The negative influence of agriculture on the natural environment, and even on the climate, is increasingly often emphasized. One of the most important current challenges is to limit this influence [1
]. The cultivation of grass and legume mixtures based on natural biological processes is a relatively small burden for the environment. These mixtures help to maintain soil fertility and biological potential, also in the context of carbon sequestration. They have minimal requirements for nitrogen fertilization, because they form efficient symbiotic systems with diazotrophic bacteria [3
]. The optimization of the fertilization of grass and legume mixtures with other macroelements, including potassium, calcium, and sulfur, remains an important problem.
Potassium is one of the most important macroelements in plant production due to the multifunctional effect of this nutrient on the physiological processes occurring in plants, and in consequence, on their yield. Potassium not only controls the water management, but also the nitrogen management in the plant, and it significantly minimizes abiotic and biotic stresses [8
]. If the amount of potassium fertilizer exceeds the metabolic capacity of plants, their yield will not increase. Instead, the yield quality may be adversely affected, including the uptake of other macronutrients such as calcium and magnesium [12
], which are particularly important for forage crops.
Sulfur is a nutrient with important structural and metabolic functions in plants. It is a component of proteins, fats, and amino acids, and it participates in their synthesis. Sulfur can be found in such amino acids as methionine, cysteine, and cystine, and in ferredoxin and vitamins (biotin, thiamine) [13
]. Additionally, sulfur is involved in redox processes and stabilizes the protein structure [14
]. If there is sulfur deficiency, the yield volume and quality decrease, e.g., the content of non-protein nitrogen in plants increases [15
]. Soil sulfur deficiency reduces the yield-generating efficiency of nitrogen [16
]. Sulfur deficiency disrupts the growth of root nodules and the process of atmospheric nitrogen fixation in alfalfa and other legumes due to lower nitrogenase activity [17
]. Sulfur affects the process of atmospheric nitrogen fixation and thus determines the yield-generating effect in legumes [18
]. Moderate doses of sulfur are the best for plants because then they exhibit the highest photosynthetic activity [19
Currently, agricultural soils tend to be increasingly deficient in sulfur. One of the reasons for this situation is the effective desulfurization of flue gases from coal combustion. Some forms of sulfur deposited in soil are difficult to access for plants. Most of the agricultural soils in Poland have a low content of sulfate sulfur [20
]. The decreasing content of sulfur in soils is a major problem of modern agriculture [21
]. The issue of fertilization of crops with sulfur has been discussed in various studies (e.g., [23
]). In order to optimize sulfur fertilization, it is necessary to select the right chemical formula and the time of application [26
The metabolic functions of calcium are also important for the normal function of plants [28
]. This element is essential for the division of cells in the growth buds of the roots and shoots. Calcium deficiency inhibits plant growth [29
]. Alfalfa is a plant species with a high demand for calcium [30
Calcium sulfate (CaSO4
) is a calcium fertilizer used in agriculture. It is natural gypsum or a product of SO2
neutralization in combined heat and power plants [31
]. When calcium sulfate is applied to soil, Ca2+
ions in the form of chemically neutral salt are available to plants, especially to the species with a high demand for calcium, such as clover and alfalfa [32
]. Calcium sulfate is also a rich source of sulfur that plants can easily access. It has positive influence on the volume and quality of the yield of plants with a high demand for this element, e.g., rape and alfalfa [34
Calcium sulfate affects plant growth and yield quality [35
]. When used as a fertilizer, it directly supplies sulfur and calcium to plants. Calcium sulfate introduced into soil as a chemically neutral salt does not form concentrated solutions and its pH is almost neutral. As a result, it does not damage leaves and it can be safely used for top dressing, even as a dust fertilizer [30
]. Calcium sulfate fertilization has positive effects on heavy clay soils because it increases the rate of water infiltration and reduces the crust on the surface of these soils [40
The content of aluminum, which is toxic to plants, increases in acidic soils and disturbs the growth of plants’ roots [36
]. Calcium sulfate only slightly modifies the soil pH, but it limits the toxic effect of aluminum in soil [43
]. When calcium sulfate is applied in the soil solution, complex AlSO4
ions or molecular Al(OH)SO4
compounds are formed, which reduce the harmful effect of aluminum on plants [45
The soil bioactivity is also manifested by the activity soil enzymes, which is a derivative of the metabolism of plants and soil organisms, especially the soil microbiome. The enzymatic activity of soil is an image and indicator of its biological condition and fertility [47
]. Dehydrogenases, and acid and alkaline phosphatases, are considered the best indicators of the general population of soil microorganisms and soil microbial activity [49
Dehydrogenases can be found in all living microbial cells, where they reflect the redox processes. They do not accumulate in the extracellular space, which prevents, for example, plant dehydrogenases from entering the soil. Therefore, the activity of soil dehydrogenases is almost exclusively related with the abundance and activity of soil microorganisms [52
]. The activity of acid and alkaline phosphatases, which can be observed in living and dead plant cells and soil microorganisms, reflects the activity of enzymes related with soil colloids and humic substances [53
]. Researchers studying the soil environment usually assess the activity of phosphomonoesterases (phosphoric monoester hydrolases), which are largely responsible for the decomposition of organic phosphorus compounds into inorganic forms—they catalyze the decomposition of organic phosphorus ester and phosphoric acid anhydrides [55
]. Their activity plays an important role in the phosphorus cycle in nature. By participating in the hydrolysis of various phosphate compounds they stimulate the transformation of organic phosphorus compounds into inorganic phosphates, which are directly available to plants and soil microorganisms [57
The soil enzyme activity is influenced by various general environmental factors (humidity, temperature, oxygen availability, pH, the presence of soil organic matter) and factors specific to a particular habitat (pesticides, heavy metals, farming methods, etc.). Organic and mineral fertilization is one of the most important factors in agrocenoses [60
The aim of this study was to assess how different doses of potassium and calcium sulfate fertilizers applied to a mixture of alfalfa and grasses influenced the yield of the sward and changes in the soil enzyme activity.
The analysis of variance (Table 2
) indicated a significant effect of CaSO4
fertilization on the yield of the sward, the soil pH, and the activity of dehydrogenases and alkaline phosphatase. The potassium fertilizer significantly influenced the yield of the fresh matter of the alfalfa and grass mixture and the dehydrogenase activity. The interaction between the CaSO4
and K fertilization was observed only for the yield. The main effect of the year was significant for all five variables (fresh matter yield, soil pH, dehydrogenase, acid phosphatase, and alkaline phosphatase activities). The year × CaSO4
fertilization interaction was statistically significant for the yield and alkaline phosphatase activity.
During the experiment, the average yield of the mixture of alfalfa and grasses (Table 3
) ranged from 30.26 to 67.92 t of fresh matter per hectare in the combination without the CaSO4
fertilizer, and from 36.48 to 70.82 t per hectare in the combinations with the CaSO4
fertilizer. The highest yields of fresh matter were noted in 2013, although it was a rather dry year. The yield of the sward in the combination with the CaSO4
fertilizer and the full dose of potassium (120 kg ha−1
) was over 70 t of fresh matter (FM) per ha. The lowest yields were noted in the last year of the experiment.
The analysis of the effect of the applied doses of the potassium fertilizer on the average yield of the sward mixture in individual combinations and years of the study showed that, in the variant without the CaSO4
fertilizer, the difference in the total yields between the K 0 and K 120 combinations was 27.52 t of fresh matter per hectare (14.93%). In the variant with the CaSO4
fertilizer, the difference between these combinations in the total yield obtained during the years of the study amounted to 31.83 t of fresh matter per hectare (15.91%) (Table 3
The analysis of the effect of the CaSO4 fertilization showed that, in the subsequent years of the study, the yields increased in all the K combinations. In comparison with the K 0 combination, the application of potassium at the doses of 30, 60, and 120 kg ha−1 in the variants without CaSO4 increased the yield of fresh matter by 8.36 t ha−1 (4.5%), 19.53 t ha−1 (10.6%), and 27.52 t ha−1 (14.9%), respectively. In comparison with the K 0 combination, the application of potassium at the doses of 30, 60, and 120 kg ha−1 in the variants with CaSO4 increased the yield of fresh matter by 6.22 t ha−1 (3.11%), 23.7 t ha−1 (11.8%), and 31.93 t ha−1 (15.9%), respectively.
The statistical analysis of the yields revealed a significant difference between the variants with and without the CaSO4 fertilizer. The potassium fertilizer also significantly increased the yields. The fertilization levels of 60 and 120 kg K ha−1 increased the yields considerably, both in the variants with and without CaSO4. It turned out that the dose of 60 kg K ha−1 was as effective as the full dose of 120 kg K ha−1, despite the lower amount of potassium applied to the soil.
The effect of CaSO4
application on the efficiency of potassium fertilization in relation to sward yield increase is shown in Figure 1
. In each year, the application of CaSO4
significantly increased the yields of fresh matter.
The assessment of the effect of the applied fertilizer combinations on the soil pH was the starting point for the interpretation of the soil enzyme activity (Table 2
and Table 4
). The CaSO4
fertilization decreased the soil pH. The effect was minimal but unambiguous. It exhibited an upward trend in the following years and it was statistically significant. The effect of different doses of potassium fertilization on soil pH was statistically insignificant (Table 4
All the analyzed factors clearly influenced the activity of dehydrogenases (Table 5
). Only the main effects Year, CaSO4
and K fertilization, separately, were significant. The CaSO4
fertilizer reduced the dyhydrogenase activity, whereas the potassium fertilizer clearly increased the activity of this enzyme as the dose of potassium increased. There was no interaction between the independent variables (Table 2
The significant differences in acid phosphatase activity were only obtained for Year (Table 2
and Table 6
). The influence of the experimental variants on the acid phosphatase activity was minimal, inconclusive, and statistically insignificant.
Unlike acid phosphatase, alkaline phosphatase is mainly of microbial origin. The significant differences in alkaline phosphatase activity were only obtained for Year, CaSO4
and the interaction Year × CaSO4
and Table 7
fertilization significantly reduced the activity of this enzyme.
In order to assess the correlations between the variables, the Pearson correlation coefficient was used for analysis (Table 8
). The strongest correlations were found between the activity of both phosphatases, the activity of phosphatases and the yield, and between the dehydrogenase activity and the soil pH. The analysis did not reveal any correlation between the dehydrogenase activity and the yield. These correlations confirmed the aforementioned explanations of the research results and observed phenomena.
There are several reasons why the year of the experiment and its interactions influenced the variables under analysis. The most important of these are the differences in the weather conditions in individual years of the experiment, and especially the differences in the amount and distribution of rainfall during the growing season, which determined the supply of water available to plants. It is a well-known fact that water is one of the main factors determining the growth and development of plants, because it influences their ability to take up soil minerals. Although alfalfa, which was the dominant plant in the sward, is relatively resistant to periodic droughts [67
], it is not indifferent to the influence of drought stress. Grasses react very strongly to periodic water shortages in the soil profile [69
]. During the growing seasons in 2012 and 2014, there was a sufficient amount of rainfall for the development of the mixture of alfalfa and grasses (Table 1
), but 2013, and especially 2015, were dry. The significant differences between the variables in individual years of the experiment were also caused by changes in the viability of the plants, which resulted from the specificity of their development in the subsequent years. In the first year, the plants fully developed and achieved their full yield potential. In the last years of the experiment this potential decreased due to the plants’ ageing [70
]. In consequence, the sward became less dense and low weeds appeared.
The doses of potassium fertilizer applied in individual experimental combinations during the years of the study had a significant influence on the plants, both in the variants with and without the CaSO4
fertilizer. Robin et al. [71
] conducted a study on white clover and observed that when the plants had a higher supply of potassium during a water deficit, the water potential and stomatal resistance in the leaves decreased, which reduced the loss in the growth of the biomass of this species. This observation was also confirmed by the yields of the plants analyzed in our experiment. In 2015, there was a very interesting reaction of the mixture of alfalfa and grasses to the CaSO4
fertilizer. The last year of the experiment was characterized by a rainfall deficit, which resulted in the lowest yields (Table 3
). However, under such unfavorable soil moisture conditions there was an interaction between CaSO4
and potassium applied at the doses of 60 and 120 kg ha−1
. The yield of fresh matter from the sward in the K 120 combination increased by 8.46 t ha−1
. It was 23.16% higher than the yield from the same combination but without the CaSO4
fertilizer. The dose of potassium reduced by half, i.e., the K 60 combined with the CaSO4
fertilizer had an even better effect. The yield of fresh matter from the sward increased by 9.55 t ha−1
, i.e., by 29.3%.
The accumulation of the effects of fertilization in the subsequent years of the experiment was undoubtedly another important reason for the changes in the variables under analysis. This particularly applied to the slow changes in the physicochemical properties of the soil, such as pH. Although the analysis did not reveal any significant effect of the potassium fertilization on the soil pH, the interaction between the year of the experiment, CaSO4 fertilization, and K fertilization had a statistically significant influence on this parameter.
The research conducted by Tirado-Corbalá et al. [72
] showed that the application of CaSO4
stimulated the growth of the root system of alfalfa in soils with limited content of water and minerals. It also improved soil fertility and the growth of alfalfa. The application of CaSO4
did not have a measurable effect on the yield of alfalfa, nor did it have a major effect on the content of macronutrients, except for the plants’ increased uptake of sulfur. CaSO4
is an excellent source of sulfur, so the fertilization of the soils where the availability of this element is limited improves the production of alfalfa biomass.
Most authors indicate that CaSO4
has either no or a very poor deacidification effect [73
]. However, there have also been reports, including our study, which documented the slightly acidifying effect of this fertilizer on the soil [75
]. The effect is caused by the sulfate ions formed after the decomposition of the salt (CaSO4
), which react with water to produce sulfuric acid. Potassium fertilization may also reduce the soil pH [73
The experimental combinations had a diversified influence on the biological properties of soil in individual years of the study, which was manifested by the differences between the mean values and their significance. In some cases, the lack of statistical significance of the differences was caused by the considerable difference in the results between individual dates of analyses. However, the same trends were observed in each year, as can be seen in the charts (Table 4
, Table 5
, Table 6
and Table 7
It is most likely that the lower soil pH reduced the dehydrogenase activity in the combinations fertilized with CaSO4
. According to Swędrzyńska et al. [78
], even slight changes in pH may strongly modify the qualitative and quantitative composition of the soil microbiome, and thus affect the activity of soil enzymes. The negative effect of CaSO4
on the dehydrogenase activity should not be attributed to sulfur alone, because the experiments conducted by Niewiadomska et al. [79
] clearly showed that the dehydrogenase activity increased after the application of both elemental sulfur fertilizer and potassium fertilizer.
The lack of the effect of the applied experimental factors on the acid phosphatase activity may be explained by the fact that the activity of this enzyme is largely the plant’s response to phosphorus deficiency in soil [80
]. In our experiment, the amount of phosphorus in the fertilizer met the nutritional demand of alfalfa and grasses in the sward. The usefulness of acid phosphatase as an indicator of soil bioactivity was also limited by the high pH of the soil in our experiment.
It is noteworthy that the course of the alkaline phosphatase activity, which is mostly affected by the activity of soil microorganisms, was similar to the course of the dehydrogenase activity—it was reduced by the CaSO4
fertilizer. Also in this case, it is most likely that the decreased activity of this enzyme was caused by the lower pH of the soil in the combinations fertilized with CaSO4
. Other authors observed a similar dependence between the soil pH and the alkaline phosphatase activity in their studies [76
Symanowicz et al. [81
] conducted a study on barley and observed that balanced nitrogen and potassium fertilization should be applied both to the forecrop and the succeeding crop in order to maintain the optimal activity of soil enzymes. Higher doses of potassium improved the dehydrogenase activity, whereas lower doses improved the activity of acid and alkaline phosphatases. The same authors observed that the fertilization of pea plants with nitrogen at a dose of 20 kg ha−1
and potassium at a dose of 166 kg ha−1
increased the urease and dehydrogenase activity in the soil [82
]. Swędrzyńska et al. [78
] found that soil bioconditioners based on calcium carbonate stimulated the activity of dehydrogenases, phosphatases, and urease. As shown in most studies discussing fluctuations in the soil enzyme activity during the growing season, it is most likely that they are influenced by variable weather conditions, which consequently affect the soil temperature and moisture [83