applied sciences Article Influence of Plasma Activated Water Generated in a Gliding Arc Discharge Reactor on Germination of Beetroot and Carrot Seeds Piotr Terebun 1 , Michał Kwiatkowski 1 , Karol Hensel 2 , Marek Kopacki 3, * 1 2 3 *



 Citation: Terebun, P.; Kwiatkowski, M.; Hensel, K.; Kopacki, M.; Pawłat, J. Influence of Plasma Activated Water Generated in a Gliding Arc Discharge and Joanna Pawłat 1, * Institute of Electrical Engineering and Electrotechnologies, Lublin University of Technology, Nadbystrzycka 38a, 20-618 Lublin, Poland; p.terebun@pollub.pl (P.T.); m.kwiatkowski@pollub.pl (M.K.) Department of Environmental Physics, Comenius University, Mlynská dolina F2, 842 48 Bratislava, Slovakia; hensel2@uniba.sk Department of Plant Protection, University of Life Sciences in Lublin, Leszczyńskiego 7, 20-069 Lublin, Poland Correspondence: marek.kopacki@up.lublin.pl (M.K.); j.pawlat@pollub.pl (J.P.) Abstract: One of the new methods of protecting and supporting plant growth is the use of lowtemperature plasma. The aim of this study is to evaluate the feasibility of using plasma activated water produced in an atmospheric pressure gliding arc reactor for germination of beetroot (Beta vulgaris) and carrot (Daucus carota) seeds. The study was carried out for different plasma treatment times of water (5, 10 and 20 min) and with fixed geometry and power of the discharge system, using air as the working gas. The effect on germination was evaluated based on the fraction of germinated seeds and their length at 7 and 14 days after treatment. Analysis of fungi present on the seed surface and imaging of the seed surface using scanning electron microscopy (SEM) were auxiliary methods to evaluate the type of treatment effect. In the case of beetroot, a positive effect on the number and length of germinated seeds was observed, which increased with increasing treatment time. This effect can be attributed, among other things, to the surface changes observed on microscopic photographs. In the case of carrot seeds, a more significant positive effect on germination was observed. Fungal decontamination effect was relatively weaker than with the use of the chemical method with sodium hypochlorite. Reactor on Germination of Beetroot and Carrot Seeds. Appl. Sci. 2021, 11, 6164. https://doi.org/10.3390/ Keywords: plasma activated water (PAW); gliding arc discharge (GAD); germination; carrot seed; beetroot seed app11136164 Academic Editor: Joachim Müller 1. Introduction Received: 24 May 2021 Accepted: 25 June 2021 Published: 2 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Currently, one of the main tasks of plant protection is the implementation of innovative and safe methods to reduce the occurrence of agrophages in crops [1,2]. This is related to the implementation of the concept of sustainable agriculture, promoting the production of high quality food in a socially responsible manner, rational use of natural resources and a reduction in the use of chemical plant protection products [3–6]. Excessive and careless use of pesticides contributes not only to resistant breeds of agrophages, but also to environmental pollution and residues in the raw materials produced [7]. One alternative method of pesticide-free crop protection may be plasma treatment. Plasma can affect living cells through the action of active particles generated in the plasma (mainly reactive oxygen and nitrogen species—RONS), the action of charged plasma particles, radiation over a wide range of wavelengths, and shear stresses and drying [8–10]. The mechanism of plasma action on harmful microorganisms is multistage and mainly involves permanent damage of the cell wall, cytoplasmic membrane, and then intracellular structures, genetic material and the enzyme apparatus [11–13]. Plasma can be used to decontaminate seed and seedling material [14–22] and have positive effects on physiological processes in plants and plant seedlings [23–32]. For example, the work of Jiang et al. indicated the effects of low-temperature plasma on seed germination, seedling growth, root morphology, and nutrient uptake in tomato [33]. Many researchers confirm the positive effect of plasma Appl. Sci. 2021, 11, 6164. https://doi.org/10.3390/app11136164 https://www.mdpi.com/journal/applsci i. 2021, 11, x FOR PEER REVIEW 2 of 16 be used to decontaminate seed and seedling material [14–22] and have positive effects on 2 of 15 processes in plants and plant seedlings [23–32]. For example, the work of Jiang et al. indicated the effects of low-temperature plasma on seed germination, seedling growth, root morphology, and nutrient uptake in tomato [33]. Many researchers confirm the positive effect of plasma on the process of plant rooting especially on increasing the on the process of plant rooting especially on increasing the length and number of roots length and number of roots formed [34,35]. formed [34,35]. One method of using plasma treatment may be to use plasma activated water (PAW), One method of using plasma treatment may be to use plasma activated water (PAW), which may contain active (such particles as dissolved ozone, hydrogen peroxide, nitrates, which may particles contain active (such as dissolved ozone, hydrogen peroxide, nitrates, nitrites, peroxynitrites, OH radicals, etc.) produced directly in the discharge or their nitrites, peroxynitrites, OH radicals, etc.) produced directly in theby discharge or by their reaction with water, depending on depending the type of on discharge the working gas [36]. gas used [36]. reaction with water, the typeand of discharge and theused working Then, PAW canThen, be used to can remove harmful organisms [37–45], either [37–45], directly either on growing PAW be used to remove harmful organisms directly on growing plants [46,47] or by aiding in the germination process [48,49]. From the perspective ofperspective of plants [46,47] or by aiding in the germination process [48,49]. From the stability over time, hydrogen peroxide and lower pH and in PAW stability over time, hydrogen peroxide lowerare pHprobably in PAW the are most probably the most influential factors in fungicidal activity. The treatment effect depends on both the the discharge influential factors in fungicidal activity. The treatment effect depends on both discharge parameters and the type of plant being treated. The purpose of this study is to is to evaluate parameters and the type of plant being treated. The purpose of this study evaluate the feasibility of using PAW generated by a gliding arc discharge (GAD) to aid the feasibility of using PAW generated by a gliding arc discharge (GAD) to aid in the in the germination of carrotof and beetroot In seeds. additionIntoaddition the results on the fraction germination carrot and seeds. beetroot to the results on the fraction of of germinated germinated seeds and the length sprouts, obtained from surface imaging and seeds andofthe length those of sprouts, those obtained from surface imaging and the the analysis of analysis the type and amount of fungi present on the seeds, on conducted viaconducted scanning via scanning of the type and amount of fungi present the seeds, electron microscopy (SEM), were also usedwere to evaluate thetoeffect of plasma. Theofresults electron microscopy (SEM), also used evaluate the effect plasma. The results were comparedwere withcompared one of thewith classic methods of plant decontamination—treatment with one of the classic methods of plant decontamination—treatment with sodium hypochlorite (NaOCl). sodium hypochlorite (NaOCl). Appl. Sci. 2021, 11, 6164 physiological 2. Materials 2. Materials and Methods and Methods 2.1. Treated Seeds 2.1. Treated Seeds Edible seeds of F1 thevariety AFALON F1 variety Moravo Seed Ożarów Edible carrot seeds of carrot the AFALON (Daucus carota,(Daucus Moravocarota, Seed Ożarów Mazowiecki, Poland) and beetroot seeds of the CYLINDRA variety (Beta vulgaris, W. Mazowiecki, Poland) and beetroot seeds of the CYLINDRA variety (Beta vulgaris, W. 1a). For Legutko, Jutrosin, Poland) were used in the research (Figure Legutko, Jutrosin, Poland) were used in the research (Figure 1a). For each treatmenteach treatment condition, 10 seeds were randomly selected, and the measurements repeated 5 times. condition, 10 seeds were randomly selected, and the measurements were repeated 5were times. Figure 1. (a)Figure Photographs of treated seeds, (b) schematic the experiment. 1. (a) Photographs of treated seeds, (b)ofschematic of the experiment. 2.2. Plasma Activated WaterActivated Water 2.2. Plasma Plasma was generated in generated a single-phase gliding arcgliding reactorarc (GAD) operating at Plasma was in a single-phase reactor (GAD) operating at atmoatmospheric pressure; this process has been described in greater detail in previous works spheric pressure; this process has been described in greater detail in previous works [50,51]. [50,51]. The discharge system system consisted of two copper 2 mm thick andthick 80 mm The discharge consisted of two electrodes, copper electrodes, 2 mm and 80 mm long, with an angle of 12◦ between them, placed in a glass tube with an inner diameter of 50 mm. The working gas (air) was blown out through the glass nozzle near the ignition area with a flow rate of 7.33 dm3 /min, adjustable with glass tube variable area flow meter (Zakłady Automatyki “ROTAMETR”, Gliwice, Poland). The reactor was powered by an Appl. Sci. 2021, 11, 6164 3 of 15 RMS (root-mean-square) voltage of 680 V (3.8 kV peak voltage), frequency of 50 Hz and apparent power of 40 VA. To obtain plasma activated water, 20 mL of distilled water, at 22.6 ◦ C, was placed in a glass vessel with a diameter of 60 mm and with a distance of 20 mm between the electrodes and the water surface (Figure 1b). The water temperature after plasma treatment of 5, 10 and 20 min, measured with a K-type thermocouple connected to the DT-847U meter (Yu Ching Technology, Taipei, Taiwan), was 27 ◦ C, 29.1 ◦ C and 30.7 ◦ C, respectively. The colorimetric method using TiOSO4 (Titanium(IV) oxysulfate) solution (Merck, Darmstadt, Germany) and colorimetric Griess assay (Cayman Chemicals, Ann Arbor, USA) were used for the measurement of hydrogen peroxide (H2 O2 ) and nitrites (NO2 − ), respectively. The obtained concentrations of active species increased with the plasma treatment time and are summarized in Table 1. The GAD plasma source is able to generate high concentrations of nitrogen reactive species but H2 O2 concentration was low, which is in a good accordance with previous data [50]. Table 1. Concentration of selected RONS. Plasma Treatment Time [min] H2 O2 [µM] NO2 − [mM] pH 5 10 20 6±1 7±3 12 ± 5 1.9 ± 0.4 2.4 ± 0.3 2.9 ± 0.6 4.2 ± 0.2 3.7 ± 0.1 3.3 ± 0.3 2.3. Germination Rate Immediately after the plasma treatment of water, the experiment with seeds was carried out in three parts: first, the seeds were treated with PAW, e.g., water was poured over 10 seeds of each species placed in glass flasks and left to soak for one hour. Then, seeds were transferred to the Petri dishes with the mineral nutrient medium solution (the composition of the solution is shown in Table 2). In the last step, mycological analysis was performed. Table 2. Composition of mineral medium. Substance Quantity Saccharose Agar NH4 NO3 MgSO4 × 7 H2 O NH2 PO4 FeCl3 × 7 H2 O ZnSO4 × 7 H2 O CuSO4 × 7 H2 O MnSO4 × 7 H2 O 38 g 20 g 0.7 g 0.3 g 0.3 g trace amounts trace amounts trace amounts trace amounts In order to compare plasma treatment with traditional decontamination methods, a series of measurements was performed using a sodium hypochlorite. For this purpose, 10 seeds were sterilized in 10% NaOCl solution for 60 s and then washed 3 times for 3 min in distilled water. NaOCl (POCH Odczynniki Chemiczne, Avantor, Poland) was diluted to proper concentration on-site immediately before application. Seed samples from the same batch were used for the tests. Then, the seeds were placed on the plates, similarly to the plasma treatment and control. All seeds were in sterile growth mineral medium, in the cultivation chamber with controlled atmosphere, under the following conditions: 20–22 ◦ C without light with 65% humidity. The amount of germinated beet and carrot seeds Appl. Sci. 2021, 11, 6164 4 of 15 was measured after 7 days for germination energy GEN and after 14 days for germination capacity GC [52]. Both coefficients were then calculated from the following equation: G= n · 100% nT (1) where: n—the number of seeds germinated at time t; nT —the total number of sown seeds. 2.4. Fungi on the Seed Surface For additional series with the same treatment conditions, in the germination study a mycological analysis was performed using the method of artificial cultures [53]. The medium with the composition presented in Table 1 was supplemented with distilled water to the volume of 1000 mL, and then sterilized for 20 min in an autoclave, at a temperature of 121 ◦ C and a pressure of 1 atmosphere. The dishes with the seeds lined up were placed at 20–22 ◦ C for 7 days without light. After one week, the plates were inspected, and the grown fungus colonies were cleaved on slants with potato-glucose agar (PDA). Then, segregation was performed on the basis of microscopic and macroscopic features. The obtained fungal isolates were sorted and labeled according to species using studies, keys and monographs [54–57]. 2.5. Surface Imaging Using an Optical Microscope and SEM Morphology of the seeds was evaluated using a KEYENCE VHX-5000 digital microscope (Osaka, Japan). Images of the samples were taken immediately after one hour of the seeds’ imbibition in plasma activated water. For more detailed visualization of the surface of Beta vulgaris seeds after PAW treatment, the treated samples were naturally dried for one hour and applied to the mounting surface (carbon disc). A layer of gold (~15 nm) was then sputtered onto the samples for imaging using a Phenom SEM microscope (Thermo Fisher Scientific, USA) at a voltage of 10 kV. 2.6. Statistical Analysis StatSoft’s Statistica 8.0 software was used for the analysis of the experimental data. Statistical differences between groups were examined with use of two-way analysis of variance (ANOVA). Tukey’s test was used to analyze the significance of differences between mean values (α ≤ 0.05). For the data obtained with measurement of the same objects at different times, the results are correlated due to the use of the ANOVA Repeated Measures Analysis of Variance. 3. Results and Discussion 3.1. Germination Rate Beta vulgaris germination results for control, NaOCl and PAW (using 5, 10 and 20 min of GAD treatment of water as PAW5′ , PAW10′ and PAW 20′ ) are summarized in Figure 2. Compared to the control, which had a rather high variability within samples, PAW treatment allowed for an increase in the average value of both GEN and GC . The difference in germination energy was evident for PAW after 5 min of plasma treatment, for which the fraction of germinated seeds increased by 16%, performing better than sodium hypochlorite treatment. Similar GEN results were obtained for a time of 10 min plasma treatment of water; however, this treatment time also allowed for an increase in GC , as opposed to PAW5’, which had a shorter treatment time that accelerated initial growth followed by no germination of the remaining seeds. The best results were obtained for PAW with a treatment time of 20 min, for which all seeds from all samples germinated after 7 days. In the case of beetroot seeds, the highest ratio was obtained with the application of water treated with plasma for 20 min; this was also the case for carrot seeds in combination with sodium hypochlorite. Appl. Sci. 2021, 11, x FOR PEER REVIEW Appl. Sci. 2021, 11, x FOR PEER REVIEW Appl. Sci. 2021, 11, 6164 5 of 16 5 of 16 after 7 days. In the case of beetroot seeds, the highest ratio was obtained with the after 7 days. In the case of beetroot seeds, the highest ratio was obtained with the application of water treated with plasma for 20 min; this was also the case for carrot seeds 5 of 15 application of water treated with plasma for 20 min; this was also the case for carrot seeds in combination with sodium hypochlorite. in combination with sodium hypochlorite. Figure 2. Germination values and standard deviation). Figure Germinationofof ofBeta Betavulgaris vulgaris(mean (mean Figure 2. 2. Germination Beta vulgaris (mean values values and and standard standard deviation). deviation). The germination results obtained for Daucus carota are shown in Figure 3. The The germination germination results results obtained obtained for for Daucus Daucus carota carota are are shown shown in in Figure Figure 3. 3. Figure3.3.Germination GerminationofofDaucus Daucuscarota carota(mean (meanvalues valuesand andstandard standarddeviation). deviation). Figure Figure 3. Germination of Daucus carota (mean values and standard deviation). Forthe thetested testedseeds, seeds,the thegermination germinationof ofthe thecontrol controlsamples sampleswas waslow lowand and did did not not For For10% theeven tested seeds, the germination oftreatment the control samples wasanlow and did not exceed after 14 days. As the plasma time increased, increase in G EN exceed 10% even after 14 days. As the plasma treatment time increased, an increase in GEN exceed 10% even after 14 days. As the plasma treatment time increased, an increase in G EN was evident, which more than doubled for a time of 20 min. However, the best results at was evident, which more than doubled for a time of 20 min. However, the best results at wasend evident, more than doubled for for a time of which 20 min.undergone However, plasma the besttreatment results at the ofthe thewhich test(14 (14 days) were obtained PAW, the end of test days) were obtained for PAW, which undergone plasma treatment the shorter end of the test (14 days) were obtained for PAW, which undergone plasma treatment for times (5 and 10 min), where the average G value was almost three times higher for shorter times (5 and 10 min), where the average GCCvalue was almost three times higher for shorter times (5 and 10 min), where the average G C value was almost three times higher thanthe thecontrol controlsample. sample.For Forthe thetested testedseeds, seeds,the thebest bestresults resultswere wereobtained obtainedfor forthe thesodium sodium than than the control sample. For the tested seeds, the best results obtainedsamples for the sodium hypochlorite treatment, although the results obtained from were the different varied hypochlorite treatment, although the results obtained from the different samples varied hypochlorite treatment, although the results obtained from the different samples varied quite significantly. quite significantly. quite significantly. 3.2. Sprout Length In the case of the total length of sprouts, the highest results were obtained in both plant species for seeds treated with sodium hypochlorite. Appl. Sci. 2021, 11, x FOR PEER REVIEW Appl. Sci. 2021, 11, 6164 3.2. Sprout Length 6 of 16 6 of 15 In the case of the total length of sprouts, the highest results were obtained in both plant species for seeds treated with sodium hypochlorite. The experimental condition condition for for Beta Beta vulgaris vulgaris are Theaverage average sprout sprout lengths lengths from from each each experimental are shown in Figure 4. shown in Figure 4. Figure4.4.Sprout Sproutlength lengthofofBeta Betavulgaris vulgaris(mean (meanvalues valuesand andstandard standarddeviation). deviation). Figure Whencomparing comparingmean meansprout sproutlengths, lengths,after after77 days, days, each each treatment treatment type type allowed allowed When greater lengths, with a slightly greater advantage for PAW with 20 min plasma treatment greater lengths, with a slightly greater advantage for PAW with 20 min plasma treatment 46%increase increaseover overthe thecontrol). control).After After14 14days, days,the thedifferences differencesfor forthe theplasma plasmatreatment treatment (a(a46% are less noticeable (4 to 17% increase over the control), with a large increase in length for are less noticeable (4 to 17% increase over the control), with a large increase in length for the sodium hypochlorite treatment (32% increase with a relatively large difference between the sodium hypochlorite treatment (32% increase with a relatively large difference the individual samples).samples). As for the at the 7th day of observation, between the individual Assprouts’ for theproduction sprouts’ production at the 7th day of application of PAW5’ produced a visible improvement in the germination process. With an observation, application of PAW5' produced a visible improvement in the germination extension in the observation time, the stimulation effect became weaker, and for 14 days Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 16 process. With an extension in the observation time, the stimulation effect became weaker, was clearly visible only for 20 min-treated PAW and NaOCl. and for 14 days was clearly visible only for 20 min-treated PAW and NaOCl. The results obtained for Daucus carota are shown in Figure 5. The results obtained for Daucus carota are shown in Figure 5. Figure Figure 5. 5. Sprout Sprout length length of of Daucus Daucus carota carota (mean (mean values values and and standard standard deviation). deviation). These seeds react in a slightly different manner than Beta vulgaris as a very weak stimulative effect can be observed after 7 days of treatment; the best results were obtained for sodium hypochlorite (250% increase), while PAW treatment was again the most effective, with the longest water treatment time with plasma (40% increase compared to the control). Such a PAW still had relatively low concentration of H2O2, with Appl. Sci. 2021, 11, 6164 7 of 15 These seeds react in a slightly different manner than Beta vulgaris as a very weak stimulative effect can be observed after 7 days of treatment; the best results were obtained for sodium hypochlorite (250% increase), while PAW treatment was again the most effective, with the longest water treatment time with plasma (40% increase compared to the control). Such a PAW still had relatively low concentration of H2 O2 , with decontaminative properties and a reasonably high concentration of nitrites with known antimicrobial properties, especially at the low pH, which together boosted plant’s growth and inhibited the development of certain fungal colonies. Nitrites are known for their fertilizing properties and support further plant development. After 14 days, a very significant increase in sprout length was noticeable for all types of treatment. For NaOCl, this was more than a thirteenfold increase over the control. For PAW treatment, the best effect was obtained for the shortest time of PAW treatment with plasma (650% increase), followed by 20 and 10 min of treatment (505% and 300% increase, respectively), which could be explained by the attaining of a kind of balance between RONS and pH, with the location of the above factors in a plant environmental tolerance zone. On the other hand, the germination boost that was clearly visible for NaOCl treatment could be explained by a strong correlation between the germination process and fungal decontamination, which is described in the next chapter. 3.3. Statistical Analysis Results of statistical analysis are depicted in Tables 3 and 4. The best results, with a statistically significant change in the case of Beta vulgaris, were obtained for PAW, with a treatment time of 20 min, for which all seeds, from all samples, germinated after 7 days. For the tested Daucus carota seeds, the best results, with a statistically significant change, were obtained for the sodium hypochlorite treatment, although the results obtained from the different samples varied quite significantly. Table 3. Statistical analysis of germination results. The letter indicators next to the means determine the statistically homogeneous groups. Beta Vulgaris Daucus Carota GEN GC GEN GC Control 0.78 b 0.9 ab 0.08 abcd 0.08 abcd NaOCl 0.9 ab 0.94 ab 0.4 ef 0.42 f 5′ 0.94 ab 0.94 ab 0.06 ab 0.22 cdef PAW 10′ 0.94 ab 0.98 a 0.1 ac 0.22 bdef PAW 20′ 1.0 a 1.0 a 0.18 abcde 0.18 abcde PAW 3.4. Fungi on the Seed Surface Fungal infestations constitute a persistent problem for improperly stored seeds, which brings losses of the seeding material, generates additional costs and is inappropriate from a sustainable ecology point of view. During our observation of basic microbiota present in the examined seeds, certain trends were observed when assessing the infestation of seeds of selected species by fungi. The seeds were highly contaminated. With longer GAD application times to water, a reduction in the number of fungi inhabiting the seeds can be observed. A total of 13 fungal species were identified in Beta vulgaris. Colony counts for each species and treatment condition are summarized in Table 5. ′′′ ′ ′′′ ′ ′′′ ′ Appl. Sci. 2021, 11, 6164 8 of 15 Table 4. Results of statistical analysis. Beta Vulgaris Daucus Carota Germination rate Sprouts’ length Table 5. Fungal species on Beta vulgaris. Species of Fungus Control NaOCl GA5′ GA10′ Alternaria alternata (Fr.) Keissl. Aspergillus niger Tiegh Botrytis cinerea Pers. Chaetomium cochliodes Palliser Clonostachys rosea (Link) Schroers Epicoccum nigrum Link Fusarium solani (Mart.) Sacc. Penicillium expansum Link Penicillium nigricans K.M. Zal. Truncatella truncata Lev. Trichoderma harzianum Rifai Trichoderma koningii Oudem. Trichothecium roseum (Pers.) Link Total 2 3 1 0 0 0 2 0 1 2 0 2 0 13 0 0 0 0 16 0 0 0 0 0 4 2 0 22 1 2 0 5 0 10 0 0 2 0 1 0 0 21 0 0 0 0 0 8 0 1 0 0 0 3 0 12 GA20′ 11 0 0 2 0 0 0 2 0 0 ′′′ ′ 1 0 1 17 Total ′′′ ′ 14 5 1 7 16 18 2 3 3 2 ′′′ 6′ 7 1 85 In total, the largest numbers of colonies were observed for Epicoccum nigrum, Clonostachys rosea and Alternaria alternata. Compared to the control, all PAW treatments eliminated fungi such as Botrytis cinerea, Fusarium solani and Truncatella truncata with the same efficiency as the sodium hypochlorite treatment. For Aspergillus niger, the reduction occurred only for PAW Appl. Sci. 2021, 11, 6164 9 of 15 with a treatment time of 10 min. On the other hand, for some treatment times, more colonies of Alternaria alternata, Epicoccum nigrum and Penicillium expansum were observed. The results obtained for Daucus carota are summarized in Table 6. Table 6. Fungal species on Daucus carota. Species of Fungus Control NaOCl GA5′ GA10′ GA20′ Total Alternaria alternata (Fr.) Keissl. Alternaria radicina Meier, Drechsler & E.D. Eddy Aspergillus niger Tiegh Chaetomium cochliodes Palliser Cladosporium sp. Clonostachys rosea (Link) Schroers Epicoccum nigrum Link Fusarium avenaceum (Fr.) Sacc. Mucor mucedo Fresen. Penicillium expansum Link Stemphylium botryosum Wallr. Trichoderma harzianum Rifai Trichoderma koningii Oudem. Total 49 7 2 0 0 0 0 0 0 0 0 2 0 60 0 4 6 0 15 2 1 0 0 0 12 2 0 42 48 0 2 1 0 0 0 0 0 0 0 0 0 51 48 0 0 0 0 0 0 0 2 0 0 0 1 51 48 0 2 0 0 0 0 2 0 3 0 0 0 55 193 11 12 1 15 2 1 2 2 3 12 4 1 259 The highest number of colonies was observed for the Alternaria alternata species, which was completely resistant to PAW treatment but was entirely removed by NaOCl. On the other hand, high numbers of colonies were observed for the species Cladosporium sp. and Stemphylium botryosum, which were only detected for the treatment with sodium hypochlorite. This may result from the surface effect of PAW, the relatively low concentration of disinfective hydrogen peroxide, and also from high primary contamination of seeds, especially carrots, as a result of improper storage. PAW treatment resulted in a complete reduction in Alternaria radicina and Trichoderma harzianum, which were present in both control and NaOCl treated samples. It also caused a slight increase in Chaetomium cochliodes, Fusarium avenaceum, Mucor mucedo, Penicillium expansum and Trichoderma koningii, which usually occurred for longer processing times. In the case of the cheapest treatment option for the feed gas (air), the RONS composition present in plasma activated water had mild fungitoxic effects. Such a treatment strongly depended on the fungal species; thus, positive point is a kind of treatment selectivity, which can be achieved using PAW. It has to be pointed out that PAW reveals basic surface activity. However, in some cases, high contamination, with the presence of pathogens that are located in the deeper zones of seeds, also cannot be removed by traditional chemical treatment techniques such as NaOCl. 3.5. Surface Imaging Using an Optical Microscope and SEM In order to emphasize the differences between two types of tested seeds and also the differences visible in the obtained results, the seeds’ structures were described on the basis of the literature data, and microscopic analyses were performed. Beta vulgaris belongs to the complex Eudicots, with a perispermic seed structure. In this case, water and oxygen penetration can be limited by thick, hard pericarp tissue surrounding the internal botanical seed. Morphologically, an ovary cap with the remnants of the stigma covers the pericarp’s upper part. On the opposite side of the seed, the basal pore is located in a position that allows for water intake. Pericarp consists of three layers with different sizes of crystalized chemical compounds, which tend to be bigger with depth. The first of two dense internal sclerenchymal layers is formed of small, multilayer-wall sclereids, followed by the second layer of thinner cell wall sclereids with crystal clusters inside, and the rather loose parenchyma cells located externally. The analysis of water extract from the pericarp revealed predominating cations such as potassium, sodium, magnesium, calcium, chlorine, sulphur and anions with nitrate, phosphate, chloride and sulphate oxalate ions dominating, pericarp’s upper part. On the opposite side of the seed, the basal pore is located in a position that allows for water intake. Pericarp consists of three layers with different sizes of crystalized chemical compounds, which tend to be bigger with depth. The first of two dense internal sclerenchymal layers is formed of small, multilayer-wall sclereids, followed by the second layer of thinner cell wall sclereids with crystal clusters inside, and the rather Appl. Sci. 2021, 11, 6164 10 of 15 loose parenchyma cells located externally. The analysis of water extract from the pericarp revealed predominating cations such as potassium, sodium, magnesium, calcium, chlorine, sulphur and anions with nitrate, phosphate, chloride and sulphate oxalate ions whichform form the osmotic potential within pericarp, which may cause delays in the seed dominating, which the osmotic potential within pericarp, which may cause delays germination process [58–63]. in the seed germination process [58–63]. When both the opticaland microscope and electron the scanning electron microscope When using both theusing optical microscope the scanning microscope (Figures 6 and 7), imaged samples are characterized by oval shaped parenchymal cells, (Figures 6 and 7), imaged samples are characterized by oval shaped parenchymal cells, of of random size, with a very developed, concaved structure. The cells are not densely random size, with a very developed, concaved structure. The cells are not densely packed packed and spaces observed. After PAW treatment, and intercellular spaces canintercellular be observed. After can the be PAW treatment, thethe structure became the structure became and folded wrinkled, and revealed signs of rupture in comparison to the folded and wrinkled, alsoand revealed signs of also rupture in comparison to the control control sample. Changes arewith noticeable forplasma PAW with 5 min of plasma treatment. On the sample. Changes are noticeable for PAW 5 min of treatment. On the other other hand, besides the undulating structure, longer times resulted in the flattening of the hand, besides the undulating structure, longer times resulted in the flattening of the outer outer edges of the cells. edges of the cells. i. 2021, 11, x FOR PEER REVIEW 11 of 16 ′ ; (c) PAW 20′ . Figure microscope 6. Optical microscope images of Beta vulgaris at 500× magnification. Control(b) (water); Figure 6. Optical images of Beta vulgaris at 500 × magnification. (a) Control(a)(water); PAW 5(b) PAW 5′; (c) PAW 20′. Figure 7. SEM images of Beta vulgaris at 1000× magnification. Control (water); PAW 5′; PAW (c) 20′ . Figure 7. SEM images of Beta vulgaris at 1000 × magnification. (a) (a) Control (water); (b) (b) PAW 5′ ; (c) PAW 20′. Carrot seeds are relatively small and they belong to the schizocarpic fruits. Their Carrot seeds are relatively small and to the fruits. Their external layer is formed by athey thinbelong seed coat andschizocarpic it has overhanging beards. Pericarp is external layer is formedjoined by a thin it hasofoverhanging beards. Pericarp is partially to theseed seedcoat andand consists the epicarp, mesocarp and endocarp. The bulk partially joinedoftothe theseed seedincorporates and consiststhick-walled of the epicarp, mesocarptissue and endocarp. The endosperm embodying anbulk embryo [64–66]. of the seed incorporates thick-walled endosperm tissue embodying embryoof[64–66]. As shown in Figure 8, on the weavy-ribbed top an structure the coat, the distinctive As shownelongating in Figure 8,beards on theadjoined weavy-ribbed of in thethe coat, the distinctive to the top ribsstructure are visible control sample. PAW treatment caused visibletochanges enhanced the ribsPAW and removal of beards. Beard elongating beards adjoined the ribssuch are as visible in theswelling control of sample. treatment removalsuch is also a common processofduring processBeard of the carrot seeds, caused visible changes as enhanced swelling the ribsthe andscarification removal of beards. takes place in during order tothe enhance germination to save space during the seed removal is alsowhich a common process scarification processand of the carrot seeds, packaging process. which takes place in order to enhance germination and to save space during the seed packaging process. of the seed incorporates thick-walled endosperm tissue embodying an embryo [64–66]. As shown in Figure 8, on the weavy-ribbed top structure of the coat, the distinctive elongating beards adjoined to the ribs are visible in the control sample. PAW treatment caused visible changes such as enhanced swelling of the ribs and removal of beards. Beard removal is also a common process during the scarification process of the carrot seeds, Appl. Sci. 2021, 11, 6164 which takes place in order to enhance germination and to save space during the seed packaging process. 11 of 15 ′ ; (c) PAW 20′ . Figure 8. Optical microscope of Daucus carota at 500× magnification. (a)(water); Control(b) (water); Figure 8. Optical microscope images of images Daucus carota at 500 × magnification. (a) Control PAW 5(b) PAW 5′; (c) PAW 20′. Previous studies with the GAD reactor indicated that high concentrations of NOx can be generated theGAD gas phase andindicated further transported to the liquid phase, where compounds such Previous studies withinthe reactor that high concentrations of NO x can as the H2Ogas also appear through secondary reactions [50]. In the case of PAW treatment of seeds, be generated in phase and further transported to the liquid phase, where 2 these can cause an increase in germination, e.g.,[50]. through H2O accumulation [48,49]. compounds such as compounds H2O2 also appear through secondary reactions In the case of 2 The increase in germination may also be related to other effects of plasma treatment on seeds, PAW treatment of seeds, these compounds can cause an increase in germination, e.g., such as surface etching and increased water absorption [67–69], or inactivation of harmful through H2O2 accumulation [48,49]. The increase in germination may also be related to these also affect seeds through [16,70,71]. other effects of organisms plasma treatment on Many seeds, of such as effects surfacecan etching and increased water active particles activating [16,70,71]. the catalase enzyme for effects the synthesis of new found PAW, e.g.,ofvia H2 O2 organisms absorption [67–69], or in inactivation harmful Many of these proteins [26], increases in chlorophyll content of hard seeds, can also affect seeds through active particles found in PAW,[72], e.g.,the viacracking H2O2 activating the thus allowing them to absorb moisture [73] or the enhancement of nitrogen contents in the seeds through catalase enzyme for the synthesis of new proteins [26], increases in chlorophyll content adsorption, diffusion and trapping of RNS produced in plasmas [74]. Their [72], the cracking of hard seeds, thus allowing them to absorb moisture [73] or the effect can not be ancontents increaseininthe germination, but adsorption, also an increase in theand quality of the plants grown enhancement ofonly nitrogen seeds through diffusion trapping and their levels of disease tolerance [75,76]. of RNS produced in plasmas [74]. Their effect can not only be an increase in germination, Wethe speculate the results of seeds’ especially with the presence but also an increase in quality that of the plants grown and imbibition their levelsinofPAW, disease tolerance of RONS and low pH, changed the osmotic potential and made the seed surface more [75,76]. prone to structural changes. On the basis of the results, it can be concluded that when using the same reactor and the same treatment conditions, the results largely depend on the type of treated seeds. In the case of Beta vulgaris, the effect of PAW treatment is evident in both the change of microflora, germination parameters and surface structure of the tested seeds. Due to the higher rate of germination (GEN ), sprouts of greater length were obtained in addition to a larger fraction of germinated seeds. Because of the relatively small decontamination effect of fungi whose specific species were restricted or stimulated, the gradual increase in treatment efficiency with increasing treatment time may be related to the change in surface structure observed via optical and scanning electron microscopy. In the case of Daucus carota, characterized by other types of fungi, a complete reduction in Alternaria radicina and Trichoderma harzianum species, which were not removed during the classical decontamination method with sodium hypochlorite, can be observed. Compared to the control, significantly better germination and sprout length parameters were obtained even for shortest time, but these were still worse than the results obtained for traditional treatment with NaOCl. In combination with the overall better decontamination effect of sodium hypochlorite, it can be thought that for carrot seeds, the decisive influence on germination was connected with fungi on the surface, which is better removed by the traditional method. However, the use of PAW may still offer a competitive alternative due to the ecology and economy of plasma treatment with the investigated device, for which only electricity and commonly available air are needed. PAW can be also generated in larger quantities in devices of different constructions [77–79], which can further improve the cost factor. 4. Conclusions Plasma activated water positively influenced the seeds germination process; however, differences between the plant species, in terms of the seeds’ response, were observed. Longer PAW imbibition times resulted in better germination parameters in comparison Appl. Sci. 2021, 11, 6164 12 of 15 to the control sample. For Beta vulgaris, treatment times of 10 and 20 min also allowed a higher fraction of germinating seeds than NaOCl treatment, with only slightly shorter average sprout lengths. For Daucus carota, the germination rate and sprout length were both noticeably lower than for sodium hypochlorite solution, but still several times higher compared to the control. Plasma activated water had an impact on the composition of fungal species inhabiting Beta vulgaris and Daucus carota seeds. Fungal species responded to the PAW treatment in different manners; some of them were unaffected. The highest counts isolated were assigned to the harmful producers of mycotoxins such as Alternaria alternata and moulds of the genus Penicillium. The beneficial species were dominated by Epicoccum nigrum, Clonostachys rosea and Trichoderma sp. Author Contributions: Conceptualization: J.P., M.K. (Marek Kopacki) and K.H.; methodology: J.P., M.K. (Michał Kwiatkowski), P.T., M.K. (Marek Kopacki) and K.H.; investigation: J.P., P.T., M.K. (Michał Kwiatkowski), M.K. (Marek Kopacki) and K.H. data curation: P.T., M.K. (Marek Kopacki) and J.P., writing—original draft preparation: P.T. and J.P.; writing—review and editing: P.T., J.P. and M.K. (Marek Kopacki); visualization: J.P., M.K. (Michał Kwiatkowski) and P.T.; supervision: J.P. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Polish–Slovak Bilateral Cooperation Programme (PlasmaBioAgro) PPN/BIL/2018/1/00065+SK-PL-18-0090 and the LUT research fund (FD-20/EE-2/418). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: We would like to thank Eng. Łukasz Kaca for the technical assistance during selected research tasks and Łukasz Remez from PIK INSTRUMENTS Sp.z.O.O for the SEM documentation. We acknowledge fruitful discussion with COST Action PlAgri CA19110 and CEEPUS CIII-AT-0063 members. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Barzman, M.; Bàrberi, P.; Birch, A.N.E.; Boonekamp, P.; Dachbrodt-Saaydeh, S.; Graf, B.; Hommel, B.; Jensen, J.E.; Kiss, J.; Kudsk, P.; et al. Eight Principles of Integrated Pest Management. Agron. Sustain. Dev. 2015, 35, 1199–1215. [CrossRef] Jamiolkowska, A.; Hetman, B.; Skwarylo-Bendarz, B.; Kopacki, M. Integrowana ochrona roślin w Polsce i Unii Europejskiej oraz prawne podstawy jej funkcjonowania. Praca przegladowa. ˛ Ann. Univ. Mariae Curie-Skłodowska Sect. 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