Sampling and sample preparation
The samples were randomly collected between January 2015 and April 2016 from different canola fields (Hyola cultivar) of 5 provinces under the supervision of the agricultural organization in western Iran (Khuzestan, Ilam, Lorestan, Ardabil, and Hamedan). All samples (120 healthy plants with adhering soil that were at the flowering stage) were placed in paper bags, transported to the laboratory, and used for isolation. Five samples from each region were randomly grouped to obtain a representative composite sample. To extract epiphytic bacteria, composed samples (i.e., stems, leaves, and flowers) were separated and their dust was cleaned with cotton. Next, they were placed in 100-ml sterilized distilled water and kept on a 120-rpm shaker for 1 h. A loopful of the resulting suspension was plated on nutrient agar medium and was supplemented with sucrose (5 g l−1) (EPPO 2011). To isolate endophytic bacteria, plant parts (roots, stems, leaves, and flowers) were surface sterilized and washed in running tap water, followed by washing with sodium hypochlorite solution (0.3 to 2%, v/v) for 1 to 3 min and 3 serial rinses in sterilized distilled water. Plant parts were sliced in 100 ml sterilized distilled water. The suspensions were placed on a 120-rpm shaker for 1 h to release bacteria from the plant tissues. A loopful of the resulting suspension was streaked on Petri plates containing the above-mentioned medium. To extract rhizosphere microorganisms, roots with adhering soil (rhizospheric soil) were placed in 100 ml sterile distilled water and placed on a 200-rpm shaker at 25 °C for 1 h. This solution was serially diluted, of which 0.1 to 1 ml was spread on a selective nutrient agar medium (Hammami et al. 2013). Petri dishes were incubated at 25–28 °C for several days to allow the bacterial growth. Different colony types in terms of shape, size, texture, pigmentation, margin, and elevation picked up were subcultured on fresh Petri plates. The purified colonies were individually stored at – 20 °C in 30% glycerol.
Protein extraction and SDS-PAGE
To extract protein of Gram-negative bacteria, bacterial strains grown on nutrient agar containing 0.5 g l−1 NaCl for 24 h at 25–28 °C were suspended in 1 ml sample buffer (0.062 M Tris-HCl pH 6.8, 15% glycerol, 7.5% mercaptoethanol, 0.6% SDS) and then, they were placed in boiling water for 10 min. After centrifugation at 10,000 rpm for 10 min, 300 μl batches of supernatant containing soluble proteins were loaded into each well of the gel (Laemmli 1970). To extract the proteins of Gram-positive bacteria, bacterial isolates grown on nutrient agar were suspended in 1 ml of distilled water. Next, 50 μl of 1 M Tris-HCl pH 8.2 and then, 100 μl of lysozyme solution (10 mg of lysozyme per ml of 0.05 M Tris-HCl buffer containing 0.02 M EDTA pH 8.2) were added to the vial and were thawed and frozen in triplicate. Subsequently, SDS was added to a final concentration of 2% and vials were placed in water at 60 °C for 10 to 15 min. After centrifugation at 12,000 rpm for 2 min, 300 μl batches of supernatant were transferred to new vials and loaded into each well of the gels (Chaasy and Giuffrida 1980). Electrophoresis was performed according to a modified Laemmli method in a constant current of 20 mA for 7 h using 5% stacking gel (pH 6.8) and a 12% running gel (pH 8.8). After electrophoresis, Coomassie brilliant blue solution was added to the mix for gel staining using 45% v/v methanol, 10% v/v glacial acetic acid, and 0.01% Coomassie brilliant blue R250. Afterward, it was kept for 24 h at room temperature and then, was destained in the same solution without Coomassie brilliant blue (45% v/v methanol and 10% v/v glacial acetic acid) until the gel was rehydrated (Laemmli 1970).
DNA extraction
The DNA extraction was prepared by the alkaline lysis method (Arabi et al. 2006). The suspension of bacterial cells was prepared in 1 ml of sterile distilled water (optical density at 600 nm, 1). Then, 30 μl of KOH 5% was added to the cells suspension, followed by placing it in boiling water for 10 min. Centrifugation was conducted at 12,000 rpm for 2 min and 60 μl of the supernatant was removed carefully (DNA extract). An appropriate amount of DNA of genomic was determined and used in PCR reactions.
PCR amplification of the 16S rRNA gene and analysis
Two conserved primers fD1 (5-AGA GTT TGA TCC TGG CTC AG-3) and rP2 (5-ACG GCT ACC TTG TTA CGA CTT-3) were used to amplify the almost complete 16S rRNA gene sequence (1506 bp). The polymerase chain reaction (PCR) was performed using a PCR thermocycler (Techno-TC-512; UK). About 25 μl of reaction volume contained 1 μl of bacterial DNA as a template for PCR procedures, 0.1 mM of primers, 1 mM MgCl2, 20 mM of each dNTP, and 0.5 U Taq DNA polymerase (Cinna Gen, Iran). The thermal cycling used for this reaction was as follows: initial denaturation at 94 °C for 5 min, 94 °C for 45 s followed by 30 cycles of 58 °C for 1 min, 72 °C for 90 min, and a final extension at 72 °C for 10 min. PCR products were sent to a sequencing service (Macrogen, Seoul, South Korea). The nucleotide sequences were aligned in the NCBI database using nucleotide BLAST software (NCBI BLAST® homepage). Phylogenetic analysis was performed using MEGA 7.0.26 software and neighbor-joining method with 1000 repetitions bootstrap to confirm the stability of the relationships (Kumar et al. 2016). The nucleotide sequences of 16S rRNA gene segments from different bacterial samples determined in this research have been deposited in the GenBank database under accession numbers (MW703484 and MW712709 to MW712715).
In vitro screening for antagonism (dual test)
The antagonistic effects of all 362 isolates were evaluated against the causative agent of rapeseed blackleg disease (Leptosphaeria maculans) using the dual-culture method and the inhibition percentage was calculated using the following formula (Vincent 1947):
$$ \%\mathrm{Inhibition}\ \mathrm{of}\ \mathrm{mycelial}\ \mathrm{growth}=\left[\left(C-D\right)/C\right)\Big]\times 100 $$
(1)
Where C is mycelial growth of pathogen in absence of antagonists and D is mycelial growth of pathogen in presence of antagonists. The experiments were conducted in a completely randomized design with three replicates.
Evaluation of plant growth-promotion properties
Regarding the ability of some Pseudomonas isolates (18 strains) to biologically control the blackleg disease, these isolates (13 leaf epiphytes, 1 stem epiphyte, 1 flower epiphyte, 1 root endophyte, and 2 leaf endophyte) were selected for the next laboratory experiments (plant growth-promotion properties and greenhouse assays). Siderophore production (Schwyn and Neilands 1987), phosphate solubilization (Lynn et al. 2013), and nitrogen fixation (Döbereiner 1998) activities were performed for the selected isolates.
Effect of bacterial seed priming on germination and growth in greenhouse
For greenhouse assays, seeds of B. napus (cv. Hyola) obtained from Agriculture and Natural Resources Research and Education Center of Hamedan were surface sterilized by 1% sodium hypochlorite for 1 min and then were washed in triplicate in sterile distilled water (Nezaret and Gholami 2009). Canola seeds were then inoculated with each bacterial suspension to a final concentration of 106 cfu ml−1 containing 1% Carboxymethylcellulose in 170 rpm for 12 h at 28 °C. In the control treatment, seeds were inoculated with distilled water. The treated seeds were exposed to sterile air for about 6 h to dry completely (Kumar et al. 2011) and then were sown in pots with 10 seeds per pot. The soil used for the experiment was made by combining sand and compost (1:1 v/v) and was sterilized by autoclaving. Seedlings were grown in a greenhouse with 12 h of light at 25 °C and were irrigated with distilled water. Thirty days after sprouting, plants were harvested and vegetative parameters were measured according to Orozco-Mosqueda et al. (2013) as follows:
$$ GR=\left( SG/ NS\right)\times 100\kern0.5em GR=\frac{SG}{NS}\times 100 $$
(3)
Where VI is Vigour index, GR is Germination rate (%), L is the total plant length, SG is the number of seeds germinated from total number of seeds, and NS is the total number of seeds.
A completely randomized block design with three replicates was used to perform this experiment. The data were analyzed using SAS9 software and the mean data were compared using Duncan’s multiple range tests at 5% level (P ≤ 0.05).
