Isolation of bacterial strains
Soil samples were collected from the agricultural area of the Faculty of Agriculture (45
o 82' 56.36" N, 16
o 032' 31.80" E). In greenhouse pot experiment, surface sterilized soybean seeds (cultivar Gabriela) were sown directly into soil samples collected. Each experimental pot contained about 3 kg of soil sample. These plants were grown under controlled conditions; day temperature 26
oC, night temperature 22
oC, moisture was continiously 65%. Daylight period was setup for 16 h and night period for 8 h. The soybean tissue was collected at the flowering stage. Ten healthy plants were carefully removed, washed under tap water to remove vermiculit and they were then separated into stems, roots and nodules. Stems and roots were cut into sections 2-3 cm long. The tissue was rinsed in 70% ethanol for 30 seconds and then sterilized with 3% NaClO 3 minutes for roots and nodules and 5 minutes for stems. The tissue was then washed ten times with sterile water (
Hung and Annapurna, 2004). Surface-disinfected tissue was crushed with a sterile glass rod in a sterile test tube. One loop full of the nodule, stem or root content suspension was streaked on yeast mannitol agar (YEM) plates containing 0.0025% (w/v) Congo red. After incubation for 3 to 5 days at 28
oC, single colonies were selected and restreaked on YEM agar for purity (
Vincent 1970). Pure cultures were preserved in 20% glycerol at -20°C until further use. The study involved 19 selected isolates and this population was partially characterized on the basis of their morphology, 16S rRNA gene sequencing and screened for different PGP traits and biocontrol properties.
Screening of isolates for potential plant growth promoting properties
Phosphate solubilisation
Cells from each isolate in the LOG growth phase (10
8 CFU/ml) were spotted on Pikovskaya’s agar
(Bhutani et al., 2018). All the bacterial isolates were spot inoculated (15 µl bacterial culture previously grown on Yeast Mannitol Broth medium) at the centre of the Pikovskay agar plates and incubated for 5-6 days at 28°C. After incubation at 30
oC a clear zone around the inoculation spot indicated positive result.
Organic acid production
For testing production of organic acids 24-h-old cultures produced in YMB were transferred to Methyl red - Voges-Proskauer (MR-VP) liquid medium
(Senko et al., 2024) and incubated for five days at 30
oC on a shaker (150 rpm). After incubation for visualization of results Methyl red test according to
Senko et al., (2024) was performed using 0.02% Methyl red solution.
Indole-acetic-acid (IAA) test
IAA production was detected as described by
Sherpa et al., (2021). 1 ml of the overnight culture of bacteria inoculated to a 250 ml Erlenmeyer containing 100 ml nutrient broth fortified with L-tryptophan (0.1 mg/ml). After incubation in shaker incubator for 48 h at 30
oC with 150 rpm (Biosan ES-20, Latvia), bacterial culture was centrifuged at 10000 rpm for 30 min. Then 2 ml of the supernatant was mixed with 2 ml Salkowski reagent (0.5 M FeCl
3 in 35% per chloric acid) and incubated for 30 min at room temperature in darkness. The optical density (OD) was recorded at 530 nm using a UV-VIS spectrophotometer (Lambda EZ 210, Perkin Elmer, USA) and the produced IAA was measured by standard curve graph.
N2- fixation test
The capability of nitrogen fixation was checked by allowing the bacterial strains to grow in medium devoid of nitrogen. The qualitative estimation of N-fixation was checked using N-free Jensen’s agar medium. Jensen’s Medium is used for detection and cultivation of nitrogen fixing bacteria. Bacterial strains were streaked on Jensen’s medium and incubated at 28±2
oC for 4-5 days. After incubation plates were observed for the bacterial growth
(Sherpa et al., 2021). The growth of the milk colonies indicates a positive result.
EPS production
EPS producing activity of the bacterial isolates was done according to the method described by
Jain et al., (2016). Volume of 200 µl of overnight grown cultures of all bacterial isolates were inoculated in 100 ml of NB medium and incubated at 28±2
oC for 72 h at 150 rpm in shaking incubator (Biosan ES-20, Latvia). After incubation, supernatant was collected with centrifugation at 12000 rpm for 10 min at 4
oC and pre-chilled acetone was added to supernatant in 3:1 ratio. Formation of precipitation was considered as positive result for EPS production.
Screening bacterial isolates for hydrolytic enzyme production
Bacterial isolates were screened for their hydrolytic enzyme production like protease and amylase.
Bacterial isolates were screened for their ability to produce protease onto skim milk agar or SMA (3 % v/v) medium. After 48 hours of incubation at 30
oC, bacterial isolates with a clear halo zone on skim milk agar showed a positive result for protease synthesis
(Tsegaye et al., 2019).
Amylase production by bacterial isolates was determined using starch agar as described in
Mir et al., (2021) and incubated at 30
oC for 48 h. At the end of the incubation period, the plates were flooded with iodine solution. Iodine reacts with starch to form a blue color compound. Hence the colorless zone surrounding colonies indicates the production of amylase.
Molecular characterization of plant growth promoting bacteria
Total DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, USA) according to manufacturer’s instructions. After extraction of genomic DNA, it was stored at -20
oC for further studies. The 16S rRNA genes were polymerase chain reaction (PCR) amplified by using two universal bacterial primers fD1 (5'-CCGAATTCGTCGACAACA GAGTTTGATCCTGGCTCAG- 3') and rD1 (5'- CCCGGGATC CAAGCTTAAGGAGGTGATCCAGCC- 3') (
Sikora and Redzepović, 2003). The reaction mixture with a total volume of 25 µl, is composed of bacterial DNA (100 ng), buffer, dNTP (100 pmol), TaKaRa TaqTM polymerase (1.25U) and sterile Milli-Q water. PCR conditions were: an initial cycle of denaturation at 95oC for 3 min; 35 cycles of denaturation at 94
oC for 1 min, annealing at 57
oC for 1 min, 72
oC for 1.5 min; and a final extension at 72
oC for 10 min (Sikora and Redzepovic). The PCR products were checked by horizontal gel electrophoresis (1% w/v agarose) in Tris-Acetate-EDTA (TAE) buffer. The PCR products were visualised under UV illumination after staining with ethidium bromide and photographed with Cannon Powershot A640 camera. The PCR products were purified and sequenced by Macrogen (Seoul, South Korea) using an ABI3730 XL automatic DNA sequencer and the primers fD1 and rD1. A similarity search for so generated sequence was performed using National Centre of Biotechnology Information (NCBI) BLAST program. The phylogenetic tree was constructed by Neighbour-joining (NJ) method using software MEGA X.
