*3.2. Counts and Diversity of Microorganisms*

The best conditions for microorganism proliferation were offered by the soil from field pea cultivation (Figure 1). The number of organotrophic bacteria in the field pea rhizosphere was 30% higher than in the rhizospheres of winter wheat and winter rape, whereas the number of actinobacteria was 98% and 110% higher than in the respective rhizospheres. The total bacteria count in the rhizosphere of winter wheat, winter rape, and field pea was not necessarily reflected in the proliferation rate and diversity of particular microorganisms. The analysis of the colony development index (CD) values demonstrated the fastest development of organotrophic bacteria in the rhizosphere of filed pea, and

the fastest development of actinobacteria in the rhizosphere of winter wheat. Within the first four days, as many as 87% of the total organotrophs and 49% of the total actinobacteria grew in the soil from winter wheat cultivation (Figure 2). The values of the ecophysiological diversity index (EP) determined for organotrophic bacteria ranged from 0.887 (winter wheat rhizosphere) to 0.715 (field pea rhizosphere), and those calculated for actinobacteria from 0.843 (field pea rhizosphere) to 0.741 (winter rape rhizosphere). Analyses of CD and EP values allowed us to conclude that regardless of the plant species, higher values of both indices were determined for organotroph bacteria than for actinobacteria (CD = 34.27 vs. CD = 22.50; EP = 0.82 vs. EP = 0.79, respectively).

**Figure 1.** Microbiological properties of soil sown *Triticum aestivum* (T), *Brassica napus* (B), Pi*sum sativum* ssp. *arvense* (P); (**a**) count of soil bacteria; (**b**) physiological diversity index of bacteria (EP); (**c**) colony development index (CD). Homogeneous groups denoted with letters (a, b, c) were calculated separately for each microorganism. Org—organotrophic bacteria, Act—actinobacteria.

The crop and its cultivation regimes have a significant impact on the soil microbiome. The prevailing phylum in the rhizosphere of all plants turned out to be *Proteobacteria*, which accounted for 33.05% in the soil from winter rape cultivation and 35.24% in the soil from winter wheat (Figure 3). Other phyla identified in all soils were *Actinobacteria* and *Firmicutes.* The greatest differences in OTU numbers were determined in the case of phylum *Actinobacteria*, i.e., the OTU number in the soil from field pea cultivation was higher by 6.39% than in the soil from winter wheat cultivation, whereas the OTU number in the soil from winter rape cultivation was higher by 4.87% than in the soil from winter wheat cultivation.

**Figure 2.** The growth and development index (Ks); (**a**) organotrophic bacteria; (**b**) actinobacteria.

When comparing the effects of the discussed plant species on individual bacterial classes, it was found that the number of *Actinobacteria* OTUs determined in the field pea rhizosphere was higher by 6.82% than in the rhizosphere of winter wheat, and by 3.15% compared to the rhizosphere of winter

rape (Figure 4). The second largest phylum was that of *Alphaproteobacteria,* whose OTU abundance was similar in soils from the cultivation of all of the studied plants.

**Figure 4.** The relative abundance of dominant class bacteria in soil. Data on the number of readings greater than 1% of all OTUs. B—*Brassica napus*; T—*Triticum aestivum*; P—*Pisum sativum* ssp. *arvense*.

The greatest OTU abundance in order of rank, on average, and regardless of the cultivated plant species, was as follows: *Actinomycetales* (34,738 OTU) classified in the class *Actinobacteria*, phylum Actinobacteria; *Bacillales* (8083 OTU) classified in the class *Bacilli*, phylum *Firmicutes*; *Sphingomonadales* (11846 OTU); *Rhizobiales*(6897 OTU); *Rhodospirillales*(5897 OTU) classified in the class *Alphaproteobacteria*, phylum *Proteobacteria*; and for *Xanthomonadales*(5154) classified in the class *Gammaproteobacteria*, phylum *Proteobacteria* (Figure 5). Differences in the OTU abundance of particular classes in the soils from

the cultivation of field pea, winter wheat, and winter rape usually did not exceed 3%. An exception was the order *Actinomycetales*, whose OTU number in the soil from field pea cultivation was higher by 6.94% than in the soil from winter wheat cultivation, and by 3.25% than in the soil from winter rape cultivation.

**Figure 5.** The relative abundance of dominant order bacteria in soil. Data on the number of readings greater than 1% of all OTUs. B—*Brassica napus*; T—*Triticum aestivum*; P—*Pisum sativum* ssp. *arvense*.

The cultivation of plants modified the soil microbiome also at the family level (Figure 6). In the soil sown with winter wheat, the highest numbers of OTUs were found for the following families:

**Figure 6.** The relative abundance of dominant family bacteria in soil. Data on the number of readings greater than 1% of all OTUs. B—*Brassica napus*; T—*Triticum aestivum*; P—*Pisum sativum* ssp. *arvense*.

*Paenibacillaceae* (4.81%), *Xanthomonadaceae* (4.43%), and *Rhodospirillaceae* (3.71%), and in the soil from winter rape cultivation, for the families: *Nocardioidaceae* (10.37%), *Sphingomonadaceae* (7,40%), *Paenibacillaceae* (3,70%), *Gemmatimonadaceae* (3,31%), *Intrasporangiaceae* (3.06%), and *Xanthomonadaceae* (3.01%); and finally, in the soil from field pea cultivation, for the families: *Nocardioidaceae* (9.36%), *Sphingomonadaceae* (7.08%), and *Paenibacillaceae* (3.43%). At this taxonomic level, the greatest differences in the effects of individual plant species were noticeable in the abundance of OTUs from the *Nocardioidaceae*.

In the soils from the cultivation of winter rape and field pea, there were by 7.93% and 6.91% more OTUs, respectively, than in the soil from winter wheat cultivation. It is worthy of notice that the highest number of genera were classified in the soil sown with winter wheat, and the lowest in the soil sown with winter rape (Figure 7). In the soils from winter rape and field cultivation, the prevailing genus turned out to be the *Nocardioides*, which accounted for 7.45% and 6.63% of all identified bacteria, respectively, whereas in the soil from winter wheat cultivation, it was the *Kaistobacter* genus (6.32%).

**Figure 7.** The relative abundance of dominant genus bacteria in soil. Data on the number of readings greater than 1% of all OTUs. B—*Brassica napus*; T—*Triticum aestivum*; P—*Pisum sativum* ssp. *arvense*.
