2.2.1. Bacterial Count

Counts of organotrophic bacteria (Org) and actinobacteria (Act) were determined in individual soil samples from the cultivation of each of the studied plant species (winter wheat, winter rape, field pea) with the serial dilution method, in three replications. Microbial counts were performed according the media and procedure described by Borowik et al. [50]. The composition of the microbiological media was as follows: organotrophic bacteria (Bunt and Rovira medium): agar medium (peptone 1.0 g, yeast extract 1.0 g, (NH4)2SO4 0.5 g, CaCl2, K2HPO4 0.4 g, MgCl2 0,2 g, MgSO4 7H2O 0.5 g, salt Mo 0.03 g, FeCl2 0.01 g, agar 20.0 g, soil extract 250 cm3, distilled water 750 cm3, pH 6.6–7.0; Actinomycetes (Parkinson medium): soluble starch 10.0 g; casein 0.3 g; KNO3 2.0 g; NaCl 2.0 g; K2HPO4 2.0 g; MgSO4·7H2O 0.05 g; CaCO3 0.02 g; FeSO4 0.01 g; agar 20.0 g; H2O 1 dm3; 50 cm3 aqueous solution of nystatin 0.05%; 50 cm3 aqueous solution of actidione 0.05%; pH 7.0. Microorganisms were cultured on petri dishes at a temperature of 28 ◦C. The colony forming units (cfu) were counted every day for ten days using a colony counter. Counts of bacteria and actinobacteria isolated from the rhizosphere of particular plant species allowed us to compute the colony development index (CD), the ecophysiological diversity index (EP), and the microbial growth indexes at specific time intervals (Ks). Descriptions of the indexes and calculations are presented in De Leij et al. [51] and Tomkiel et al. 2015 [52]. The CD and EP were calculated from the following formulas [51]: CD = [N1/1 + N2/2 + N3/3 ... N10/10] × 100, where N1, N2, N3, ...N10 are the sum of ratios of the number of colonies of microorganisms identified on particular days (1, 2, 3, ...10) to the total number of colonies identified throughout the study period, and EP = −Σ(pi·log10 pi), where pi is the ratio of the number of colonies of microorganisms identified on particular days to the total number of colonies identified throughout the study period. KS was determined with the use of the following formula [52]: Ks = (Nx/Nt) × 100, where Ks is the percentage of microbes cultured at specific time intervals, Nx is the number of colonies cultured at two-day intervals counted for ten days, and Nt is the total number of colonies cultured within ten days.

2.2.2. DNA Extraction and Bioinformatic Analysis of Specific Bacterial Taxa

DNA was isolated from the rhizosphere of the three plant species using a "Genomic Mini AX Bacteria+" kit. According to the manufacturer's instructions, the PCR reaction was carried out using Q5 Hotstart Hight-Fidelity DNA Polymerase (NEBNext). In the case of amplicon libraries, the data were pooled and normalized in the final stage of the library preparation. Afterwards, a 16S amplicon sequencing encoding gene was conducted for each DNA sample based on the hypervariable region V3–V4. The selected region was amplified and the library was developed using specific sequences of 341F (5 -CCTACGGGNGGCWGCAG-3 ) and 785R (5 -GACTACHVGGGTATCTAATCC-3 ) primers. The 16S library sequencing was performed on a MiSeq Reporter sequencer ver. 2.6 in the paired-end (PE) technology, 2 × 250 bp, using a v2 Illumina kit. The Illumina 16S Metagenomics workflow with MiSeq Reporter (MSR) software (San Diego, CA, USA) and Greengenes v13\_5 software (South San Francisco, CA, USA) were used [53]. Preparation of the reference database included filtering low-quality, degenerate, and incomplete sequences, and then combining paired sequences based on the reference sequence database. The algorithm Uclust was assigned taxonomy, taking into account the ChimeraSlayer algorithm. Sequencing was performed by the Genomed S.A. Company (Warsaw, Poland).
