*4.3. Biochemical Activity*

Enzymatic activity is one of the best indicators for assessing biological activity in the soil, and the following enzymes are widely used: dehydrogenase, phosphatase, urease and protease [64]. In the literature, numerous authors [65–67] report that dehydrogenases do not accumulate in the soil, but only in the cytoplasm and characteristic structures formed from the cytoplasmic membranes of living microorganisms. Being intracellular enzymes, they can be indicative of the presence of physiologically active soil microorganisms, which provides information on the respiratory activity of the entire soil microbiota, especially bacteria and Actinomycetes [68]. Numerous studies on dehydrogenases and their association with soil factors indicate that they are useful and sensitive indicators of soil changes [69–71].

In our study, a significant stimulating effect from the use of biostimulators and foliar fertilizers on dehydrogenase activity was observed. High dehydrogenase activity was observed during the full growing season of the pea crop (onset of flowering), and may be associated with increased secretion by

the root system during this period, which subsequently leads to increased numbers of microorganisms. Similar results were obtained by Siwik-Ziomek and Szczepanek [72], who analyzed the impact of inorganic fertilization (NPK and S) and the Kelpak biostimulator on dehydrogenase activity in the soil during the cultivation of winter rapeseed. In their study, they observed an increase in dehydrogenase and catalase activity in the period from flowering to ripening. In addition, they showed that the use of the Kelpak biostimulator and sulphur application resulted in a significant stimulating modification of catalase and dehydrogenase activity. Moreover, Brzezi ´nska et al. [68] noted that dehydrogenase activity is associated with the activity of other soil enzymes, for example catalase and β-glucosidase, as well as with the presence of nitrogen. Prashantha [73] indicated that, among other elements, boron increases the activity of dehydrogenases. In contrast, Niewiadomska et al. [29] reported the positive e ffect of molybdenum on the production of these enzymes by white lupin plant root nodules, and discussed the possibility of a positive correlation between titanium concentration and soil biochemistry.

Phosphatases are also important enzymes for the soil environment. Many authors report that phosphatases are good indicators of the potential for organic phosphorus mineralization and soil biological activity [74,75], and these enzymes are characterized by high susceptibility to changing soil conditions [76]. The main sources of phosphatase in the soil environment are mainly soil microorganisms, plant roots and soil fauna.

Aon and Colaneri [77] observed correlations between organic matter content and acid/alkaline phosphatase activity. Margalef et al. [78] noted that phosphatase activity is higher in soils with a low phosphorus content. A lack of phosphorus in our fertilization treatments increased acid phosphatase activity. A study conducted by Niewiadomska et al. [79], which assessed the e ffect of fertilizer application (PRP SOL) containing phosphorus, potassium, zinc, boron and molybdenum in the cultivation of yellow lupin, showed that the decrease in acid phosphatase activity was caused by the activation of compounds inaccessible to the plants. Fukuda et al. [80] reported that plants typically produce acid phosphatases when the amount of available phosphorus in the soil is low.

In our study, the largest significant decrease in the level of acid phosphatase activity was observed after application of the Bolero Mo biostimulator in 2016 and 2017, and by the Adob Zn IDHA foliar fertilizer in 2017. The results observed for most of the other experimental treatments at the beginning of the flowering phase were also promising in terms of phosphorus availability due to the action of fertilizers/biostimulators used, as evidenced by the reduced level of the enzyme activity in the soil compared to the control treatment.

In our study, di fferent levels of alkaline phosphatase activity were observed after the application of foliar fertilizers and biostimulators. The increase in the activity of this enzyme could have been the result of the increased activity of soil microorganisms on organic phosphorus compounds (e.g., phytins) secreted into the soil by the pea plants. Phosphatase activity in the soil depends on available phosphorus content, which suggests that alkaline and acid phosphatase participate in the regulation of the nutrient economy [81].

The results observed in this study in regard to catalase activity show the significant stimulating effect of the treatments. Catalase is a well-documented enzyme in scientific literature and has the best-known chemical structure. It is an enzyme from the group of oxidoreductases (E.C.1.11.1.6), found in microorganisms, plants and animals. In the soil environment, it is present in the cells of all microorganisms that use oxygen for respiratory processes (aerobes, facultative anaerobes). Obligatory anaerobes show very little or no catalase activity. It is considered one of the main enzymes with antioxidant activity and works mainly to remove excess hydrogen peroxide (H2O2) by converting water and oxygen in all aerobic organisms [82]. Brzezi ´nska et al. [72] showed a significant relationship between catalase activity and the oxygenation of soils. The stimulating e ffect of biostimulators on the activity of this enzyme was demonstrated by Niewiadomska et al. [29] in a study conducted during the cultivation of soybean and white lupin, while St ˛epniewska et al. [83] showed significant positive correlations between soil catalase activity and organic matter content, biomass, oxygen absorption, carbon dioxide secretion, as well as dehydrogenase, glucosidase, amidase, and phosphodiesterase activity.

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As the results of our study on the activity of all enzymes tested in the experiment show, it is noteworthy that in all years of the experiment the highest dehydrogenase activity was observed during the flowering period of the plants, regardless of the biostimulators/fertilizers used. There were similar dependencies observed for the acid and alkaline phosphatases and catalase in 2016 and 2018. There were no such dependencies in 2017, when the highest phosphatase activity was noted during the period of full growth of the plants, i.e., at phases BBCH 35–40, whereas the highest catalase activity was observed after the plants had been harvested.

It is very likely that the significant increase in the activity of the enzymes tested in the experiment during the flowering of pea plants was caused by secretions of the root system. The substances contained in root secretions and in the dying cells of root tissues are rich sources of nutrients and energy for various groups of microorganisms. Hupe et al. [84] proved that the phase of pea development had significant influence on the dynamics of nutrients in the plant's root zone, and consequently, on the soil enzyme activity. The researchers observed strong rhizodeposition of carbon and nitrogen in the period ranging from the emergence of plants to their flowering. They noted significant inhibition of nitrogen rhizodeposition after the flowering period and attributed this e ffect to the displacement of nitrogen in the plants. The reduced amount of organic nitrogen substances in relation to carbon in the rhizosphere after the flowering of the plants explains the reduced metabolic level of the enzymes analyzed in the experiment.
