1.1. Collection of plant material and isolation of bacteria from rhizosphere
Five rape plants (variety of Arazzo) were collected from a field cultivated with rape in Górsk (Poland 53°01'46.1"N 18°26'59.4"E). Firstly, the soil surrounding the root was removed, then the separated roots of the plant were washed with sterile distilled water to remove the remaining soil. The cleared roots were cut with a sterile scalpel into 1-2 cm pieces and prepared in the appropriate decimal dilutions. Subsequently, surface spreading was carried out on nutrient agar medium (Biomaxima, Poland) supplemented with (40 µg/ml) antifungal agent amphotericin B to prevent the growth of mold and fungi. The plates were incubated at 28 °C for 72 hours. Fifty colonies were selected and used for further study. The selection was made based on their color, shape and size.
1.2. Determination of PGPR traits
1.2.1. Determination of IAA production
To determine the amounts of indole-3-acetic acid (IAA), a colorimetric technique was performed with Salkowski's method using Van Urk Salkowski reagent (Ehmann 1977). The isolates were grown in medium containing (g/l): peptone 5.0; yeast extract 3.0; L-tryptophan 1.0, and were incubated at 28°C for 4 days. After incubation, the cultures were centrifuged (10 000 rpm/ 10 min). The supernatant was reserved and 1 ml was mixed with 2 ml of Salkowski's reagent (2 % 0.5 FeCl3 in 35 % HCLO4 solution), then kept in the dark. After 30 minutes of incubation, IAA concentration was measured using Hitachi U-2500 spectrophotometer at a wavelength of 530 nm, relative to a standard curve.
1.2.2. Determination of ACC deaminase activity
The ACC deaminase activity was determined by the modified Honma and Shimomura (1978) method. In the first step, 10 ml of liquid medium (nutrient broth) was inoculated with isolated bacteria and incubated at 30 °C for 24 hours with shaking. The samples centrifuged (6000 rpm / 10 min / 4 °C). The pellet was suspended in 5 ml of DF fluid medium (Dworkin and Foster 1958) and centrifuged. Then, the pellets were resuspended in 5 ml DF with 30 μl ACC (0.5M). The tests were incubated under the same conditions as above. The samples were centrifuged twice (6000 rpm / 10 min / 4 °C) washed with 5 ml of 0.1M Tris-HCl (pH 7.6). Then, the pellet was suspended in 1 ml of 0.1M Tris-HCl (pH 7.6) and placed in eppendorff (1.5 ml). Samples were centrifuged (10 000 rpm / 5 min) once again and after separation of the supernatant, the bacteria were suspended in 600 μl 0.1M Tris-HCl (pH 8.5). Then 30 μl of toluene was added to destroy the cells and release the enzyme. The resulting slurry (200 μl) was taken for further stages. The remaining volume was used to determine the content of the bacterial protein. 20 μl substrate – ACC was added to the mixture and the samples were incubated at 30 °C for 30 min. After incubation, 1 ml HCl (0.56M) was added to the tubes and the samples were centrifuged (10 000 rpm / 5 min). After separation of the remaining lysed cells, 1 ml of supernatant and 800 μL of HCl (0.56M) and 300 μl of 2,4-dinitrophenylhydrazine reagent (0.2 %). Samples were incubated again at 30 °C for 30 min in a water bath. In the last step of the reaction, 2 ml NaCl (2M) was added. Positive samples that acquire a red color, which intensity is proportional to the amount of product (α-ketobutyrate), were measured spectrophotometrically (absorbance at λ = 540 nm). The ACC deaminase activity was reported as the amount of α-ketobutyrate (mM) per hour per milligram of bacterial protein. Protein was determined by the Bradford method (1976).
1.2.3. Determination of phosphate solubilization
Phosphate (P) solubilization was determined on Pikovaskaya’s agar medium (g/l: glucose 10; Ca3(PO4)2 5; (NH4)2SO4 0.5; NaCl 0.2; MgSO4 x 7H2O 0.1; KCl 0.1; yeast extract 0.5; MnSO4 and FeSO4 trace; agar 15; pH 7.0). Isolates were transferred on the medium and incubated for 7 days at 26 °C. Production of clear halos around the colonies indicated a positive result for phosphate solubilization (Ahmad et al. 2008).
1.2.4. Determination of siderophores production
Siderophores were assayed according to Schwyn and Neilands (1987) method. Tested strains were transferred on Chrome Azurol S agar medium (Alexander and Zuberer 1991), and incubated at 26 °C for 4 days. The bacteria, which produce siderophores, gave orange zones around the colonies. Siderophores production index was evaluated as the ratio between the halo diameter and the diameter of the colony growth.
1.2.5. Determination of salicylic acid
Production of salicylic acid (SA) was determined according to Meyer et al. (1992). The tested bacteria were grown in 100 ml of medium containing (g/l): succinic acid 4.0; K2HPO4 6.0; KH2PO4 3.0; (NH4)2SO4 1.0; MgSO4 x 7H2O 0.2. Incubation was performed at 26 °C for 48 hours on a rotary shaker (120 rpm). After incubation, the culture was centrifuged (6000 rpm/5 min) and 4 ml of the supernatant was acidified with 1N HCl to pH 2.0 and SA was extracted in CHCl3 (2×2 ml). To the pooled CHCl3 phases, 4 ml of distilled water and 5 µl of 2M FeCl3 were added. The absorbance of the purple iron-SA complex, in the aqueous phase was measured at 527 nm in a Hitachi F 2500 spectrophotometer. A standard curve was prepared with SA dissolved in succinate medium. The quantity of SA in the culture was expressed as µg/ml.
1.2.6. Determination of ammonia production
The production of ammonia (NH3) was detected in the nutrient broth (Biomaxima, Poland). After incubation at 26 °C for 72 hours, the Nessler’s reagent (0.5 ml) was added to each tube. The orange color of the suspension indicated the presence of ammonia. All analyses were made in triplicate.
1.2.7. Determination of HCN production
Hydrogen cyanide (HCN) production was detected according to Lorck (1948) method. The bacterial isolates were streaked on nutrient agar amended with glycine (4.4 g/l). Filter paper discs soaked in picric acid (0.5 %) prepared in Na2CO3 solution (2 %) were kept inside the lid of the Petri plates. Petri dishes were sealed with parafilm and incubated in the dark at 28 °C for 3–4 days. The paper discs changed color from yellow to brown indicating HCN production.
1.2.8. Determination of fungal cell lytic enzymes production
The studies examined the production of enzymes associated with their antifungal activity. Chitinase production of all bacterial isolates was studied using the medium composed (g/l): peptone 1.0, FeSO4 × 7H2O 0.1, iron gluconate 0.1, yeast extract 0.1, colloidal chitin – 7.0 g dry mass, agar 15.0 g. After 14 days of incubation at a temperature of 22 °C, the bright halo diameter around the colonies was measured, as the ability of the bacteria to produce chitinase (Swiontek Brzezinska et al. 2013). The colloidal chitin was prepared according to Lingappa and Lockwood (1962).
Moreover, three bacterial isolates B14, B16 and B19 were further used to determine quantitative assessment of chitinase and 1,3-b-glucanase activity. Chitinase activity was studied using the medium composed of (g/l): peptone 1.0, yeast extract 0.1, colloidal chitin – 2.0 g. pH of medium was 6.8–7.2. After 4 days of incubation at a temperature of 26 °C, the chitinase activity was measured (Swiontek Brzezinska et al. 2013). The fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide [4-MU-GlcNAc] was used to detect chitinase activity (Sigma-Aldrich). The reaction mixture contained: 1 ml crude enzymes, 0.125 ml substrate 4MU- GlcNac solution (the final concentration in a sample was 50 µM/l and 0.125 ml of phosphate buffer (50 mM, pH 7). The control sample, prior to addition of the substrate, was treated with 0.1 ml solution of HgCl2 in order to deactivate the enzymes present in the sample (final concentration: 4 mM/L. After incubation (1 hour, 40 °C), enzymatic reactions were stopped by adding HgCl2. The released 4-methylumbelliferone (MU) was measured fluorimetrically (318 nm excitation and 445 nm emission) using the Hitachi F 2500 spectrofluorometer. One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 nM MU/ml.
1,3-b-glucanase was measured by mixing 100 µl of sample, 50 µl of 50 mM phosphate buffer (pH 5.9), containing 1 % laminarin (Sigma). After incubation (1 hour, 40 °C), the reducing sugar produced was determined by Miller (1959). One unit of enzyme activity (U) was defined as the amount of enzyme that produced 1 µmol of reducing sugar min-1.
1.3. Determination of antifungal activity
Bacteria cultures were tested for growth inhibitory effect on the mycelium growth of Fusarium solani 25, Fusarium oxysporum 872, Fusarium culmorum 2333, Botrytis cinerea 873, Alternaria alternata 783, Phytophthora megasperma 404, Phytophthora cactorum 1925, Penicillium verrucosum 1681, Colletrotrichum acutatum 2153, Sclerotina sclerotium 2242, and Phoma lingam 2284. Molds were obtained from the Pathogens Bank of the Institute of Plant Protection in Poznań (Poland). Molds were grown on PDA at 26 °C for 96 hours. Then the cultures were stored at 4°C. The tested bacterial strains were grown 24 hours at 28 °C in PDA. In the next step, agar disc of each molds (with diameter of 5 mm) was placed in the agar discs containing the spread bacteria. Cultures were incubated at 26 °C for 7 days. After incubation, the diameter of the fungal mycelium was measured. At the same time, for each molds species control experiments were made (without bacteria). Inhibition rate was calculated from the following formula (Oldal et al. 2002):
Inhibition (%) = C-B/C, where C is the diameter (mm) of the control molds and B is the diameter of the molds grown in the presence of the bacteria. Each assay was performed in triplicate.
1.4. Identification of the rhizobacterial strains
The three rhizobacterial strains, B14, B16, and B19, were identified based on the analysis of the gene sequence encoding 16S rRNA. Amplification of the 16S rRNA gene was carried out with the 27F and 1401R primers (Watanabe et al. 1990). The matrix for the PCR was the genomic DNA separated from bacterial cells harvested during logarithmic growth stage by the method according to Kutchma et al. (1998). After purification, the PCR product was sequenced by the DNA Sequencing and Oligonucleotides Synthesis Laboratory at IBB (PAN—Polish Academy of Sciences, Poland). The obtained nucleotide sequence was identified using the EzBioCloud (https://www.ezbiocloud.net/ Yoon et al. 2017).
1.5. Whole genome sequencing and analysis
Genomic DNA extracted from the three PGPR’s isolates, B14, B16, and B19 was sent for whole-genome sequencing at the University of Birmingham, United Kingdom. Whole genome libraries were prepared using a Nextera XT Library Prep kit (Illumina, San Diego, USA) according to the manufacturer’s protocol with the following modifications: 2 ng of DNA instead of 1 ng was used as input, and PCR elongation time was increased to 1 min from 30 s. DNA quantification and library preparation were carried out on a Hamilton Microlab automated liquid handling platforms. Pooled libraries were quantified using the KAPA Library Quantification Kits for Illumina platforms on a Roche LightCycler 96 real-time PCR machine.
Libraries were sequenced on the Illumina HiSeq 250 bp paired-end HiSeq protocol. Reads were adapter trimmed using Trimmomatic 0.30 with a sliding window quality cutoff of Q15 (Bolger et al. 2014). De novo assembly was carried out with SPAdes version 3.7 (Bankevich et al. 2012), and contigs were annotated using Prokka 1.11 (Seemann 2014). Further annotation was made using NCBI's Prokaryotic Genome Annotation Pipeline (PGAP).
To get insight into the molecular mechanisms encoded in the genomes of rhizobacterial strains B14, B16, and B19 amino acid sequences predicted by Prokka were used as input to GenBank Trans Extractor (http://www.bioinformatics.org/sms2/genbank_trans.html) for prediction of protein translations of the DNA sequence. These sequences were assigned KO identifiers (KEGG Orthology) via “annotate sequence” (https://www.kegg.jp/kegg/tool/annotate_sequence.html), and were used to perform KEGG pathway analysis (https://www.kegg.jp/kegg/tool/map_pathway.html). The annotated genes of three PGPRs were inspected for identifying those involved in PGP functions, improvement nutrient availability, pathogenic fungi suppression, abiotic stress tolerance, quorum sensing and other important relevant functions.
1.6. Genome data availability
Sequencing data were deposited in the Sequence Read Archive at NCBI under BioProject accession PRJNA614621. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAAUVM000000000, JAAUVN000000000, JAAUVO000000000, and the version described in this paper in version JAAUVM010000000, JAAUVN010000000, JAAUVO010000000, respectively for B14, B16, and B19 strains.
1.7. Compatibility assay
Three strains (B14, B16 and B19) were tested for their antagonistic effect for consortium development. Two colonies of three different strains were resuspended with sterile water (107 CFU/ml) and 2 µl inoculum of each strain was placed on a Petri dish containing nutrient agar medium (Biomaxima, Poland). Each strain was inoculated 1 cm apart from other strains and then the plates were incubated at 28 °C for 48 h (Hashmi et al. 2019; Kumar et al. 2011). The presence of the inhibition was tested for each strain. Each assay was performed in triplicate.
1.8. Effect of rhizobacteria inoculation on rape growth
The plant growth promotion ability of the isolates on rape growth was conducted in pot experiment. Rape cultivar seeds were sterilized (2 min) in a 1:1 mixture of 30 % hydrogen peroxide and 96 % ethanol, and washed three times with sterile distilled water (Piernik et al. 2017). A sterility test of water after the last washing was done on Petri dishes with R2A medium (DifcoTM) in three replicates and incubated at 20 °C for 5 days. Bacterial and fungal colonies were checked. Before sowing, sterilized seeds were incubated with strains B14, B16, B19 and consortium of these bacteria by dipping in bacterial suspensions (10 ml of 109 cfu per ml) for 30 min. A control was incubated with 10 ml of sterile distilled water. Seeds for sowing were prepared according to the modified method of Piernik et al. (2017). Three replicates of 10 seeds for each strain and consortium, were germinated in the palette containing a sterile mixture of sand and vermiculite (1:1). All variants were put in a growth chamber at 22 °C and 16-h light period under a sodium lighting system [100 μmol/m2/s PAR (photosynthetically active radiation)]. The palettes were watered by equal amount of Hoagland’s solution each day to ensure homogeneity of nutrient supply. Germinated seeds were counted after 48 h and then each day for 2 weeks. Plants were grown for 6 weeks. Then plants were washed up from the substrate, and the content of chlorophyll in rape leaves was measured (as the CCI - chlorophyll content index). The measurements were carried out using a chlorophyll meter CCM-200plus. Then, live (LL) and dead leaves (DL) were counted. Each plant was divided into parts: leaves, petioles, shoots, epicotyls, roots and the lengths of shoots (shoot), epicotyls (epic) and roots (root) were measured. The leaves were scanned and their photosynthetic surface (Al) was measured using the digiShape 1.3 program (Moraczewski 2005). All parts of the plants were dried at 85 °C for 48 hours. After this time, they were weighed to obtain weights of leaves (Wl), petioles (Wpet), epicotyls (Wep) and roots (Wroot), respectively. Based on the obtained dry masses, two indexes for plant growth analysis were calculated (Kvét et al. 1971). These were SLA (specific leaf area, Al/Wl) and LWR (leaf weight ratio, Wl/W), where W is the plant's total biomass.
1.9. Salt stress tolerance assay
The effect of the rhizobacterial strains on rape growth under salt stress condition was tested by the modified Eida et al. (2020) method. Rape seeds were sterilized by shaking for 10 min in 70 % ethanol + 0.05 % SDS, washed two times in 96 % ethanol, and one time with sterilized H2O. Then, the seeds were incubated by dipping in bacterial suspensions (10 ml of 109 cfu per ml) with strains B14, B16, B19 and consortium of these bacteria for 30 min. A control was incubated with 10 ml of sterile distilled water. Then, seeds were sown on ½ MS plates (Murashige and Skoog basal salts, Sigma) with or without 100 mM NaCl, stratified in the dark for 2 days at 4 °C, and placed vertically in growth chambers at 22 °C with a photoperiod of 16/8 h (light/dark, 100 μmol/m2/s). After 12 days of seedlings transfer, the plant material was dried at 85°C for 48 hours and dry weight of shoots and roots were measured. Each experiment was performed at least in three biological replicates.
1.10. Statistical analysis
Statistical analysis of the data from the effect of rhizobacteria on rape growth were performed using canonical analysis (CCA). The indirect ordination analysis CCA was performed in the Canoco 5.0 program (ter Braak and Šmilauer 2012). The result of the analysis is a diagram in which the cultivars are marked with the triangle symbols and the parameters are vectors. Variable designations near the vector arrowhead are analogous to those indicated in parentheses above. At the same time, forward selection and Monte Carlo permutation test were performed. That indicated which variables were statistically significant for the variability of our data.
Statistical analysis of the data from salt stress tolerance assay were performed in Past3, v 3.25. To determine significant differences in response variables, ANOVA were performed. The assumptions for ANOVA were checked using Shapiro-Wilk test for normality, and Levene´s test for homogeneity of variances. Tukey’s multiple comparison test was performed to find means that are significantly different from each other.