Isolation and identification of endophytic bacteria
Healthy fresh roots of common bean plants (Vigna unguiculata L.) were collected in sterile plastic bags from Aswan University greenhouse. Immediately nodules were surface- sterilized using 70 % ethanol (30 sec) followed by 5 % sodium hypochlorite (3 min) and then washed three times with sterilized distilled water (Vincent 1970). Under aseptic conditions, nodules were crushed in a test tube contained one mL of sterilized saline solution. Loopful of the obtained suspension was streaked on the surface of tryptic soy agar and nutrient agar plates. Plates were incubated at 37 oC for 72 h for the appearance of colonies.
The ribosomal (16S rRNA) gene of the selected strain was amplified using 27F and 1492R primers (Frank et al. 2008) in Applied Biotechnology lab at Ismailia, Egypt. PCR product was sent to SolGent Co., Ltd., South Korea for sequencing. Then, the similarity of the obtained sequence was evaluated based on BLAST outputs using NCBI reference sequence database. Neighbor-joining phylogenetic tree of the strain was constructed using MEGA X 10.1.7 software (Kumar et al. 2018).
Cd tolerance of the strain
Maximum tolerable concentration (MTC) of cadmium by the strain was determined according to the method of Vashishth and Khanna (2015), with slight modification. Briefly, 10 mL of yeast extract-mannitol broth (YMB) in glass tubes was supplemented with different concentrations of CdCl2 i.e., 0 (control), 50, 100, 150, 200, 250 and 300 mg L-1. 10 mL of YMB without CdCl2 was used as control. Tubes were inoculated with 1 mL of inoculum (107 CFUmL-1), and incubated for 48 h at 28 oC and 150 rpm. The optical density (OD) was measured at 600 nm. The highest concentration of cadmium (CdCl2) that allowed visible bacterial growth after 48 h of incubation was considered as the maximum tolerable concentration (MTC).
Evaluation of Cd- adsorption potential of the strain
The ability of the whole culture of the present strain (cells and supernatant) for adsorping cadmium was evaluated using the method of Du et al. (2016 b). 100 mL of the whole culture broth contained 50 and 100 mg L-1 of CdCl2 was shaken at 120 rpm and 28 oC for 24 h. Cells were then removed by centrifugation. Concentration of the residual, non-adsorbed metal ion in the solution was estimated by atomic absorption spectrophotometer (Thermo Scientific™ iCE™ 3000). Experiment was performed in triplicate. The adsorption efficiency (%) was calculated according to the following formula:
where Cdi and Cde are the concentration of initial and equilibrium Cd ion in the solution (mgL-1) respectively.
Seed inoculation and pot experiment
Seeds of safflower (cv. Giza-1) were obtained from Faculty of Agriculture and Natural Resources, Aswan University. Seeds were surface sterilized with 70 % ethanol for 3 min, rinsed three times with sterilized distilled water. Seeds thereafter were soaked in a freshly prepared bacterial suspension (1×108 CFU mL-1) for 1 h, and left to dry before sowing. Seeds used for control were soaked in sterilized distilled water.
Seeds were sown in pots containing an autoclaved mixture of clay and sand (1:1 w/w), with maintaining field capacity at 90 %. Pots were kept under normal climatic conditions. After three weeks of sowing, five homogenous plants in each pot were subjected to three Cd treatments including 0 (control), 50 and 100 mg L-1 of CdCl2. After three weeks of cadmium exposure, healthy expanded leaf samples were collected, frozen and then used for measuring the defensive non-enzymatic and enzymatic antioxidant activities. The experiment was repeated twice.
Estimation of hydrogen peroxide (H2O2) content
To evaluate the H2O2 content of the leaves, the method of Velikova et al. (2000) was followed. One gram of fresh leaves was homogenated in 10 mL trichloroacetic acid (0.1 %) using a mortar and pestle, and then centrifuged. To 0.5 mL of the supernatant, 0.5 mL of potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI were added. The mixture was vortexed, and the absorbance was read at 390 nm. A calibration curve of different concentrations (µmol) of 30 % (v/v) H2O2 was used as standard.
Assessments of non-enzymatic antioxidants
The Folin-Ciocalteu assay described by Singleton et al. (1999) was followed to determine the total phenolic compounds in the leaves extracts. Absorbance was read at 700 nm, and the content of total phenolics was expressed as mg gallic acid equivalents per gram of fresh weight using gallic acid as a reference.
Aluminum chloride method according to Chang et al. (2002) was used for quantifying the total contents of flavonoids of the extracts. The absorbance was recorded at wavelength 510 nm. The concentration of flavonoids was calculated from quercetin calibration curve as mg quercetin equivalents per gram of fresh weight.
Pigments were extracted from fresh leaves and their contents were estimated as described by Lichtenthaler and Wellburn (1983). One gram of fresh leaves was macerated in 80 % acetone, the supernatant was filtered and makeup to 50 mL with the solvent. The total contents of chlorophylls a (Chl a), chlorophylls b (Chl b) and carotenoids were measured by reading the absorbance at wavelengths 646, 663 and 440.5 nm respectively. The content of each pigment was calculated in mg per gram of fresh weight using the following equations:
Chl a (mg g f.wt.-1) = (12.21×A663) - (2.81×A646)
Chl b (mg g f.wt.-1) = (20.13×A646) – (5.03×A663)
Carotenoids (mg g f.wt.-1) = (4.69×A440.5) - 0.268× (Chla + Chlb)
Total antioxidant capacity
Total antioxidant capacity of the ethanolic extracts of the leaves was measured per gram of fresh weight as mg ascorbic acid equivalents using ascorbic acid standard curve, according to phosphomolybdnum assay (Prieto et al. 1999).
Assessments of enzymatic antioxidants
Antioxidant enzymes were extracted from fresh leaves according to Cavalcanti et al. (2004) with slight modification. One gram of fresh leaves was homogenized using a mortar in 10 mL of extraction buffer containing 0.2 M of potassium phosphate buffer (pH 7.2), 0.1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride as proteinase inhibitor. The homogenate was filtered. The obtained filtrate was used for enzymatic assays.
Catalase (CAT) activity
Catalase activity was estimated by the method of Kato and Shimizu (1987). To 3 mL of the reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0) and 20 mM H2O2, 100 µl of enzymatic extract was added. The decrease in H2O2 was followed as decline in optical density at 240 nm. Catalase activity was calculated with the extinction coefficient of H2O2 (40 mM−1 cm−1), and expressed as 1μmol of H2O2 decomposed per minute under assay conditions.
Guaiacol peroxidase (POX) activity
The activity of guaiacol peroxidase enzyme was determined following the method of Kim and Yoo (1996). Briefly, the reaction mixture contained 0.2 mL of enzyme extract, 0.8 mL of phosphate buffer (0.2 M, pH 7.2), 1 mL of guaiacol (15 mM) and 1 mL of hydrogen peroxide (3 mM) was incubated for 10 min at 30 °C. Reaction was terminated using 0.5 mL of H2SO4 (5 %), and the absorbance was read at 470 nm. POX activity was calculated using the extinction coefficient of oxidation product (tetraguaiacol), (ε470= 26.6 mM cm-1) as follow:
U/mL = [Change in absorbance min−1× Reaction mixture volume (mL) × Dilution factor]/ [ε470× Enzyme extract volume (mL)]
Ascorbate peroxidase (APX) activity
Ascorbate peroxidase activity was evaluated according to Senthilkumar et al. (2021). To 0.8 mL of a reaction mixture contained potassium phosphate buffer (50 mM), ascorbic acid (0.5 mM), H2O2 (1.0 mM) and EDTA (0.1 mM), 0.2 mL of the enzyme extract was added. After 30 s the decrease in absorbance at 290 nm was followed up to 60 s with an interval of 15 s. One unit of enzyme activity was expressed as the amount of enzyme required to oxidize 1 μmoL of ascorbic acid per minute with absorbance coefficient 2.8 mM cm at 290 nm.
Superoxide dismutase (SOD) activity
Superoxide dismutase activity was estimated according to Van Rossun et al. (1997). Three mL of reaction mixture contained 50 mM sodium phosphate buffer (pH 7.6), 0.1 mM EDTA, 50 mM sodium carbonate, 50 μM nitroblue tetrazolium (NBT), 10 μM riboflavin, 12 mM L-methionine and 100 μl of crude extract. Tubes contained the same reaction mixture without enzyme extract used as control. The tubes were placed under two 15 W fluorescent lamps for 15 min to start the reaction. The absorbance was recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme which reduced the absorbance to 50 % compared with the control.
Experimental data were compared using one-way analysis of variance (ANOVA) with Tukey's HSD test. Values were expressed as means ± standard errors (SEs) of three biological replicates from two independent experiments. Differences were considered significant if p ≤ 0.05.