*3.3. Soil Enzymatic Activity*

Soil enzymatic activity analysis, which was closely related to the plant development phase (BBCH) significantly influenced the level of dehydrogenase activity in the soil. In all study years, the lowest value was recorded during the emergence of the plants, while the greatest value was observed at the beginning of the flowering phase (Table 15).

**Table 15.** The influence of biostimulator and fertilizer applications on dehydrogenase activity (μmol triphenyl formazane (TPF) 24 h−<sup>1</sup> g<sup>−</sup>1dm of soil.


Different letters denote significant differences at level α = 0.05, (1) control—no biostimulators or foliar fertilizers applied to the plants; (2) Tytanit; (3) Optysil; (4) Metalosate potassium; (5) Rooter; (6) Bolero Mo; (7) Adob Zn IDHA; (8) Adob B; (9) Adob 2.0 Mo.

In addition, the greatest dehydrogenase activity was observed in all years after Bolero Mo application (compared to the control). In contrast, the lowest value was noted after the application of Optysil foliar fertilizer in 2016 and 2018, as well as in 2017 after the application of the Rooter biostimulator. It was also observed across all study years that, dehydrogenase activity was always greater after the application of the treatments (compared to the control) in the second term of the analysis, when the plants were in full vegetation phase. Similar relationships were also observed during the flowering period for most treatments, however, reduced activity was noted after the application of Optysil in 2016, Adob B in 2017, and Optysil, Metalosate potassium or Adob Zn IDHA in 2018.

Analyses of acid phosphatase activity showed that the function of this enzyme was also closely related to the developmental phase of the plant, and its greatest activity was observed during the third term of analysis; at the beginning of the flowering phase in 2016 and 2018 (Table 16). At this time, a greater level of enzyme activity was only observed (compared to the control) after the application of Adob B foliar fertilizer. In 2017, the greatest values were recorded after the application of Metalosate potassium during the second term of analyses (the development phase BBCH 35–40). It should be noted that the lower level of activity of phosphatase (compared to the control) at the beginning of the flowering phase (i.e., when demand for phosphorus is greatest) was observed after the application of most of the treatments, Specifically, the lowest activity level was observed after the application of the Bolero Mo biostimulator in 2016 and 2017, and the foliar fertilizer Adob Zn IDHA in 2018.


**Table 16.** The influence of biostimulator and fertilizer applications on acid phosphatase activity μmol (*p*-nitrophenol) PNP h−<sup>1</sup> g<sup>−</sup>1dm of soil.

Different letters denote significant differences at level α = 0.05, (1) control—no biostimulators or foliar fertilizers applied to the plants; (2) Tytanit; (3) Optysil; (4) Metalosate potassium; (5) Rooter; (6) Bolero Mo; (7) Adob Zn IDHA; (8) Adob B; (9) Adob 2.0 Mo.

The greatest alkaline phosphatase activity level was observed in 2016 (Table 17). Similar relationships were observed as with acid phosphatase. In 2016 and 2018, the greatest activity was also noted at the beginning of the flowering phase (BBCH 59), while the greatest activity in 2017 was observed during the BBCH 35–40 phase.


**Table 17.** The influence of the biostimulators and fertilizers on alkaline phosphatase activity μmol (*p*-nitrophenol) PNP h−<sup>1</sup> g<sup>−</sup>1dm of soil.

Different letters denote significant differences at level α = 0.05, (1) control—no biostimulators or foliar fertilizers applied to the plants; (2) Tytanit; (3) Optysil; (4) Metalosate potassium; (5) Rooter; (6) Bolero Mo; (7) Adob Zn IDHA; (8) Adob B; (9) Adob 2.0 Mo.

The lowest level of activity occurred during the emergence of the plants (BBCH 5–10). In both 2016 and 2018, the highest phosphatase activity level was observed after the application of Adob B fertilizer; 3.299 and 0.225 μmol PNP h−<sup>1</sup> g<sup>−</sup>1dm of soil, respectively, while in 2017 the greatest value was observed after the application of Metalosate potassium; 0.485 μmol PNP h−<sup>1</sup> g<sup>−</sup>1dm of soil (Table 17).

The greatest catalase enzyme activity value was also recorded in 2016 and 2018 at the beginning of flowering phase. In 2017, the highest metabolic activity occurred in the period after plant harvest (Table 18). The biostimulators and foliar fertilizers used in most of the experimental treatments stimulated the level of catalase activity in relation to the control treatment. The highest level of catalase enzyme activity was recorded after the application of the Tytanit biostimulator in 2016, and after the application of the Adob B foliar fertilizer in 2018. In 2017, the greatest activity was observed after the applications of Tytanit and Bolero Mo (Table 18).



Different letters denote significant differences at level α = 0.05, (1) control—no biostimulators or foliar fertilizers applied to the plants; (2) Tytanit; (3) Optysil; (4) Metalosate potassium; (5) Rooter; (6) Bolero Mo; (7) Adob Zn IDHA; (8) Adob B; (9) Adob 2.0 Mo.

The BIF value was determined based on dehydrogenase and catalase activity. A high value was observed during the flowering phase of the plants compared to the emergence phase, in all experimental treatments. However, the highest level was recorded in 2016 and 2017 after the harvest of the plants; 33.907 and 33.306, respectively (Table 19). In 2018, the highest BIF value was 55.813 and was observed at the beginning of the flowering phase.

Principal Component Analysis (PCA) in Figure 2a–c shows that the applied biostimulators/fertilizers di ffered in their influence on the agronomic and microbiological parameters in individual years. Figure 2d illustrates the influence of all the factors under analysis (treatment) on the indicators in a given year. In 2016 all the treatments applied in the experiment had strong positive influence on the activity of catalase (CAT) and alkaline phosphatase (PAL), as compared with the other parameters. In 2017 the biostimulants/fertilizers significantly influenced the BNF, H, PDN, Y and BIF, whereas in 2018 they influenced only the dehydrogenase activity (DHA). PCA explained a significant part of the variability in each study year, and also over the three-year study period. In 2016, approximately 60% of the total variability was explained by the first two principal components (Axis 1: 30.4%, Axis 2: 28.5%) (Figure 2a). It was observed that, to a greater or lesser extent, each of the biostimulators/fertilizer treatments a ffected the studied parameters (Figure 2a). In addition, there was a

strong correlation between the basic soil biochemical parameters studied here [dehydrogenase activity (DHA), alkaline phosphatase activity (PAL), biological index fertilizer BIF, acid phosphatase activity (PAC) and soil moisture (M), and the strong e ffect of the applied Adob B (t8) on the parameters indicated above. In turn, BNF was closely correlated with catalase activity (CAT), LAI and share of seed in whole plant weight (SSPW). In 2016, the foliar fertilizers Optysil (t3) and Metalosate Potassium (t4), and the Rooter biostimulator (t5) had the greatest impact on the above-mentioned parameters.


**Table 19.** The influence of the biostimulators and fertilizers on the biological indicator of fertility (BIF).

Different letters denote significant differences at level α = 0.05, (1) control—no biostimulators or foliar fertilizers applied to the plants; (2) Tytanit; (3) Optysil; (4) Metalosate potassium; (5) Rooter; (6) Bolero Mo; (7) Adob Zn IDHA; (8) Adob B; (9) Adob 2.0 Mo.

In 2017, PCA explained approximately 50% of the variation (Axis 1: 25%, Axis 2: 24.4%) (Figure 2b). A strong relationship between BIF, catalase activity and share of seed in whole plant weight (SSPW) was noted. The level of activity of these parameters was influenced by the Tytanit and Rooter biostimulators. In 2017, a strong relationship was also observed between biochemical soil activity parameters and agronomic parameters, such as plant dry mass (PDM), seed weight per plant (SW), seed moisture (M) and the physiological parameter Fv/Fm.

In 2018, as in 2016, all tested parameters were more or less a ffected by the applied foliar fertilizer and biostimulator treatments. In that year, PCA explained almost 60% of the total variability (Axis 1: 31.4%, Axis 2: 26%) (Figure 2c). The studied biochemical soil parameters (DHA, PAL, PAC, BIF) were closely correlated with each other. The most important influence on soil metabolism was the

application of Bolero B (t6), Adob B and Adob 2.0 Mo fertilizers. In turn, Adob Zn IDHA and Adob 2.0 Mo. foliar fertilizers influenced agronomic parameters, such as yield (Y) and seed moisture (M).

**Figure 2.** Principal components analysis for pea fertilization treatments in: (**a**) 2016, (**b**) 2017, (**c**) 2018, (**d**) 2016–2018 years. Abbreviations: Y—Yield, M—Seed moisture %, TSW—1000 seed weight, SW—Seed weight per plant, SW/P—seed weight in 1 pod PW—Pod weight per plant, SSPW—Share of seed in whole plant weight, H—plant height, CCI—chlorophyll content index, Fv/Fm—maximum photosynthetic efficiency of PSII, PDM—plant dry mass, LAI—leaf area index, BIF—biological index of fertility, BNF—biological nitrogen fixation, DHA—dehydrogenase activity, CAT-catalase activity, PAL—alkaline phosphatase level, PAC—acid phosphatase level. Treatment: t1—control—no biostimulators, t2—Tytanit; t3—Optysil; t4—Metalosate potassium; t5—Rooter; t6—Bolero Mo; t7—Adob Zn IDHA; t8—Adob B; t9—Adob 2.0 Mo; PC1—first principal component; PC2—second principal component.

PCA for each pea fertilization treatment in all the study years is shown in Figure 2d, and highlights the differentiated effect of the applied fertilizers/biostimulator treatments on the tested soil biochemical and agronomic parameters during the study period. In 2018, regardless of the fertilization treatment, a high level of dehydrogenase activity was observed, while in 2016, a high level of phosphatase and catalase activity was observed.

**Figure 3.** Relationship between the biometric, physiological characteristics of the pea plants and enzymatic activity in the soil, biological index of fertility (BIF), and nitrogenase activity. Abbreviations: M—Seed moisture %, SW/P—seed weight in 1 pod, LAI—leaf area index, PW—Pod weight per plant, BIF—biological index of fertility, H—plant height, PDM—plant dry mass, SW—Seed weight per plant, Y—Yield, TSW—1000 seed weight, SSPW—Share of seed in whole plant weight, CCI—chlorophyll content index, BNF—biological nitrogen fixation Fv/Fm—maximum photosynthetic efficiency of PSII, DHA—dehydrogenase activity, PAC—acid phosphatase level, CAT—catalase activity, PAL—alkaline phosphatase level. Treatment: t1—control—no biostimulators, t2—Tytanit; t3—Optysil; t4—Metalosate potassium; t5—Rooter; t6—Bolero Mo; t7—Adob Zn IDHA; t8—Adob B; t9—Adob 2.0 Mo.

The heat map and dendrogram presented in Figure 3 illustrates the large variation in activity level of the studied parameters and clearly demonstrates the correlation between the biochemical soil activity parameters and the examined agronomic parameters. Taking into account all the parameters tested in the study, the e ffect of the fertilizers/biostimulators was similar in 2016 and 2018, but di ffered in 2017. The research conducted in 2017 indicated that the fertilizer/biostimulator treatments had a stimulating e ffect on plant yield at low soil enzymatic activity (catalase, phosphatase, dehydrogenase). Only BIF, based on the level of metabolic activity of dehydrogenase and catalase, and BNF were highest in 2017. Di fferent relationships were observed in 2016 and 2018, where a correspondingly higher level of biochemical activity in the soil was observed, and agronomic parameters exhibited lower values.

Regardless of the study year, the heat map indicates similar relationships between the soil biochemical activity parameters and the agronomic parameters. Soil fertility index (BIF) was correlated with pod weight per plant (PW) and LAI, as well as with seed weight in one pod (SW/P). In turn, yield (Y) was correlated with 1000 seed weight (TSW) and plant height (H), and BNF was correlated with the share of seed in whole plant weight (SSPW), CCI, and Fv/Fm. Lastly, phosphatase activity was correlated with catalase, dehydrogenase and acid phosphatase activity.
