3.1. The Effect of PAW on Chlorophyll Contents (N-Tester Value)
The mean content of chlorophyll (N-tester value) in the maize leaves treated by PAW was not significantly (
p ≤ 0.05) different from the values measured after the foliar treatment with DW. The N-tester values reached the level of 97.7% for the treatment variants with the foliar DW application. The mean N-tester values of individual experimental variants are presented in
Table 3. Comparing both tested water types (DW and PAW), provable differences were determined for N-tester values between the doses/intensity of water application (
p ≤ 0.05). Whereas the stepped DW application decreased the N-tester values but with no significant trend, the PAW application has enhanced the N-tester values significantly. By the comparison of variant PAW3 to PAW1, the N-tester value was enhanced significantly (
p ≤ 0.05) by 6.5%. This effect is probably related to the presence of nitrogen (in a form of nitrates and nitrites) in the PAW that has led to the increased chlorophyll production [
32,
33].
Time evaluation of N-tester values for both water types was different. Whereas the N-tester values for plants treated by DW and PAW were not remarkably varied in the first term of the measurement (T1), they were significantly (
p ≤ 0.05) different in the following terms. A decrease of the N-tester values in time was higher for variants with the enhanced dose/intensity of DW application. Chlorophyll contents of the variant DW3 expressed by the N-tester value was decreased from the original 483
c ± 6 to 316
a ± 8 within three weeks, i.e., by 34.6% (
Figure 2). By the PAW application of the same doses/intensity, the N-tester values were also decreased in time (from 471
c ± 6 to 355
b ± 3) but it represented the decrease of 24.7%, only. This phenomenon is probably related to the previously mentioned contents of nitrates in the PAW and its influence on the chlorophyll formation. This fact is amplified by the N-tester values determined for PAW variants in the last term (T4), which are enhanced by the stepped PAW application. A fertilization effect of PAW applied into the soil, that is stimulated by the presence of nitrates and which positively influences the plant growth, is mentioned in other studies [
8,
34,
35]. Adhikari et al. [
36] also noticed a change of chlorophyll contents after the PAW application in the dependence on the PAW preparation.
3.2. The Effect of PAW on Chlorophyll Fluorescence Parameters
The quantum yield of the electron transport of the photosystem II (
ΦPSII), that indicates an actual capacity of the photosystem II (PSII) for photochemical processes by availability of reaction centers of the photosystem II, was not significantly (
p ≤ 0.05) influenced by the PAW application. The stepped application of DW did not significantly (
p ≤ 0.05) influence the quantum yield of PSII. On the other hand, its value was provably increased by the dose/intensity of the PAW2 variant (
Table 4). However, further increase of the PAW doses/intensity (PAW3) did not lead to any significant change of the quantum yield values. Fryer et al. [
37] mentioned a strong linear relationship between
ΦPSII and the efficiency of carbon fixation, especially at the laboratory conditions. Kučerová et al. [
38] reported on higher content of photosynthetic pigments of wheat plants, which were irrigated with the PAW. Watering by PAW increases the concentration of photosynthetic pigments and simultaneously has no or negative impact on net photosynthesis of barley and maize [
39].
Variable fluorescence of the dark-adapted leaves (
Fv), which expresses ability of the photosystem II to absorb radiation, was significantly (
p ≤ 0.05) decreased by the PAW application. The mean value of all tested variants (PAW1−3) reached 95% of the value determined after the DW application. The ability of plants to absorb radiation was reduced by the PAW application, as it is demonstrated in
Table 4. The significantly (
p ≤ 0.05) highest values of variable fluorescence were achieved by the application of DW (DW1 and DW2).
The application of the tested water types had a different influence on the fluorescence decrease ratio (RFd), which is measured at the saturation irradiance and is directly proportional to the net CO2 assimilation rate. Whereas the stepped doses/intensity of the DW application led to a significant (p ≤ 0.05) increase of the RFd value that has reached for the variant DW3 the value of 121.7% compared to DW1, the stepped PAW application had decreased this parameter. The highest dose/intensity of the PAW application (PAW3) led to the provable reduction of the RFd value with the decrease of 19.4% comparing to the PAW1 variant.
Although the mean values of
ΦPSII were provably (
p ≤ 0.05) enhanced by the double dose/intensity application of plasma activated water (PAW2), from the value evaluation of the photosystem II capacity, it is evident for the photochemical processes that the application of DW and PAW had no provable (
p ≤ 0.05) influence on the availability of reaction centers of the photosystem II in individual observation terms, as it is demonstrated in
Figure 3.
Variable fluorescence, which is determined by the redox state of the primary quinone electron acceptors of the PSII [
40], was significantly (
p ≤ 0.05) the highest for variants DW1 and DW2 in the most of the observation terms. The values of variable fluorescence maximal yield in the dark-adapted leaves measured in all terms did not differ between the stepped PAW doses (
Figure 4), but their value was provably (
p ≤ 0.05) lower in the last two terms (T3 and T4) compared to the plants treated by DW. This decrease can be caused by the presence of hydrogen peroxide (H
2O
2) in the PAW which decreases the maximal photochemical efficiency of the PSII even at its low concentration as well as the maximal quantum yield of primary photochemistry and other photosynthetic parameters [
41].
The chlorophyll fluorescence decrease ratio (
RFd) correlates with the potential CO
2 fixation rate of leaves as was shown for several plants as well as sun and shade leaves [
30]. Values of
RFd below 1.0 suggest disruption in CO
2 assimilation [
42]. The values of the fluorescence decrease ratio were substantially different in the observation terms (
Figure 5). In the early terms (T1 and T2), the PAW application had a positive effect on the CO
2 fixation evaluated as the
RFd value. Especially in the term T2, the
RFd value was significantly (
p ≤ 0.05) enhanced when the PAW variant was used instead of DW. Specifically, the
RFd value was increased from 0.27
a ± 0.01 to 0.50
c ± 0.05 by the PAW application of 1–2 doses whereas it was increased from 0.31
ab ± 0.07 to only 0.40
bc ± 0.03 by the higher PAW application of 3 doses. However, even in this term it is evident that the
RFd value is decreased with the rising doses/intensity of the PAW application (PAW3) and this decrease is further deepened in the following terms. Based on these results it can be assumed that the intensive PAW application on the maize plants leads to the damage of the photosystem apparatus and to the subsequent reduction of the CO
2 fixation by the plants. This presented fact correlates with the weight of the dry matter determined in the last experimental term (T4). Contrary to this result, the PAW application in the low or middle intensity (PAW1 and PAW2, respectively) leads to the enhancement of the
RFd value if we omit the data obtained in the last but one term (T3). These values from the early terms were significantly (
p ≤ 0.05) the highest at the end of the experiment. An opposite trend was observed by the application of DW. Comparing to the one dose DW application (DW1), significantly (
p ≤ 0.05) higher
RFd values were achieved with the stepped DW application (DW2 and DW3) at the end of the experiment (T4).
3.4. The Effect of PAW on Dry Matter Weight
The dry weight of the aboveground organs of maize, which was determined only in the last experimental term (T4), was not influenced by the foliar PAW application (
p ≤ 0.05). Nevertheless, from the mean values it is evident that the weight of the dry matter of the aboveground mass was decreased by the increasing doses/intensity of application. The lowest value among all variants was achieved with the variant PAW3 (
Table 6). The available results indicate that the PAW used for watering of germinated plants of wheat effectively stimulate seedlings growth and positively affect their metabolism in the soil with a low nutrient content [
38]. An opposite trend was observed by the DW application when the weight of the dry matter was increased. The results of Fan et al. [
45] indicates that time of the PAW preparation determines its effect on the plant growth. While the application of the PAW exposed to the plasma for 15 min stimulated the plant weight and stem length of
Vigna radiata seedlings, the PAW exposed to the plasma for a long time decreased their weight. Sajib et al. [
47] also reports the effect of the PAW treatment of the
Vigna mungo seeds on the plant growth and weight of the aboveground mass in the dependence on the plasma treatment durations. Based on the available results dealing with the PAW effect on the plant growth we can suppose that the higher PAW dose/intensity of application (PAW2 and PAW3) could be the cause of the decreased weight of the aboveground plant mass even by its foliar application.
3.5. The Effect of PAW on Nutrients Content in Plant Tissue
Plasma activated water contains ions NO
3− and NO
2−. Therefore, its stepped application significantly (
p ≤ 0.05) increased content of nitrogen in the plant tissue between the variant PAW1 by (1.43
a ± 0.05)% and PAW3 by (1.62
a ± 0.07)%. Nitrogen increase in the dry matter of the aboveground biomass was 13.3% (
Figure 6a). Although the nitrate ion provided by the foliar application is a worse form for the plant intake [
48] and compared to another forms, its absorption through the cuticle is worse than absorption of amides (urea) or ammonia [
49], the foliar fertilization by water solutions has a promising potential to increase content of nitrogen in the plant. Peuke et al. [
50] reports that the foliar applied inorganic N, both in the form of nitrate and ammonium, was entirely assimilated in the shoots of
Ricinus communis. Our results with the enhanced nitrogen content in the maize plant were probably influenced by the lower weight of the dry matter in the variant PAW3, which decreased the dilution effect of the utilized nitrogen [
51]. Contents of the rest macro-nutrients varied within the ranges: (0.15–0.17)% of P, (2.23–2.45)% of K, (1.09–1.22)% of Ca and (0.36–0.40)% of Mg. By the PAW application, only the content of P was significantly (
p ≤ 0.05) enhanced compared to the plants treated by DW (specifically DW2). Nevertheless, the mean content of phosphorus in plants treated by the PAW (0.17
a ± 0.00)% was almost the same as the mean content in plants treated by DW (0.16
a ± 0.00)%.
Among the evaluated micro-nutrients in the plant, zinc was detected for its importance in the maize nutrition and its positive effect on the seed production is described by many authors e.g., [
52,
53]. Besides other functions, zinc represents an important cofactor for the formation of enzymes and proteins and significantly influences nitrogen metabolisms in plants [
54], likewise manganese [
55] or iron [
56]. Contents of studied micro-nutrients was not significantly (
p ≤ 0.05) influenced by the water application (DW and PAW), as it is demonstrated in
Figure 6b.
The supposed effect of the PAW application as the source of the foliar applied nitrogen, by which metabolisms of the plant influences the intake and utilization (content) of other nutrients, was not confirmed by the experiment.