*3.1. Grass Yield*

The growth and development of grasses were significantly affected by their species (Figure 1). Generally, regardless of cutting term and grass species, the yield of fodder grasses (T1) was higher by 71.68% on average than that of the lawn grasses (T2). Among the fodder grasses, in the first and third terms of harvest—the greatest biomass yield was obtained from *Lolium* x *hybridum* Hausskn (Lh), whereas in the second term—from *Festuca arundinacea* (Fa). In the case of lawn grasses, the best yield was produced by *Lolium perenne* (Lp) in all three terms of harvest. To sum up, regardless of the harvest date, the largest biomass among fodder grasses was obtained in the case of *Lolium* x *hybridum* Hausskn (Lh), and, among lawn grasses, *Lolium perenne* (Lp).

**Figure 1.** The yield of grasses in g dry matter (d.m.) per pot. T1—fodder grasses; T2—lawn grasses; homogeneous groups denoted with letters (a–d) were calculated separately for every cutting; Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra*.

#### *3.2. Counts and Diversity of Soil Bacteria*

Cultivation of grasses ambiguously modified soil bacteriobiome (Figure 2). Sowing the soil with *Lolium* x *hybridum* Hausskn (Lh) caused a significant increase in the population number of both organotrophs and actinobacteria, whereas cultivation of *Festuca arundinacea* (Fa), *Lolium perenne* (Lp), and *Festuca rubra* (Fr)—only in the population number of actinobacteria. In turn, sowing *Phleum pratense* (Pp), *Lolium perenne* (Lp), *Poa pratensis* (Pr), and *Festuca rubra* (Fr) on soils significantly reduced the proliferation of organotrophic bacteria. Regardless of grass species, but considering their functional character, it was demonstrated that the fodder grasses (T1) increased the count of organotrophic bacteria by 26.57% and that of actinobacteria by 156.49% compared to the control soil (not sown with grasses), whereas the lawn grasses (T2) increased the count of actinobacteria by 47.43% and decreased that of organotrophs by 37.91% compared to the control soil.

**Figure 2.** Count of soil organotrophic bacteria (Org) and actinobacteria (Act), cfu 109 kg−<sup>1</sup> d.m. of soil: (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Homogeneous groups denoted were calculated separately for each microorganisms, groups denoted with letters (a–d) were calculated for the species of grass and groups denoted with letters (x–y) were calculated for the type of grass. C—unsown soil; Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

The positive impact of the fodder grasses on the proliferation of soil microorganisms was not reflected in their ecophysiological diversity index (EP), because cultivation of Lh, Fa, and Pp not only did not increase the EP index of organotrophs but decreased its value by 7.9% on average, and also did not change the EP index of actinobacteria (Figure 3). Also the cultivation of the lawn grasses caused insignificant changes in the value of EP index of organotrophic bacteria, whereas Lp and Fr had the same effect also on actinobacteria. Only Pr decreased EP index of actinobacteria by 8.14%.

Sowing the soil with both fodder and lawn grasses caused a significant decrease in the values of the colony development index (CD) calculated for the organotrophic bacteria (Figure 4). A decrease in CD value calculated for organotrophic bacteria ranged from 27.80% (Fa) to 36.88% (Lh) in the case of fodder grass species, and from 30.02% (Fr) to 37.73% (Pr) in the case of lawn grass species. A lesser decrease in CD value was observed in the case of actinobacteria, i.e., from 3.97% (Fa) to 16.26% (Pp) in the soils used to cultivate fodder grasses, and from 6.23% (Fr) to 16.93% (Pr) in the soils sown with lawn grasses.

**Figure 3.** Ecophysiological diversity index (EP) of organotrophic bacteria (Org) and actinobacteria (Act): (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Homogeneous groups denoted were calculated separately for each microorganisms, groups denoted with letters (a–c) were calculated for the species of grass and groups denoted with letters (x) were calculated for the type of grass. C—unsown soil; Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

**Figure 4.** Colony development index (CD) of organotrophic bacteria (Org) and actinobacteria (Act): (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Homogeneous groups denoted were calculated separately for each microorganisms, groups denoted with letters (a–d) were calculated for the species of grass and groups denoted with letters (x–y) were calculated for the type of grass. C—unsown soil; Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

At all plots, the prevailing phyla included *Proteobacteria* and *Actinobacteria* (Figure S1). In the control soil, *Proteobacteria* accounted for 28.78% of total bacteria, whereas this fraction was 38.56% in the soil sown with fodder grasses and 32.93% in that sown with lawn grasses. In the control soil, *Actinobacteria* accounted for 23.55%, in the soil sown with fodder grasses, for 19.77%, and, in the soil sown with lawn grasses, for 21.32% of total bacteria. The OTU number of *Proteobacteria* in the soil sown with fodder and lawn grasses was higher by 9.8% than in the control soil, whereas the OTU number of *Actinobacteria* decreased by 3.4% in the soil sown with fodder grasses and by 2.9% in soil sown with lawn grasses, compared to the control soil.

Apart from *Proteobacteria* and *Actinobacteria*, taxa identified at the phylum level included: *Acidobacteria*, *Chloroflexi*, *Gemmatimonadetes*, *Firmicutes* and *Planctomycetes*, *Bacteroidetes*, *Verrucomicrobia*, *Cyanobacteria*, as well as OD1 and TM7 (Figure 5A,B). The OTU number of bacteria classified as 'others' reached 4.3% in the control soil, 3.38% in the soil sown with fodder grasses, and 3.80% in the soil

sown with lawn grasses. Cultivation of all grass species facilitated the proliferation of *Proteobacteria*, *Bacteroidetes*, and *Verrucomicrobia* bacteria, which resulted in higher OTU numbers of these bacteria compared to the control soil. The OTU number of *Actinobacteria* increased only in the sample of soil sown with *Lolium* x *hybridum* Hausskn (Lh) and *Lolium perenne* (Lp); the OTU number of *Chloroflexi* increased in the soils sown with *Lolium perenne* (Lp), *Poa pratensis* (Pr), *and Festuca rubra* (Fr); the OTU number of Firmicutes increased in the soil sown with *Poa pratensis* (Pr), whereas the OTU number of *Planctomycetes* rose in the soil sown with *Poa pratensis* (Pr), and *Festuca arundinacea* (Fa).

**Figure 5.** Bacterial communities at the phylum level (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Abundances <1% are gathered into the category "other". C—unsown soil; T1—average bacteria abundance in soils sown with fodder grasses; T2—average bacteria abundance in soils sown with lawn grasses. Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

Both, in the control soil and soils sown with grasses, the prevailing class of bacteria was *Alphaproteobacteria,* which in the control soil accounted for 19.32% of total bacteria, in the soil sown with fodder grasses—for 19.99%, and in the soil sown with lawn grasses—for 16.50% (Figure S2). The second prevailing class was *Actinobacteria,* which in the structure of all bacterial classes represented 12.98% in the control soil, 13.88% in the soil used to cultivate fodder grasses, and 13.98% in the soil used to grow lawn grasses. Sowing the soils with fodder grasses caused the greatest changes in the abundance of bacterial classes: *Gammaproteobacteria, Betaproteobacteria*, and *Acidobacteria*-6, whose OTU numbers increased by 6.37%, 2.64%, and 1.99%, respectively, compared to the control soil.

Also, sowing the soil with lawn grasses increased OTU numbers of these bacteria in the range from 3.56% (*Gammaproteobacteria*) to 1.92% (*Acidobacteria*-6). Differences in changes in the bacterial structure were also noticeable between the rhizospheres of the fodder and lawn grasses. The OTU number of *Alphaproteobacteria* in the soils sown with fodder grasses was higher by 3.49% and that of *Gammaproteobacteria* by 2.82% than in the soils sown with lawn grasses. Regardless of grass species and functional designation, apart from the two prevailing classes *Alphaproteobacteria*

and *Actinobacteria,* all soils contained also (in a descending order of OTUs): *Betaproteobacteria*, *Thermoleophilia*, *Gammaproteobacteria*, *Bacilli*, *Acidobacteria*-6, *Gemmatimonadetes*, *Deltaproteobacteria*, *Planctomycetia*, *Solibacteres*, *Acidimicrobiia*, *Acidobacteriia*, Gemm-1, C0119, Ellin6529, *Chloracidobacteria*, *Clostridia, Phycisphaerae, Saprospirae*, *Ktedonobacteria*, *Pedosphaerae, ZB2, Thermomicrobia, Spartobacteria, Sphingobacteriia, Verrucomicrobiae, Chloroflexi, Chloroplast, TM7-1, Flavobacteriia,* and *Nostocophycideae* (Figure 6A,B).

**Figure 6.** Bacterial communities at the class level (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Abundances <1% are gathered into the category "other". C—unsown soil; T1—average bacteria abundance in soils sown with fodder grasses; T2—average bacteria abundance in soils sown with lawn grasses. Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

The effect of grasses on soil bacteriobiome was also noticeable at the order level. Among the 37 identified orders with OTU numbers above 1%, the greatest abundance was demonstrated for bacteria classified to *Actinomycetales*, *Sphingomonadales*, and *Rhizobiales* (Figure S3). When comparing effects of various functional types of grasses, it was found that sowing the soils with fodder grasses increased OTU numbers of *Xanthomonadales* by 4.67%, *Sphingomonadales* by 3.18%, *Burkholderiales* by 2.41%, *Solibacterales* by 2.04%, and *Actinomycetales* by 1.12%, whereas sowing the soils with lawn grasses increased OTU numbers of *Burkholderiales* by 2.60%, *Xanthomonadales* by 1.85%, *Alteromonadales* by 1.39%, *Actinomycetales* by 1.23, and *Rhizobiales* by 1.02%, compared to the nonsown control soil.

The highest OTU number of *Actinomycetales* bacteria was determined in the soils sown with *Lolium* x *hybridum* Hausskn (Lh) and *Lolium perenne* (Lp); whereas that of *Sphingomonadales*—in the soils sown with *Lolium* x *hybridum* Hausskn (Lh), *Festuca arundinacea* (Fa), *Festuca rubra* (Fr), and *Lolium perenne* (Lp) (Figure 7A,B). Higher OTU numbers were also determined in the soils with growing grasses than in the control soil for the bacteria from the following orders: *Burkholderiales, Xanthomonadales, Saprospirales,* and *Sphingobacteriales*.

**Figure 7.** Bacterial communities at the order level (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Abundances <1% are gathered into the category "other". C—unsown soil; T1—average bacteria abundance in soils sown with fodder grasses; T2—average bacteria abundance in soils sown with lawn grasses. Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*

Differences in the abundance of bacterial populations were also observed at the family level. Compared to the control soil, the greatest changes in the structure of bacteria classified to families, after soil sowing with both fodder and lawn grasses, occurred in the families of *Gaiellaceae* (decrease by 3.16% and 2.57%, respectively), *Koribacteraceae* (decrease by 2.77% and 2.54%), *Intrasporangiaceae* (increase by 2.14% and 1.38%), *Xanthomonadaceae* (increase by 3.54% and 1.13%), and *Comamonadaceae* (increase by 2.61% and 2.43) (Figure S4).

When comparing effects of individual grass species on OTU number of bacteria classified to families, it can be concluded that they were inexplicit (Figure 8A,B). All species increased OTU numbers of the following families: *Intrasporangiaceae, Bradyrhizobiaceae, Xanthomonadaceae, Sinobacteraceae, Comamonadaceae, Pirellulaceae, Chitinophagaceae, Ellin5301, Nostocaceae,* and *Rhodocyclaceae,* but decreased OTU numbers of *Gaiellaceae, Rhodospirillaceae, Koribacteraceae, Solibacteraceae, Acetobacteraceae, Pseudonocardiaceae, Paenibacillaceae, Frankiaceae,* and *Chthoniobacteraceae*. Noteworthy is the fact that

sowing the grasses onto soils resulted in the appearance of families: *Alteromonadaceae, Methylophilaceae, Flavobacteriaceae,* and *Verrucomicrobiaceae*, that were not identified in the control soil.

**Figure 8.** Bacterial communities at the family level (**A**) depending on the species of grass, (**B**) depending on grass type (fodder or lawn). Abundances <1% are gathered into the category "other". C—unsown soil; T1—average bacteria abundance in soils sown with fodder grasses; T2—average bacteria abundance in soils sown with lawn grasses. Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra*.

Considering OTU numbers above 1%, only 11 bacterial genus were identified in the control soil not sown with grasses, whereas 17 genus in the soil sown with Pp and Pr, 16 genus in the soil sown with Fr and Lp, 15 genus in the soil sown with Fa, and 14 genus in the soil sown with Lh (Figure 9). The contribution of the identified bacteria in the genus structure ranged from 13.86% in the soil sown with Pr to 20.34% in the soil sown with Lh. The genus *Kaistobacter* was found to predominate on all plots. Regarding OTU number, it was followed by *Rhodoplanes* in the control soil and soil sown with Pr, by *Terracoccus* in the soil sown with Lh and Lp, by HB2-32-21 in the soil sown with Fa and Fr, and by *Flavobacterium* in the soil sown with Pp. Compared to the soils overgrown with grasses, no OTUs of the following genera were identified in the control soil: *Arenimonas, Dechloromonas, Flavobacterium, Methylotenera,* and *Mycoplana.*

The analysis of values of Shannon and Simpson diversity indices points to a richer microbiome of the soils sown with grasses compared to the control soil (Table 4). The greatest abundance among the fodder grasses was found in the rhizosphere of Pp, and, among the lawn grasses, in the rhizosphere of Pr.

**Figure 9.** The operational taxonomic units (OTUs) structure of identified genus of bacteria in the total number of OTUs. C—unsown soil; T1—fodder grasses; T2—lawn grasses. Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*, Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*


**Table 4.** Shannon and Simpson indices calculated from abundance of OTU.

Homogeneous groups denoted were calculated separately for each taxon groups denoted with letters (a–f) were calculated for the species of grass and groups denoted with letters (x–z) were calculated for the type of grass. C—unsown soil; Lh—*Lolium* x *hybridum* Hausskn; Fa—*Festuca arundinacea*; Pp—*Phleum pratense*; Lp—*Lolium perenne*, Pr—*Poa pratensis*; Fr—*Festuca rubra.*
