that the application of ozone and hydrogen peroxide did not significantly affect the commercial yield obtained, but the timing of the experiment was important.

3.2. Starch Content

Figure 3 shows the starch contents of the analyzed potato tubers, depending on the treatment type and the growing season. There was no significant effect of the applied ROS factors on the starch contents of tubers in 2016 and 2017. Significant differences were observed in 2018, and the starch content value ranged from 17 g∙100 g⁻¹ to 15 g∙100 g⁻¹, for the H₂O₂ and control groups, respectively. The highest average starch content of 18 g∙100 g⁻¹ was recorded in the 2016 season and the lowest at 15.7 g∙100 g⁻¹ in the 2018 season. In the 2017 season, the average starch content was 17.4 g∙100 g⁻¹. Briefly, the starch content of the analyzed potato tubers was significantly affected by the cultivation date, which may be due to the prevailing weather conditions in each growing season.

3.3. Vitamin C Content

Figure 4 shows the vitamin C contents of the analyzed potato tubers, depending on the factors used on plots. The vitamin C content determined in the tubers was a parameter that significantly influenced the range of analyses performed in subsequent seasons of conducting the study. After the determination made on material collected from plots in the 2016 season, and the observation of an increased vitamin C content in potatoes from plots where ozone fumigation was applied, it was decided to extend the yield analyses in the 2017 and 2018 seasons to include other parameters concerning the antioxidant activity of potato tubers. Unfortunately, it was not possible to extend them in the 2016 season due to insufficient research material. In 2016, significant differences were noted between the vitamin C contents of potato tubers of all variants. Specifically, its value ranged from 16.8 mg∙100 g⁻¹ to 21.7 mg∙100 g⁻¹ and 32.5 mg∙100 g⁻¹ for the control, H₂O₂ and O₃ groups, respectively. In the second year of the study, there was also a significant effect of the applied factor on the vitamin C content of potato tubers. Again, the highest vitamin C content was characterized by tubers from experimental plots where ozone fumigation was applied, but no similar phenomenon was observed as in the 2016 season. Significant differences were noted between the control and O₃. Specifically, its value ranged from 12.6 mg∙100 g⁻¹ to 21.7 mg∙100 g⁻¹ for the control and O₃ groups, respectively. This relationship was confirmed in the following year of conducting the study. Significant differences were noted between the control and O₃ groups. Specifically, its value ranged from 11.1 mg∙100 g⁻¹ to 14.4 mg∙100 g⁻¹ for the control and O₃ groups, respectively.

The results obtained from the whole experiment confirm that the average content of vitamin C in tubers obtained from experimental plots where ozone and H₂O₂ was applied was significantly higher compared to the other variants of the experiment. The effect of the timing of the experiment on the vitamin C content of the analyzed tubers was also noted. The production of antioxidant-like compounds in tubers induced by the effects of oxidative stress was confirmed in the 2017 and 2018 seasons using additional qualitative yield analyses.

3.4. Antioxidant Activity

Figure 5A,B shows changes in the total antioxidant capacity of potato tubers determined by methods using ABTS and DPPH radicals, depending on the growing season and the used factors. In 2017, the antioxidant activity (measured by the method using the ABTS radical) varied and depended on the method used on aboveground part of the potato. The highest AA (antioxidant activity) value was recorded for potatoes obtained from experimental plots where ozone fumigation was carried out, while the lowest value was obtained for potatoes obtained from plots where hydrogen peroxide was applied compared to the control. Specifically, its value ranged from 28.5 µmol Trolox∙100 g⁻¹ to 27.4 µmol Trolox∙100 g⁻¹ and 29.4 µmol Trolox∙100 g⁻¹ for the control, H₂O₂ and O₃ groups, respectively. This relationship was partially confirmed during the research conducted in 2018, but the hydrogen peroxide sample did not differ from the control. Specifically, its value ranged from 27.1 µmol Trolox∙100 g⁻¹ to 29.6 µmol Trolox∙100 g⁻¹ for the control and O₃ groups, respectively. There was no effect of the timing of the study on the value of ABTS AA in the analyzed potato tubers. In the case of the determination of antiradical activity against DPPH, similar observations were noted. In 2017, significant differences have been noted between the control and O₃ factor. Specifically, its value ranged from 14.1 µmol Trolox∙100 g⁻¹, to 17.3 µmol Trolox∙100 g⁻¹ for the control and O₃ groups, respectively. Significance differences were noted also for AA value measured against DPPH on different test dates.

3.5. Total Content of Polyphenolic Compounds

Figure 6 shows the changes in the total contents of phenolic compounds in the analyzed potato tubers, depending on the factor applied and the date of the study. In 2017, for potato tubers obtained from plots where gaseous ozone was applied, an about 50% higher content of phenolic compounds was recorded compared to the other variants of the experiment. There were no significant differences between the hydrogen peroxide-sprayed and control samples. Significant differences were noted between the control and O₃ groups. Specifically, the value ranged from 14.1 mg∙100 g⁻¹ to 17.3 mg∙100 g⁻¹ for the control and O₃ groups, respectively. In 2018, significant differences in the contents of polyphenolic compounds were characterized in tubers obtained from plots where both ROS were applied. Specifically, the value ranged from 19.6 mg∙100 g⁻¹ to 22.1 mg∙100 g⁻¹ and 21.9 mg∙100 g⁻¹ for the control, H₂O₂ and O₃ groups, respectively. Synthetically, the use of gaseous ozone significantly provided the best results compared to the other variants of the experiment. There was also a significant effect of the timing of the study on the content of phenolic compounds in potato tubers.

3.6. Total Antioxidant Capacity

Figure 7 illustrates the changes in the total antioxidant capacity of the analyzed potato tubers, depending on the factor and the date of the study. In 2017, the total antioxidant capacity was highest for potato tubers obtained from plots where gaseous ozone was used. This value was significantly higher compared to the values obtained for potato tubers from plots where other factor was applied. Specifically, its value ranged from 74 mg∙100 g⁻¹ to 70.6 mg∙100 g⁻¹ and 90.8 mg∙100 g⁻¹ for the control, H₂O₂ and O₃ groups, respectively. In 2018, the highest value of the analyzed parameter was characterized in potato tubers from plots where gaseous ozone was applied. A slightly lower value was obtained for potatoes grown in plots where hydrogen peroxide was sprayed. These values were significantly higher compared to control plots. Specifically, its value ranged from 48.3 mg∙100 g⁻¹ to 62.3 mg∙100 g⁻¹ and 68.4 mg∙100 g⁻¹ for the control, H₂O₂ and O₃ groups, respectively. There was also a significant effect of the growing season in which potato cultivation was carried out on the total antioxidant capacity of the obtained tubers.

4. Discussion

The sustainability of crop production systems depends on many factors, the most important being crop productivity, which is usually measured by yield. Yield is the end result, but many aspects of crop development and growth can be involved or responsible for its amount and quality. The amount of yield obtained can also indicate areas where production is encountering problems [74,75]. Incorporating practices such as crop rotation, cover crops, green manures, amendments, and conservation tillage can help increase crop productivity, sustainability, and environmental quality [75,76]. Potato production is most often characterized by short, two-year rotations, extensive cultivation, minimal residue return, and minimal crop diversity, often negatively affecting crop productivity over time [77]. Studies available in the literature have repeatedly discussed the comparative yields from organic and conventional production methods. Most of them point to the advantage of conventional farming. On organic farms, the yields obtained are lower. A meta-analysis conducted by Ponisio et al. [78], based on more than 115 research papers, indicated that yields in organic agriculture are on average 19.2% lower in comparison to conventional systems. In a study by Kazimierczak et al. [79], the yield of organic potatoes was on average about 50% (32–65% depending on the variety) lower compared to the yield of potatoes obtained from conventional farming. Similar conclusions were reached by Brazinskiene et al. [80]. In addition to environmental or genomic factors, potato growth and, consequently, yield are also significantly affected by the crop management system [81,82]. The study found that the use of some ROS treatments in potato cultivation affects potato yields. Although in the first year of the study there was a significantly lower (by 18.5%) yield from plots where plant fumigation with ozone was applied, modification of the field application device reduced these differences. In the next year of conducting the study, although the yield was still significantly lower, its decrease was smaller at 13%, and in the third season, the overall yield of potatoes was half that of the previous years, but the difference was not statistically significant. This was due to different weather conditions, which increased the pressure from a pathogen, potato blight, a disease that contributes to significant yield losses of up to several tens of percents. However, in the case of a yield analysis in experiments conducted on microplots, it should be considered that the obtained results may be strongly influenced by a small plant population. In the case of the conducted research, however, it was not possible to conduct them on larger objects. Ozone fumigation treatments were performed in the afternoon, when the ambient temperature was lower. This was caused by the irritating effect of ozone used under conditions of elevated ambient temperature. The statistical analysis showed a significant influence of weather conditions on the obtained results. In the individual years of the experiment, no significant effect on changes in the marketable potato yield was observed. Statistically significant differences in the comparison of individual growing seasons may be caused by different weather conditions in 2018 and high plant disease pressure. Moreover, in this case too, the obtained results may be influenced by conducting research on microplots, which, although suitable for qualitative research, may be difficult in the case of quantitative parameters.

Potato starch consists of two polymer forms, branched amylopectin and linear amylose. Starch synthesis occurs in plastids, where the two polymers form semi-crystalline, water-insoluble granules. It is generally accepted that starch is synthesized mainly from ADP-glucose through the organized actions of several enzymes located in plastids [83,84]. In potato tubers, starch accumulates during their development and is stored for a long time. It maintains the energy requirements of the dormant tuber and drives the growth of new shoots after dormancy is broken [85]. Two main factors are responsible for the differences in physical and chemical parameters of starch: environmental and genetic factors. Environmental conditions, particularly temperature, have been shown to have significant effects on the crystallinity of starch grains, their dimensions, and amylopectin structure through the deactivation of certain enzymes [86]. Differences due to genetic factors of a given variety, which determine the properties of starch, are well known and are often taken into account in food production [87,88,89,90]. In a study by Turska et al. [91], the average starch content of the potato tubers analyzed was 12.7–16.1%. The author indicates that the starch content depended significantly on the analyzed variety, which is confirmed in the studies of other authors [88,92,93,94,95]. According to Wierzbicka [96], the starch content of potato tubers ranges from 10.3 to 12.7%, while in Mystkowska’s [97] study, it is 155–165 g∙kg⁻¹. The authors also indicate that in years with excessive rainfall during the growing season, potatoes accumulated the least amount of starch in tubers, regardless of the locality and variety [98]. López-Delgado et al. [99], based on a study in which spraying on potato was perform twice a week (days 21 to 90 after planting) with two concentrations of H₂O₂ (0.02% and 0.17%), indicated that the treatments significantly increased the accumulation of starch contained in tubers by 6.7% to 30% and the stems were up to 27% thicker, mainly due to enlarged parenchymal cells in the core, in comparison with the control group. The results obtained in the 2018 season showed a significant effect of hydrogen peroxide spraying on starch content in potato tubers. However, this relationship was not confirmed in other growing seasons and in a holistic approach. These differences are probably due to the difficult 2018 growing season, with different weather conditions and significant pressure from pests and pathogens. The highest average starch content of 18 g∙100 g−1 was recorded in the 2016 season, and the lowest at 15.7 g∙100 g−1 in the 2018 season. In the 2017 season, the average starch content was 17.4 g∙100 g−1. The differences in starch content arise from the influence of atmospheric conditions on the plant growth process.

One of the most important compounds needed in the human diet is vitamin C, especially in combination with flavonoids. Its two forms—L-ascorbic acid (L-ASC) and dehydroascorbic acid (DHA)—are mainly acquired from fruits and vegetables. Increased absorption of vitamin C from the gastrointestinal tract is associated with interactions with flavonoids [100]. As estimated by Herencia et al. [101], a higher vitamin content in plant materials from organic crops compared to conventional crops was shown in 58% of studies on the impact of production methods. It has also been verified by a meta-analysis based on 343 selected publications conducted by Baranski et al. [102]. In a study by Kazimierczak et al. [79], the vitamin C content, including L-ascorbic acid, was not affected by the cropping system. Different results are presented by Wegener et al. [103]. They have discovered that in response to biotic and abiotic stresses, plants in an organic farming system produced more vitamin C. Similar results were obtained by Zardzewiały et al. [104], confirming the effect of ROS treatment of tomatoes on the vitamin C content. In the first year of the study, a phenomenon was observed consisting of an almost 100% higher vitamin C content in potatoes from plots where ozone fumigation was used. This value, although not confirmed to such an impressive degree in subsequent years, confirms that the irritating effect of ROS affects the production of antioxidant compounds in potato tubers. In the next season of the study, an increase of 50% was noted, and in the last one, the increase was 30%. In the case of hydrogen peroxide spraying, the differences were smaller, but the increases were 29%, 6%, 14% in 2016, 2017, and 2018, respectively. Statistical analysis confirmed significant differences between the vitamin C contents for both used factors, as well as the effect of weather conditions in individual years on this quality parameter.

One of the methods of determining the level of stress in plants can be the determination of antioxidant activity (AA) [105]. The production of compounds that protect against toxic effects is associated with a cascade of biochemical reactions induced by oxidants. One of such mechanisms is the mobilization of PAL (phenylalanine ammonia lyase), and it can be enhanced by the generation of polyphenols that interact with AA [106]. According to Khan et al. [107] H₂O₂ acts as a signaling molecule with tremendous effects on plant growth and development. Spraying plants with H₂O₂ and fumigation with O₃ induce oxidative stress by disrupting cellular ROS homeostasis and ROS-dependent signaling networks, which increase the accumulation of latent defense proteins such as ROS uptake enzymes and modulate physiological processes that lead to enhanced stress responses. Presumably, both compounds can act as abiotic elicitors, causing a number of intracellular interactions that can consequently induce stress resistance [108]. Literature reports have indicated that H₂O₂ stimulates a defense response in mustard seedlings, which can trigger the activation of both ROS and methylglyoxal detoxification pathways and enable seedlings to tolerate drought-induced oxidative damage [109]. In maize cultivated under water-deficient conditions, an increase in the activity of antioxidant enzymes in young plants and increases in the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were also observed after pre-dosing H₂O₂ on seeds [110]. Piechowiak and Balawejder [106] showed that O₃ action increases the activity of PAL (phenylalanine ammonium lyase), which plays an important role in the biosynthesis of phenolic compounds in plants. These compounds can act directly in the detoxification of ROS in plant cells [111]. In addition, Sarkar et al. [112] report increases in the levels of the major antioxidant enzymes ascorbate peroxidase (APX) and glutathione reductase (GR) in two rice varieties after exposure to elevated O₃ levels. Similar correlations regarding the induction of APX under O₃ stress have also been reported in different varieties of wheat [21,113,114], rice [115], and corn [116]. Castagna and Ranieri [33] showed that plants were damaged by O₃ exposure. This was manifested by the aging and yellowing of leaves, which was not observed in the present experiment. The induction of accelerated leaf aging under the influence of O₃ has also been described in studies by other authors [33,117]. Ueda et al. [39] observed damage to rice leaves after a six-hour O₃ fumigation at a concentration of 150 ppb. This situation may have been caused by lipid peroxidation due to the formation of hydroxyl radical ·(OH), which is the most reactive form of oxygen, formed in the Haber–Weiss/Fenton reaction, among others [39,42]. According to Rai et al. [118], plants allocate more carbon to produce antioxidants and secondary metabolites for repair processes to overcome O₃-induced stress. ROS used in the experimental plots also affected the total antioxidant capacity. In the 2017 season, ozone caused an increase in the content by 3% and by 9% in the 2018 season (in the method using ABTS). In the case of the method using DPPH, the increase was 23% in 2017, and in 2018, no significant differences were observed. In turn, the use of hydrogen peroxide did not contribute to an increase in the total antioxidant capacity. The observed differences confirm the irritating effect of ROS on inducing the production of antioxidant compounds in potato tubers.

Potato tubers contain significant amounts of such phenol-like compounds, including phenolic acids and flavonoids [102,119]. Phenolic compounds are considered important health-promoting compounds underrepresented in the diet of modern societies [120]. Early potato tubers contain large amounts of ascorbic acid and phenols [121,122]. As discussed earlier, ascorbic acid is an inhibitor of enzymatic browning, and so its presence helps reduce some of the quality losses in tubers [123,124], while phenols have a number of health-promoting properties [125]. The phenolic compound content is typical of the variety but also depends on habitat conditions. High humidity increases the total polyphenol content [126], and precipitation has a beneficial effect on the content of phenolic compounds in potatoes [127,128]. Lachman et al. [129] studied the relationship between the total polyphenol content and applied nitrogen fertilization. They showed that the total polyphenol content was highest in tubers in which no nitrogen fertilization was applied during production, compared to N140 and N280. In a study by Kazimierczak et al. [79], significantly larger amounts of total phenols, including phenolic acids and flavonoids, were found in crops from organic production compared to potatoes obtained from conventional cultivation. The authors also noted that most of the potato varieties grown in the organic system had higher flavonoid concentrations (total) in comparison to their conventional equivalents. These relationships were observed for individual flavonoids, among them quercetin-3-O-glucoside and quercetin. Similar conclusions were reached by Hajslova et al. [130] and Lombardo et al. [131]. From the point of view of human health, increasing the total content of polyphenols through the right choice of agrotechnology may be important in the diets of societies in which potatoes predominate. In the experiment conducted in 2017, a 107% increase in the total content of polyphenols was noted and caused by the action of ozone, while it was statistically insignificant in the case of the use of hydrogen peroxide. In the next growing season, both ROS influenced the increase in the levels of this parameter, the increase was 13% for hydrogen peroxide and 12% for ozone. The observed differences confirm the irritating effect of ROS on inducing the production of antioxidant compounds in potato tubers.

Potato is an important source of antioxidant compounds. Several studies have shown that potatoes are the third source of antioxidant compounds in the diet, after oranges and apples, due to their high daily per capita intake [132]. The positive effects of antioxidant components on human health have been reported by several authors [133,134,135,136,137]. These studies show the potential to improve the diet quality when consuming foods with high antioxidant properties. Consequently, there is a constant search for new natural compounds with antioxidant properties [138,139,140,141,142]. Many epidemiological studies link cardiovascular disease prevention to dietary polyphenol intake, as polyphenols prevent oxidative stress, cell and protein damage, and lipid peroxidation processes. Consumption of raw materials containing significant amounts of polyphenols is also associated with the prevention of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease [143,144,145,146]. The main antioxidants are phenolic compounds, vitamin C, flavonoids, and carotenoids [147,148]. Potato tubers contain four types of polyphenolic components: phenolic acids, flavonols, flavan-3-ols, and anthocyanin components. The most widespread polyphenol in potato is chlorogenic acid, accounting for about 90% of the total polyphenol content (TPC). Gumul et al. [149] described five potato varieties grown in Poland with a polyphenol content of 2.12–3.27 mg catechin∙g d.m.⁻¹, a total flavonoid content of 0.71–1.00 mg rutin∙g d.m.⁻¹, and a flavonol content ranging from 0.01 to 0.27 mg quercetin∙g d.m.⁻¹. Deusser et al. [150] described 16 potato varieties grown in Luxembourg with TPC values ranging from 0.40 mg GAE∙g s.m.⁻¹ to 5.40 mg GAE∙g s.m.⁻¹ in potato flesh. Al-Weshahy and Rao [151] described six potato varieties grown in Canada with potato skin polyphenol contents ranging from 1.51 to 3.33 mg GAE∙g∙g s.m.⁻¹. In the case of the total antioxidant activity, the increase in this parameter caused by ozone was 23% in the 2017 season, and in the 2018 season, both ROS influenced its content. The difference was 29% for hydrogen peroxide and 42% for ozone. The observed differences confirm the irritating effect of ROS on inducing the production of antioxidant compounds in potato tubers.

5. Conclusions

Controlled oxidative stress caused by the use of selected reactive oxygen species under field conditions significantly increased selected quality parameters of potato tubers. At the same time, when using a device that did not damage plants, it did not significantly reduce the yield. The use of ozone fumigation increased the content of vitamin C, antioxidant activity, total polyphenol content, and antioxidant capacity of tubers. The use of hydrogen peroxide sprays increased the content of vitamin C, total polyphenol content, and total antioxidant capacity, but did not affect the changes in the total antioxidant capacity. These factors did not affect the content of starch, but ozone caused a slightly decreased potato yield. The obtained results confirm that even in ozone-sensitive plants (such as Solanaceae) it is possible to use microdoses of the irritant to stimulate plant defense mechanisms, which contribute to the production of food with increased nutritional value.