Alleviation of cadmium stress in rice by inoculation of Bacillus cereus

Revival of industrial waste bacterial strains

Eleven bacterial strains isolated from industrial waste were obtained from the Applied Microbiology and Biotechnology Lab, COMSATS University, Islamabad, Pakistan. Revival of these bacterial strains from their glycerol stocks was made on Luria Bertani (LB) media: NaCl (5 mg), Tryptone (5 g), Agar (7.5 g), and Yeast (2.5 g) in 500 ml distilled water. Streaking of bacterial strains was performed on each plate containing 20 ml agar media. After streaking, the plates were secured with parafilm to avoid any contamination. Petri plates were then placed in an incubator at 30 °C for approximately 24 h. Afterward separate bacterial colonies were obtained on plates and stocked at 4 °C for further use.

Screening of industrial waste bacterial strains for cadmium tolerance

Cadmium (in the form of CdCl₂) tolerance of bacterial strains was assessed by growing bacteria in nutrient agar media with different concentrations of CdCl₂ (100 µM and 200 µM). Control plates were also maintained. Eleven different strains i.e. S₃D_1–1, S₃D_1–4, S₃D_4–6, S₁D_2–33, S₁D_2–34, S₆D_1–42, S_1–89, S₆D_1–105, S₂D_2–111, R₁, and R₂ were used. Streaking of these strains was performed afterwards on agar plates and incubated for 48 h at 30 °C. Growth of the tested strains was observed under both the concentrations of CdCl₂ to screen tolerant bacterial strains.

Determination of minimum inhibitory concentration (MIC) of bacteria for Cadmium

Maximum resistance of the selected bacterial isolate S₆D_1–105 against the increasing concentrations of CdCl₂ on nutrient agar plates was evaluated until the strain was unable to grow colonies on the LB agar plates. Nutrient Agar plates augmented with CdCl₂ stress at 100 µM to 2,000 µM concentrations were used to determine MIC.

DNA extraction

For genomic DNA extraction, Cetyltrimethylammonium Bromide (CTAB) based procedure was used. Samples were merged with 500 μl of CTAB buffer, 300 μl sterile dH₂O, and 20 μl proteinase K (20 mg/ml) along with 1% polyvinylpyrrolidone (PVP). Afterwards, samples were incubated at 65 °C for 2 min. The blend was then incubated in a water bath at 65 °C for 10 min. The samples were then centrifuged at 16,000×g for 10 min along with the extraction of supernatant twice with 500 μl chloroform. The upper layer was collected and incubated for 1 h at room temperature, thereafter, merging it with the twofold volume of CTAB solution. The samples were further centrifuged at 16,000×g for 5 min. The pellet was mixed in the mixture of 350 μl chloroform and 1.2 M NaCl, and centrifuged for 10 min at 16,000×g. Again, the upper layer was shifted to a new test tube, blended with isopropanol, and further centrifuged for 10 min at 16,000×g. Subsequently, supernatant was discarded, and pellet was washed with 500 μl of 70% ethanol. Following centrifugation, the supernatant was thoroughly disposed, and the pellet was dried up and was dissolved in 100 μl ddH₂O.

Identification of bacteria by 16S rRNA gene sequencing

The 16S rRNA gene of bacteria was amplified using universal primers as, F-primer MP1 (5’-CGGGATCCAGAGTTTGATCCTGGTCAGAACGAACGCT-3’) and R-primer MP6 (5’-CGGGATCCTACGGCTACCTTGTTACGACTTCACCCC-3’). Amplification was carried out by PCR with a reaction mixture of 25 µl, that consisted of PCR water, MgCl₂, 10× Taq buffer, Taq polymerase 5 U/µl, reverse primer 10 µM, forward primer 10 µM, dNTPs 10 mM, and DNA template ≥1,000 ng. The PCR was carried out in a thermal cycler manipulating cycling condition, that contained 1 cycle of initial denaturation at 95 °C for 5 min, then 35 cycles of denaturation for 45 s at 95 °C, annealing at 54–65 °C for 45 s and extension for 1 min 30 s at 72 °C for 16S rRNA gene and final extension was executed for 10 min at 72 °C. The PCR products were assessed by 1% agarose gel electrophoresis. The bacterial 16S rRNA gene sequence was identified using the Basic Local Alignment Search Tool (BLAST) algorithm at the National Centre for Biotechnology Information (NCBI) and accession number (OK310861) of the most potent bacterial sequence submitted to NCBI Gene Bank was obtained, accordingly.

Plant growth promoting traits

Plant growth promoting (PGP) traits such as: phosphate solubilization, potassium solubilization, as well as protease and siderophores production by Cd tolerant bacterial strain were examined.

Phosphate solubilization assay

The bacterial strain was tested for phosphate solubilization by an agar assay method using Pikovaskaya’s media. The bacterial strain was streaked on Pikovaskaya’s plates, and then placed in an incubator for 7 days at 30 °C. After incubation period, plates were observed for the presence of clear halo-zones surrounding the culture spot which points towards the isolate’s phosphate solubilization ability. The solubilization index was accessed by the following formula:

$$\mathrm{S}\mathrm{I}=\frac{\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}+\mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}{\mathrm{C}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}$$

Potassium solubilization assay

The bacterial strain was tested for potassium solubilization using Aleksandrov media. The media was dispensed into petri plates. After the media was solidified, the bacterial strain was streaked on the media. Plates were incubated for 3 days at 30 °C. After incubation, the presence of clear halo-zones surrounding the culture spot, indicated the potassium solubilization potential of the bacteria. Potassium solubilization index was noted by the following formula:

$$\mathrm{S}\mathrm{I}=\frac{\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}+\mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}{\mathrm{C}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}$$

Protease detection

The protease production test was performed according to the method mentioned by Hirano & Nagao (1988). Petri plates with nutrient agar medium altered with skimmed milk powder (1%) were used for protease detection. The 24 h bacterial colonies were spot inoculated on media plates and kept for 48 h in an incubator. Bacterial isolate having a clearing zone formation around the colony was taken positively for protease enzyme production (Kumar et al., 2012).

$$\mathrm{S}\mathrm{I}=\frac{\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}+\mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}{\mathrm{C}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}$$

Production of siderophores

Bacterial isolate was observed for siderophores production by nutrient agar medium added with an indicator dye. The nutrient agar media containing the bacterial culture was incubated for 7 days at 30 °C. The positive or negative reaction of bacterial strain to the assay was recorded. After incubation, plates were detected for halo-zones formation and the following formula for accessing the solubilization index was used:

$$\mathrm{S}\mathrm{I}=\frac{\mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}+\mathrm{h}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{z}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}{\mathrm{C}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}$$

Screening of rice genotypes for cadmium tolerance

Seeds of three rice genotypes were acquired from Rice Research Institute (Kala Shah Kahu), District Shaikhupura Punjab, Pakistan. Fifty seeds of each genotype were soaked for 3 min in H₂O₂ (3%) and were washed with distilled water. These seeds were grown in the germination box on wet double-layer filter paper. Seeds were germinated under different levels of Cd concentration (100 µM, 200 µM, and 300 µM). Seeds were kept in germination boxes in the dark until the plumule (shoots) and radical (roots) of the seeds emerged. After 7 days of germination fresh and dry weights, shoot, and root lengths were measured.

Pot experiment

After screening, test seeds of Super Basmati rice with high germination percentage were selected for further experimental work. After 7 days of germination, seeds of Super Basmati were grown hydroponically in Hoagland nutrient solution in plastic pots containing foam-plugged holes placed in a greenhouse. Nutrient solution composition at its full strength was mg/L (NH₄)₂SO₄ 48.2, K₂SO₄ 15.9, MgSO₄.7H₂O 154.88, KNO₃ 37, KH₂PO₄ 49.6, Ca (NO₃).2H₂O 172.36, MnCl₂.4H₂O 1.8, ZnSO₄.7H₂O 0.22, Fe-Citrate 14, CuSO₄.5H₂O 0.08, H₃BO₃ 5.8, H₂MnO₄ 0.02 and prepared according to Jabeen et al. (2021). The pH of the nutrient solution was maintained at 6.0 ± 0.5 with HCl or NaOH. Bacterial strain S₆D_1–105 was pre inoculated with 2 weeks old rice seedlings. After 5 days of inoculation, rice plants were exposed to four treatments i.e., (1) Control, (2) 100 µM CdCl₂ stress, (3) bacterial strain-105, (4) 100 µM CdCl₂ + bacterial strain-105 with three replicates.

After 14 days of treatment, rice plants were harvested. Plant roots, and leaves were separated for assessment of different growth parameters i.e. roots and shoots length, fresh and dry weight, leaf relative water content, chlorophyll content, and antioxidant enzymatic activities. Roots and leaves were collected and frozen immediately in liquid nitrogen and stored at –80 °C until further processing.

Plant morphological traits

Shoots and roots length of rice was measured. The fresh weight of roots and shoots from each treatment was recorded. For dry weight analysis, plant samples were dried for 72 h at 70 °C.

Root scanning

Roots of all the treated as well as untreated (control) plants were gathered separately and rinsed three times with deionized water. Roots taken from each treatment sample were spread carefully in a transparent tray containing distilled water and scanned with Epson Expression 16801.0 scanner for analysis.

Chlorophyll contents

Fresh leaves of rice (0.2 g) were homogenized with mortar and pestle in 20 ml (80%) acetone and samples were placed in dark for 24 h. After that, samples were centrifuged for about 10 min and Chlorophyll contents were measured by taking supernatant. Chlorophyll-a, chlorophyll-b, and carotenoids were checked by recording the corresponding absorbance at 470 nm, 647 nm, and 664 nm respectively. The quantity of photosynthetic pigments was expressed in mg/g of the fresh weight (FW) (Biczak, 2016).

Determination of malondialdehyde (MDA)

Fresh rice leaves (0.5 g) were homogenized in 5 ml (0.1%) trichloroacetic acid (TCA). Malondialdehyde (MDA) determination was carried by measuring the absorbance of supernatant at 532 nm and 600 nm and it was calculated according to its extinction coefficient of 155 nm/cm (Biczak, 2016).

Superoxide dismutase assay (SOD)

Superoxide dismutase (SOD) activity was measured by its ability to inhibit photochemical reduction of NBT on blue formazan, and then monitored by the absorbance of assay mixture spectrophotometrically at 560 nm (Habib, Kausar & Saud, 2016).

Catalase (CAT) activity

Activity of catalase (CAT) in the leaves and roots of rice was assessed with spectrophotometer at 30 °C by monitoring the absorbance resulting from H₂O₂ decomposition at 240 nm_. The CAT activity was measured in accordance with protocol followed by Habib, Kausar & Saud (2016).

Peroxidase (POD) activity

Peroxidase (POD) activity was determined by analysis of POD ability for oxidizing guaiacol using H₂O₂. Reaction mixture was composed of 2.5 ml of 50 mmol/L potassium phosphate buffer (pH 6.1), 1 ml (1%) hydrogen peroxide, 1 ml (1%) guaiacol and 10–20 μl enzyme extract (Marszałek, Mitek & Skąpska, 2015).

Ascorbate peroxidase (APX) assay

Ascorbate peroxidase (APX) activity was measured according to Habib, Kausar & Saud (2016). Three ml reagent solution containing potassium-phosphate (50 mM) buffer (pH 7.0), H₂O₂ (0.1 mM), ascorbate (0.5 mM), and enzyme extract (20 μl) was used. The APX activity was observed at 290 nm, using the extinction coefficient (2.8 mM/cm).

Polyphenol oxidase (PPO) activity

Determination of polyphenol oxidase (PPO) activity was carried out by adding 100 μL of the enzymatic extract into 3 mL catechol (0.07 M) in sodium phosphate buffer (0.05 M) having pH 6.5. Assay mixture absorbance was measured in the kinetic mode at 420 nm (25 °C for 10 min) using the UV-visible spectrophotometer (Zareei, Javadi & Aryal, 2018).

Phenylalanine ammonia lyase (PAL)

PAL activity was determined according to Gill et al. (2016). The reaction mixture contained 3 mM/L-phenylalanine, 150 mM of tris-HCL and enzyme extract. PAL activity was measured at 290 nm. PAL activity was measured by using formula:

$$\mathrm{P}\mathrm{A}\mathrm{L}\mathrm{A}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{U}/\mathrm{m}\mathrm{l})=\frac{\mathrm{O}\mathrm{D}270-\mathrm{O}\mathrm{D}270\mathrm{B}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\times \mathrm{a}\times \mathrm{d}\mathrm{f}}{19.73\times 0.1}$$where, 3 represents total volume of assay, 19.73 represents milli-molar extinction co-efficient, and df represents dilution factor.

Relative water content (RWC)

Relative water content of rice leaves was determined according to Weatherley (1950). Fresh weight of control and treated plants was measured immediately after harvesting. The leaves were then soaked in distilled water container for 24 h. Then, weight of completely turgid leaves was measured again. The leaves were subsequently dried in a conventional oven to a constant weight at about 60 °C for 72 h. RWC of leaves was measured by the following formula:

$$\mathrm{R}\mathrm{W}\mathrm{C}\mathrm{\%}=[(\mathrm{F}\mathrm{W}-\mathrm{D}\mathrm{W})/(\mathrm{T}\mathrm{W}-\mathrm{D}\mathrm{W})]\times 100$$

FW, fresh weight; DW, dry weight; TW, turgid weight.

Statistical analysis

Statistical analysis of data was done by applying One-way analysis of variance (ANOVA). Variability in data was expressed as standard error and the assessment of level of significance among different treatments was carried out by Least Significance Difference (LSD) at a significance level of P value ≤ 0.05 (Yang et al., 2016). All data are the means ± SD calculated from three replicates per treatment.

Screening, identification, and morphology of bacterial strains isolated from industrial waste for CdCl₂ tolerance

For screening, 11 strains of bacteria i.e. S₃D_1–1, S₃D_1–4, S₃D_4–6, S₁D_2–33, S₁D_2–34, S₆D_1–42, S_1–89, S₆D_1–105, S₂D_2–111, R1, and R2 were used. Only three strains S₆D_1–42, S₆D_1–105 and S₂D_2–111 showed growth at 100 µM and 200 µM Cd concentrations (Fig. 1A). We selected Cd tolerant S₆D_1–105 strain for further experiments and determined its morphological features such as surface, colony shape, elevation edge/margin, size, and opacity (Table 1). Based on 16S rRNA gene sequence analysis, the bacterial isolate S₆D_1–105 (OK310861) showed maximum similarity with B. cereus (97.30%).

Identification of PGPR

Minimum inhibitory concentration (MIC) of Cd resistant bacteria

Bacterial strain S₆D_1–105 was able to grow up to 1,000 µM CdCl₂ concentration which was determined by steadily increasing CdCl₂ concentration on growth media till the colony failed to propagate on agar plates (Figs. 2A–2H).

Plant growth promoting (PGP) traits of Cd resistant bacteria

The bacterial strain S₆D_1–105 was capable of protease and siderophores production and phosphate and potassium solubilization in plate-based test, supported by the appearance of a clear halo zone around the bacterial colony. Strains showed positive results for all tested PGP traits (Fig. 3; Table 2).

Table 2:

Protease, phosphate and potassium solubilization assays of metal tolerant strains.

Strain	PGPR traits	+ive/–ive	Solubilization index (SI)
S6D1–105	Protease	+ive	2.7 cm
S6D1–105	Phosphate	+ive	1.2 cm
S6D1–105	Potassium	+ive	1.1 cm
S6D1–105	Siderophore	+ive	1.4 cm

DOI: 10.7717/peerj.13131/table-2

Screening of rice genotypes for CdCl₂ tolerance

Three rice genotypes (Basmati 515, Super Basmati, and Chenab) were exposed to 100 µM, 200 µM, and 300 µM CdCl₂ stress for 6 days (Fig. 4A). On the sixth day of germination Basmati 515 showed 98% growth under the control condition, 96% growth under 100 µM, 98% growth under 200 µM and 95% growth under 300 µM CdCl₂ stress, Super Basmati showed 100% growth at control and all three levels of CdCl₂ stress. Chenab showed 100% growth under control, 98% growth under 100 µM, 200 µM and 300 µM of CdCl₂ stress (Figs. 4A and 4B). Therefore, we selected Super Basmati for further pot experiments.

Growth and biomass of rice inoculated with bacterial strain S₆D_1–105 under cadmium stress

After 7 days of exposure to Cd stress, plants inoculated with bacterial strain showed enhanced growth as compared to uninoculated seedlings (Fig. 5). Cadmium stressed rice plants exhibited a substantial reduction in the shoot length and shoot fresh and dry weight as compared to the control plants. A reduction of 20%, 21%, and 77% were observed respectively (Table 3). The inoculation of bacterial strain, in the presence of Cd stress, showed a rise of 11% in the shoot length and a decline of 14% in the shoot fresh weight while 31% reduction was detected in the shoot dry weight, while bacteria alone showed 15% increase in shoot length, 57% in fresh weight, and an 8% increase in dry weight relative to control. Cd + bacteria showed a 27% increase in root length. Stressed plants showed 28% decrease in the root length relative to control plant. Cd + bacteria showed 27% increase in root length. The fresh and dried weight of roots was considerably improved in the bacterial inoculated plants as compared to the non-inoculated control plants. The stressed plants showed 67% and 65% reduction in the fresh and dry weight of roots as compared to the control plant. Bacteria (alone) showed an increase of 67% in the root fresh and 50% increase in root dry weight as compared to the control plants. Combination (Cd + bacteria) showed 33% increase in fresh and dry root weight as compared to the control plants (Table 3).

Table 3:

Effect of bacterial inoculation on shoot length, shoot fresh and dry weight, root length, root fresh and dry weight of rice under Cd stress.

Treatments	Shoot length (cm)	Shoot fresh weight (g)	Shoot dry weight (g)	Root length (cm)	Root fresh weight (g)	Root dry weight (g)
Control	31.00 ± 1b	0.14 ± 0.01b	0.13 ± 0.05a	10.5 ± 0.44b	0.03 ± 0.01b	0.02 ± 0.01a
CdCl2	24.8 ± 1.6c	0.11 ± 0.00c	0.03 ± 0.02c	7.50 ± 0.88c	0.01 ± 0.01c	0.007 ± 0.0b
Bacteria	35.6 ± 1.4a	0.22 ± 0.02a	0.14 ± 0.01a	12.3 ± 1.02a	0.05 ± 0.01a	0.03 ± 0.02a
CdCl2 + Bacteria	34.5 ± 1.0a	0.16 ± 0.01b	0.09 ± 0.01b	13.3 ± 1.53a	0.04 ± 0.01ab	0.03 ± 0.01a

DOI: 10.7717/peerj.13131/table-3

Note:

Different letters indicate a significant difference (P < 0.05) among the treatments.

Root scanning

Plants grown with bacterial strain exhibited a significant increase in the total root volume, root length, root area, and average diameter as compared to the Cd-treated plants (Fig. 6A). An 11% and 14% decrease was observed in total root length when plants were exposed to bacteria alone and Cd + bacteria treatment as compared to the control plants. Moreover, 20% decrease in total root length was observed in Cd stressed plants as compared to the control (Fig. 6B). A pronounced reduction of 17% in total root volume was noticed under Cd stress as compared to the control, while 4% and 17% increase in total root volume was observed in bacteria alone and Cd + bacteria treatment as compared to the control (Fig. 6C). Compared to control, the total root area of Cd-treated plants was decreased by 9% while 11% and 12% increase was noticed under alone bacteria and Cd + bacteria treatment (Fig. 6D). About 1% and 2% increase were observed for average diameter as compared to the control. Cadmium stressed plants showed 12% decrease in average root diameter as compared to the control plants (Fig. 6E).

Effect of bacterial inoculation and Cd stress on chlorophyll contents, malondialdehyde and relative water content

Chlorophyll a, b, and carotenoid content was increased in bacteria treated plants by 6%, 39%, and 26% respectively, while Cd treated plants showed a decrease of 50%, 58%, and 44%, respectively. In Cd + bacteria inoculated plants, a decrease of 44%, 32%, and 43% was observed as compared to the control plants (Figs. 7A–7C). An increase of 229% in roots and 187% in leaves was observed in Cd treated plants while MDA levels in bacteria inoculated plants showed a reduction of 53% in roots and 18% in leaves. Whereas Cd + bacteria treated plants showed a 100% increase in MDA level in roots and 84% in leaves in comparison to the control plants (Figs. 7D and 7E). Under Cd stress, relative water content (RWC) of leaves was decreased. Inoculated plants with bacteria showed an increase in RWC in comparison to the control plant. Plants treated with bacteria (alone) displayed a 78% increase in RWC as compared to the control plants (Fig. 7F). Cadmium + bacteria treated plants had a higher (83%) leaves water content as compared to the control plants. Plants grown under Cd stress showed a 31% decrease in RWC as compared to the control.

Effect of bacterial inoculation and Cd stress on antioxidant enzymes

A significant impact of bacterial strain S₆D_1–105 was observed on the PPO, PAL, and SOD activity (Figs. 8A–8F). Under Cd stressed conditions, PPO content increased by 766% in roots and 361% in leaves as compared to control plants, while PPO content in bacteria inoculated plants increased by 1,566% in roots and 361% in leaves as compared to the control, in Cd + bacteria inoculated plants an increase of 1,133% in roots and 223% in leaves was observed as compared to control plants (Figs. 8A and 8B). The PAL activity under Cd stress showed a 200% and 300% increase in roots and leaves respectively. In bacteria inoculated plants, an increase of 11,150% in both roots and leaves, was observed, while Cd + bacteria treatment caused a significant increase of 700% in roots and 657% in leaves in comparison to the control plants (Figs. 8C and 8D). In Cd stressed plants, SOD activity was increased by 215% in the leaves and 142% in the roots relative to the control plants. The SOD activity was considerably greater in bacteria inoculated plants in comparison to the control plants. Under Cd + bacteria treatment, SOD level was increased by 230% in leaves and 276% in roots, while under alone bacteria treatment its level was increased by 144% in leaves and 215% in roots (Figs. 8E and 8F).

Effect of bacterial inoculation and Cd stress on antioxidant enzymes (CAT, POD and APX) in rice plants

Antioxidant enzymes (CAT, POD, and APX) activities were increased in stress-treated plants compared to the control (Figs. 9A–9F). CAT activity showed a 251% increase in Cd stressed plants in roots and a 15% increase in leaves. In bacterial inoculated plants, the CAT level was increased by 575% in roots and 175% in leaves. Under the Cd + bacteria treatment, CAT activity was increased by 5,981% in roots and 208% in leaves (Figs. 9A and 9B). POD level was increased by 400% in roots and 200% in leaves under Cd stress. In bacteria inoculated plants, POD level was increased by 650% in roots and 1,000% in the leaves in comparison to the control plant, while in Cd + bacteria inoculated plants, POD level was increased by 600% in roots and 533% in leaves compared to the control plants (Figs. 9C and 9D). APX activity was decreased in Cd treated plants by 84% in roots while an increase of 100% was recorded in leaves. Bacteria inoculated plants showed a 638% rise in roots APX and a 5% decrease in leaves APX, while Cd + bacteria inoculated plants showed a 359% increase in roots and a 350% increase in leaves APX as compared to their control plants (Figs. 9E and 9F). Physiological parameters of roots showed a strong positive correlation with antioxidant enzymatic activities, while MDA content showed a negative correlation with physiological and enzymatic parameters as depicted in Fig. 10. Leaves RWC showed a strong positive correlation with CAT, PAL, and PPO. Chlorophyll a content showed a strong positive correlation with chlorophyll b and carotenoids while a negative correlation was observed with MDA, CAT APX, and SOD. Chlorophyll b showed a positive correlation with carotenoid while chlorophyll b and carotenoid showed a negative correlation with MDA, APX, and SOD. CAT showed a positive correlation with POD, PAL, and PPO. POD showed a positive correlation with PAL and PPO, whereas PAL showed a positive correlation with PPO (Fig. 11).