*Article* **Using Waste Sulfur from Biogas Production in Combination with Nitrogen Fertilization of Maize (***Zea mays* **L.) by Foliar Application**

**Petr Škarpa 1, Jiˇrí Antošovský 1,\*, Pavel Ryant 1, Tereza Hammerschmiedt 1, Antonín Kintl 1,2 and Martin Brtnický <sup>1</sup>**


**Abstract:** In Europe, mainly due to industrial desulfurization, the supply of soil sulfur (S), an essential nutrient for crops, has been declining. One of the currently promoted sources of renewable energy is biogas production, which produces S as a waste product. In order to confirm the effect of the foliar application of waste elemental S in combination with liquid urea ammonium nitrate (UAN) fertilizer, a vegetation experiment was conducted with maize as the main crop grown for biogas production. The following treatments were included in the experiment: 1. Control (no fertilization), 2. UAN, 3. UANS1 (N:S ratio, 2:1), 4. UANS2 (1:1), 5. UANS3 (1:2). The application of UAN increased the N content in the plant and significantly affected the chlorophyll content (N-tester value). Despite the lower increase in nitrogen (N) content and uptake by the plant due to the application of UANS, these combinations had a significant effect on the quantum yield of PSII. The application of UANS significantly increased the S content of the plant. The increase in the weight of plants found on the treatment fertilized with UANS can be explained by the synergistic relationship between N and S, which contributed to the increase in crop nitrogen use efficiency. This study suggests that the foliar application of waste elemental S in combination with UAN at a 1:1 ratio could be an effective way to optimize the nutritional status of maize while reducing mineral fertilizer consumption.

**Keywords:** chlorophyll content; fluorescence parameters; plant weight; plant nutrient content; nitrogen use efficiency

#### **1. Introduction**

One of the principles of the European Green Deal is the proposal of greenhouse gas emissions cut by at least 55% by the year 2030, which should set Europe to a path to becoming climate-neutral by the year 2050 [1]. According to the European Biogas Association (EBA), biogas, biomethane, and other renewable gases will play a key role in helping Europe's transition to a clean energy system [2], and the European Commission's strategies promise targeted support for biogas in the revised Renewable Energy Directive and gas legislation. EBA, Eurogas, and the Gas for Climate consortium are calling for an EU-wide renewable target of at least 11%. The annual production of biogas in Europe reaches 15.8 bcm and is relatively stable with a total of 18,943 biogas plants according to the EBA [3].

A biogas plant produces biogas, which can then be used for the cogeneration of electricity and heat. Biogas is a mixture of methane, carbon dioxide, and other components such as hydrogen sulfide (H2S) [4]. The biogas must be pretreated before use. The first step of the purification process is the removal of H2S, which is corrosive and harmful to health [5,6]. Biogas production is thus associated with the production of waste products.

**Citation:** Škarpa, P.; Antošovský, J.; Ryant, P.; Hammerschmiedt, T.; Kintl, A.; Brtnický, M. Using Waste Sulfur from Biogas Production in Combination with Nitrogen Fertilization of Maize (*Zea mays* L.) by Foliar Application. *Plants* **2021**, *10*, 2188. https://doi.org/10.3390/ plants10102188

#### Academic Editors:

Przemysław Barłóg, Jim Moir, Lukas Hlisnikovsky and Xinhua He

Received: 19 September 2021 Accepted: 13 October 2021 Published: 15 October 2021

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**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/).

The utilization of waste sulfur obtained from the purification process seems to be promising from the point of view of plant nutrition and especially from the economic aspect of biogas production [7,8] and sulfur deficiency in the environment.

European SO2 emissions have been reduced by 70–80% since 1990 [9,10]. According to results of Engardt et al. [11], sulfur deposition in Europe will decrease until, at least, 2050. For example, in the Czech Republic, atmospheric sulfur deposition is about 5 kg/ha per year [12], so there is a shortage of sulfur in the soil, as it has been presented by many authors [13–18]. According to Zbíral et al. [19], a statistically highly significant decrease in the soil S content caused by reduction of SO2 emissions in the long-term field experiments in Czech Republic from 33 mg/kg in 1981 to 8 mg/kg in 2017. Therefore, it is necessary to pay special attention to fertilization by sulfur in addition to the other essential nutrients, especially because of the increased cultivation of crops with high sulfur requirements [19,20]. Sulfur in plants is essential for the synthesis of cysteine, methionine, and some vitamins [21]. The deficiency of sulfur in maize as the main crop for biogas production not only reduces yield but also quality parameters such as the content of starch, carbohydrates, and proteins [22]. Sulfur is usually applied in the form of mineral fertilizers, and co-application with nitrogen is recommended by many authors as these nutrients have been proven to have good synergy [23,24]. Salvagiotti and Miralles [25] showed that S addition increased the biomass and grain yield of cereal and the positive interaction of N and S, which resulted in a greater nitrogen use efficiency. A shortage of S supply also lowers the utilization of nitrogen and results in a deterioration in crop quality [26]. As sulfur is an essential constituent of enzymes involved in nitrogen metabolism, its deficiency could lead to a decrease in N assimilation [27,28]. Some reports have shown the accumulation of nitrates in S-deficient plants [29]. In addition, Haneklaus et al. [30] reported that each kg of S deficit causes 15 kg of nitrogen to be lost in the environment. Maize is an important crop that, despite its relatively low sulfur requirements, is severely affected by its deficiency [31,32].

Nitrogen is essential for plants in terms of biomass and yield production [33]. In addition to the conventional nitrogen fertilization of the soil, the nutritional status of the plants can be optimized by foliar fertilization during the plant growth [34]. Foliar fertilization could be used under farming conditions as a quick correction for unexpected nutrient deficiencies, for the late supply of N (and another nutrients) during advanced growth stages, and as a preventive measure against unsuspected (or hidden) deficiencies [35–38]. The foliar application of nutrients is also recommended when the soil or the plant conditions limit the availability of some nutrients [39] and is appropriate under conditions when high loss rates of soil-applied nutrients may occur [40]. For example, the foliar application of nitrogen has significantly improved the grain yield of maize [41] and other cereal crops [42].

The aim of this study was to verify the effect of the foliar application of waste elemental sulfur from biogas production in combination with conventional liquid fertilizers UAN applied in different ratios. Such a reutilization of waste sulfur from biogas plants back in agriculture is suitable from the economic aspect of biogas purification and waste management. The application of this sulfur could help to reduce the consumption of mineral fertilizers and, at the same time, address the deficient sulfur content in the soil and plants.

#### **2. Results and Discussion**

The application of UAN fertilizer alone and in combination with sulfur increased the chlorophyll content (N-tester value) in maize leaves compared to the unfertilized control. The increase in chlorophyll was evident at both monitoring terms (t1 and t2), while the differences between the control (N-unfertilized treatment) and the N (UAN) and NS (UANS1-3)-fertilized treatments increased over time (Table 1).


**Table 1.** The effect of the foliar fertilizer application on chlorophyll contents (N-tester value).

The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation). The same letters next to the numbers describe no statistically significant differences between the treatments (Fisher's LSD test, *p* < 0.05). The relative expression of the values is shown in the column marked Rel. % (Control = 100%). The measurements were performed at two growth stages, t1 (5th true leaf) and t2 (6th true leaf).

The N-tester values were significantly correlated with the rate of nitrogen applied in fertilizers at both terms, as presented in Figure 1. The results agree with several studies that have reported a strong correlation between chlorophyll content and the amount of nitrogen in leaves [43–47].

**Figure 1.** Dependence of N-tester value on nitrogen dose. The measurements were carried out on the 1st (t1) and 2nd (t2) growth stages of maize.

Nitrogen is part of the enzymes associated with chlorophyll synthesis [48] and the chlorophyll concentration reflects relative crop N status. Statistically significant highest N-tester values were found for the treatment fertilized with UAN applied without sulfur (UAN) on both measurement terms (t1; t2). The highest nitrogen dose was applied on this treatment. The application of UAN in combination with elemental sulfur (UANS1–3) significantly increased the N-tester value compared to the unfertilized (control) treatment, but the level of chlorophyll content did not reach the values found in plants fertilized with UAN alone. While, in the first measurement term (t1), the highest N-tester value was found for the UANS2 treatment (Table 1), in term t2, the N-tester values were in direct dependence on the nitrogen doses contained in the UAN–sulfur mixture. The N-tester values found at both terms (t1; t2) were significantly correlated with the plant nitrogen content detected at term t3 (r = 0.711, *p* < 0.001; r = 0.707, *p* < 0.001, respectively). Evaluation of the nutritional status after the joint application of nitrogen and sulfur using the N-tester was also performed on several dates by Lacroux et al. [49], and their results showed a significant increase in measured values compared to the control, with the highest values achieved by the joint foliar application of N and S.

The ability of the photosystem II to absorb radiation is expressed by the variable chlorophyll fluorescence for dark-adapted leaves (*Fv*). The more radiation a plant can absorb, the more radiation the plant can use for photosynthesis. Although the ability of the plant tissue to absorb radiation decreased over time (comparison of *Fv* levels between t1 and t2), this decrease was not significant for the UAN and UAN combination with sulfur. A significant reduction in *Fv* values was only observed in the unfertilized treatment (Figure 2). Even though the treatment with the highest sulfur dose (UANS3) showed the lowest *Fv* values, the results showed that the decrease in *Fv* between terms t1 and t2 was smallest on this treatment. Nitrogen deficiency decreases the photosynthetic assimilation capacity of CO2 of plant leaves, leading to decreases in light-saturated photosynthetic rates [50]. In addition, Ciompi et al. [51] and Jin et al. [52] reported a positive correlation between the nitrogen content in the plant tissue of leaves and photosynthetic capacity.

**Figure 2.** Variable fluorescence (*Fv*) value after the foliar application of fertilizers. The measurements were carried out on two growth stages of maize (t1 and t2). The values represent the arithmetic mean (*n* = 8); the bars represent the standard deviation of the mean. There are no statistical differences between columns with the same letters (Fisher's LSD test, *p* < 0.05).

After dark adaptation of the maize leaves, the maximum photosynthetic capacity (*ΦPSII*) was estimated as the quotient between variable and maximum fluorescence (*Fv*/*Fm*). The quantum yield, which indicates the actual capacity for photochemical processes by the availability of reaction centers of the photosystem II (PSII), was significantly (*p* ≤ 0.05) influenced by the fertilizer application (Figure 3). It is clear that nitrogen significantly affects photosynthesis and chlorophyll fluorescence of the plant. This was demonstrated by the response of maize to nitrogen fertilization in a study by Ahmad et al. [53], in which the effect of nitrogen application increased the electron transport rate, photochemical quenching coefficient, variable fluorescence, maximal quantum yield, and effective quantum yield of PSII photochemistry. A significant increase in *ΦPSII* values in three maize varieties due to a high nitrogen dose was demonstrated by Jin et al. [52]. Reductions in the quantum yield of PSII electron transfer due to nitrogen deficiency were also described by Nunes et al. [54] and Verhoeven et al. [55]. In our study, the values of *ΦPSII* were decreased over time regardless of fertilization treatment. The highest value of *ΦPSII* was determined after the application of UAN with the highest elemental sulfur content (UANS3). These results contradict the above studies, but, on the other hand, they show a positive effect of applied sulfur on nitrogen utilization and its use by the plant. A high linear dependence between the efficiency of carbon fixation and quantum yield value was presented by Fryer et al. [56].

The rate of fluorescence decline (*RFd*), an empirical parameter for the quantification of plant vitality under tested conditions, was measured. In contrast to the values of the variable chlorophyll fluorescence (*Fv*) and quantum yield of PSII (*ΦPSII*), the rate of fluorescence decline was not statistically significantly affected by foliar fertilization. Only at term t2 did the *RFd* value of plants grown on the UANS2 treatment decrease significantly

below the control level, but no trend in the decrease in *RFd* due to UAN fertilization in combination with elemental sulfur was observed (Figure 4).

**Figure 3.** The effect of the foliar application of fertilizers on the quantum yield of PSII photochemistry (*ΦPSII*). The measurements were carried out on two growth stages of maize (t1 and t2). The values represent the arithmetic mean (*n* = 8); the bars represent the standard deviation of the mean. There are no statistical differences between columns with the same letters (Fisher's LSD test, *p* < 0.05).

**Figure 4.** Fluorescence decrease ratio (*RFd*) in maize leaves after the foliar application of fertilizer. The measurements were carried out on two growth stages of maize (t1 and t2). The values represent the arithmetic mean (*n* = 8); the bars represent the standard deviation of the mean. There are no statistical differences between columns with the same letters (Fisher's LSD test, *p* < 0.05).

The average dry weight of the above-ground biomass (AGB) of plants determined on the 35th day after the foliar application of fertilizer (t3) is shown in Figure 5. The highest plant dry weight was found for the treatment fertilized with UAN, which provided the most nitrogen to the plants. The dry weight of plants produced on this treatment was 2.4 times higher compared to the unfertilized Control. The dry weight of plants fertilized with the UANS fertilizer combination ranged from 17.44 to 17.84 g/plant and was not statistically different from the UAN treatment (Figure 5). A significant effect of foliar nitrogen application on plant dry matter yield has been demonstrated in the available literature [57–59], in agreement with our results. The increase in plant weight due to foliar sulfur fertilization was also documented. Perveen et al. [60] observed a significant increase in root and shoot biomass and root and shoot length of maize grown under salinity conditions due to the foliar application of different sulfur compounds. An increased barley yield after elemental sulfur application was described by Grzebisz and Przygocka-Cyna [61] in their long-term experiment. A positive effect of the foliar application of sulfur on canola pods formation and subsequent seed yield was demonstrated by Khalid et al. [62].

**Figure 5.** Weight of dry matter above-ground biomass of maize after the foliar application of fertilizer. The measurements were taken at the end of experiment (t3). The values represent the arithmetic mean (*n* = 8); the bars represent the standard deviation of the mean. There are no statistical differences between columns with the same letters (Fisher's LSD test, *p* < 0.05). AGB—above-ground biomass.

The UAN fertilizer application significantly increased the nitrogen content of maize leaves. The highest N content, 10.3 g/kg DM, was found in leaves after the application of UAN fertilizer alone (Table 2). There was no significant difference in plant N content among treatments fertilized with a mixture of UAN and elemental sulfur (UANS1-3), but the data showed a relative increase in N content with sulfur rate. An increased leaf N concentration following sulfur fertilization has also been described [31,63]. An increase in the nitrogen content of wheat grain, due to the foliar application of sulfur, was observed by Tea et al. [64] and Rossini et al. [65]. This effect could be due to a better assimilation of foliar-applied N and S compared to their soil-applied counterparts.

**Table 2.** Nutrient content and nutrient uptake by DM of AGB and the N:S ratio.


The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation). The same letters next to the numbers describe no statistically significant differences between the treatments (Fisher's LSD test, *p* < 0.05). DM—dry matter, AGB—above-ground biomass.

Sutar et al. [22] described the critical sulfur concentration in dry matter of maize leaves as 1.5 g/kg DM. The sulfur content in the ABG of maize plants ranged from 2.8 to 3.6 g/kg DM (Table 2). Its content in the ABG of plants grown on the treatments fertilized with a mixture of UAN and elemental sulfur (UANS1–3) was identical to that of unfertilized plants (Control). Only in the nitrogen-fertilized treatment (UAN) was the amount of sulfur significantly lowest (Table 2). This fact is not only related to the absence of sulfur in the fertilizer, but it can also be explained by the dilution of nutrients in the maize plant tissue that occurred as a result of the increase in DM weight of AGB on this treatment (Figure 5). Therefore, the nutrient uptake by the plant was calculated as a more appropriate parameter expressing the nutritional status of the plants (Figure 6). Nutrient uptake is the relationship between the DM weight of AGB and its nutrient content, expressed in g of nutrient per plant (g/plant). Logically, the highest nitrogen uptake was recorded in the UAN-fertilized treatment, i.e., the treatment with the highest applied nitrogen rate. Even though nitrogen uptake by plants was not significantly different among the treatments fertilized with UAN and elemental sulfur mixtures, plants fertilized with fertilizers containing a higher proportion of elemental sulfur (UANS2 and UANS3) showed a higher uptake of nitrogen by plant AGB. A positive significant interaction between nitrogen and sulfur uptake and utilization was confirmed.

**Figure 6.** Nitrogen and sulfur uptake by above-ground plant biomass (mg/plant). The measurements were taken at the end of the experiment (t3). The values represent the arithmetic mean (*n* = 8); the bars represent the standard deviation of the mean. There are no statistical differences between columns with the same letters (Fisher's LSD test, *p* < 0.05).

The N:S ratio of the plant may also be an interesting indicator of nutritional status, as reported by some authors [31,66]. The principle behind this assessment is the fact that plants need a balanced amount of nitrogen and sulfur for proper amino acid synthesis. Therefore, nitrogen-to-sulfur ratios above a N:S ratio threshold indicate S deficiency [67]. A possible disadvantage of this assessment is the decreasing value of the N:S ratio during the growing season, as reported, for example, by Calvo et al. [68,69] or Scherer [70]. A 15-19:1 N:S ratio has been reported as a limiting ratio for cereals at the time of tillering [71], and an ideal N:S ratio for the optimum growth and development of maize is 15:1 [72]. The observed N:S ratio (Table 2) indicated that the sulfur contained in maize was not deficient in any of the fertilization treatments. From the ratios obtained, it is possible to observe the already described trend, where the highest ratio of nitrogen and sulfur was logically found on the treatment fertilized only with UAN fertilizer. In contrast to our study, significant changes in the N:S ratio after sulfur application were observed [73,74]. However, they agreed that

an increase in the sulfur content of the plant does not necessarily predict increased yield. Sutradhar et al. [31] also confirmed the same conclusion.

The previously mentioned synergism between nitrogen and sulfur can be documented by crop nitrogen use efficiency. The nitrogen supplied by foliar nutrition from fertilizer applied without sulfur addition (UAN) was utilized by the plant at 30.5% (Table 3). A similar level of NUECrop was found on the treatment fertilized with the lowest sulfur fertilizer mixture (UANS1), whereas an increase in the proportion of sulfur in the fertilizer mixture increased nitrogen use efficiency. The relationship between nitrogen recovery from applied fertilizers and the dose of sulfur applied by the fertilizer mixture was statistically significant (NUECrop = 22.9 + 0.171 × sulfur dose, r = 0.709; *p* = 0.002).

**Table 3.** Crop nitrogen use efficiency.


The values in the table represent the arithmetic mean (*n* = 8) ± SD (standard deviation). The same letters next to the numbers describe no statistically significant differences between the treatments (Fisher's LSD test, *p* < 0.05).

In agreement with our results, several studies showed that sulfur fertilization may increase NUE [75–77]. As sulfur is an essential constituent of enzymes involved in nitrogen metabolism [78], its deficiency may lead to ineffective utilization of the nitrogen content in plant [79,80]. An increase in nitrogen uptake by maize plants due to graded doses of foliar sulfur application was presented by Sarfaraz et al. [81].

#### **3. Materials and Methods**

#### *3.1. Experimental Methodology, Plant Material, and Growth Conditions*

The pot vegetation experiment was established in the vegetation hall of the Biotechnological house at Mendel University in Brno located at 49◦21 03 N and 16◦61 38 E. Mitscherlich pots (STOMA GmbH, Siegburg, Germany) were filled with 6.5 kg of air-dried and sieved soil (2 cm diameter sieve). Properties of the soil used in the pot experiment are shown in Table 4.


**Table 4.** Properties of soil used in pot experiment.

The maize (*Zea mays* L.), cultivar SY ORPHEUS (Syngenta Czech s.r.o., Prague, Czech Republic), was chosen for this study. Four seeds of maize were sown to a 4 cm depth

in each pot. The number of plants in each pot was reduced to two plants per pot two weeks after the sowing.

The pot experiment was carried out under seminatural conditions in the outdoor vegetation hall under a rain shelter. The air temperature, air humidity, and solar radiation during the maize growing season are shown in Figure 7. After a cooler April (11.8 ◦C) and May (12.6 ◦C), a warming period occurred at the beginning of June (22.6 ◦C), which lasted until the end of the experiment (average air temperature in July was 19.6 ◦C). The relative air humidity fluctuated evenly between 40 and 90% during the experiment. Global solar radiation also fluctuated over time depending on weather conditions, with levels increasing slightly during the experiment (April: 16.9; July: 22.1 MJ/m2). A controlled watering regime identical for all treatments (pots) was used in the experiment. Plants were watered to 70% of the maximum water holding capacity throughout the growing season. The pots were watered by hand with demineralized water on the soil surface.

**Figure 7.** The average daily temperature (◦C), relative humidity (%), and global solar radiation (MJ/m2) in the vegetation hall during the experiment.

Liquid urea ammonium nitrate fertilizer (UAN; 30% total N–15% N-NH2, 7.5% N-NO3, 7.5% N-NH4) was applied to maize plants in combination with waste elemental sulfur suspension (12% S0 suspension) in the ratios shown in Table 5. The sulfur suspension was obtained by the desulfurization of biogas using the Thiopaq® scrubber (Paques, Balk, The Netherlands), which works by washing the raw biogas with a slightly alkaline solution (pH 8–9) and the subsequent biological oxidation of sulfides to elemental sulfur. The elemental sulfur particle size in the suspension was less than 60 μm (96.9% of the particles). Each of the treatments was established in eight replicates (pots). The pots were placed randomly in the vegetation hall under the rain shelter.

The foliar application was carried out on the plant development stage of the 4th true leaf unfolded. The application of 3 mL of fertilizer mixture per pot of each treatment was used. Fertilizers were evenly applied using a pressurized hand pump sprayer (DPZ 1500, ProGlass, Weilheim an der Teck, Germany). The mixture of the waste elemental sulfur with the UAN fertilizer was mixed prior to application to ensure that the elemental sulfur was evenly distributed in the fertilizer mixture and applied uniformly.

During the maize vegetation, chlorophyll content (N-tester value) and chlorophyll fluorescence parameters were evaluated. The measurements were performed 7 (t1) and 21 days (t2) after the foliar application. The weight of dry matter (DM) of maize plant AGB, the content and ratio of nutrients (N and S) in maize plant AGB, and their uptake by plants were determined 35 days after the foliar application of fertilizer mixtures (t3). The schedule of the experiment is shown in Table 6.


**Table 5.** Experimental treatments of foliar application in pot experiment.

**Table 6.** Timetable of the experiment.


*3.2. Determination of Plant Growth and Development Parameters*

3.2.1. Chlorophyll Content in Plant Leaf (N-Tester Value)

The chlorophyll content of maize leaves was measured using a Yara N-tester (Yara International ASA, Oslo, Norway). The chlorophyll content was expressed as "N-tester value." Measurement was performed at a wavelength range of 650–940 nm [85]. Eight plants were assessed in each treatment in both terms. The measurement of chlorophyll content was performed on the 5th (t1) and 6th true leaves (t2), and the value of the chlorophyll content of each plant was the mean of 60 measurements.

#### 3.2.2. Chlorophyll Fluorescence Parameters

To determine the photochemical efficiency of photosystem II, selected fluorescence parameters of chlorophyll were measured in maize plant. The tested parameters were measured with the PAR-FluorPen FP 110-LM/S (Photon Systems Instruments, Drásov, Czech Republic) and evaluated using the FluorPen 1.1 software [86]. Measurements of fluorescence parameters were carried out on identical leaves (5th and 6th true leaves) at identical terms (t1 and t2) as for chlorophyll content determination. Maize leaves were dark-adapted for 25 min before measurement. The protocol for measuring the fluorescence parameters of chlorophyll is shown in Table 7.

The variable fluorescence of the dark-adapted leaves (*Fv*), quantum yield of photosystem II (*ΦPSII*), and chlorophyll fluorescence decrease ratio (*RFd*) were determined (Table 8).


**Table 7.** Measurement protocol of the chlorophyll fluorescence parameters.

The measured at wavelength (*λ*) of 454 nm, L—light, DR—dark recovery, *ΦPSII*—quantum yield of photosystem II, *RFd*—chlorophyll fluorescence decrease ratio, *Fv*—variable fluorescence of the dark-adapted leaves.

**Table 8.** The photochemical quenching parameters.


*F0*—minimal fluorescence from the dark-adapted leaves, *ΦPSII*—quantum yield of photosystem II, *RFd*—chlorophyll fluorescence decrease ratio, *Fm*—maximal fluorescence from the dark-adapted leaves; *Fd*—fluorescence decrease from *Fm* to *Fs*; *Fs*—steady-state chlorophyll fluorescence.

3.2.3. Determination of Weight Biomass, Nutrient Contents and Uptake, and Nitrogen Use Efficiency

The ABG of maize plants was harvested on 22 July 2019 (t3). The ABG was then ovendried at 60 ◦C for the first two hours. The temperature was then reduced to 45 ◦C where the samples were kept for 72 h. The dry weight of ABG was determined using a laboratory-scale PCB Kern (KERN & Sohn GmbH, Balingen, Germany). Then, the dried ABG was crushed and homogenized by the grinder Grindomix GM200 (Retsch GmbH, Haan, Germany). The HNO3/H2O2 [90] digestion of biomass was achieved using a microwave digestion system in ETHOS 1 (Milestone Srl, Sorisole, Italy). Subsequently, the nutrient content (Table 9) and nutrient ratio were determined, and crop nitrogen use efficiency (NUECrop) was calculated by the relationship NUECrop = N yield/N input · 100 (N yield = nitrogen uptake by plants (mg/pot); N input = nitrogen applied by foliar fertilizers (mg/pot)) [91]. In the calculation of NUECrop, the nitrogen uptake by plants grown on the fertilized treatments (N yield) was subtracted from the nitrogen uptake observed on the control treatment (this amount of nitrogen characterized the natural soil supply).

**Table 9.** The methods for the determination of nutrients in maize AGB.


#### *3.3. Statistical Data Analysis*

The effect of the foliar application of fertilizer mixtures on the plant growth and development parameters was statistically analyzed by the STATISTICA 12 program (TIBCO Software, San Jose, CA, USA) [94]. The effect of the foliar application on the N-tester value, chlorophyll fluorescence parameters, dry weight of ABG, and nutrient content ABG of maize was analyzed separately for each treatment of the experiment. The normality was checked using the Shapiro–Wilk test, and the homogeneity was verified by the Levene test at *p* ≤ 0.05. The effect of fertilization was analyzed using ANOVA. Fisher's LSD test (*p* ≤ 0.05) was used to determine any statistically significant differences between the means of treatments.

#### **4. Conclusions**

Nitrogen plays an important role in maize nutrition, contributes to chlorophyll formation, and significantly influences the photosynthetic activity of plants. The result of the vegetation experiment showed that the efficiency of mineral nitrogen fertilization can be increased by the foliar application of liquid fertilizers with sulfur addition. The optimal adjustment of the ratio of applied nutrients (N and S) improves the nutritional status of the plants and allows the reduction in their doses while minimizing the environmental risks associated with fertilization. The foliar application of UAN fertilizer in combination with elemental sulfur from biogas production in a 1:1 ratio seems to be a sensible way to optimize the nutritional status of maize, both for the economics of biogas purification, when the waste sulfur is reused as a fertilizer, and for environmental reasons. However, verification of the results obtained from the pot experiment in field trials will be necessary.

**Author Contributions:** Conceptualization, P.Š. and J.A.; methodology, P.Š., J.A. and M.B.; validation, J.A. and P.Š.; investigation and data analyses, J.A. and P.Š.; resources, J.A., M.B., A.K. and P.Š.; writing—original draft preparation, P.Š. and J.A.; writing—review and editing, P.R., M.B., A.K. and T.H.; project administration, M.B.; funding acquisition, M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Technology Agency of the Czech Republic, grant TH04030142: Utilization of Biogas Waste to Improve Soil Properties and Increase Sulphur Content of Plants.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. Due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data are not available.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Jarosław Potarzycki, Witold Grzebisz \* and Witold Szczepaniak**

Department of Agricultural Chemistry and Environmental Biogeochemistry, Poznan University of Life Sciences, Wojska Polskiego 28, 60-637 Poznan, Poland

**\*** Correspondence: witold.grzebisz@up.poznan.pl

**Abstract:** Wheat fertilized with Mg, regardless of the method of application, increases nitrogen fertilizer (Nf) efficiency. This hypothesis was tested in 2013, 2014, and 2015. A two-factorial experiment with three doses of Mg (i.e., 0, 25, and 50 kg ha−1) and two stages of Mg foliar fertilization (without; BBCH 30; 49/50; 30 + 49/50) was carried out. Foliar vs. in-soil Mg fertilization resulted in a comparable grain yield increase (0.5–0.6 t ha<sup>−</sup>1). The interaction of both fertilization systems increased the yield by 0.85–0.9 t ha<sup>−</sup>1. The booting/heading phase was optimal for foliar fertilization. Mg accumulation by wheat fertilized with Mg increased by 17% compared to the NPK plot. The recovery of foliar Mg was multiple in relation to its dose. The recovery of the in-soil Mg applied ranged from 10 to 40%. The increase in yield resulted from the effective use of N taken up by wheat. In 2014 and 2015, this amount was 21–25 kg N ha<sup>−</sup>1. The increase in yield resulted from the extended transfer of N from vegetative wheat parts to grain. Mg applied to wheat, irrespective of the method, increased the efficiency of the N taken up by the crop. Mg fertilization resulted in higher Nf productivity, as indicated by the increased nitrogen apparent efficiency indices.

**Keywords:** in-soil Mg application; foliar Mg fertilization; Mg uptake; N uptake; nutrient use efficiency indices

#### **1. Introduction**

Wheat is a basic source of staple food for the world's human population. Currently, the largest producers are China, India, the Russian Federation, the USA, Canada, France, and Ukraine. The mean yields in these countries in 2018–2021 were 5.6 ± 0.17, 3.4 ± 0.12, 2.8 ± 0.15, 3.9 ± 0.23, 3.4 ± 0.01, 7.1 ± 0.58, and 3.3 ± 0.14, respectively [1]. The yield variability, as shown by the coefficient of variation (CV), in leading producers such as Canada, China, and India was 3.5%, 3.0%, and 2.5%, respectively. This low CV indicates an extensive system of wheat production. Therefore, in these countries, the yield gap (YG), as a measure of the ineffectiveness of the applied means of production, is low [2]. For Germany, the average yield in 2018–2020 was 7.3 ± 0.58 t ha<sup>−</sup>1, compared to 4.5 ± 0.65 t ha−<sup>1</sup> for Poland. The CV for Germany was 7.9% and for Poland 14.4%.

A realistic determination of the maximum attainable yield, as the basis for calculating the YG, requires local data that reflect both the short-term variability in weather conditions and the variability in management factors affecting the actual yield. The order of these factors for wheat in Poland, in descending order, is nitrogen (N), fore-crop, soil class, available potassium content, crop protection level—fungicide and foliar fertilization, and available phosphorus content [3]. Another method for the calculation of the YG, which is, in fact, simpler to use, is the concept of the nitrogen gap [4,5]. Both methods allow for the discrimination of factors, which is critical for the maximum wheat yield in a well-defined geographical area [6].

The nutrient requirements of wheat compared to other cereals is high, including, firstly, N, P, and K [7]. The yield-forming function of N is widely discussed in both science and

**Citation:** Potarzycki, J.; Grzebisz, W.; Szczepaniak, W. Magnesium Fertilization Increases Nitrogen Use Efficiency in Winter Wheat (*Triticum aestivum* L.). *Plants* **2022**, *11*, 2600. https://doi.org/10.3390/ plants11192600

Academic Editors: Dimitris L. Bouranis and Christian Dimkpa

Received: 24 July 2022 Accepted: 29 September 2022 Published: 2 October 2022

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**Copyright:** © 2022 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/).

agricultural practice. Some of the most important data on the impact of N on yield in wheat concern the *critical window* that extends from the beginning of stem elongation to flowering. N supply to wheat during this period is critical for both the number of ears and the number of grains per ear. Both yield components determine the aggregate component of the yield, i.e., the number of grains per unit area (grain density (GD) [8,9].

New, high-yielding wheat varieties developed to exploit their yield potential, require, first, the development of efficient technologies aimed at the effective use of nitrogen fertilizer (Nf). The price of Nf increased many times in 2021 and 2002 [10]. The 4R Stewardship approach, the keywords of which are right source, right rate, right time, and right place, is actually limited by Nf [11].

Magnesium (Mg) cannot be treated as a *forgotten* nutrient, especially in modern, intensive, and effective agriculture [12,13]. The physiological and yield-forming functions of Mg are well known [14–16]. The increase in the yield of cereals in response to the application of Mg ranges from 5 to 10% [17]. The Mg content in the flag leaf of winter wheat at the onset of plant flowering can be used to forecast the yield [18]. The amount of Mg accumulated in crop plants is relatively low compared even to phosphorus [14]. In agricultural practice, farmers use two basic Mg fertilization systems, differing in the method of Mg fertilizer application to crops. The first, a classic method, is the in-soil application of Mg fertilizer using lime (Mg oxide or Mg carbonate) for acidic soils and magnesium sulfate (Kieserite) for soils with an optimum pH [19]. Mg availability to plants is not a problem, provided the content of its available form in the soil is in the medium class, at least [19]. The second method of Mg supply to crop plants is foliar fertilization. Magnesium is widely used in this way throughout the world [20,21]. The key challenge for farmers using soluble Mg fertilizers (i.e., sulfate or chloride) is to determine the (i) rate, (ii) methods, and (iii) time—the plant growth stage of fertilizer application. A well-developed Mg fertilization system should be oriented toward an increase in the efficiency of Nf.

The interaction between Mg and N occurs at all levels of a plant's organization. The importance of N for a plant's growth and yield results from its presence in key biological molecules such as chlorophyll and the ribulose bisphosphate carboxylase–oxygenase enzyme, simply called Rubisco (RuBP) [22]. The latter is a key N-dependent enzyme, decisive in the survival of life on Earth. Its key function is to capture and then fixate the CO2 molecule, which is the basic substrate for the production of elementary sugar compounds [23,24]. The key function of Mg is to maintain Rubisco activation, which results in the stabilization of the net photosynthetic rate, as was demonstrated for wheat by Shao et al. [25]. Crop plants well supplied with Mg since the beginning of their growth increases N uptake, resulting in an increase in its unit productivity [26,27]. For example, Mg concentration in maize leaves during the grain filling period is the critical factor affecting the grain yield of this crop. The adequate nutrition of plants with Mg increases N productivity, in turn decreasing the required Nf dose [28,29].

Two main questions remain open. The first, and most important, concerns the relationship between N and Mg from the point of view of the impact of Mg on the management of N by arable crops. The N/Mg ratio during the early growth of potato tubers or sugar beets is crucial for the final yield of both crops [20,27,30,31]. The second, more minor one concerns the efficiency of the applied Mg, depending on the method of its application. The main objective of this study was to evaluate (i) the effectiveness of applied Mg depending on the fertilization method and (ii) the relationship between Mg and N uptake by wheat and its use efficiency. A secondary objective was to evaluate Mg fertilization systems including soil and foliar fertilization of wheat fertilized with an optimum dose of Nf.

#### **2. Results**

#### *2.1. Magnesium Fertilization Systems and Yield Increment*

The yield gain due to the application of Mg to winter wheat was the result of the interaction between the soil and the foliar treatment (Figure 1). Detailed information on the grain yield and the elements of the yield structure can be found in an article by Grzebisz and Potarzycki [32]. In all years of the study, Mg application, regardless of the Mg fertilization system, increased the grain yield. The recorded increase ranged from approximately 0.58 to 0.74 t ha<sup>−</sup>1. The lower yields were due to unfavorable weather conditions during the spring growing season (2013 and 2015). Nevertheless, the effect of the Mg fertilization system (Mg-FS) on the yield gain in the consecutive years of the study was highly stable (Figure A1). A strong interaction between the Mg and FSs was observed (Figure 1). The increase in yield due to the soil-applied Mg (Mgs) in comparison with the absolute Mg control (the plot treated only with NPK) amounted to approximately 0.52 t ha<sup>−</sup>1. The effect of a single stage of Mg foliar treatment (Mgf), regardless of the growth stage of wheat, was 0.57 t ha<sup>−</sup>1. A double stage of Mg foliar application (at BBCH 30 and repeated at BBCH 49/50) resulted in a yield increase of 0.74 t ha<sup>−</sup>1. The effect of the interaction of both Mg and FSs with the yield gain was dependent on the dose of Mgs. The increase in yield was 0.77 t ha−<sup>1</sup> for the plot fertilized with 25 kg Mg ha<sup>−</sup>1, but it provided Mg foliar application at BBCH 30. The same level of increase in the yield was recorded on the plot with 50 kg Mg ha−1, regardless of the growth stage of wheat treated with Mg. In-soil and double, two-phase foliar feeding of wheat with Mg resulted in a yield gain of 0.92 t ha<sup>−</sup>1.

**Figure 1.** The net increase in the yield of winter wheat in response to the interaction between Mg fertilization systems. a, b. Similar letters mean a lack of significant differences using Tukey's test. Mgs and Mgf—soil and foliar Mg fertilization systems, respectively. \* Doses of applied Mg, kg ha<sup>−</sup>1; \*\* stages of Mg. Wheat stages of Mg foliar fertilization: I—BBCH 30; II—BBCH 49/50.

#### *2.2. Magnesium Accumulation and Indices of Efficiency*

Magnesium accumulation in wheat at maturity was significantly dependent on the course of the weather during the growing season (Table 1). The effect of the Mg fertilization system was not significant for grain, but it was significant for wheat residues (straw + chaffs). The total Mg accumulated by wheat at harvest resulted from the interaction between the Mg fertilization systems and years (Figure 2). In 2013, the effect of the Mgs was visible only on the Mgs50 plot. The effect of Mgf was the strongest on the Mgs25 main plot. Plants foliar fertilized at the BBCH 49/50 stage increased Mg accumulation by 2 kg ha−<sup>1</sup> compared to its uptake on the Mgs control plot. In 2014, the uptake of Mg was, in general, higher than in 2013 and 2015. In the Mgs control plot, the double-stage foliar fertilization of wheat increased the uptake of Mg by 2.0 kg ha−<sup>1</sup> compared to the Mg absolute control. The effect of the Mgs was only slightly stronger on the Mgs50 than on the Mgs25 main plot. The interaction between both fertilization systems was the strongest

on the Mgs50 plot and the double-stage Mgf treatment. Mg extra uptake was 2.9 and 2.5 kg ha−<sup>1</sup> higher compared to the Mg absolute control and the Mgs50 control, respectively. In 2015, the average Mg accumulation by wheat was 30% and 21% lower compared to 2014 and 2013, respectively. The positive effects of Mgf on the uptake of Mg were observed only on the Mgs plots. The strongest increase in Mg accumulation by wheat in response to Mgf was observed on the Mgs50 plot, reaching the maximum for the double-foliar-treated plants (BBCH 30 + 49/50). The greatest amount of Mg taken up by wheat during the growing season (i.e., slightly above two-thirds) was accumulated in grain as indicated by the Mg harvest index (Mg–HI).

Only two indices of Mg productivity (i.e., the Mg unit accumulation in grain (MgUA– G) and the total Mg unit accumulation (MgUA–T) of the nine), showed a significant but negative relationship with the yield (Table A1). Four of the five indices (Mg–HI, MgUA–G, MgUA–T, and Mg unit productivity–grain (MgUP–G)) showed year-to-year variability, but their response to the interaction Y × Mgs × Mgf was not significant. The only significant dependence was noted for the Mg total unit productivity (MgUP–T). However, this index was negatively correlated with the total Mg accumulation (*r* = –0.78 \*\*\*). Moreover, its highest values were recorded in the dry year of 2015, when they were 46% and 28% higher compared to 2013 and 2014, respectively.

Four classic efficiency indices of the applied nutrients (PFP–Mg, Mg–AE, Mg–R, and Mg–PhE) responded significantly to all studied factors including Y × Mgs × Mgf (Table 1). However, none of them showed a significant relationship with the yield (Table A1). All these indices displayed a steady yearly trend, clearly highlighting much higher values in 2014, the year with the highest yield. A significant effect of the experimental treatments on these indices resulted from the increase in the applied doses of Mg from 2.4 to 56.4 kg ha<sup>−</sup>1. This trend can be described by a power regression model. The general trend in the data obtained is presented in detail for Mg recovery (Mg–R) (Figure A2). Its values, regardless of the year of the study, were the highest for the plots with only foliar-applied Mg. The recovery of the applied Mg for these treatments exceeded 100%. The differences between years were the clearest for the Mgf dose of 2.4 kg ha<sup>−</sup>1, reaching 383% in 2014, 300% in 2013, and 260% in 2015. The developed regression models clearly showed that the yearly pattern, observed for the lowest dose of Mg, was maintained with an increase in the Mg dose (Figure A3). The impact of the Mgs × Mgf interaction on the Mg-R values was observed only on the main Mgs25 plot.

#### *2.3. Nitrogen Accumulation and Indices of Efficiency*

The total amount of N accumulated in winter wheat at maturity (TN) significantly depended on the experimental factors. In fact, they influenced the accumulation of N, but the interaction with years was only noted for wheat residues (Table 2). The amount of N in grain (NaG) was the result of independent interactions of the year with Mg fertilization systems (Y × Mgs and Y × Mgf). The first were revealed in 2014 and 2015. In both years, the Mgs caused a significant increase in NaG compared with the Mg absolute control. A significant effect of Mgs50 was only observed in the dry year of 2015 (Figure 3). The effect of the Mgf was most evident in 2013 (Figure 4). In 2014, it was only slightly marked at BBCH 30. In 2015, it was much more visible, but a significant difference was only noted between the Mgs control and the plot with double Mg foliar fertilization (BBCH 30 + 49/50). The same principles were noted for the nitrogen harvest index (NHI). The values of this index were very high, approaching almost 90% in 2014.



respectively;

crop residues, and total, respectively;

MgUP–G and

magnesium apparent efficiency;

MgUP–T—magnesium

 30% n.s.—not significant.

 I—BBCH 30; II—BBCH 49/50. Mgf—Mg fertilizer doses; MgaG, MgaCR, and

 unit productivity:

Mg–R—magnesium

 recovery;

MgHI—magnesium

 harvest index; MgUA–G and

 grain and total, respectively;

 physiological

 efficiency.

Mg–PhE—magnesium

PFP–Mg—partial

 factor productivity

MgaT—magnesium

MgUA–T—magnesium

accumulation:

 grain and total, respectively;

 of fertilizer magnesium;

 MgAE—

accumulation:

 grain,

**Figure 2.** Magnesium accumulation by winter wheat in response to Mg fertilization systems and years. a, b, c, d, e, f, g, h, i, j, k, l, m Similar letters mean a lack of significance; differences using Tukey's test. Mgs and Mgf—soil and foliar Mg fertilization systems, respectively. \* Doses of applied Mg, kg ha−1; \*\* stages of Mg foliar fertilization to wheat: I—BBCH 30; II—BBCH 49/50.


**Table 2.** Nitrogen uptake by winter wheat at maturity and indices of nitrogen use efficiency.

a, b, c Similar letters mean a lack of significant differences using Tukey's test. \*\*\*, \*\*, and \* indicate significant differences at *p* < 0.001, *p* < 0.01, and *p* < 0.05, respectively; n.s. = not significant. I—BBCH 30; II—BBCH 49/50. Nf–nitrogen dose; NaG, NaCR, and NaT—nitrogen accumulation: grain, crop residues, and total, respectively; NHI—nitrogen harvest index; NUA–G, NUA–T—nitrogen accumulation: grain and total, respectively; NUP–G and NUP–T—nitrogen unit productivity: grain and total, respectively; PFP–N—partial factor productivity of fertilizer nitrogen; NAE—nitrogen apparent efficiency; N–R—nitrogen recovery; N–PhE—nitrogen physiological efficiency.

**Figure 3.** Effect of soil-applied magnesium to winter wheat on nitrogen accumulation in grain. a, b, c, d Similar letters mean a lack of significant differences using Tukey's test. Mgs—soil Mg fertilization system. \* Doses of applied Mg, kg ha<sup>−</sup>1.

Of the nine nitrogen use efficiency (NUE) indices studied, only three (i.e., nitrogen unit accumulation in grain (NUA–G), nitrogen unit productivity for grain (NUP–G), and total nitrogen unit productivity (NUP–T)) responded significantly to the Y × Mgs × Mgf interaction. The NUP–G was ultimately the most sensitive index (Figure 5). In 2013, its values showed no response to experimental factors, oscillating around 46 ± 0.8 kg grain kg-1 Na. In 2015, the NUP–G was significantly lower, on average, reaching only 37 ± 1.5. In 2014, the highest N productivity was found for the Mgs control. The highest NUP–G of 50 kg grain kg-1 Na was recorded on the plot with Mgf at the BBCH 49/50 stage. On plots

with soil-applied Mg, the NUP-G indices were slightly lower than for the Mgs control but at the same time significantly responded to the Mgf treatments. Despite that, the NUP–G did not show a significant impact on the yield (Table A2). Moreover, it was negatively correlated with Na–G, Na–CR, TN, and, as a rule, with NUA–G and NUA–T, and also with N–R. At the same time, this particular index was significantly and positively correlated with N-PhE (r = 0.98 \*\*\*).

**Figure 4.** Effect of foliar fertilization of winter wheat with magnesium on nitrogen accumulation in grain. a, b, c, d, e Similar letters mean a lack of significant differences using Tukey's test. Mgf—foliar Mg fertilization system. \* Stages of Mg foliar fertilization to wheat: I—BBCH 30; II—BBCH 49/50.

The classic NUE index, the partial factor productivity of fertilizer N (PFP–Nf), responded to the Mgs × Mgf interaction, and the nitrogen physiological efficiency (N-PhE) responded to the Y × Mgf interaction. The application of Mg significantly increased the productivity of Nf. The differences between the Mg absolute control plot and plots fertilized with Mg were significant. The strongest increase in PFP-Nf was recorded for Mgs50, which provided double-foliar fertilization with Mg. The highest variability in N–PhE indices was recorded in 2013 (Figure 6). Nitrogen utilization by wheat increased in response to Mg foliar fertilization, peaking when applied at BBCH 49/50. In the remaining two years, N–PhE showed significantly lower variability in response to Mgf, especially on plots with double Mg treatments. Two other NUE indices (i.e., nitrogen apparent efficiency (NAE) and nitrogen recovery (N–R)) responded significantly to the studied factors but did not interact with each other. The NAE indices were highly sensitive to the weather, having the highest values in 2013 (31% higher than in the dry year of 2015). A reverse trend was recorded for N–R, which was high in all years but exceeded 100% in 2015. The soil-applied Mg increased the values of both indices. The same effect was observed with Mg foliar application.

**Figure 6.** Effect of winter wheat foliar fertilization with magnesium on nitrogen physiological efficiency. a, b, c Similar letters mean a lack of significant differences using Tukey's test. Mgf—foliar Mg fertilization system. \* Stages of Mg foliar fertilization to wheat: I—BBCH30; II—BBCH 49/50.

The wheat yield showed the highest correlation with N–HI and then with NAE (Table A2). The yield formula based on NHI, despite a statistically proven linear model, can be represented as a quadratic function:

$$\text{GY} = 0.26\text{NHI} - 11.54 \text{ for } n = 36, \text{R}^2 = 0.82 \text{ and } p \le 0.01 \text{ (t ha}^{-1}\text{)}\tag{1}$$

$$\text{GY} = 0.016\text{NHI}^2 + 2.9\text{NHI} - 122.7\text{ for } n = 36, \text{R}^2 = 0.85, p \le 0.01 \text{ (t ha}^{-1}\text{)}\tag{2}$$

The second equation indicates that an N–HI of 92.4% would give a yield of 11.22 t ha<sup>−</sup>1. The maximum yield of 11.18 t ha−<sup>1</sup> was obtained when the NHI reached 88%. This effect was recorded in 2014 on the Mgs25 plot and Mgf at BBCH 49/50. N–HI was negatively correlated with the amount of N in crop residues and with NUA–G and NUA–T. The highest positive relationships were recorded with PFP–N (*r* = 0.91 \*\*\*) and NAE (*r* = 0.81 \*\*\*).

Magnesium fertilization, as previously documented, significantly affected its accumulation in wheat, but its total accumulation (MgaT) was sensitive to the interaction of experimental factors with years (Table A3). The most significant impact of MgaT was recorded for NaCR:

$$\text{CN}\_{\text{a}}\text{CR}=-4.16\text{Mg}\_{\text{a}}\text{T}+111.4 \text{ for } n=36,\text{R}^{2}=0.70 \text{ and } p \le 0.01 \text{ (kg ha}^{-1}\text{)}\tag{3}$$

The increase in MgaT decreased the N accumulated in crop residues. The response of the NUE indices to MgaT, as well as to MgaG as a major part of MgaT, was very specific. A decrease was noted for NUA–G, NUA–T, and N-R (Table A3). An increase was recorded for NUP–G, NUP–T, and N–PhE. PFP–N and NAE were significantly correlated with each other and deserve special attention. PFP–N responded positively but weakly to the amount of MgaG or MgaT. In contrast, NAE showed a highly positive, linear response to the MgaT (Figure A4):

$$\text{NAE} = 1.34 \text{Mg}\_{\text{a}} \text{T} + 8.93 \text{ for } n = 36, \text{R}^2 = 0.67 \text{ and } p \le 0.01 \text{ (grain kg kg}^{-1} \text{ Nf)} \tag{4}$$

At the same time, NAE was significantly correlated with both NUP indices but especially with NUP–T (*r* = 0.84 \*\*\*) (Table A2).

#### **3. Discussion**

#### *3.1. Magnesium Use Efficiency*

An increase in the yields of crop plants in response to the applied nutrient, regardless of the method of application, is the essence of the application of any fertilizer [33]. An assessment of wheat's response to Mg fertilization requires answers to three basic questions:


The net increase in the yield of winter wheat due to the different systems of Mg fertilization was significant, regardless of the course of the weather over the studied years. In general, the increase in the yield resulting from the single-stage foliar application of Mg was only slightly higher than that of soil-applied Mg (0.52 vs. 0.57 t ha<sup>−</sup>1). The increase in wheat yield as a result of Mg foliar fertilization increased up to 0.7 t ha−<sup>1</sup> but provided its double application at BBCH 30 and repeated at BBCH 49/50. The end of booting/beginning of heading is considered the optimal stage for wheat foliar Mg fertilization [26,27]. This stage is crucial for the number of grains per unit area (GD) [34]. In the presented case, the recorded increase in the yield resulted directly from the increase in the GD [32]. The best effect of Mg application on wheat, resulting in a yield gain of 0.92 t ha<sup>−</sup>1, was due to the interaction of both Mg fertilization systems. The course of weather during the growing season did not markedly change the trend of the wheat response to the tested Mg fertilization systems. On this basis and according to the data in the literature, three strategies for wheat fertilization with Mg during growth can be identified:


The first strategy was very efficient in years with some disturbances in the weather course during the spring growing season of winter wheat (Figure 1). It requires, however, a higher dose of the in-soil applied Mg and its frequent application to wheat foliage [14,19,20]. The yield-forming effect of the applied Mg can be explained both by the increase in the GD and the maintenance of the photosynthetic activity of leaves during the grain-filling period [24,25,32]. The second Mg fertilization strategy can be recommended in the years or regions of the world with favorable growth conditions for winter wheat. A sufficiently high yield increase can be achieved by applying relatively low in-soil Mg doses, provided a high solubility of fertilizer is used as well as one-stage foliar fertilization with Mg [19,26]. The third fertilization strategy of winter wheat with Mg is based on a double-stage foliar fertilization, regardless of the weather and soil conditions [27].

Mg foliar fertilization was superior to its soil application, but provided a double-stage application, as supported by the obtained data from its extreme efficiency. The values of the Mg recovery indices (Mg–R) on the main Mgf plot ranged from 100% to 300%. In contrast, the Mg recovery on the Mgs plots were in the range of 18–38% for the Mgs25, and 10–19% for the Mgs50 main plots. Moreover, the highest Mg–R indices, regardless of the method of Mg application, were recorded in 2014, the year with the highest grain yield. The extremely high Mg–R values of the Mgf plot were due to the higher Mg uptake by plants and its accumulation in vegetative wheat biomass. The increase in Mg uptake, averaged over treatments, was 16.6% higher compared to the absolute Mg control. It is well documented in the scientific literature that chlorophyll Mg, despite being a stable plant trait, is the main source of N for the growing seed/grain [35]. The obtained results suggest an extension of the grain-filling period (GFP), which resulted in the higher yield. This conclusion is confirmed by Ahnadi-Lahijani and Emam [36], who showed that the

higher the chlorophyll content in wheat leaves during GFP and the longer the leaf surface is kept green, the greater the increase in wheat yield.

#### *3.2. Impact of Magnesium Uptake on Nitrogen Management by Wheat*

The fourth question concerns the impact of the Mg fertilization system on N management, i.e., N uptake by wheat and its net utilization. The conducted study clearly showed that wheat treated with Mg, regardless of the method of its application, significantly increased the amount of N in wheat at harvest (TN). It should be emphasized that the N taken up by wheat in response to Mg application was mainly accumulated in the grain (Table 2). Both experimental factors contributed to this but without interaction between the systems. The soil-applied Mg increased the amount of N in wheat grain by 24 and 21 kg ha−<sup>1</sup> in 2014 and 2015. Assuming a protein concentration in the grain at a level of 13%, the corresponding yield increase would be at a level of 1.1 and 0.93 t ha<sup>−</sup>1. These values are almost equal to the yield increment as a result of the interaction of both mg fertilization systems (Figure 1). Thus, it can be concluded that regardless of weather conditions, the increase in yield is directly related to the efficiency of N utilized by wheat.

The strong relationship between the amount of extra N accumulated in wheat grain, as a result of the of Mg fertilization, was due to the critical role of both nutrients in photosynthesis. Nitrogen is the limiting component of Rubisco, the key plant enzyme responsible for CO2 fixation [24,25]. The influence of Mg on the activity of Rubisco increases in conditions of water shortage, which is often accompanied by elevated temperatures [36,37]. Rubisco activity increases during the GFP of wheat growth, as recently documented by Shao et al. [25]. This specific effect was probably observed in 2015 on the main plot fertilized with 50 kg Mg ha−<sup>1</sup> and double Mg fertilization. The stabilizing effect of the soil-applied Mg on the yield of crops is well documented for various biologically different plants such as maize and sugar beets [28–31]. The assumed stabilization results from a plant's accessibility to the readily available Mg, even from the beginning of growth [19,26]. In wheat, the critical period of yield formation starts with the beginning of the elongation phase [8,9]. However, *the critical window* for wheat grain density takes place during the booting and heading stages [8,38]. Foliar Mg fertilization at these stages results in a higher grain density in cereals and maize [27].

Out of the nine studied NUE indices, only four were significantly related to the yield of wheat. The highest relationship was found for the nitrogen harvest index (N–HI). This index is a rule treated as a conservative wheat trait [39]. The study clearly showed its significant dependence on the total amount of Mg in wheat at harvest. The effect of Mg on N–HI was indirect, i.e., it reduced the amount of N in wheat residues. This trend means a greater transfer of N during GFP from the vegetative parts of wheat to the growing grains. The proposed explanation confirms an earlier study by Potarzycki [40,41]. The author showed that foliar Mg applied to wheat at the beginning of the booting phase increased the remobilization of N from vegetative tissues to grain.

Nitrogen apparent efficiency (NAE) was the second-most important (*p* ≤ 0.01) NUE index, showing a significant relationship with the yield. It also depended on the total amount of Mg in the wheat biomass or grain (Figure A4). The positive relationship between this index and wheat grain yield indirectly indicates a higher yield from plots fertilized with Mg. Moreover, this index was inversely correlated with the amount of N accumulated in wheat residues but positively with N–HI, NUP–G, NUP–T and, finally, N–PhE. On the basis of the obtained relationships, it can be concluded that, regardless of the fertilization system, Mg had a strong, significant effect on N utilization by winter wheat (Figure A4). The positive effect of Mg on the productivity of Nf was confirmed by the increased productivity of applied fertilizer N, as confirmed by the response of the PFP–Nf index. Its values ranged from 45 kg grain kg−<sup>1</sup> Nf in 2013, an unfavorable year for wheat growth, to 58 kg grain kg−<sup>1</sup> Nf in 2014, the year with the highest yields. The highest PFP-Nf values for an Nf rate of 190 kg N ha−<sup>1</sup> are comparable to those presented by Szczepaniak et al. [42] for wheat fertilized with 160 kg N ha−1. The effective use of Nf by winter wheat through the use of

Mg is emphasized by the values of the nitrogen recovery index (N–R). It was very high on the Mg absolute control plot (NPK only), well above 80%. The use of Mg raised its values above 90%. Moreover, the impact of Mg on this index was very stable, regardless of the weather conditions during the growing season and the method of Mg application.

#### **4. Materials and Methods**

#### *4.1. Experimental Site*

A field experiment was carried out at Jarosławiec (52◦15 N, 17◦32 E, Poland) on soil originated from sandy loam, classified as Albic Luvisols (Neocambic) [43]. The content of organic matter (Corg) in a 0.0–0.3 m layer during the study ranged from 21 ± 0.1 to <sup>25</sup> ± 0.9 g kg−<sup>1</sup> soil (losses on ignition). Soil reaction (pH) was in the neutral range (1 M KCl). The content of available nutrients, measured before the application of fertilizers was, in general, good for P and sufficient for K and Mg. The amount of the mineral N (Nmin), determined in a 0.0–0.9 m layer, was high in the first two growing seasons and medium in the third (Table 3).


2013/2014 0–30 6.7 91.6 <sup>1</sup> <sup>H</sup> 168.0 M 30.2 M <sup>74</sup> 30–60 91.6 <sup>1</sup> <sup>H</sup> 153.6 M 24.7 M 2014/2015 0–30 6.6 87.2 <sup>1</sup> <sup>H</sup> 182.6 H 24.1 M <sup>57</sup> 30–60 95.9 <sup>1</sup> <sup>H</sup> 149.9 M 30.2 M

**Table 3.** Soil characteristics of the experimental plots during the 2012–2015 growing seasons.

<sup>1</sup> Egner–Riehm method; <sup>2</sup> Schachtschabel method; <sup>3</sup> classes of the available nutrient content: M—medium, H—high; <sup>4</sup> layer: 0–90 cm (measured in 0.01 M CaCl2).

#### *4.2. Weather Conditions*

The weather conditions were very variable in the consecutive growing seasons (Figure 7). The beginning of spring, with the exception of 2012/2013, favored the growth of wheat. In 2013, negative temperatures in the first two decades of March arrested plant growth. In all years of the study, temperatures during flowering and grain filling were within the ranges optimal for yield development. The sum of rainfall during the spring growing season was as follows: 2013—299.4, 2014—285.2, and 2015—265 mm. In 2015, a shortage of rainfall was revealed, which covered three main phases of wheat development, ranging from shooting to early flowering. The sum of rainfall in this period was 37.6 mm, while in 2013, it reached 88.8 mm, and in 2014, 92.6 mm.

#### *4.3. Experimental Design*

The field experiment was arranged as a two-factor split-plot design, replicated 4-fold:

	- a. Without application, i.e., Mgf control;
	- b. Applied at the BBCH 30 stage (I) (I–BBCH 30);
	- c. Applied at the BBCH 49/50 stage (II) (II–BBCH 59/50);
	- d. Applied at the BBCH 30/31 and BBCH 49/50 stages; double-stage application (I + II).

**Figure 7.** Weather conditions during the consecutive growing seasons.

Spring barley was the fore-crop for winter wheat. The *Tobak* wheat variety was sown annually on 20–25 September. The soil was fertilized with Mg in the form of Kieserite (MgSO4 · H2O), containing 25% MgO and 50% SO3. Kieserite was applied to the soil three weeks before wheat sowing. Foliar fertilization of wheat with Mg was carried out using Epsom salt (MgSO4 · H2O) containing 16% MgO and 37.5% SO3. The amounts of the applied nutrients are shown in Table 4. Sulfur applied together with the Mg fertilizer was balanced in the first dose of N. It was used as a mixture of ammonium sulfate and ammonium saltpeter (17.5% SO3). The first N dose of 80 kg ha−<sup>1</sup> was applied just before the beginning of the growing season in spring. The second dose of 50 kg ha−<sup>1</sup> was applied, and the third one of 60 kg ha−<sup>1</sup> at BBCH 45–47. Phosphorus at a rate of 30.1 kg P ha−<sup>1</sup> as triple superphosphate (46% P2O5) and K at a rate of 66.4 kg K ha−<sup>1</sup> as muriate of potash (KCl) were applied together with the soil Mg. The total area of a single plot was 30 m2, and the harvested area was 15 m2. Plant protection was conducted in accordance with the codex of good practice.


**Table 4.** Fertilization schedule.

#### *4.4. Plant Material Sampling and Analysis*

The plant material used for dry matter determination was collected at BBCH 89 from an area of 2.0 m2. The sampled material was then divided, depending on the wheat stage, into subsamples of grain (G) and crop residues (CRs) composed of leaves (LE), stems (ST), ears (EA), and chaffs (CH). The results are expressed on a dry weight basis.

The N content was determined in both parts of the plant, using the standard macro– Kjeldahl procedure. For determination of the Mg content, the plant sample was dried at 65 ◦C and then mineralized at 550 ◦C. The obtained ash was then dissolved in 33% HNO3. The concentration of Mg was determined using atomic absorption spectrometry—flame type. The results are expressed on a dry matter basis.

#### *4.5. Parameters and Indices of Nitrogen Use Efficiency*

The equations used to calculate the amount of N in the grain or crop residues and the N use efficiency (NUE) indices are presented below. The corresponding Mg indices were calculated in the same way.

1. Nitrogen accumulation in wheat grain, *NaG:*

$$N\_{\rm d}G = GY \times N \text{ kg ha}^{-1}$$

2. Nitrogen accumulation in crop residues, *Nr:*

$$N\_a \text{CR}\_s = \text{CR}\_s \times N \text{ kg ha}^{-1}$$

3. Total accumulation of nitrogen in wheat biomass, TN:

$$TN = N\_{\rm a}G + N\_{\rm a}CR\_{\rm s} \text{ kg ha}^{-1}$$

4. Nitrogen harvest index, *NHI:*

$$NHI = \frac{\text{N}\_aG}{TN} \times 100\%$$

5. Nitrogen unit accumulation in grain, *NUA-G:*

$$NIAA = \frac{N\_aG}{GY} \text{ kg } N \times t^{-1}$$

6. Nitrogen unit accumulation in total wheat biomass, *NUA-T:*

$$Nulia = \frac{TN}{GY} \text{ kg } N \times t^{-1}$$

7. Nitrogen unit productivity—grain, *NUP*−*G:*

$$NUP - G = \frac{GY \times 1000}{N\_aG} \text{ kg } grain \times \text{kg}^{-1} \text{ N}$$

8. Nitrogen unit productivity–total, *NUP-T:*

$$NUP - T = \frac{GY \times 1000}{TN} \text{ kg } grain \times \text{kg}^{-1} \text{ N}$$

9. Partial factor productivity of fertilizer *N*, *PFP-N:*

$$PFP - N = \frac{GY \times 1000}{N\_f} \text{ kg grain kg}^{-1} \text{ N}\_f$$

10. Nitrogen agronomic efficiency, *NAE*:

$$NAE = \frac{GY\_f \times 1000 - GY\_{Nc} \times 1000}{N\_{if}} \text{ kg } grain \times \text{kg}^{-1} \text{ N}\_f$$

11. Nitrogen recovery, *N–R:*

$$N - R = \frac{TN\_{N\_f} - TN\_{N\_c}}{N\_f} \times 100\%$$

12. Nitrogen physiological efficiency, *N-PhE:*

$$N - PhE = \frac{GY\_f \times 1000 - GY\_{Nc} \times 1000}{TN\_{N\_f} - TN\_{N\_c}} \text{ kg } grain \text{ kg}^{-1} \text{ N}$$

where:

*NHI*—nitrogen harvest index, %;

*CRs*—crop residues, t ha<sup>−</sup>1;

*N*—*N* content in grain or crop residues, %, g kg−<sup>1</sup> DW;

*Nf*—plots fertilized with N;

*Nc*—nitrogen control;

*Nf*—treatment fertilized with nitrogen.

#### *4.6. Statistical Analysis*

The collected data were subjected to an analysis of variance using STATISTICA® 13 (StatSoft, Inc., Krakow, Poland, 2013). The distribution of the data (normality) was checked using the Shapiro–Wilk test. The homogeneity of variance was checked by the Bartlett test. Means were separated by honest significant difference (HSD) using Tukey's method, where the *F*-test indicated significant factorial effects at a level of *p* < 0.05. To determine the wheat grain yield, stepwise regression was used to define the optimal set of wheat components. In the computational procedure, a consecutive variable was removed from the multiple regressions in a step-by-step manner. The best regression model was chosen based on the highest F–value for the model and the significance of all variables.

#### **5. Conclusions**

Magnesium applied to wheat resulted in a significant yield gain with respect to the effect of NPK, treated as the Mg control. The method of application was of secondary importance. A slightly higher increase in the yield was caused by foliar fertilization, preferably performed at the booting/heading stages of wheat growth. The yield gain, as a result of foliar fertilization with Mg fertilization, ranged from 0.6 to 0.9 t ha<sup>−</sup>1, while in the soil, its application resulted in a yield gain in the range of 0.4–0.7 t ha<sup>−</sup>1. Magnesium accumulation by wheat, averaged for the fertilization treatments, increased by 17% compared to the NPK plot. The recovery of foliar-applied Mg was multiple in relation to the applied dose. The recovery of soil-applied Mg depended on the dose, ranging from 18 to 38% on the 25 kg Mg ha−<sup>1</sup> main plot and from 10 to 19% on the 50 kg Mg ha−<sup>1</sup> plot. The main effect of wheat fertilization with Mg was its impact on the uptake and then partitioning of the accumulated N in wheat biomass between the grain and crop residues. The amount of extra accumulated N was effectively converted into grain yield. This process manifested itself in an increase in the value of the nitrogen harvest index and in a decrease in the N content in crop residues.

The main action of Mg, regardless of the weather and the method of its application, was an increase in the productivity of fertilizer nitrogen, which was confirmed by a set of various tested indices such as NUP–G, NUP–T, N–PhE, and PFP–Nf. The yield-forming effect of the applied Mg fertilizer to winter wheat was revealed by the increased N transfer to the grain, which indicates its impact on the nitrogen utilization efficiency.

**Author Contributions:** Conceptualization, J.P. and W.G; methodology, J.P. and W.G.; software, J.P. and W.Sz.; validation, J.P and W.G.; formal analysis, J.P, W.G. and W.S.; investigation, J.P.; resources, J.P and W.S.; data curation, J.P.; writing—original draft preparation, J.P and W.G.; writing—review and editing, J.P and W.G; visualization, J.P. and W.G.; supervision, W.G.; project administration, J.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Table A1.** Matrix of the correlation indices for magnesium accumulation, efficiency indices, and yield, *n* = 36.


\*\*\*, \*\*, and \* indicate significant differences at *p* < 0.001, *p* < 0.01, and *p* < 0.05, respectively; n.s.—not significant. MgaG, MgaCR, and MgaT—magnesium accumulation: grain, crop residues, and total, respectively; Mg–HI—magnesium harvest index; MgUA-G and MgUA-T—magnesium accumulation: grain and total, respectively; MgUP–G and MgUP–T—magnesium unit productivity: grain and total, respectively; PFP–Mg—partial factor productivity of fertilizer magnesium; MgAE—magnesium apparent efficiency; Mg–R—magnesium recovery; Mg–PhE—magnesium physiological efficiency.

**Table A2.** Matrix of the correlation indices for nitrogen accumulation, efficiency indices, and yield, *n* = 36.


\*\*\*, \*\*, and \* indicate significant differences at *p* < 0.001, *p* < 0.01, and *p* < 0.05, respectively; n.s.—not significant. <sup>1</sup> I—BBCH 30; II—BBCH 49/50. NaG, NaCR, and NaT—nitrogen accumulation: grain, crop residues, and total, respectively; N–HI—nitrogen harvest index; NUA-G and NUA-T—nitrogen accumulation: grain and total, respectively; NUP–G and NUP–T—nitrogen unit productivity: grain and total, respectively; PFP–N—partial factor productivity of fertilizer nitrogen; NAE—nitrogen apparent efficiency; N–R—nitrogen recovery; N–PhE—nitrogen physiological efficiency.

**Table A3.** Correlation matrix of magnesium accumulation and indices of nitrogen use efficiency.


\*\*\*, \*\*, and \* indicate significant differences at *p* < 0.001, *p* < 0.01, and *p* < 0.05, respectively; n.s.—not significant. MgaG, MgaCR, and MgaT—magnesium accumulation: grain, crop residues, and total, respectively; NUA–G and NUA–T—nitrogen accumulation: grain and total, respectively; NUP–G and NUP–T—nitrogen unit productivity: grain and total, respectively; PFP–N—partial factor productivity of fertilizer nitrogen; NAE—nitrogen apparent efficiency; N–R—nitrogen recovery; N–PhE—nitrogen physiological efficiency.

**Appendix B**

**Figure A3.** General course of magnesium recovery in consecutive years of study.

**Figure A4.** Effect of magnesium total accumulation in winter wheat on indices of nitrogen utilization.

Legend: NUP-G and NUP-T—nitrogen unit productivity: grain and total, respectively; N-PhE—nitrogen physiological efficiency.

#### **References**


## *Article* **Impact of Foliar Fertilization on Growth, Flowering, and Corms Production of Five** *Gladiolus* **Varieties**

**Endre Kentelky \* and Zsolt Szekely-Varga**

Department of Horticulture, Faculty of Technical and Human Sciences, Sapientia Hungarian University of Transylvania, Sighis,oarei 1/C, 540485 Targu Mures, Romania; szekelyvarga.zsolt@gmail.com

**\*** Correspondence: kentelky@ms.sapientia.ro

**Abstract:** Degraded and salt affected soils are appearing more often in cultivated areas. These specific problems could reduce nutrient uptake, which can result in quality and yield loss of the cultivated plants. In order to cope with this pedo-climatic condition growers are applying fertilizers; however, due to inadequate application, soil degradation will continue. Five *Gladiolus* varieties were subjected to foliar fertilization treatments to assess the effect on the plant's growth parameters, vase durability and daughter corm production. Our results indicate that plants treated with foliar fertilization show significant increase in the measured parameters, flower stem length, vase durability and daughter corm production. In conclusion, our study suggests that application of foliar fertilization can increase *Gladiolus* plants decoration and propagation, even with a smaller footprint on nature.

**Keywords:** corms; foliar fertilization; *Gladiolus*; vase durability

**Citation:** Kentelky, E.; Szekely-Varga, Z. Impact of Foliar Fertilization on Growth, Flowering, and Corms Production of Five *Gladiolus* Varieties. *Plants* **2021**, *10*, 1963. https:// doi.org/10.3390/plants10091963

Academic Editors: Jim Moir, Lukas Hlisnikovsky, Xinhua He and Przemysław Barłóg

Received: 24 August 2021 Accepted: 16 September 2021 Published: 20 September 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/).

#### **1. Introduction**

Currently in the world 20% of the cultivated land is degraded and salt affected, which is affecting nutrient uptake and resulting in quality and yield reduction of the cultivated plants. More importantly, these factors are contributing to crop losses worldwide [1,2]. According to several studies, in order to cope with these conditions and increase production, chemical fertilizers are applied, but due to inappropriate application, soil degradation (acidification; salinization; nutrient imbalance; and irregular accumulation of nitrogen, phosphorus and potassium) occurs in the cultivated lands [3,4].

Fertilization can be divided into two main methods: root and foliar fertilization [4]. Foliar fertilization can be absorbed directly through the leaves and can be transported more quickly and efficiently to the other plant organs compared to root fertilization [5,6]. Moreover, foliar fertilization can be sprayed at optimum times and concentrations, according to the requirement of different plants, at different growth stages. This type of fertilization can be more suitable to the plant's needs, in contrast to root fertilization [4,7,8].

*Gladiolus* genus is a perennial, monocotyledonous, geophyte, semi-rustic ornamental plant and includes about 260 species [8,9]. The *Gladiolus* originates in Mediterranean Europe, Asia, and South and Tropical Africa [10–13]. It can be found as an ornamental garden plant and also as cut flowers used for bouquets and arrangements [13]. These majestic plants are found in different shapes, colours, and sizes, and can be cultivated almost everywhere, but should be considered for regions where spring and summer conditions are favourable [14].

The main aim of the present research was to test the responses to foliar fertilization of five highly cultivated *Gladiolus* varieties. 'Black Beauty', 'Green Star', 'Nova Lux', 'Zizane', and 'Frizzled Coral Lace' were analysed in the study. The effect of three foliar fertilizers— Fitofolis, Bionat Plus and Cropmax—and the mixture of the three on the flower quality and the amount of new daughter corms produced by the selected gladioli was investigated. We expected to establish whether any of the five varieties was more suitable for cultivation in the climate conditions of the Carpathian Basin.

#### **2. Results**

#### *2.1. Plant Growth*

Considering the shoot growth, almost all *Gladiolus* varieties showed small increases in shoot length (Figure 1). However, in the case of 'Green Star' (Figure 1a) the Fitofolis and the F + B + C (the mixture of Fitofolis, Bionat Plus and Cropmax) treatments influenced the shoot growth significantly compared to the control treatments. The effect of the treatment was evident, especially for the 'Black Beauty' gladioli, which showed an increase in growth in all treatments with respect to the controls, with ~10 cm (Figure 1b).

**Figure 1.** Effect of foliar fertilization on the shoot growth parameters in *Gladiolus* varieties: 'Green Star' (**a**), 'Black Beauty' (**b**), 'Nova Lux' (**c**), 'Zizane' (**d**), and 'Frizzled Coral Lace' (**e**). Plants shoot growth under control conditions, in the presence of the indicated foliar fertilization: Fitofolis, Bionat Plus, Cropmax and the mixture of Fitofolis–Bionat Plus–Cropmax (F + B + C). Shoot growth was measured in all plants just before starting the treatments (13 May), and before the harvesting of the inflorescences (9 August). Bars represent the means ± SE (*n* = 10). Different letters above the bars indicate significant differences between the treatments, according to Tukey test (α = 0.05).

Regarding 'Nova Lux' (Figure 1c), only for the Bionat Plus and F + B + C treatments were significant differences reported; however, all treatments showed small increases (between 5–10 cm) compared to the control plants. An increase in mean shoot growth was also reported in 'Zizane' for the Fitofolis treatment (Figure 1d); for 'Frizzled Coral Lace' (Figure 1e), an increase of approximately 4 cm, was observed for the Fitofolis and F + B + C treatments.

Percentage increases in shoot growth of *Gladiolus* varieties influenced by the foliar fertilization were as follows: for 'Green Star', 'Zizane', and 'Frizzled Coral Lace' the highest percentage increase was recorded with Fitofolis (3.92%, 13.58%, and 15.54%) compared to the control. In contrast, the smallest percentage increases for the same three varieties were observed with Cropmax (0.02%, 0.61%, and 4.86%). The 'Black Beauty' reported the highest increase with Bionat Plus (23.81%) and the smallest percentage increase at F+B+C (16.27%). For 'Nova Lux', a 30.6% increase was found with Bionat Plus compared to only a 9.8% increase with Cropmax fertilization.

The results of the present study indicated that a variety-specific response exists to foliar fertilization; in most cases the treatments significantly, positively influenced the flower stem growth (Figure 2) compared to the controls.

**Figure 2.** Effect of foliar fertilization on flower stem growth parameters in *Gladiolus* varieties: 'Green Star' (**a**), 'Black Beauty' (**b**), 'Nova Lux' (**c**), 'Zizane' (**d**), and 'Frizzled Coral Lace' (**e**). Plant flower stem growth shown under control conditions and in the presence of the indicated foliar fertilization: Fitofolis, Bionat Plus, Cropmax and the mixture of Fitofolis–Bionat Plus–Cropmax (F + B + C). Flower stem growth was measured in all plants just before starting the treatments (13 May), and before the harvesting of the inflorescences (9 August). Bars represent the means ± SE (*n* = 10). Different letters above the bars indicate significant differences between the treatments, according to Tukey test (α = 0.05).

'Green Star' (Figure 2a) showed significant growth of the flower stem under the Bionat Plus fertilization, with a 19 cm increase; the other fertilization treatments influenced the flower stem growth, but at smaller percentages.

All types of foliar fertilization increased flower stem length for 'Black Beauty' (Figure 2b), compared to the control plants, in some cases by almost 20 cm. Regarding 'Nova Lux', 'Zizane' and 'Frizzled Coral Lace' *Gladiolus* (Figure 2c–e), similar results were found for all four fertilization treatments. For these three *Gladiolus* varieties, growth increase was between 5 and 30 cm depending on the treatment.

Comparing the stem growth between varieties in all treatments, the highest increase was observed in 'Nova Lux'—almost 30 cm; the least growth was in 'Frizzled Coral Lace'. This could be explained by the variety morphology.

When comparing the flower stem growth to the control, the greatest percentage increases in 'Black Beauty', 'Frizzled Coral Lace', and 'Zizane' were recorded with Cropmax (27.96%, 19.76%, and 34.35%), in 'Nova Lux' and 'Green Star' with Bionat Plus with 37.91% and 25.07%, respectively. The lowest percentage increases were observed with Fitofolis ('Nova Lux'–11.82%, 'Frizzled Coral Lace'–9.18%, and 'Zizane'–18.02%) and F + B + C ('Black Beauty'–11.27% and 'Green Star'–2.66%) fertilizers.

#### *2.2. Vase Durability*

Under our experimental conditions, significant differences between the varieties and the treatments were observed in the vase durability of the *Gladiolus* (Figure 3). When comparing the varieties, it could be concluded that 'Green Star', 'Black Beauty' and 'Nova Lux' had the longest vase durability, whereas 'Zizane' and 'Frizzled Coral Lace' had shorter vase durability, with fewer points.

**Figure 3.** Effect of foliar fertilization on vase durability of *Gladiolus* varieties: 'Green Star', 'Black Beauty', 'Nova Lux', 'Zizane', and 'Frizzled Coral Lace'. Vase durability of floral stems produced under control conditions and in the presence of the indicated foliar fertilization: Fitofolis, Bionat Plus, Cropmax and the mixture of Fitofolis–Bionat Plus–Cropmax (F+B+C). Bars represent the means ± SE (*n* = 5). Different lowercase letters above the bars indicate significant differences between the five varieties for each foliar fertilization, and different uppercase letters indicate significant differences between treatments, according to Tukey test (α = 0.05).

Foliar fertilizations influenced vase durability in a positive way, although with small differences. Almost all types of fertilization affected the gladioli durability, supporting the general conclusion of the individual experiments that all varieties responded to fertilization. The average vase durability was 7.45 days.

#### *2.3. Daughter Corms Production*

It was concluded that foliar fertilization had a positive effect on the increase in number of daughter corms production.

Under our experimental conditions 'Green Star' and 'Black Beauty' showed significant increases from all types of foliar fertilization, compared to the controls (Figure 4a,b).

**Figure 4.** Effect of foliar fertilization on increment in daughter corms in *Gladiolus* varieties: 'Green Star' (**a**), 'Black Beauty' (**b**), 'Nova Lux' (**c**), 'Zizane' (**d**), and 'Frizzled Coral Lace' (**e**). Increase in daughter corms under control conditions and in the presence of the indicated foliar fertilization: Fitofolis, Bionat Plus, Cropmax and the mixture of Fitofolis–Bionat Plus–Cropmax (F + B + C). Bars represent the means ± SE (*n* = 10). Different letters above the bars indicate significant differences between the treatments, according to Tukey test (α = 0.05).

For 'Nova Lux' (Figure 4c), there was no effect from the Fitofolis treatment. In contrast, the Bionat Plus, Cropmax and F + B + C treatments increased the corms production. Increases in the number of corms were also observed in 'Zizane' (Figure 4d) with the Bionat Plus and F + B + C treatments.

'Frizzled Coral Lace' (Figure 4e) showed a high increase from the F + B + C treatment, as daughter corms production was five times higher compared to the controls. This result could be influenced also by the variety: comparing the five different *Gladiolus* varieties, the greatest daughter corm production occurred in this variety.

In the cases of 'Green Star' (60.97%), 'Black Beauty' (92%), 'Nova Lux' (77.14%), and 'Zizane' (63.63%) the smallest percentage increase in relation to the controls were reported from Fitofolis. For 'Frizzled Coral Lace', the Cropmax fertilizer recorded the smallest increase in percentage with a 228.12% compared to control. The greatest increases were observed with Bionat Plus ('Green Star'–143.9% and 'Black Beauty'–184%) and the mixture of the three foliar fertilizers ('Nova Lux'–185.71%, 'Zizane'–281.81%, and 'Frizzled Coral Lace'–714.06%).

#### **3. Discussion**

The results of this experiment show proper foliar fertilization can support and influence the growth, vase durability and daughter corms production of some *Gladiolus* varieties. Saima et al. [15] found that application of foliar spray affected flower production and it was the best method to getting maximum flower production in *Gladiolus*. Furthermore, it has a potential effect on the nutrient uptake and on the stimulation of growth parameters and flowering characteristics [5,16]. Foliar fertilization increases micronutrient uptake and physiological and biochemical indexes [17,18]. Many studies suggest that foliar fertilization may help to stimulate the uptake of soil applied fertilizers, which could provide a solution to salt accumulation in the soil [4].

Foliar fertilization was more effective and significantly enhanced the shoot and flower stem growth compared to the control plants. Similar to our study, some researchers reported that foliar fertilization promoted the flower stem growth to the maximum levels in gladioli, which could have a constructive role in the development of the flowers [15,19,20]. Similar findings have been described where the administration of foliar fertilization treatments influenced the *Calendula* inflorescence yield, but not the chlorophyll parameters, where no significant differences were observed between the treatments [21,22].

The data obtained clearly show that foliar fertilization can affect shoot growth in a positive way. Furthermore, the Fitofolis fertilizer obtained the best results compared to the control, which in some cases increased the growth up to 5 cm. The mixture of the three fertilizers (F + B + C) influenced shoot growth of gladioli in a positive way. In some varieties increases of 3 cm were shown compared to treatments with only Cropmax or Bionat Plus.

Like the shoot growth parameters, flower stem growth was influenced in a positive way by the foliar fertilization in all five varieties. Generally, the highest increases were observed in the plants fertilized with Bionat Plus, followed by Cropmax and Fitofolis. The mixture of foliar fertilizers in this case did not record as high an increase compared to the other three treatments. The macronutrients (N, P and K) are known to have effect on plant growth [23]. Nitrogen, phosphorus and potassium influenced the shoot growth and the flower stem length in a positive way. NPK used at an optimal dose can supplement sufficient nutrient uptake, which foster conditions for plants growth and development [24]. In some studies, it was also reported that B (boron) could increase–stimulate nutrient uptake, maintaining cell integrity and intensify respiration rate, which could promote growth and flower development [25–27].

Vase durability of *Gladiolus* is one of the most important considerations for consumers. Foliar fertilizer effects on vase durability have been reported on *Rosa* [28], *Lilium* [29], *Anthurium andreanum* [30] and *Gladiolus* [31,32]. In the present experiment, vase durability of the five gladioli varieties was improved compared to the control in almost all treatments. The longest vase durability was obtained under the Bionat Plus treatment, and the longest vase durability among the five varieties was observed for 'Green Star'. *Gladiolus* fading or wilting are important signalling factors of senescence [33]. Calcium (Ca) has an important role in regulating the senescence in gladioli cut flowers [34]. Ca increases membrane

stability and reduces the level of reactive oxygen species, which could delay senescence in *Gladiolus* cut flowers [35]. However, in a study conducted by Dhakal et al. [31] it was concluded that phosphorus could also improve vase durability of gladioli cut flowers.

Daughter corm production is an important part of the gladioli propagation; our study results clearly indicate foliar fertilization has an important role in this sequence of the cultivation. Previous reports have also shown an increase in daughter corms production under foliar fertilization treatments [16,36–38]. Under our experimental conditions the highest increase was recorded in 'Frizzled Coral Lace' compared to the other four *Gladiolus varieties*. The mixture foliar fertilization (F + B + C) improved the daughter corm production. This behaviour could be explained also with the variety characteristics, where previous results reported 'Frizzled Coral Lace' has a high yield of daughter corms. Daughter corm production could be influenced by nitrogen dose [39,40], and some studies have shown that daughter corm production is also influenced by applying a higher potassium dose [15,16,41].

#### **4. Materials and Methods**

#### *4.1. Experimental Site and Plant Material*

Open field experiments were conducted between April and September 2018 at the Sapientia Hungarian University of Transylvania, Târgu Mures, (46◦31 17" N 24◦35 54" E). The gladioli corms were obtained from Sieberz Garden Centre (Gödöll˝o, Hungary) and planted in five rows/block, each row containing 10 gladioli corms, with sizes of 12–14 cm in circumference. According to the soil analysis and of the analysis of its profile we can state that the type of soil at the experiment location is gley chernozem, carbonated in depth and clayish in the alluvial deposits (Epiaquic Hapludalfs) (Table 1).


**Table 1.** Planted soil proprieties.

The average temperature during the experiment was 17.99 ◦C, the minimum was recorded in April (15.4 ◦C), and the maximum temperature in August (21.83 ◦C) (Figure 5). From Figure 5, it can be concluded that the average precipitation amount was 54.83 mm during the experimental months. The minimum precipitation was recorded in April at 15.40 mm, and the maximum in June was 129.40 mm. The precipitation and temperature data were collected using Delta–T devices WS–GP2 Automatic Weather Station (Delta-T Devices Ltd., Burwell, UK).

Morphological description of the five selected *Gladiolus* varieties:


*Gladiolus* corms were planted on 18 April 2018 with a row length of 25 cm and 15 cm between the plants. The plant growth was already observed at the end of the planting month.

**Figure 5.** Meteorological conditions, precipitation and temperature during the field experiment (April–September 2018).

#### *4.2. Application of the Foliar Fertilization*

On 13 May, two weeks after sprouting, first shoot measurements and the first foliar fertilization were made, according to the experimental design: A–Control, B–Fitofolis (Chemtech, Târgu Mures, , Romania), C–Bionat Plus (Panetone, Timis,oara, Romania), D– Cropmax (Blondy, Târgu Mures, , Romania) and E–the mixture of the three foliar fertilizers (first it was fertilized with Fitofolis, the second fertilization was made with Bionat Plus, the third with Cropmax, and the last one with the mixture of the three fertilizers in 1:1:1 proportion) (Table 2).

**Table 2.** Planting design: A: Control; B: Fitofolis; C: Bionat Plus; D: Cropmax; E: The mixture of Fitofolis, Bionat Plus and Cropmax (F + B + C).


The used foliar fertilizers content:


The application of the foliar fertilizers was done with a hand sprayer; for each product a 2% solution was prepared. To prevent the fertilization from getting into the wrong row, we placed a plastic film between the rows to protect them from the other treatments.

On 2 June the second foliar fertilization was applied; the only difference was that the gladioli on bed E were sprayed with the Bionat Plus. The third fertilization was made on 20 June, for the last (E) gladioli bed, Cropmax foliar fertilizer was applied.

The last fertilization was done on 5 July; E bed was fertilized with the mixture of the three foliar fertilizations (Fitofolis, Bionat Plus, Cropmax) in 1:1:1 proportions. The first flower buds appeared on 13 July. We measured the flower stem length of each plant. The last shoots measurements were made before harvesting the five *Gladiolus* varieties (9 August).

#### *4.3. Vase Durability*

When almost all gladioli started flowering (11 August) we randomly harvested five flowered stems, at a cut distance of 5 cm above the soil, from each treatment/*Gladiolus* variety. The vase durability was studied for seven days, and the gladioli cut flowers were kept under the same conditions: at room temperature, in clean-fresh water, and monitored and noted every three hours.

The classification criteria for the vase durability were determined as follows:


The above was used to determine the maximum value of vase durability.

#### *4.4. Corms Propagation*

At the end of August, all inflorescences were harvested from the plants; however, we left two leaves on each *Gladiolus* plant, thus contributing to the growth of the corms. In this way most of the photoassimilation is destined for the growth of the daughter corms. By the end of September, when the plants had stopped nutrient uptake and the remaining leaves withered, we gathered corms of five *Gladiolus* varieties. We dried them and cleaned the remaining soil of the corms.

#### *4.5. Statistical Analysis*

All data were tested for normality of errors and homogeneity of variance. As all data were normally distributed, ANOVA followed by Tukey test were used to compare variances. The significance of the differences between the treatments was tested by applying two-way ANOVA, at a confidence level if 95%. When the ANOVA null hypothesis was rejected, Tukey's Post hoc test was carried out to establish the statistically significant differences at *p* < 0.05.

#### **5. Conclusions**

Gladioli growers strive to achieve the greatest possible stem length, vase durability and daughter corm production. The present study provides new experimental data on the responses of five *Gladiolus* varieties to foliar fertilization. For flower stem length and vase durability increase we recommend the use of Bionat Plus fertilizer, and for cut flowers the 'Green star' variety, which in our experiments had the best increases. The highest yield of daughter corm production was observed with the mixture of the three foliar fertilizations (F + B + C). These approaches/results will help to enhance the production of *Gladiolus*, with a smaller footprint on the degradation and salinization of the cultivated lands. Due to the frequent changes in cultivated *Gladiolus* varieties, we propose repeating this experimental design in a few years to examine the effect of foliar fertilization on new and requested varieties on the market.

**Author Contributions:** Conceptualization, E.K.; methodology, E.K.; formal analysis, E.K. and Z.S.-V.; writing—original draft preparation, E.K. and Z.S.-V.; writing—review and editing, E.K. and Z.S.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to express their special thanks to Cecilia Kurko, Kázmér Kovács and László Ferencz for the help with this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Petr Škarpa 1, Dominika Mikušová 1, Jiˇrí Antošovský 1, Milan Kuˇcera <sup>2</sup> and Pavel Ryant 1,\***


**Abstract:** Fertilizer coating can increase the efficiency of N fertilizers and reduce their negative impact on the environment. This may be achieved by the utilization of biodegradable natural coating materials instead of polyurethane-based polymers. The aim of this study was to detect the effect of calcium ammonium nitrate (CAN) fertilizer coated with modified conventional polyurethane enhanced with vegetable oils on the yield and quality of *Brassica napus* L. compared to CAN fertilizer with a vegetable oil-based polymer and to assess the risks of nitrogen loss. Three types of treatments were tested for both coated fertilizers: divided application (CAN, coated CAN), a single application of coated CAN, and a single application of CAN with coated CAN (1:2). A single application of coated CAN with both types of coating in the growth stage of the 9th true leaf significantly increased the yield, the thousand seed weight, and oil production compared to the uncoated CAN. The potential of using coated CAN may be seen in a slow nitrogen release ensuring the nitrogen demand for rapeseed plants throughout vegetation and eliminating the risk of its loss. The increased potential of NH4 + volatilization and NO3 − leaching were determined using the uncoated CAN fertilizer compared to the coated variants. Oil-based polymer coatings on CAN fertilizer can be considered as an adequate replacement for partially modified conventional polyurethane.

**Keywords:** control release fertilizer; yield; oiliness; nitrogen losses; nitrate leaching

#### **1. Introduction**

With the world's exponential population growth and diminishing of arable lands, the agriculture industry has faced a great challenge of crop and food resources for the past decades [1,2]. Predictions are that the earth's population could approach 9.5 billion by 2050, which may result in an almost double increase in food demand and crop production. In one specific example, cereal production is expected to increase from 940 million tons to 3 billion tons a year [3,4]. Satisfying increasing grain yield demands has been achieved by enhancing the use of mineral fertilizers to cropland soil. However, the excessive application of fertilizers presents one of the main sources of polluting soil (heavy metals), water (nitrates leaching into groundwater), and air environments (emission of greenhouse gases), which could be a threat to human health [5,6].

Nitrogen occupies a unique position among essential plant nutrients. Nitrogen and water availability are considered the two major limiting factors in plant growth and development of metabolic processes—nutrient distribution, photosynthesis, biomass, and ultimately yield building [7–9]. The deficiency of nitrogen strongly decreases chlorophyll content, enzymatic activity, photosynthesis, respiration rate, and yield of crops [10]. Nitrogen can be directly absorbed by plant roots in inorganic forms (mineral nitrogen) as ammonium (NH4 +) and nitrate (NO3 −). These forms are the key components of nitrogen fertilizers such as ammonium nitrate (AN) and urea included in the two most widespread nitrogen fertilizers [11].

**Citation:** Škarpa, P.; Mikušová, D.; Antošovský, J.; Kuˇcera, M.; Ryant, P. Oil-Based Polymer Coatings on CAN Fertilizer in Oilseed Rape (*Brassica napus* L.) Nutrition. *Plants* **2021**, *10*, 1605. https://doi.org/ 10.3390/plants10081605

Academic Editors: Przemysław Barłóg, Jim Moir, Lukas Hlisnikovsky and Xinhua He

Received: 26 June 2021 Accepted: 3 August 2021 Published: 5 August 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/).

According to FAO, world demand of nitrogen (N), phosphate (P2O5), and potash (K2O) fertilizers were reported to be in total up to 184.0 mil tons in 2015. The forecast for 2022 could be up to 200.9 mil tons (nitrogen fertilizers present up to 111.6 mil tons) [12,13]. Despite the fact that the use of nitrogen fertilizers plays an essential role in meeting the demand for crop production, nitrogen use efficiency (NUE) is relatively low due to their excessive use (in general between 25–50%) and often leads to losses of redundant nitrogen from agroecosystems [14]. Nitrogen losses due to gaseous emissions of ammonia (NH3), nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) along with leaching and runoff in the forms of ammonium (NH4 +), nitrite (NO2 −), and nitrate (NO3 −) present an alarming threat to the environment [15].

Leakage prevention of nitrates may present one of the greatest environmental challenges in terms of nitrogen fertilizer use. Nitrogen losses, caused by NO3 − leaching from soil into water, represent a loss of soil fertility and also pose a threat to the environment and subsequently to human health [5,16]. Increased nitrate levels present in drinking water create a risk of cancer, heart disease, and methemoglobinemia in babies [17]. According to calculations by Grizzetti et al. [17], up to 50% of the European population live in areas with a concentration of nitrates in water exceeding 25 mg·L<sup>−</sup>1, and up to 20% live in areas where nitrates exceed the recommended level of 50 mg·L<sup>−</sup>1. As already mentioned, nitrate coming from agriculture is the most common contaminant in the world's groundwater aquifers [18]. In the European Union, up to 38% of water bodies are significantly under pressure from agricultural pollution [19].

The application of controlled-release fertilizers (CRFs) is one way to improve nutrient use efficiency, reduce nitrogen loss, and contribute to minimizing environmental pollution, providing a better compromise among soil fertility, yield, and grain quality [20–22]. CRFs prove the potential to decrease the fertilizer application rate by 20% or 30% of the recommended value to achieve the same yield [23]. According to Trenkel [23] and Shaviv [24], CRFs can be defined as coated or encapsulated fertilizers by water-insoluble, semipermeable, or impermeable-with-pores materials, for which the factors determining the rate, pattern, and the duration of release have been known and regulated during the fabrication. Coating materials can be divided into two categories—inorganic materials (e.g., sulfur, bentonite, phosphogypsum) and organic polymers consisting of synthetic polymers derived from petroleum-based derivates (polyurethane, polyethylene, alkyd resin, etc.) or natural polymers (e.g., vegetable oil, starch, chitosan, cellulose) [23,25]. One of the most effective methods of preparing CRFs and thus the reduction of nutrient losses is by coating the surface of fertilizer with polyurethane materials. However, these coating materials are commonly linked with high costs and come from non-renewable petrochemical productions [26,27]. Furthermore, studies have shown that the residue of polyurethane shells in soils is difficult to degrade and may cause potential environmental risk [28]. This is one of the reasons why the agricultural industry has been searching for cheap, degradable, and renewable bio-based materials [29]. Vegetable oil is considered to be the most significant material for bio-based polymers, and polymeric material preparation to be an adequate substitution for polyurethanes [30]. Recent studies have shown that the use of oil-based polymers as coating materials led to gradual, uniform nutrient release and proved a high rate of biodegradability [31,32]. The most widely used vegetable oils to produce bio-based polymers are castor, linseed, canola, sunflower, palm, tobacco, corn, soy, and rapeseed [33].

The positive effect of CRFs in rapeseed cultivation has been described in several studies. The data show that the addition of coated N-fertilizers significantly increases the yield and quality of rapeseed [34,35]. Increased yield rates can be a consequence of advanced root volume and the improvement of plant biomass accumulation (especially during the growth stages of stem elongation, flowering, and harvest), extending the photosynthetic lifespan of pods [36–38].

The aim of our study was to evaluate the differences in the effectiveness of two newly developed coated fertilizers in nutritional status and yield of rapeseed and to assess their environmental impact (especially nitrate leaching). We assumed that the CAN fertilizer coated with a polymer-based on vegetable oil might provide comparable results with the same fertilizer coated with a modified conventional polyurethane.

#### **2. Results and Discussion**

The evaluation of the effect of coated fertilizers was created by comparing the data within the groups using the treatments with the same fertilizer application system (divided, single, and blends). Each method of fertilization was assigned with a control treatment (the treatments D and S). D served as the control variant for the group with a divided application, and S served as a control for the group with a single application and blends.

#### *2.1. Yield and Oiliness of Rapeseed and N Content in Plant Biomass*

The appropriate type of fertilizer and method of fertilization is important for the high yield production of rapeseed. Several studies describe the increase in yield and qualitative parameters of crops after using coated fertilizer application [38–41]. Our study showed that the use of coated CAN fertilizers has no negative effect on the yield and qualitative parameters of winter rapeseed. Statistical evaluation of the data shown in Figure 1 revealed no significant differences between the treatments in the groups with divided application (D, D-opu, D-o) and blends (S, Bl-opu, Bl-o). A significant positive effect was recorded in the group of treatments with a single application of coated CAN fertilizers (opu-CAN-oil-based polyurethane-coated CAN; o-CAN-oil-based polymer-coated CAN) in seed yields and oil contents. Seed yields of this group showed a trend of opu-CAN > o-CAN > CAN with opu-CAN up to 18% higher in comparison to the uncoated CAN. Similar results were recorded in the study by Tang et al. [42], in which a single basal application of coated nitrogen fertilizers contributed to the increase of the yield and rice quality in comparison to the divided application. A different trend was recorded in the case of the oil content that reached up to 5.5% higher after a single application of oil-coated CAN fertilizer compared to the use of the uncoated CAN fertilizer. The presumption was that the total nitrogen applied in the single application of coated fertilizers was released over a longer period of time and thus was present in the phase of the seed formation confirmed by Tian et al. [38]. In this study, the increase was recorded by an average of 17.3% after the application of coated fertilizers in rapeseed yield rates compared to the control. This study also proved that lower doses of the total N applied in coated fertilizers contributed to a yield increase of 14.2%, which confirmed their environmental potential in terms of nitrogen release. The study by Lu et al. [43] showed the positive effects of CRFs application on rapeseed yield manifested in the increase of rapeseed pods from 27 to 32% in comparison to non-coated urea. In comparison to the treatments with coated CAN fertilizers, a single application of the uncoated CAN (treatment S) proved the decline in the parameters of oil production and thousand seed weight (TSW) shown in Table 1. Similar positive effects of coated CAN fertilizers were proved on yield and qualitative parameters of rapeseed. It can be concluded that o-CAN may be a proper alternative instead of opu-CAN.



Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer, TSW—thousand seed weight. The same letters next to the numbers depict no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (divided, single, blend) was evaluated separately. The values represent the mean (*n* = 4) ± standard deviation (SD).

**Figure 1.** Rates of yield and oiliness of rapeseed. The groups of the treatments D—divided application; S—single application; Bl—blend. The columns represent the mean (*n* = 4), error bars present the mean standard deviation (SD). The same letters at the top of the columns describe no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (D, S, Bl) was evaluated separately.

> The data from Figure 2 indicate a connection between the yield rates and the nitrogen concentration in aboveground plant biomass. In general, plants can only consume a part of nutrients (in our case nitrogen) from conventional fertilizers, and the rest may be subject to losses to the environment [44]. This trend is mainly visible in the treatment with the application of conventional uncoated CAN fertilizer in a single dose (S), resulting in a significantly lower concentration of nitrogen in plant biomass in the growth stage of flower bud emergence (*t2*) compared to the growth stage of stem elongation (*t1*). This decrease indicates that the overdose of quickly released nitrogen in uncoated CAN fertilizer led to N-loss available for direct plant consumption and ultimately caused the lowest yield and oil content. The declining trend in the supply of the available form of N, released from conventional uncoated CAN, during the period and the increased supply of mineral N released from coated CAN is also evident from the assessment of N content in aboveground biomass (Table 2). The nitrogen content in the plant shows a gradual release of the available forms of this nutrient from the coated CAN that is particularly evident in the group of singly applied fertilizers (S). While the nitrogen content detected in the aboveground mass of rapeseed fertilized with uncoated CAN (S) was detected almost 4 and 2 times higher in the term *t1* compared to the treatments with coated CAN (S-opu, S-o) in the term *t2,* the nitrogen content of the treatments fertilized with coated fertilizers was increased. These values show that the oil-coated CAN is able to release nitrogen more rapidly than the oil-based, polyurethane-coated CAN and thus may supply the plant's demand for this nutrient. Nitrogen contents in plants, treated with coated fertilizers applied in blends with conventional CAN (Bl-opu, Bl-o), can confirm this trend.

> The relationship between the optimal nitrogen supply and its impact on the yield and oil content of rapeseed is described in many studies [45,46]. A similar trend was recorded in the treatments with coated CAN fertilizers applied in blends with the uncoated CAN fertilizer (Bl-opu, Bl-o). Nitrogen content in plant biomass in the growth stage of stem elongation decreased about 1.3% and 0.9% compared to the uncoated CAN fertilizer applied in a single dose. The N content in plant rapeseed showed the most even N pumping during vegetation in the variant with divided application and a single application of coated CAN fertilizers.

**Figure 2.** Nitrogen concentration (% DM) in aboveground plants dry matter collected in two growth stages *t1* and *t2* of rapeseed. The groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The columns represent the mean (*n* = 4), error bars present the mean standard deviation (SD). The same letters at the top of the columns describe no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (D, S, Bl) was evaluated separately.


**Table 2.** Content of nitrogen in aboveground plant dry matter (mg/plant).

Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The same letters next to the numbers depict no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (divided, single, blend) was evaluated separately. The values represent the mean (*n* = 4) ± standard deviation (SD).

#### *2.2. Mineral Nitrogen Content in the Soil*

The release of nitrogen from coated CAN fertilizers significantly affected the dynamic change of the soil mineral N (Nmin) content in the growth process of rapeseed. Contents of Nmin and its ionic forms (NO3 −, NH4 +) were determined in the soil in three experimental phases (*t1*–*t3*,). Although, enough of the available nitrogen can be essential for direct plant consumption. The excessive content may inevitably increase its loss in soil [47]. Average contents of Nmin in soil (without differencing into layers), shown in Table 3, serve as an overview of nitrogen release development in the treatments during the rapeseed vegetation.


**Table 3.** Contents of mineral nitrogen (Nmin) in soil (mg/kg) on selected experimental phases (*t1–t3*).

Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The same letters next to the numbers describe no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (divided, single, blend) was evaluated separately. The values represent the mean (*n* = 4) ± standard deviation (SD).

One of the important aspects of coated fertilizers is the longevity of nutrient release in sufficient levels for plant uptake. The use of coated CAN fertilizers in each form of the application (D, S, and Bl) has shown a positive effect on Nmin release pattern, as can be seen from Figure 3. The effect was visible, especially in the period between the first (*t1*) and the second term (*t2*) of soil samples collection that was significantly milder compared to conventional uncoated CAN.

**Figure 3.** Contents of mineral nitrogen (Nmin) in soil (top, middle and bottom layer of pot) in three experimental phases (*t1, t2, t3*). Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The columns represent the mean (*n* = 4).

The relatively accelerated release of nitrogen was observed in high Nmin concentration after the application of fertilizers (*t1* single application, *t2* divided application) in the treatments with conventional uncoated CAN shown in Table 3. Rapid release Nmin was visible mainly in the single application in which Nmin concentration decreased rapidly up to 65.4% between *t1* and *t2* (up to 22 days). Our assumption was that although the part of the soil Nmin was obtained from the soil through plant roots, the great contrast in Nmin concentration was due to N loss (NH4 <sup>+</sup> volatilization and NO3 − leaching) between *t1* and *t2*. On the contrary, the data of the soil samples, collected in the harvest time (*t3*), showed relatively high levels of Nmin in the treatments with divided (especially D-o) and single (S-opu, S-o) application of fertilizers in comparison with conventional CAN treatments. Dynamic of gradual Nmin release was most visible after a single application of both coated CAN (S-opu, S-o) with no definite decrease in Nmin content in *t3*. A single application of oil-based polyurethane-coated CAN fertilizer (S-opu) caused an increase by 14.2% in *t3* in mineral nitrogen content compared to *t2* in soil. These findings corresponded to the data of yield and qualitative parameters (Figure 1), in which a single application of coated CAN fertilizer (S-opu) proved to be the most effective. The assumption was that the amount of released nitrogen reached sufficient levels for the plant demand in the time of the experiment duration from these treatments, thus leading to the increased nitrogen use efficiency and subsequently to a more positive environmental impact (lower risk of N loss). Our data are consistent with the findings of Xiao et al. [48], who described that the total Nmin content continued gradually to an increase in the top layer of soil on the ninetieth day after the application of coated fertilizers, while high levels were maintained in the middle and bottom layer of soil.

The positive effect of coated CAN fertilizers on Nmin content was also visible in the nitrogen distribution between soil layers during the experiment (Figure 3). The application of conventional uncoated CAN fertilizer (D and S treatment) showed high Nmin concentrations mainly in the top and middle layers of the soil right after fertilization. The treatments with coated CAN fertilizers showed that Nmin content was, in general, focused mainly on the top layer of the soil during *t1* and *t2.* Nmin content was evenly distributed between each layer of the soil in the harvest time (*t3*). This indicates that both coated CAN fertilizers (opu-CAN and o-CAN) proved a high ability of gradual nitrogen release leading to more efficient nitrogen use by the plant and a reduction in the environmental risk. A gradual Nmin release by coated fertilizers was also described in the study by Zheng et al. [49], who found that the application of coated fertilizers resulted in enhanced Nmin concentration in soil, especially during later crop stages.

Considering the placement of the fertilizers (the placement on the soil surface without incorporation to the soil), the highest potential for the NH4 <sup>+</sup> volatilization is most likely to be closest to the soil surface [50]. Ammonium nitrate (used CAN in our experiment), depending on N dose and irrigation, belongs to the conventional nitrogen fertilizers with a high potential of NH4 <sup>+</sup> volatilization [51].

This assumption was confirmed by the data obtained from the top layer of the soil samples (Figure 4). The data showed the greatest potential for NH4 <sup>+</sup> volatilization in the treatments with conventional uncoated CAN (D and S treatments) expressed in significantly high NH4 <sup>+</sup> concentrations in *t1* and *t2*. Analogous to Nmin, the uncoated CAN potential of volatilization was visible between *t1* and *t2,* in which the NH4 <sup>+</sup> concentration decreased up to 39.8% in soil. Higher NH4 <sup>+</sup> concentrations were accountable to the use of conventional uncoated CAN (1/3 of the total N dose) after the application of blend fertilizers (Bl-opu, Bl-o). Similarly, the S variant (a single application of uncoated CAN) was resolved in its rapid release. NH4 <sup>+</sup> contents in Bl-opu and Bl-o were detected almost over half lower in *t1* than in the S treatment; therefore, major risks of NH4 <sup>+</sup> losses were not found. In addition to the volatilization, a rapid NH4 <sup>+</sup> release also presents the risk of the increased concentration of nitrates as an initial component of nitrification in soil and thus increased the risk of NO3 − leaching [52].

The positive effect of coated fertilizers was expressed by significantly lower NH4 + concentrations during *t1*–*t3* in comparison to conventional uncoated CAN. The data were indirectly consistent with the findings of Xiao et al. [48], who mentioned that the application of coated fertilizers resulted in lower NH4 <sup>+</sup> rates in soil samples in comparison to conventional uncoated nitrogen fertilizer. A gradual NH4 <sup>+</sup> release was also expressed by the increase of NH4 <sup>+</sup> concentration in the top layer of the soil in *t2*. This fact was noticeable in the treatments of D-opu (up to 41.3%), D-o (up to 58.8%), and S-o (up to 29.7%). The treatments with a divided and single application of fertilizers were proved to be the most efficient in terms of the longevity of NH4 <sup>+</sup> release. These types of fertilizer applications

showed significantly higher NH4 <sup>+</sup> contents in *t3* treatments compared to the treatments with conventional uncoated CAN. On the contrary, NH4 <sup>+</sup> contents showed no significant difference in the S treatment in Bl-opu and Bl-o. This led to an assumption that all nitrogen contained in coated fertilizers and applied in blends was released during the rapeseed vegetation, predetermining the blend application as the most suitable alternative.

**Figure 4.** Contents of NH4 <sup>+</sup> in the top layer of the soil samples collected in three experimental phases (*t1*–*t3*). Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The columns represent the mean (*n* = 4), error bars present the mean standard deviation (SD). The same letters at the top of the columns depict no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (D, S, Bl) was evaluated separately.

Contents of NO3 − were monitored as the main potential source of N loss in the soil samples due to their high leaching ability. One of the first studies by Liegel and Walsh from 1976 [53] proved that the application of controlled-release N fertilizers was the most effective technique in sandy irrigated soils with a high risk of nitrate leaching. Preventing the leaching of nitrates presents one of the greatest environmental challenges in terms of nitrogen fertilizer use. The estimation of the potential for N losses due to the NO3 − leaching from the experimental treatments were provided by the isolation of the data from the bottom and middle layers of soil. The data obtained from the middle layer (ML) of the soil (Figure 5) served for the evaluation of potential NO3 − migration to the lower layers of the soil, which might consequently lead to its leaching into the groundwater. The data obtained from the bottom layer (BL) of the soil (Figure 6) served to evaluate the potential of nitrates leaching to the groundwater during the rapeseed vegetation and directly after its harvest.

As predicted, significantly, the highest potential for NO3 − leaching was due to rapid nitrogen release from conventional uncoated CAN fertilizers recorded in single or divided CAN application. The potential for NO3 − leaching after uncoated CAN application was possible to confirm from the data of NO3 − concentrations in *t1* and *t2* shown in Figures 5 and 6. The NO3 − content of ML and BL was detected over three times higher (>3.3) in the treatment fertilized with a single application of uncoated CAN in *t1* compared to the treatments with coated CAN fertilizers. The data showed that the NO3 − decrease was found up to 73.9% in ML and up to 75.5% in BL in the S treatment between *t1* and *t2*. Considering the amount and duration (up to 14 days), it is most likely that nitrates of the uncoated CAN fertilizer were lost due to the nitrate leaching. These findings corresponded with the data by Zhang et al. [54], who discovered that the rates of the leached nitrates

in water samples were detected significantly higher in comparison to coated urea in the treatments with conventional urea.

**Figure 5.** Contents of NO3 − in the middle layer of soil samples collected in three experimental phases (*t1*–*t3*). Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The columns represent the mean (*n* = 4), error bars present the mean standard deviation (SD). The same letters at the top of the columns depict no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (D, S, Bl) was evaluated separately.

**Figure 6.** Contents of NO3 − in the bottom layer of soil samples collected in three experimental phases (*t1*–*t3*). Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer. The columns represent the mean (*n* = 4), error bars present the mean standard deviation (SD). The same letters at the top of the columns depict no statistically significant differences between the treatments (Fisher's LSD test, *p* ≤ 0.05). Each group of the treatment (D, S, Bl) was evaluated separately.

Identical to Nmin and NH4 +, the positive effect of coated CAN fertilizers was recorded in the form of gradual NO3 − release over the course of the whole experiment. Gradual release of nitrates was discovered to be the most visible between *t2* and *t3* in coated fertilizers. The increased NO3 − contents were observed up to 64.7% in ML and up to 119.9% in BL. While the NO3 − amount was decreased in ML and BL in the treatments fertilized with uncoated CAN, the coated CAN fertilizers were able to supply the plants with nitrogen even in the later stages of the development. Compared to the low levels of NO3 − content in the treatments with conventional CAN fertilizers (due to rapid nitrogen release and subsequent N loss). This increase correlated with the data of seed yield and qualitative parameters (Figure 1) and can be used as a potential supply of available nitrogen for the next crops. The data correlated with the findings of Xiao et al. [48]. Similar Nmin release (especially NO3 −) was proved using oil-based polymer-coated CAN, which can be a proper alternative for oil-based polyurethane-coated CAN. This fact is not suitable for future use due to polyurethane's lower biodegradability. The positive effect of coated fertilizers on nitrates leaching was recorded in several studies [55–58].

#### **3. Materials and Methods**

The pot experiment was performed under controlled conditions in the vegetation hall of Mendel University in Brno, Brno, Czech Republic (49◦12 36.94" N and 16◦36 49.95" E).

#### *3.1. Plant Material and Growth Conditions*

Rapeseed (*Brassica napus* subs. *napus*) cv. DK Exception (Bayer s.r.o, Prague, Czech Republic) was used in this study. Mitscherlich pots (STOMA GmbH, Siegburg, Germany) were filled with 6 kg of air-dried and <2 cm sieved soil and placed in the vegetation hall. The properties of the used soil for the pot experiment are shown in Table 4. Ten seeds of rapeseed were sown in 2 cm depth in each pot. Three weeks after sowing, the number of rapeseed plants was adjusted to three plants per pot.

#### **Table 4.** Agrochemical properties of used soil.


Mehlich III—soil test extractant.

#### *3.2. Experimental Design*

In the experiment, coated CAN fertilizers were compared with a conventional uncoated CAN. The same total dose of nitrogen was applied in all treatments using different N sources such as calcium ammonium nitrate (CAN, up to 13% N-NH4 <sup>+</sup> and 13% N-NO3 −, Lovochemie a.s., Lovosice, the Czech Republic), oil-based polyurethane-coated CAN (opu-CAN) and oil-based polymer-coated CAN (o-CAN). Coated fertilizers were prepared by spreading the coating on conventional fertilizer CAN using the LDP-3 fluidized bed granulating machine (Changzhou Jiafa Granulating Drying Equipment Co., Ltd., Changzhou, China). The coating consisted of oil-based polyurethane polymer (opu-CAN—coating up to 7.6 wt.%, up to 13% N-NH4 <sup>+</sup> and 13% N-NO3 −, VUCHT a.s., Bratislava, Slovakia) and

oil-based polymer (o-CAN—coating up to 6.1 wt.%—triglycerides of fatty acids, up to 75 wt.% of which unsaturated were up to 45 wt.%, polylactic acid up to 10 wt.%, up to 13% N-NH4 <sup>+</sup> and 13% N-NO3 −, VUCHT a.s., Bratislava, Slovakia). The composition of the polyurethane-based coating (opu-CAN) differed from the conventional polyurethanes prepared by the reaction of the diisocyanates with the polymeric diols. The polymeric diols were replaced with the vegetable oil having hydroxy groups in its structure. The prepolymer, obtained by this way, was finally applied in the crosslinking. These modifications led to a substantial increase in the biodegradable fraction of the coating. The prepolymer was completely replaced with a more biodegradable component in the oil-based coating (o-CAN). The biodegradable fraction of the coating material is further increased in this way.

The individual treatments and fertilizer addition are detailed in Table 5. The fertilizers were applied to the soil surface. Each treatment was replicated 8 times in a complete randomized block design in the vegetation hall (Figure 7).

**Figure 7.** Schematic illustration of the layout of the pots (and their repetition) in the vegetation experiment. Treatment D (1), D-opu (2), D-o (3), S (4), S-opu (5), S-o (6), Bl-opu (7), Bl-o (8).

The treatments of fertilizer were divided into 3 groups according to the term of application and the type of fertilizer chosen. The first group was the divided application of fertilizers (the designation of the treatments with D). The total nitrogen dose was divided into two parts; the first was applied by the conventional uncoated CAN in the 1st term (1st Fertilization), the second dose was applied by uncoated CAN (treatment D) and coated CAN (treatments D-opu and D-o) in 2nd term (2nd Fertilization). The second and third groups consisted of treatments with a single application of total nitrogen dose in one term (1st Fertilization), where fertilizers of one type (the designation of the treatments with S) and fertilizers of a mixture (the designation of the treatments with Bl) were applied. The fertilizer mixtures (Bl) were created by mixing conventional CAN and coated CAN in a 1:2 ratio (converted to N rate).

The pot experiment was carried out under semi-natural conditions (under a rain shelter) in the vegetative hall. Figure 8 shows the average daily temperature and the average daily relative humidity during the experiment. A controlled watering regime, used identical for all treatments (pots), was in the experiment. Plants were watered to 70% of maximum water holding capacity throughout the growing season. The pots were hand-watered with demineralized water on the soil surface.

Rapeseed plants were harvested manually by cutting above the soil surface from each pot. The rapeseed was threshed using a laboratory thresher (HALDRUP LT-20, Haldrup GmbH, Ilshofen, Germany).

The rape seeds were purified from coarse impurities by repetitive sifting. Rapeseed yield was measured in three plants within each pot, and the value was adjusted to 9% of moisture. Seed yield was determined by weighing (laboratory scale PCB Kern, KERN & Sohn GmbH, Balingen, Germany) and exceeded as gram per pot (g/pot). Seeds were then counted and hand-ground in mortar for further analysis of the oil content.



Groups of treatments D—divided application, S—single application; Bl—blend; opu—oil-based polyurethane polymer, o—oil-based polymer.

**Figure 8.** The average daily temperature (◦C) and relative humidity (%) in the vegetation hall during the experiment.

#### *3.3. Plants and Soil Sampling*

The evaluation of soil mineral nitrogen content (NO3 −, NH4 +) and nutritional plant properties was provided in the soil samples and plant biomass collected in the specific experimental phases shown in Table 6. The collection of the soil samples was carried out by a probe with the aligned tip. After the collection, the soil profile was divided in three zones for the observation of mineral nitrogen movement in soil and subsequently frozen for further analysis (Figure 9).

The plant biomass was dried at 50 ◦C and homogenized to determine the nitrogen content in the dry matter.


**Table 6.** Experimental phases and dates.

#### *3.4. Analytical Methods*

The Nmin determination was provided according to the methodology by Zbíral et al. [62], who described that nitrate and ammonium nitrogen was extracted from the soils with a solution of neutral salt (1% of K2SO4). The NH4 <sup>+</sup> determination was carried out spectrophotometrically (λ660 nm). The NO3 − contents were determined by ISE (Ion Selective Electrode) [63].

The nitrogen determination was provided in aboveground plant biomass according to the methodology by Zbíral et al. [64]. Nitrogen contents were determined by the Kjeldahl method using the Kjeltec 2300 device (Foss, Hillerød, Denmark).

The thousand seed weight (TSW) determination was performed using a laboratory counter MK (MEZOS spol. s r.o., Hradec Králové, the Czech Republic). The determination was carried out by weighing the number of 2 × 500 seeds to prevent possible measurement errors.

The determination of seed oil content was provided according to the methodology of the Central Institute for Supervising and Testing in Agriculture [65]. The oil content was determined gravimetrically after the extraction of the samples with diethyl ether using the Soxhlet method based on the NMR extraction of rapeseeds in a continuous flow extractor Minispec mq series TD-NMR (Bruker Corporation, Ettlinger, Germany).

#### *3.5. Statistical Analysis*

The effect of the treatment on the evaluated parameters was statistically analyzed in the STATISTICA 12 program (TIBCO Software, San Jose, CA, USA) [66]. The effect of the treatment on the seed yield, oiliness, oil production, thousand seed weight, nitrogen concentration and content in aboveground plant biomass and the content of mineral nitrogen (ammonium and nitrate) in soil were analyzed separately for each group of the treatment (divided, single and blend application of fertilizers). The normality and homogeneity of variances were verified, respectively, by Shapiro-Wilk and Levene values at *p* ≤ 0.05. The influence of the monitored factors was analyzed via analysis of variance (level of significance *p* ≤ 0.05). The effect of the treatment on the mentioned parameters was analyzed using two-way analyses of variance with the treatment such as fixed effect and the pot used as the random effect to take into account the grouping of individuals in the same pot. The differences between the means were evaluated by the Fisher's (*LSD*) test.

#### **4. Conclusions**

The use of coated CAN fertilizer proves the potential to gradually release acceptable nitrogen during the growing season in winter rape nutrition and thus continuously meet the needs of plants. Compared to the effect of conventional CAN, the use of coated CAN fertilizers has been shown to increase the efficiency of nitrogen fertilization and reduce its losses. A suitable method seems to be the application of a mixture of conventional CAN and coated CAN in a ratio of 1:2 during spring fertilization, ensuring a sufficient amount of rapidly releasing N during the regeneration of rapeseed and its slower release during further developmental stages. The CAN fertilizer coated with a biodegradable oil-based polymer proves the ability to release the optimum amount of nitrogen for canola nutrition. The use does not pose a risk of rapid release of mineral N in quantities potentially polluting the atmosphere (ammonia volatilization) and hydrosphere (nitrate leaching). According to these results, the CAN fertilizers coated with a polymer-based on vegetable oils could be used as a replacement for commonly used synthetic polymers based on polyurethane confirming the initial hypothesis.

**Author Contributions:** Conceptualization, P.Š., and P.R.; methodology, P.Š. and P.R.; investigation and data analyses, D.M. and J.A.; writing—original draft preparation, D.M.; writing—review and editing, P.Š. and P.R.; supervision, P.R. and M.K.; project administration, P.Š., P.R., and M.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Technology Agency of the Czech Republic, grant TH03030220: Environmentally acceptable solid controlled release fertilizers, and Internal Grant Agency of the Faculty of AgriSciences, Mendel University in Brno, grant AF-IGA2020-IP031: Use of coated nitrogen fertilizers in the nutrition of oilseed rape (*Brassica napus* L.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

